ISOTOPES AND RADIATION IN ENTOMOLOGY

P R O C E E D I N G S O F A S Y M P O S I U M ,V I E N N A , 4 -8 D E C E M B E R 1 9 6 7J O IN T L Y O R G A N I Z E D B Y T H E I A E A W A N D F A O

ISOTOPES AND RADIATION IN ENTOMOLOGY

PROCEEDINGS SERIES


P R O C E E D IN G S O F A jS Y M P O S IU M ON T H E USE O F IS O T O P E S A N D R A D IA T IO N IN E N T O M O L O G Y

J O IN T L Y O R G A N IZ E D B Y T H E IN T E R N A T IO N A L A T O M IC E N E R G Y A G E N C Y

A N D T H EF O O D A N D A G R IC U L T U R E O R G A N IZ A T IO N O F T H E U N IT E D N A TIO N S

A N D H E L D IN V IE N N A , 4 - 8 D E C E M B E R 1967

IN T E R N A T IO N A L A T O M IC E N E R G Y A G E N C Y , 1968

ISOTOPES AND RADIATION IN ENTOMOLOGY (Proceedings Series)

ABSTRACT. Proceedings o f a Sym posium join tly organ ized by the IAEA and the F ood and Agriculture O rgan iza tion o f the U nited Nations and held in V ien n a , 4 -8 D ecem b er 1967. T h e m eetin g was attended by 82 participants from 29 countries and six in ternational organ ization s.

Contents: Isotope applications - e co lo g y (7 papers); Radiation e ffe c t studies - n on -gen etic (8 papers);Isotope applications - physiology and biochem istry (2 papers); Isotope applications - chemosterilants (2 papers); S terile -m ale technique (9 papers); Radiation e ffect studies - genetic (5 papers); Isotope applications - genetic (1 paper).

Each paper is in its orig inal language (21 English, 11 French, 1 Russian and 1 Spanish) and is preceded by an abstract in English with a second one in the orig ina l language i f this is not English. Discussions are in English.

(428 p p . , 16 x 24 cm , paper-bound, 104 figures)(1968) Price; US $ 9 .0 0 ; £ 3 .1 5 .0

IS O T O P E S A N D R A D IA T IO N IN E N T O M O L O G Y IA E A , V IE N N A , 1968

S T I /P U B /1 6 6Printed by the IAEA in Austria

June 1968

FOREWORD

In se c ts that a re h a rm fu l to an im als and plants con tin u e to p o se one o f the m ost se r io u s p ro b le m s in a g r icu ltu re . D esp ite sop h istica ted equipm ent and the con tin u ou s d e v e lo p m e n t o f new te ch n iq u e s , o n ly a fe w s p e c ie s o f h a rm fu l in s e c ts can be sa id to be 'c o n t r o l le d 1. A g r ic u ltu r a l l o s s e s fr o m the a c t iv ity o f in s e c t p e s ts am ount to m illio n s o f d o lla r s annually, and the sp re a d in g o f plant d is e a s e b y in s e c t v e c t o r s is one o f the m a jo r c a u se s o f fa m in e .

In s e c t ic id e s have m e t w ith on ly lim ite d s u c c e s s in p e s t e r a d ic a t io n p ro g ra m m e s . M any in s e c ts d ev e lop r e s is ta n c e so that new in s e c t ic id e s o r s tro n g e r con ce n tra tio n s o f th ose a lre a d y in e x is te n ce m ust be u sed . Som e in se c tic id e s a d v erse ly a ffe ct other com ponents in food chains, thus upsetting e c o sy s te m s . The p o ss ib le accum ulation of tox ic in se c tic id e resid u es, which m igh t r e a c h le v e ls h a rm fu l to the b io ta , r e p r e s e n ts an oth er p oten tia l u n d e s ira b le e f fe c t . N e v e r th e le s s , su ch p e s t ic id e s p la y an im p o rta n t r o le in the co n tro l o f arth rop od p e s ts . I s o to p ica lly tagged p e s t ic id e s p erm it the s c ie n t is t to fo l lo w th e ir d is tr ib u tio n and c o n ce n tra tio n d u rin g m e ta b o lis m in the o rg a n is m o r in h ig h e r le v e ls o f e c o lo g i c a l c la s s i f i c a t io n .

The n u c le a r e r a h as p r o v id e d m an w ith new and v e r s a t i le to o ls f o r co n tro llin g in se c t p ests and fo r studying v a r iou s a sp e cts o f en tom ology that w ere h itherto unheard o f o r con s id e re d im p ra ct ica l o r im p o ss ib le . Radiation and is o to p e s con trib u te s ig n ifica n tly to o u r kn ow ledge and u nderstanding of v a r io u s d is c ip lin e s r e la te d to e n to m o lo g y , su ch as g e n e t ic s , p h y s io lo g y , behaviour and e c o lo g y . T h is know ledge is e sse n tia l f o r the sc ie n tis t to plan the m ost e ffe c t iv e and e c o n o m ic a l w ays, not on ly to e lim in a te in se c t p ests , but a lso to propagate b e n e fic ia l in s e c ts .

T he v e r s a t i l ity o f th e se new tech n iq u es a p p ea rs in a lm o s t a ll a sp e cts o f applied and b a s ic en tom ology . At the m o le cu la r le v e l, cod in g and r e p l i ca tio n o f D NA have b e e n fo l lo w e d b y is o to p e a p p lica t io n . A t the c e l lu la r le v e l, n u c le a r ra d io s e n s it iv ity re la t iv e to c y to p la s m ic ra d io r e s is ta n c e has been d eterm in ed by ir ra d ia tin g in se c t e g g s . At the tissu e and organ le v e ls , the concen tra tion and d istribu tion of tagged in se c tic id e s have been determ ined, th ereby co n tr ib u tin g to o u r u n d erstan d in g o f the p h y s io lo g ic a l m e ch a n ism o f t o x ic o lo g y . A t the le v e l o f the o r g a n is m it s e l f , the p e r fo r m a n c e and b eh a v iou r o f tagged in d iv id u a ls , both ca g e d and f r e e - l iv in g , have b een in vestig a ted . Thus o u r u nderstan d in g o f the abundance and d is tr ib u tion o f in s e c t s p e c ie s h as been g re a t ly in c r e a s e d .

Iso top es and ra d ia tio n s a re b e c o m in g in c r e a s in g ly im p ortan t in e n to m ology , and the d issem in a tion and exchange o f in form ation on th eir m anifold u s e s r e p r e s e n t an im p o rta n t fa c t o r in the s u c c e s s o f p e s t e r a d ic a t io n p rogram m es throughout the w orld . The International A tom ic E n ergy A gency and the F o o d and A g r ic u ltu r e O rg a n iz a t io n o f the U nited N a tion s jo in t ly convened a S ym p osiu m on the U se o f Iso to p e s and R adiation in E n tom ology , w hich w as h e ld at the IA E A H e a d q u a rte rs in V ienna fr o m 4 to 8 D e ce m b e r 1967. T h is w as the th ird S y m p o s iu m to be h e ld on th is s u b je c t ; the tw o p r e v io u s o n e s had taken p la c e in B o m b a y in 1962 and in A th en s in 1963 .

The ran ge o f s u b je c ts w as in ev ita b ly w ide ow in g to the o v e r la p p in g o f v a r io u s f ie ld s o f study. The p a p e rs a re re p re s e n ta t iv e o f p r e se n t tre n d s , although o n ly a few p a p e rs d e s c r ib e d a p p lic a t io n s o f r a d io is o t o p e s in p h y s io lo g y o r b io c h e m is t r y .

T he h e te ro g e n e ity o f the in form a tion p re se n te d at the S y m p osiu m w as then to be ex p ected . It is by the in teg ra tion o f sp e c ia liz e d in form a tion , h o w e v e r , that a b a s is f o r s ig n ifica n t s c ie n t i f i c a d v a n ces is p r o v id e d . An e x a m p le o f th is p r o c e s s is in the s u c c e s s fu l d e v e lo p m e n t o f e r a d ic a t io n p r o ce d u r e s that in vo lve the re le a s e o f la rg e n u m bers o f in se c ts p r e v io u s ly s t e r i l iz e d by rad ia tion : th is dem ands an understan d in g not on ly o f the b a s ic b io lo g y and o f rad ia tion b io lo g y but a lso o f the te ch n o lo g y in v o lv e d in m a ss r e le a s e s at op tim a l t im e s and in op tim a l qu an tities .

The use o f radiation in the s te r i le - in s e c t re lea se m ethod is being applied o r c o n s id e r e d by m any M e m b e r States o f the IA E A . The m eth od is h igh ly sop h is tica ted . It is n e ith er e a sy n or a p an acea . It is s u c c e s s fu l on ly when d e v e lo p e d and s u p e r v is e d b y co m p e te n t s c ie n t is t s . T h e d if f i c u lt ie s o f applying la b ora tory resu lts in the fie ld are obviou s, e sp e c ia lly since the highly s e le c t e d la b o r a to r y o r g a n is m m a y d if fe r c o n s id e r a b ly f r o m the w ild type e n co u n te re d in the f ie ld .

The p resen t vo lu m e conta in s the com p le te p roceed in g s o f the S ym posium . It is hoped that it w ill be a va lu ab le s o u r c e o f in form a tion to s c ie n t is ts and w ill stim ulate fu rth er r e s e a r c h in the fascinatin g and rapid ly grow ing subject o f radiation en tom ology .

EDITORIAL NOTE

The p a p e r s and d is c u s s io n s in co r p o ra te d in the p r o c e e d in g s pu blish ed by the In tern a tion a l A to m ic E n e rg y A g e n c y a r e ed ited b y the A g e n c y 's e d i to r ia l s ta ff to the e x te n t c o n s id e r ed n e c e s s a r y fo r the r e a d e r 's a s s is ta n c e . The v iew s e x p r e s s e d and the g e n e r a l s ty l e adopted rem a in , h o w e v er , the r e s p o n s ib i l i ty o f the n am ed a u th ors o r p a r tic ip a n ts .

F o r the sa k e o f s p e e d o f pu blica tion the p r e s e n t P r o c e e d in g s h ave b een p rin ted by co m p osition typing and p h o to -o ffs e t lithogra phy. Within the l im ita tion s im p o sed b y th is m eth od , e v e r y e f fo r t has b e en m a d e to m aintain a high ed ito r ia l stand ard ; in p a r ticu la r , the units and s y m b o ls em p lo y ed a r e to the fu l le s t p r a c t ic a b le e x te n t th o se s ta n d a rd ized o r r e c o m m e n d e d b y the co m p e ten t in tern a tion a l s c ie n t i f i c b o d ie s .

T he a ffilia tio n s o f a u th o rs a r e th o s e g iv e n at the t im e o f n om in a tion .The u se in th e s e P r o c e e d in g s o f p a r t ic u la r d es ig n a tion s o f c o u n tr ie s o r

t e r r i t o r i e s d o es not im p ly any ju d g em en t by the A g e n c y a s to the leg a l sta tu s o f su ch c o u n tr ie s o r t e r r i t o r i e s , o f th e ir a u th o r itie s and in stitu tion s o r o f th e d elim ita tion o f th e ir b o u n d a r ie s .

The m en tion o f s p e c i f i c co m p a n ies o r o f th e ir p ro d u cts o r bran d-n am es d o e s not im p ly any en d o rs e m e n t o r r eco m m en d a tio n on th e p a r t o f the In tern a tion a l A to m ic E n e r g y A g e n c y .

CONTENTS

A . ISOTO PE A P P L IC A T IO N S : E C O LO G Y (S e ss io n s I and II)

R a d io tra ce r study o f the p re d a to rs on D istan tie lla th e o b ro m a (D istant)(H em ip tera : M ir id a e ) (S M -1 0 2 /1 ) ................................................................... 3H. M a rch a rtD iscu ss io n ....................................................................................................................... 14

Etude, au m oyen de l 'o r ra d io a c t if 198Au, du rayon d 'a c t io n de co lo n ie s de bou rdon s (B om bus sp . ) en vue de la p o llin isa tio n desplantes cu lt iv é e s (S M -1 0 2 /3 ) ............................................................................... 17J. L e c o m te e t A . P ou vrea uD iscu ss io n ...................................................................................................................... 20

R a d io iso to p e s in the in vestig a tion o f in te rre la tio n sh ip s betw eenaphids and h o st-p la n ts (S M -1 0 2 /4 ....................................................................... 23W. K lo ft, P . E h rh ard t and H. K unkelD is c u s s io n ......................................................................................................................... 28

R a d io iso to p e s in stu d ies on the e c o lo g y o f tick v e c t o r s o f d is e a s e(S M -1 0 2 /5 ) ......................................................................................................................... 31D . E , S onensh ineD iscu ss io n ...................................................................................................................... 51

Etude é co lo g iq u e de deux p u ceron s v e cte u rs des v ir o s e s de lab ettera v e (S M -102/6 ) ................................................................................................ 53E . F r a n ç o is , A . Higa e t /?. Im p en s

. D is c u s s io n ............................................................................................................................... 67E ffe c ts o f ir ra d ia tio n on in se c t h o s t -p a ra s ite re la tion sh ip

(S M -1 0 2 /7 ) .......................................................................................................... ........... 69G. W. R ahalkar and V. R am akrishnanD iscu ss io n ........................................................................................... .......................... 73

In vasion o f c o n ife r p lantations by ra d io a ct iv e ly la b e lledH ylobius a b ie tis L . (S M -1 0 2 /9 ) .......................................................................... 75H. H. Eidm annD is c u s s i o n . ...................................................................................................................... 84

B . RADIATIO N E F F E C T STUDIES: N O N -G EN ETIC (S e ss io n s III and IV)

C o rre la t io n s , in te ra c tio n s and d iffe r e n c e s betw een rad ia tion e ffe c tson lon gev ity and natural aging (S M -1 0 2 /1 0 ) ............................................... 87H. NSthel

D estru ction d es c e llu le s p o la ir e s de l 'œ u f du d ory p h ore parirra d ia tio n (S M -1 0 2 /1 1 ) ........................................................................................... 103R. B lu zatD iscu ss io n ...................................................................................................................... 107

Q uelqu es e ffe ts de l 'ir r a d ia t io n aux rayon s gam m a su r Spodoptera (L aphygm a) ex igu a Hb. (L e p id o p te ra : N octu idæ )(S M -1 0 2 /1 2 ) .................... ................................................................................................ 109M . A nw arD iscu ss io n .................................................................................... . ................................ 120

S en sib ilité des d iv e r s stad es de dévelop pem en t de Sitophilusze a m a is M ots (S. o r y z æ L . ) aux rad ia tion s io n isa n te s : Etude des stad es en d ogés par ra d iog ra p h ie et e n re g is tre m e n t actograp h iqu e(S M -1 0 2 /1 4 ) .................................................................................................................... 123P . P e s s o n e t G. K. G irish ' •D isc u ss io n ................................................................' . ................ ................................ 137

S té r ilisa tio n de la p y ra le du m a ïs (O str in ia n u b ila lis Hb. ) parl 'ir r a d ia t io n aux ra y on s gam m a (S M -1 0 2 /1 5 ) ................................... .. 139M . A n w a rD isc u ss io n ..................................................................... ................................................. 14 7

In flu en ce o f d iapause on the r a d io s e n s it iv ity o f Khapr’a bee tlela rv a e ( S M -1 0 2 /1 6 ) ..................................................' ............................................... .. 149G. W. R ah alka r and К . K . N a irD isc u ss io n ....................................................... ................................................... ............ 154

E ffe ts d es rad ia tion s gam m a su r la fe r t il i té et la lon gév ité des c o lo n ie s de D o lich o d e ru s quadripunctktus (H ym en op tère :F o r m ic o id æ D o lic h o d e r id æ ) (S M -1 0 2 /1 7 ) .................................................... 155C. T orossia ri e t R. C a u sseD isc u ss io n ....................................................................................................................... 162

R e a rin g tech n ique, b io lo g y and s te r i liz a t io n o f the c o f fe e le a f m in e r , L e u co p te ra c o f fe e l la G u er . (L e p id o p te ra :L yon etiid a e ) (S M -1 0 2 /1 8 ) .................................................................................... : 165K. P . K a tiya r and F . F e r r e rD isc u ss io n .................................................................................................. ................... 173

C. ISO TO PE A P P L IC A T IO N S : PH YSIO LO GY AND BIO CH E M ISTRY(S e ss io n V , P art 1) ' ■

Uptake o f is o to p ic a lly la b e lle d in s e c t ic id e s in re la t io n totem p era tu re and o th er p h y s ica l fa c to r s (S M -1 0 2 /1 9 ) ............................ ' 179L . K . Cutkom pD isc u ss io n ..................................... ............................................................... ................. 184

M etab o lism o f m alath ion in the d e s e r t lo cu s t S c h is to ce r cag re g a r ia F o r s k a l : d is tr ib u tion and d egra d ation o f 32P -la b e lle dm alath ion in v iv o (S M -1 0 2 /2 1 ) ............................................................................. 187K. N. M eh ro tra , G. R. Sethi and Y . P ra sa dD iscu ss io n ....................................................................................................................... 197

D. ISOTO PE A P P L IC A T IO N S : CH E M O STE RILAN TS (S e ss io n V , P a rt 2)

C h em oster ila n ts in en tom ology and the s t e r i le -m a le technique(S M -10 2 /2 4 ) ......................................................................................................................... 201A .B . B o r k o v e cD iscu ss io n ................................................................................................... ................... 205

A ction du T epa su r le d év e lop p em en t nym phal de D acus o le æ G m el.(D ip tera : T e p h ritid æ ) (S M -1 0 2 /4 4 ) ................................................................ 209E . F y t iz a s e t A . B a coya n n isD iscu ss io n .................................................................................................................... .. 215

E . S T E R IL E -M A L E TECHNIQUE (S e ss io n s VI and VII)

Т рудности и успехи м а с с о в о г о разведения н асеком ы х в лаборатории и возм ож н ости сам ои стреблен и янекоторы х вредны х видов (S M -1 0 2 /2 5 ) ........................ ............ .. 219Е . М . Ш умаковD iscu ss io n ................................................................... ................................................... 232

T h e o re t ica l and p r a c t ic a l stu d ies on a p o s s ib le g en etic m ethod 'fo r tse tse fly c o n tro l (S M -1 0 2 /2 6 ) ...................................................................... 233C .F . C u rtis and W .G . H illD iscu ss io n ....................................................................................................................... 245

Irra d ia tion e ffe c ts on re s p ir a t io n and b lood d ig e s t io n in the tick O rn ith od oros th o loza n i and th e ir im p o rta n ce fo r the s t e r i le -m a letechnique (S M -1 0 2 /2 7 ) .............................................................................................. 249R a ch el Galun and M . W a rbu rgD iscu ss io n .......... ............................................................................................................ 257

New site fo r the e ra d ica t io n o f a white grub popu lation(M elolon th a v u lg a r is F a b r . and M elolon th a h ip pocastan i F a b r . )by r e le a s e o f ir ra d ia te d adults (S M -1 0 2 /3 2 ) ............................................... 259E . H o rb erD iscu ss io n .............................. ..................... .................................................................. 270

E x p lo ra to ry stu d ies on the e ra d ica t io n o f the K orean pinec a te r p il la r D en d ro lim u s sp e c ta b ilis B u tler by m eans o f rad ia tion(SM - 102/2 9 ) ................................................................................... ................................ 273К. B . L e e , U. K . H w ang and K. S. L e eD iscu ss io n ....................................................................... .................I ........................... 285

C ria m a sa l de D y sd e rcu s peruv ianu s G. y su e s te r i l iz a c ió nm ediante ra y o s gam m a ( S M - 1 0 2 /3 0 ) .................................................................. 287J. E . Sim ónD iscu ss io n ............. ....................................................... .............. .................................. 299

A p p lica tion aux O rc.ytes (C o lé o p tè r e s : S ca ra b æ id æ ) de la m éthodedu lâ ch e r de m â les s t é r i le s (S M -1 0 2 /3 1 ) .................................................... 301B . H urpinD iscu ss io n .............................................................. ........................................................ 315

E ffe c t o f r e le a s in g s t e r i le S cirpoph aga n ive lla F . on c r o p dam age and in se c t popu lation den sity in su g a r can e plantations(SM - 10 2 /2 8 ) .................................................................................................................... 317S. H atrn osoew arnoD iscu ss io n ....................................................................................................................... 329

C on tro l o f adult p e s ts by the ir r a d ia t io n -o f -m a le m ethod(S M -1 0 2 /3 4 ) .................................................................................................................... 331R. C. von B o rs te l.D iscu ss io n .................................................................................................................. .. . 336

F . RADIATIO N E F F E C T STUDIES: G EN ETIC (S ess ion VIII, P art 1)

R adiation sen s itiv ity and r e p a ir o f c h r o m o s o m a l dam age(S M -102/3 5 ) .................................................................................................................... 341p . S trom naesD iscu ss io n ...................................................................................................................... 354

E ffe ts c o m p a ré s d 'ir ra d ia t io n s aux rayon s gam m a et aux neutrons su r le s org a n es re p ro d u cte u rs de la m ou che m éd iterra n éen n e des fru its C e ra titis cap itata W ied . (D ip tera T ry p e t id æ ) (S M -1 0 2 /3 6 ) . . 355R. C a u sse , M . F éro n e t P. l ’ é r e a u -L e r o yD is c u s s i o n : ....................................................................................................................... 363

In cre a se d y ie ld o f g a m m a -in d u ced eye c o lo u r m utations fromc h ro n ic v e rs u s acute e x p o su re s in D ah lbom inus ( S M -1 0 2 /3 7 ) ............. 365W ..F . BaldwinD iscu ss io n .................................................................................................................... 375

R ep rod u ctiv e p e r fo rm a n ce o f fem a le B ra co n id s co m p a re d a fte r (A ) b r ie f and (B ) p ro tra c te d e x p o su re s to ion iz in g rad ia tion s(S M -10 2 /38 ) .................................................................................................................... 377D. S. G ro schD iscu ss io n ....................................................................................................................... 389

G en etic and cy to g e n e tic b a s is o f ra d ia tion -in d u ced s te r i lity in theadult m a le cabbage lo o p e r T r ich o p lu s ia ni ( S M - 1 0 2 /3 9 ) ...................... 391D. T. N orth and G. G. H oltD iscu ss io n ....................................................................................................................... 402

G. ISOTO PE A P P L IC A T IO N S : G EN E TIC (S e ss io n VIII, P a rt 2)

La syn th èse d 'A D N dans le s noyaux géants des in s e c te s — M od a lités de c o n trô le et s tru c tu re s o b s e r v é e s dans la g lande s é r ic ig è n e de .B om byx m o r i (S M -1 0 2 /4 0 ) ................................................................... ................. 407V. N igon, J. D a illie , S. G illo t, M .M . N e u la t -P o r t ie r e t M . d e T urenn e

C h airm en o f s e s s io n s and s e c r e ta r ia t o f the S ym posium ......... .............. 417L is t o f p a r t ic ip a n t s .......................................................................................................... 419A uthor index . . ..................................................................................................................... 427

ISOTOPE APPLICATIONS: ECOLOGY

(Sessions I and II)

C h a irm a n : H . M A R C H A R T

G H A N A

RADIOTRACER STUDY OF THE PREDATORS ON Distantiella theobroma (DISTANT) (HEMIPTERA: MIRIDAE)

H. MARCHARTCOCOA RESEARCH IN STITU TE,

GHANA ACADEMY OF SCIENCES,TAFO, GHANA

Abstract

RADIOTRACER STU D Y OF THE PREDATORS ON D istantiella theobrom a (DISTAN T) (HEMIPTERA: MIRIDAE). T h e paper describes a m ethod for la b e llin g the c o c o a capsid D istantiella theobrom a (Distant) w ith 32 P in sucrose solution . T h e label was excreted w ith a b io lo g ic a l h a lf - l i fe o f six days. When taken up from a la b e lled natural substrate (c o c o a pods) the 32 P activ ity in the insect attained an equilibrium va lu e after nine days and was excreted w ith the sam e h a lf - l i fe . Labelled larvae in the fie ld w ere exposed to the natural predator popu lation , w h ich was sam pled by pyrethrum k n ock down after 48 hours. N one o f the predators iden tified is sp ec ia lized as a capsid predator. T h e broad range o f predatory species re flects the high species diversity within the trop ica l forest zon e , w hich is considered to b e an im portant fa ctor in integrated capsid control.

1. IN TR O D U C TIO N

C o c o a c a p s id s (M ir id a e )a r e p e s ts o f m a jo r e c o n o m ic im p o rta n ce and a con tin u al th reat to W est A fr ic a n c o c o a p ro d u ctio n . In G hana, D ista n tie lla th e o b ro m a (D istant) is the m o s t im p ortan t s p e c ie s . C h e m ica l c o n t r o l o f th is in se c t w as a ch ie v e d w ith lin d a n e , but the p r o b le m o f r e s is ta n c e to th is in s e c t ic id e and the im p lic a t io n s w hich a r is e fr o m the con tin u ou s use o f b ro a d sp e ctru m p e s t ic id e s n e c e s s ita te d an e c o lo g i c a l a p p ro a ch w hich co u ld even tu a lly lea d to m o r e s e le c t iv e m eth od s o f c o n t r o l . T he p re se n t study a c c o r d in g ly d e a ls w ith the id e n tifica tio n o f the p r e d a to r s o f D is ta n t ie l la .

A n u m ber o f p r e d a to ry in s e c ts a s s o c ia te d w ith c o c o a c a p s id s w as l is te d by S q u ire [1 ], but e v id e n ce b a se d on f ie ld o b s e rv a t io n w as m e n tion ed on ly fo r fiv e s p e c ie s o f s p id e r s and th re e s p e c ie s o f m a n tid s , the n a m es o f w h ich w e re not g iven . W illia m s [2] o b s e r v e d the ants P h e id o le m e g a ce p h a la (F . ), on e C a m pon otu s s p e c ie s , and O e co p h y lla lon g in od a (L . ) con su m in g m ir id la rv a e . He c ite d in d ir e c t e v id e n ce that, on se e d lin g c o c o a , an ts , m antids and re d u v iid s a r e o f about equ a l im p o rta n ce fo r n atu ra l c o n t r o l o f the c a p s id , but he did not m en tion w h ich s p e c ie s w e re con trib u tin g . In a r e ce n t study, G e ra rd [3] c o n f ir m e d p re d a t io n by O e co p h y lla lon g in oda and m en tion ed on e s p e c ie s o f A sca la p h id a e w hich fe e d s on D ista n tie lla in the la b o r a to r y .

P r e y -p r e d a t o r in te r re la t io n s h ip s w e re eva lu ated w ith r a d io t r a c e r s by Jen k in s et a l. [4 , 5], B aldw in et a l. [С] and J a m e s [7] fo r v a r io u s s p e c ie s o f m o s q u ito e s ; C la rk [8] d e te cte d p r e d a to rs o f 32P - la b e l le d pink b o l l w o rm m oth s . T h e u se o f the tech n iq u e fo r the quan tita tive d e term in a tion o f h e rb iv o r e con su m p tion by p r e d a c io u s in s e c ts w as d e m o n stra te d by C r o s s le y [9].

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2. E X P E R IM E N T A L

2 .1 . M a te r ia l

D ista n tie lla th eob rom a w as la b o r a to r y r e a r e d in sm a ll n u m bers by Raw [10] and P r in s [11 ], but an e ffe c t iv e m eth od fo r m a ss r e a r in g has not yet b een d ev e lo p e d . T h is is due to (a) the fr a g ile n ature o f the m ir id , w h ich m a k es m anipulation d iff icu lt , and (b) the fa ct that th is su ck ing in s e c t is a h igh ly d e s tru c tiv e fe e d e r re q u ir in g la rg e qu an tities o f fr e s h c o c o a t issu e d a ily . T h e r e fo r e , f i e ld - c o l le c t e d 4th and 5th in sta r la rv a e w e re u sed , w hich , b e fo r e la b e llin g , w e re kept in the la b o r a to r y fo r24 h ou rs on fr e s h c o c o a sh oots at 24°C . .There w as a da ily m o r ta lity ra te o f about 15% o f the bu gs so trea ted .

2. 2. L a b e llin g o f la rv a e

F o r p re d a to r stu d ies the p r e y sh ou ld be la b e lle d in te rn a lly , s in ce s o m e p r e d a to rs , n otab ly s p id e r s , w ill not co n su m e the ch it in ou s p a rts o f the p re y .

P r e lim in a r y e x p e r im e n ts sh ow ed that la b e llin g fr o m a n atu ra l d iet, e . g . ra d io a ct iv e c o c o a s h o o ts , w as r e la t iv e ly in e ffic ie n t , s in c e on ly a s m a ll fra c t io n o f the ra d io p h o sp h o ru s ap p lied w as tra n s lo ca te d into the plant t is s u e s on w hich the in s e c ts feed . C on seq u en tly , a tech n iqu e s im i la r to that u sed by L ew is and W a lo ff [12] on the m ir id O rth oty lu s v ir e s c e n s w as adopted . The s e t -u p show n in F ig . 1 w as d e v ise d w ith the a im o f m in im iz in g m anipulation o f the fr a g ile in se c t .

The la b e llin g m ed iu m w as 32P , o rth op h osp h ate , w ith a s p e c i f i c a c t iv ity o f 1 0 -3 0 ju C i/m l in 5% s u c r o s e so lu tion con ta in in g 0.1% p o ta ss iu m d ih y d rog en phosphate as a c a r r ie r .

E a ch 'fe e d in g b e l l 1 con ta in ed ] m l so lu tion on a 9 -c m (W hatm an N o. 1) f i lt e r p a p e r d is c fo r la b e llin g 25 la rv a e . The m ethod has the advantage o f a v o id in g the n e c e s s ity o f COa a n a e sth e s ia (w hich a d v e r s e ly a f fe c ts D ista n tie lla la rv a e ) fo r handling the la b e lle d in s e c ts , as they can c o n v en ien tly be t r a n s fe r r e d by in vertin g the d e v ice so that they s l id e into the tra n sp o rt co n ta in e r . T he la rv a e w e re kept on the ra d io a c t iv e d iet fo r 1C h o u rs .

2 .3 . L a b e llin g o f c o c o a tr e e s

T o c o m p a re the in s e c t s ' uptake o f 32P fr o m su g a r so lu tio n w ith that fr o m a n atu ra l su b stra te , bugs w e re fed on la b e lle d c o c o a t r e e s .C o r n w e ll [13] d em on stra ted that a u n ifo rm d is tr ib u tion o f 32P is d ifficu lt to obta in by trunk im p lan ta tion . T h e r e fo r e , the ba rk in fu s ion m eth od w as u sed (F ig . 2). T h is re su lte d in a su ff ic ie n t ly u n ifo rm d is tr ib u tio n o f a c t iv ity o v e r the e n tire tr e e (5 y e a r -o ld A m a zo n ), but r e q u ire d a high d o sa g e (1 0 -2 0 m C i p e r t r e e ) b e c a u se the uptake th rough b a rk w as s lo w and in co m p le te even a fte r r e m o v a l o f the dead ou ter c o r t e x la y e r .

2 .4 . D eterm in a tion o f 32P in c a p s id s

T o d e te rm in e the b io lo g ic a l h a l f - l i f e o f 32P in D ista n tie lla th e o b r o m a , the la b e lle d bu gs w e re w e t-a s h e d and cou n ted in a G e ig e r -M ü lle r liqu id

SM-102/1 5

Perspex cap

polythenefunnel

silicone oil film

petri dish FIG. 1. Set-up for la b e llin g D istantiella larvae.

glasscapillary

thermoplasticParafilm

Í9 -

plasticine / rings

P solution

lft above ground

trunk of cocoa tree

FIG. 2. Bark infusion o f S2P into c o c o a tree (cross-section ).

co u n te r tube. F o r l iv e cou n ts a w h o le -b o d y cou n tin g c e l l w as u sed , p la ce d u nder an en d -w in d ow co u n te r (1 .5 m g /c m 2 ).

2. 5. R e le a se and sa m p lin g

F ifty 4th and 5th in sta r la rv a e w e re r e le a s e d on each tr e e onto a c lu m p o f le a v e s p inned at the m ain fo r k . T he bugs w e re a llo w e d to d is p e r s e fo r 48 h ou rs .

Sam pling o f the e n tire in s e c t fauna on m a tu re c o c o a w as done by sa tu ra tin g the ca n op y with an aqu eou s p y re th ru m e m u ls io n (0 .1% a c tiv e in g red ien t) at a ra te o f 1 g a l / t r e e by m ea n s o f a m o t o r -p o w e r e d kn apsack m is tb lo w e r . T he in s e c ts k n ock ed down w e re c o l le c te d a fte r 1 h ou r fr o m co tton sh eets sp re a d on the g rou n d u n der the t r e e s , and the ra d io a ct iv e s p e c im e n s w e re s o r te d out.

3. RE SU LTS AN D DISCUSSION

3 .1 . Uptake and b io lo g ic a l h a lf - l i fe o f 32P

A lthough the feed in g co n d itio n s in the 'la b e llin g b e l l ' w ere, quite d if fe re n t fr o m th o se in the f ie ld (D ista n tie lla is a p h lo e m fe e d e r ) , 32 p w as r e a d ily taken up by the in s e c t . T h e to ta l r a d io a c t iv ity taken up within a

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p e r io d o f 16 h ou rs v a r ie d g re a t ly am ong in d iv id u a ls , the c o e f f i c ie n t o f v a r ia t io n b e in g 3 5%.

L a rv a e that had a b s o r b e d 0.25 ¿¡Ci sh ow ed in cr e a s e d m o r ta lity when co m p a re d w ith la rv a e kept on fr e s h c o c o a sh o o ts . It is , h o w e v e r , not c le a r to what extent th is w as ca u se d by rad ia tion e ffe c t on the on e hand and the a r t i f i c ia l d iet on the o th e r . A t a co n ce n tra tio n o f 0 .08 ¿ iC i/in s e c t , w h ich w as the a v e ra g e le v e l ch o se n fo r the f ie ld e x p e r im e n ts , the m o r ta lity w as s lig h tly but not s ig n ifica n tly in c r e a s e d o v e r that o f u n la b e lled in s e c ts .

T o e s ta b lish the p e r io d that the la rv a e w ould rem a in la b e lle d , the b io lo g ic a l h a lf - l i fe w as d e term in ed by cou n tin g sa m p le s fr o m a ba tch o f la b e lle d la rv a e at v a r io u s in te rv a ls a fte r ta gg in g . F ro m F ig . 3, cu rv e (a), it can be seen that the ra d io p h o sp h o ru s w as e x c r e te d at a ra te w hich gave a h a lf - l i fe o f 6 .2 d a y s . T h e s c a t te r o f the m e a su re m e n ts r e f le c t s the v a r ia tio n in the 32P uptake am ong in d iv idu a l la rv a e .

T o d e te rm in e how m u ch o f the a c t iv ity o f the la b e lle d in s e c ts w as due to s u r fa c e con tam in a tion , a ba tch o f 10 la rv a e w as kept on a ra d io a ct iv e c o c o a p od fr o m a la b e lle d tr e e and the uptake and e x cr e t io n o f the la b e l m e a su re d by counting the liv e in d iv idu a l in se c ts in the w h o le -b o d y co u n te r c e l l .

T he 32P le v e l r o s e ra p id ly during the f ir s t fiv e days o f feed in g and th e r e a fte r le v e lle d o f f as the e x cr e t io n b a la n ce d the uptake (F ig . 3, c u r v e (b)). A fte r nine d a ys , the in s e c ts w e re tr a n s fe r r e d to a n o n -r a d io a c t iv e d iet and 32P w as e x cr e te d w ith a h a lf - l i fe o f 6 d a ys . A s th is did not d if fe r s ig n ific a n tly fr o m the h a l f - l i f e m en tion ed a b o v e , it w as co n c lu d e d that s u r fa c e con tam in a tion w as o f m in o r im p o rta n ce w ith the stan d ard la b e llin g p r o c e d u r e . T he b io lo g ic a l h a lf - l i fe o f 6 d a ys , co m b in e d w ith the p h y s ic a l is o to p e h a lf - l i fe o f 14.3 d a ys , g iv e s an e f fe c t iv e h a lf - l i fe o f 4 .2 da ys . T h is m ea n s that the la b e l sh ou ld rem a in d e te cta b le fo r about th re e w eek s a fte r ta gg in g . In the fie ld , h o w e v e r , d is p e r s a l o f the in se c ts w as the fa c to r lim itin g du ration o f the e x p e r im e n ts , not the p e r s is te n c e o f the la b e l.

L a b e llin g c o c o a c a p s id s in the f ie ld on tagged t r e e s w as im p r a c t ic a l f o r p r e d a to r stu d ies b e ca u se o f in te r fe r e n c e by o th e r ph ytophagou s in s e c t s (m e a ly b u g s , p s y ll id s , le a fh o p p e rs ) and the r e la t iv e ly low le v e l o f a c t iv ity a cq u ire d by the plant t is su e .

3. 2. S p ectru m o f p re d a to rs

T h e la rv a e r e le a s e d in the fie ld w e re e x p o se d to a ll n atu ra l p r e d a to rs p r e se n t . T h e se co u ld be id en tified in the k n ock -d ow n sa m p le s b y m ean s o f the r a d io a c t iv ity they had a cq u ire d b y feed in g on the la b e lle d D ista n tie lla la rv a e .

T he d is tr ib u tion o f the ra d io p h o sp h o ru s w ithin the k n ock -d ow n in se c t fauna in ]2 can op y re p lic a t io n s in w hich a to ta l o f 3000 D ista n tie lla la rv a e had been r e le a s e d is show n in T a b le I.

T he p o s s ib il it y o f a con tr ib u tion fr o m the th ird and h igh er lin k s o f the fo o d -ch a in s w as n e g le c te d . T h is a p p ea red ju s t if ie d on the b a s is o f s iz e d if fe r e n c e s betw een the r e s p e c t iv e p r e d a to r s , the high ra tio o f the n u m ber o f la b e lle d c a p s id s to that o f the ra d io a ct iv e p r e d a to rs r e c o v e r e d , and the sh o rt t im e o f in tera ction betw een p r e y and p r e d a to rs .

SM-102/1 7

lo a count ra te

FIG. 3. 52 P activ ity in D istantiella theobrom aCurve (a ): excretion after u p -tak e from sucrose Curve (b ): U p-take and excretion from

solution on filter paper. labelled c o c o a pods.

S om e s ligh tly a c t iv e s p e c im e n s , o th e r than an ts, w e re p re su m e d s u r fa c e -co n ta m in a te d th rough con ta ct w ith the la b e lle d la rv a e in the k n ock -d ow n sa m p le s and a r e not in clu d ed .

T he tro p h ic p o s it io n o f the ra d io a ct iv e te ttig on id s (lon gh orn g r a s s h o p p e rs : A fr o g r y l la c r is a fr ica n a (B ru n n er), A m yttin a q u ad rim acu la ta (K arn y ), A m yttin a aliqu an tu la (K a rsch ) is not known, but as th e se had a cq u ire d d is t in c tly le s s 32P than o th e r s p e c ie s th ey w e re r e g a rd e d as s c a v e n g e r s . T he sa m e a p p lie s to the few ra d io a ct iv e s p e c im e n s o f b la ttids and D íp tera . T h e 32P a c t iv ity o f the ants w as u su a lly c o m p a ra t iv e ly low , s in ce the la b e l w as d is tr ib u te d o v e r a la rg e n u m ber o f in d iv id u a ls th rough fo o d exch an ge w ithin the c o lo n ie s .

T o d istin gu ish p r e d a c io u s fr o m s c a v e n g e r an ts, o b s e rv a t io n s w ere m ade in the f ie ld and on ly s p e c ie s found to attack liv e c a p s id s on the c o c o a tr e e w e re c la s s i f ie d a s p r e d a to rs .

T he fo llo w in g s p e c ie s o f p r e d a to rs w e re id en tified :

F o r m ic id a e : P la ty th y re a co n ra d t i E m e ryO e co p h y lla lon g in oda (L a tr e i l le )C r e m a to g a s te r d e p r e s s a (L a tre ille )C r e m a to g a s te r a fr ica n a (M ayr)C a m pon otu s ’b a rb a r u s 'M a c r o m is c h o id e s a cu lea tu s (M ayr)

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R ed u v iid a e : R h in o co r is c a r m e lita (Stal)R h in o co r is a lb o p ilo su s (S ign oret)V estu la lin e a t ice p s (S ign oret)R h in o co r is obtu su s (P . de B ea u v o is )E n dochu s a fr ica n u s B e rg ro th

M an todea : Sp h od rom an tis lin e ó la (B u rm e is te r )C a ta sp ilo ta m isaria (G ig l io -T o s )P a n u rg ica c o m p r e s s ic o l l is (S au ssu re)P r o h ie ro d u la o rn a tip en n is (B o liv a r )T a ra ch o d e s g e r s ta e c k e r i (W ern er)

A ra ch n id a : V ic ir ia e q u e s tr is Sim onV ic ir ia o c e lla ta (T h o re ll )H yllus h o lo ch a lce u s Sim on T e la m o n ia sp . n.C h iracan th iu m sp .

and nine m o r e s p e c ie s , so fa r unnam ed, in clu d in g a s a lt ic id o f a new genu s and two s p e c ie s w hich p ro b a b ly b e lo n g to C isp iu s and A p o ch in o m m a .

P la ty th y rea co n ra d t i, a la rg e p on erin e ant, w as h ith erto not r e g a rd e d a s p re y in g on c a p s id s [3 ]. By tra c in g w o rk e r s c a r r y in g la b e lle d p r e y w ith a f ie ld co u n te r , the n ests o f th is s p e c ie s w e re found in the dead w ood o f h a lf -r o tte n tr e e stu m p s. T h e c o lo n ie s con ta in ed on ly up to one hundred in d iv id u a ls . T he w o rk e r s a re h igh ly a c t iv e s o l ita r y fo ra g e rs ', and s y s te m a t ica lly s e a r c h the e n tire su r fa c e o f the c o c o a t r e e . T h is b eh a v iou r m igh t p a rt ly co m p e n sa te fo r the low p opu la tion as fa r a s th is s p e c ie s ' e f f ic ie n c y as a ca p s id p re d a to r is c o n c e rn e d . S in ce it is a g rou n d n e s te r , th is ant is p a rt ia lly p r o te c te d from in s e c t ic id e s a p p lied to the c o c o a t r e e .

O e co p h y lla lon g in od a and the two C r e m a to g a s te r s p e c ie s a r e a r b o r e a l an ts , w id e sp re a d and o c c u r r in g in la rg e n u m b e rs . T h ey a re known to be m u tu ally e x c lu s iv e s p e c ie s . O e co p h y lla is the m o r e d e s ir a b le p r e d a to r fr o m the v iew p oin t o f the c o c o a g r o w e r , a s it d o e s not attend P s e u d o c o c c u s n ja le n s is , the m ost im p ortan t v e c t o r o f the Sw ollen Shoot v ir u s . It is , h o w e v e r , m o r e lik e ly to b e a ffe c te d by sp ra y in g , a s it n ests in the c o c o a ca n o p y , w h erea s C r e m a to g a s te r m o s tly n ests on high fo r e s t t r e e s w e ll b e y o n d the re a ch o f the s p ra y d r ift .

M a c r o m is c h o id e s a cu lea tu s and C a m p o n o tu s 'b a r b a r u s 1 w e re o c c a s io n a l ly o b s e r v e d attack in g c a p s id s on c o c o a . T h ey s e e m e d to b e o f l it t le im p o r ta n ce as ca p s id p r e d a to r s .

T h e o th e r C a m pon otu s s p e c ie s w e re not o b s e r v e d p re y in g and w e re th e r e fo r e c o n s id e r e d s o le ly as s c a v e n g e r s .

T h e re d u v iid s l is te d , w ith the e x ce p t io n o f E n dochu s a fr ic a n u s , w e re c o l le c t e d by hand fr o m young c o c o a . In th e se p lo ts a con tin u ou s ca n op y had not yet fo r m e d , and the abundance o f w eed s su p p orted a r ic h fauna c h a r a c t e r is t ic o f the h erb and shrub le v e l . E n dochu s a fr ica n u s w as the on ly ra d io a c t iv e re d u v iid ob ta in ed fr o m fo r e s t c o c o a .

Out o f a to ta l o f 78 m a n tid s , 15% w e re r a d io a c t iv e . T h is su g g e s te d that m an tids w e re the m o s t a c t iv e p r e d a to r s . S p h od rom a n tis l in e ó la a cco u n te d fo r one th ird o f the ra d io a ct iv e s p e c im e n s .

O f the s p id e r s , 1.2% o f the s p e c im e n s had fed on c a p s id s . B e ca u se o f th e ir high popu lation le v e l th ey w e re r e g a rd e d as im p orta n t in c a p s id

SM-102/1 9

T A B L E I. D IS T R IB U T IO N O F 32p щ K N O C K -D O W N S A M P L E S

T o ta l n u m ber o f

sp ec im en s

N u m b er o f r a d io a c t iv e sp ec im en s

Ants

O e co p h y lla lo n g in o d a 3 410 284 8

C a m p on otu s barbarus 505 10

C a m p on otu s spp. 7963 6813

M a cro m isch o id e s a cu lea tu s 366 134

C rem atoK aster a fr ica n a 15 15

C rem a toga ster depressa 776 6 3 4

P latythyrea con ra d ti 33 16

R eduviids 2 1

Spiders 1263 15

M antids 78 12

T ettig on id s 461 13

Blattids 188 4

D iptera 523 о

c o n t r o l . T w o th ird s o f the ra d io a c t iv e s p e c im e n s b e lo n g e d to the s a lt i - c id s . A s r e la t iv e ly l it t le is known about W est A fr ic a n s p id e r s , 9 s p e c ie s c o u ld not be n am ed . T h ey w e re sen t to the B r it is h M useum fo r id e n tifica tio n .

A s r e g a rd s the im p a ct o f p re d a tio n on the popu la tion d y n a m ics o f the c o c o a c a p s id in the fie ld , it b e c a m e c le a r du ring the study that the e x p e r im e n ta l s e t -u p , w hile b e in g h igh ly e ffic ie n t fo r eva lu ation o f the q u a lita tive co m p o s it io n o f the p r e d a to r s p e c tru m , w as o f l im ite d v a lu e fo r a quan tita tive in te rp re ta t io n . T h is w as fo r the fo llo w in g r e a s o n s :

T h e r e c o v e r y o f 32P in the sa m p le s w as o f the o r d e r o f 10% o f the input a c t iv ity , in clu d in g r e c o v e r e d la b e lle d c a p s id s . T h us it w as c le a r that the e x p e r im e n t w as fa r fr o m ap p rox im a tin g a c lo s e d s y s te m . T h e r e la t iv e ly high m o r ta lity o f the la rv a e a v a ila b le fo r the e x p e r im e n ts w as a co m p lica t io n m a sk in g the e f fe c t o f p re d a t io n . T h e r e le a s e o f

10 MARCHART

50 D ista n tie lla la rv a e on on e t r e e r e p re s e n te d an a r t i f ic ia l s itu a tion , a s it e s ta b lis h e d a h igh lo c a l pop u la tion d en sity se ld o m o c c u r r in g in the fie ld .A s p re d a t io n in ten sity o ften is a fu n ction o f the p opu la tion d en s ity o f the p r e y , th is w ould le a d to an u n r e a lis t ic e s t im a te o f the p r e d a to r a c t iv ity .T h e la b e lle d c a p s id s fo r s o m e t im e a fte r r e le a s e w e re not fu lly a d ju sted to th e ir habitat. W ith an in se c t w h ich is c h a r a c te r iz e d by its c r y p t ic b e h a v io u r , the re su lt in g in c r e a s e d lo c o m o t o r y a c t iv ity n eed ed in s e a rc h o f s h e lte r and su itab le feed in g s ite s w as bound to e x a g g e ra te the e f fe c t o f p re d a tio n .

T he r e la t iv e im p o rta n ce o f an ts , m a n tid s and re d u v iid s w as d is cu s s e d by W illia m s [2 ]. A c c o r d in g to h is e s t im a te s , th ese th re e taxa w e re about e q u a lly e ff ic ie n t in ca p s id c o n t r o l . T he r e s u lts o f the p r e se n t study su g g e s t that the con tr ib u tion fr o m the re d u v iid s is o f m in o r s ig n if ic a n c e at the ca n o p y le v e l o f m a tu re t r e e s . On young t r e e s , w h ere a high ligh t in ten sity on the grou n d fa v o u rs a r ic h w eed f lo r a , re d u v iid s can be im p orta n t. P re d a t io n by s p id e r s - w h ich h as b e e n n e g le c te d so fa r - m ust be c o n s id e r e d co m p a ra b le to that o f m a n tid s . T he r e la t iv e im p o rta n ce o f ant p re d a t io n w as im p o s s ib le to e s t im a te b e ca u se on ly the w o rk e r ca s te co u ld be sa m p led , but an unknown p a rt o f the p r e y w ould end up ou ts id e the sa m p lin g a re a in the n e sts in the g rou n d in a d jacen t c o c o a stan ds and on high shade t r e e s . T h e re is no in d ica tion o f any o f the nam ed s p e c ie s b e in g s p e c ia liz e d o r dependent on c a p s id s , a s a ll o c c u r r e d in sa m p le s f r o m n o n -in fe s te d c o c o a .

T h e la rg e n u m ber o f s p e c ie s in v o lv e d in ca p s id p red a tion r e f le c t s the g e n e r a l ph enom enon o f h igh s p e c ie s d iv e r s ity c h a r a c t e r is t ic o f the t r o p ic s , p a r t ic u la r ly the fo r e s t zon e . T h is is o f so m e p r a c t ic a l s ig n if ic a n c e fo r the esta b lish m e n t o f an in teg ra ted c a p s id c o n tr o l p r o g r a m m e , s in ce the p o s s ib i l i t y o f s e r io u s in te r fe re n c e o f c h e m ic a l c o n t r o l w ith n atu ra l c o n tr o l by p re d a tio n w ill be re d u ce d by a h igh s p e c ie s d iv e r s ity am ong the p r e d a to r s .

A C K N O W L E D G E M E N T S

T he au th or w ould lik e to thank M e s s r s D. L eston and D. G. G ibbs fo r th e ir help in the id e n tifica tio n o f the in s e c ts c o l le c t e d and A . D. P ick ett fo r v a lu ab le d is c u s s io n and su p p ort. He is fu rth e r in debted to M e s s r sD. J . C la rk and J. H uxley o f the B r it is h M useum fo r the id e n tifica tio n o f the s p id e r s and te ttig o n id s .

T h is w ork w as sp o n s o re d by the In tern ation a l O ff ic e o f C o c o a and C h o co la te and is p u b lish ed by p e r m is s io n o f the D ir e c to r o f the C o co a R e s e a r c h Institu te.

R E F E R E N C E S

[ II SQUIRE, F. A . , Revta. E n t., Rio d e J. 18 (1947) 219.[2 ] WILLIAMS, G . . Bull. ent. Res. 45 4 (1954) 723.[3 ] GERARD, B. M . , H a lf-Y early Progress Report, C o co a Res. Inst. Ghana N o. 4 (19Ü4) 28.[4 ] JENKINS, D. W . , HASSET, C. C . , N ucleon ics 6 (1950) 5.[5 ] JENKINS, D .W . , KNIGIIT, K. L . , Proc. ent. S oc . Wash. 52 (1950) 203.[6 ] BALDWIN, W .F ., JAMES, H. G . , WELCH, H. E ., Can. Ent. 87 (1955) 350.

S M -102/1 1 1

[7 ] JAMES, H .G . , Can. J. Z o o l . 43 (1965) 155.[8] CLARK, E. W . , GLICK, P. A . , J. e con . Elit. 54 (1961) 815.[9 ] CROSSLEY, D. A . ,J r . , in Radiation and Radioisotopes Applied to Insects o f Agricultural Im portance,

IAEA, Vienna (1963) 43.[10 ] RAW, F . , Bull. ent. Res. 50 1 (1959) 11.L ll] PRIMS, G . , Proc. M irid C ontrol C o n f . , T a fo (19.63) 33.[12] LEWIS, C . T . , WALOFF, N ., E ntom ología exp . appl. 7 1 (1964) 15.[13] CORNWELL, P. B . , T rop . A grie . Trin. 34 2 (1957) 132.

APPENDIX

CO CO A R E SE A R C H IN GH ANA: TH E R O L EO F RA D IO ISO TO PES IN TH E STU D Y O F IN SECT PESTS

C o co a fa rm in g is the m ost im portan t s in g le fa c to r in G h an a 's e c o n o m y , and th is is r e f le c t e d by the fa ct that c o c o a a cco u n ts fo r one h a lf to tw o th ird s o f G hana 's e x p o rt e a rn in g s . Ghana is the w o r ld 's la rg e s t c o c o a p r o d u c e r , and th ere a re a p p ro x im a te ly 4 .5 m illio n a c r e s (1 .8 m illio n h e c ta r e s ) u nder c o c o a , y ie ld in g o v e r 400 000 ton s o f raw c o c o a annually and av era g in g 36% o f the w o r ld 's p ro d u ctio n .

T h e c o c o a fa r m e r fa c e s two m a jo r th rea ts fr o m in se c t p e s ts : ca p s id s (M ir id a e ) and m e a ly b u gs (C o c c id a e ) . Both a ffe c t the tr e e ra th e r than the c r o p : ca p s id s d e s tr o y the p h loem tis su e , ca u sin g d ie -b a c k o f the t r e e ,w h e re a s the m ea ly bu gs sp re a d the Sw ollen Shoot d is e a s e , a v iru s in fe c tion w hich u su ally r e s u lts in the death o f the c o c o a t r e e . T h e se two p é s ts have been devastatin g vast a r e a s o f the r ic h e s t c o c o a r e g io n s , the c a p s id s a lon e cau sin g lo s s e s e st im a te d at 20 -30% o f the n ationa l y ie ld . In add ition , v a r io u s m in o r le p id o p te ro u s p e s ts , p en ta tom id s , p s y ll id s , th rip s and le a fh o p p e rs ca u se fu rth e r lo s s e s . The study o f in se c t p e s ts w ithin the e c o s y s te m o f the c o c o a fa rm has th e r e fo r e been , and s t i l l is , a m a jo r c o n c e rn o f c o c o a r e s e a r c h , and r a d io is o to p e s a re c o n s id e r e d an in va lu ab le to o l fo r this w ork .

E a r ly w ork by C o rn w e ll w as co n ce n tra te d on the s p e c ie s P s e u d o c o c c u s n ja le n s is L aing , the dom inant v e c t o r o f the Sw ollen Shoot v ir u s . Uptakeand tra n s lo ca tio n o f 32P w as stud ied in the la b o r a to r y and in the f ie ld on se e d lin g s and m a tu re T h e o b ro m a c a c a o . T e ch n iq u e s o f la b e llin g with32P b y trunk im p lan ta tion , r o o t im m e r s io n and s o i l a p p lica tion w e re c o m p a re d [11.

T he p r a c t ic e o f c o n tro ll in g Sw ollen Shoot by cutting out in fe cte d tr e e s h as to take into a ccou n t the d is p e r s a l p a ttern o f v ir u s -c a r r y in g m ea lybu g la r v a e . D is p e r s a l stu d ies w ith 32P -la b e lle d P s e u d o c o c c u s n ja le n s is sh ow ed that u nder Ghana fa rm co n d it io n s la rv a e can m ig ra te to ad jacen t t r e e s v ia con ta ctin g b ra n ch e s [2 ], and b e tte r c o n t r o l can th e r e fo r e be a ch ie v e d by cutting out a p p aren tly healthy t r e e s w hich a re in co n ta ct w ith the d is e a s e d t r e e s [3 ].

In an oth er study , C o r n w e ll [4] w o rk e d on the d is p e r s a l o f la b e lle d m e a ly b u gs on the g rou n d , sh ow in g that m ov em en t fr o m h eaps o f v i r u s - in fe cte d t im b e r to n e a rb y healthy stands co n s titu te s no s e r io u s , r is k o f sp rea d in g the in festa tion .

It w as a lso d e m o n stra te d that P s e u d o c o c c u s n ja le n s is is attended by ants o f the genus P h e id o le [4] and by C r e m a to g a s te r s tr ia tu la E m e ry [1].

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T h e la tte r s p e c ie s a ccu m u la ted 32P to a g re a te r extent than its h ost. A n t-m e a ly b u g re la t io n sh ip s p r o v e d to be o f so m e p r a c t ic a l s ig n if ic a n c e in so fa r a s adult m e a ly b u gs can b e c a r r ie d to , and e s ta b lis h e d on , a d jacen t t r e e s by attending ants.

R oiva in en [5] w ork ed on so m e a s p e c ts o f fo o d exch an ge in ant c o lo n ie s o f the s p e c ie s C re m a to g a s te r s tr ia tu la w ith the a im o f in vestig a tin g the p o s s ib i l i t y ô f c h e m ic a l ant c o n t r o l by m ea n s o f in s e c t ic id e b a its . He d e m o n stra te d that 32P a p p lied in a honey bait so lu tion is d is tr ib u te d into a l l d e v e lo p m e n ta l s ta g e s o f the c o lo n ie s within fou r w e e k s , pupae and young w o rk e r s b e in g the la s t s ta g e s to a c q u ir e the la b e l.

T he sa m e author a lso c a r r ie d out stu d ies on the feed in g b eh a v iou r o f P s e u d o c o c c u s n ja le n s is on c o c o a s e e d lin g s , with the o b je c t o f obta in in g in fo rm a tio n on v iru s tra n s m is s io n by th is sucking in se c t . He w as ab le to show that the la b e llin g o f v ir u s - in fe c te d se e d lin g s w ith 32P is an e f fe c t iv e m ethod o f a s s e s s in g the extent to w hich m ea ly b u g s that have fed on in fe cte d plants a re in fe c t iv e . F eed in g w as show n to be a fu n ction o f te m p e ra tu re .

32P -la b e lle d D ista n tie lla th e o b ro m a (D istan t), the b la ck c o c o a ca p s id , w as u sed fo r tes tin g sa m p lin g tech n iq u es in e c o lo g ic a l s tu d ie s . It w as show n that the p y re th ru m knockdow n tech n ique u sed at the C o c o a R e s e a r ch Institu te fo r sa m p lin g the can opy p opu la tion o f c o c o a ca p s id s g iv e s a r e c o v e r y o f a p p ro x im a te ly 85% o f the la rg e la rv a e and a b ov e 90% o f the adult in s e c ts [6].

T h e a b so lu te popu la tion d en sity o f c o c o a c a p s id s in in fe ste d fa r m s had been e st im a ted h ith erto by m ean s o f hand c o l le c t io n . T h is m ethod is l ia b le to be quite in a ccu ra te . By r e le a s e and re ca p tu re o f la b e lle d la rv a e the author found that p r e v io u s e s t im a te s o f the m a xim u m le v e l o f in festa tion w e re low by a fa c to r o f at le a s t tw o [7 ]. On the b a s is o f th ese t r a c e r e x p e r im e n ts , the sa m p lin g p r o c e d u r e has been m o d ifie d to g iv e a m o r e r e lia b le index o f the le v e l o f in festa tion .

T o a cq u ire in fo rm a tio n on the fa c to r s in v o lv ed in n atural c o n t r o l o f c o c o a c a p s id s , a study o f the ca p s id p r e d a to rs am ong the c o c o a e n to m o - fauna w as c a r r ie d ou t. M eth ods o f la b e llin g D ista n tie lla th e o b ro m a with 32P w e re e v o lv e d , and by r e le a s e o f ta gged la rv a e in the f ie ld it w as p o s s ib le to iden tify m o r e than tw enty s p e c ie s o f an ts , m a n tid s , s p id e r s and re d u v iid s as c a p s id p r e d a to rs [8 , 9]. L eston flO ] stu d ied p re d a tio n on la b e lle d D ista n tie lla by O e co p h y lla lon g in od a and d e c id e d that th is ant is an im portan t p r e d a to r . In a c o lo n y he found the h igh est a c t iv ity a s s o c ia te d with the fe m a le a la te s ; m a le s con ta in ed fiv e t im e s le s s , and la r g e w o rk e r s fo r ty tim e s le s s , 32P than the qu een s. S m a ll w o r k e r s had a c q u ir e d lit t le o r no a c t iv ity .

A study o f the d is p e r s a l o f D ista n tie lla r e a r e d on 32 p - la b e l le d tre e s u nder f ie ld co n d itio n s gave so m e in fo rm a tio n on the fligh t a b ility o f the in s e c t , but, b e ca u se o f the low r e c o v e r y o f ra d io a c t iv e ad u lts , th is w as not c o n c lu s iv e [11 ].

137C s w as e ffe c t iv e as a t r a c e r fo r the sap s tre a m in the c o c o a tr e e du rin g feed in g e x p e r im e n ts . T he v e r y sh o rt b io lo g ic a l h a lf - l i fe o f th is is o to p e in the c o c o a ca p s id (4 h o u rs ) su g g e s ts that it co u ld b e a p p lied fo r d iffe re n t ia tio n o f s h o r t - t e r m fr o m lo n g -t e r m m ig ra t io n by u sin g the t r a c e r a s a c lo c k to m a rk the tim e o f d ep a rtu re fr o m the point o f r e le a s e in d is p e r s a l stu d ies [11].

S M -102/1 13

F u rth e r w ork a lon g the fo llo w in g lin e s w ould be p r o m is in g :

B a th y co e lia th a la ssin a ( I le r r i c h -S c h a e f fe r ) , a pen ta tom id w hich can ca u se h eavy lo s s e s to the c r o p by in h ib itin g bean d ev e lop m en t [12 ], sh ou ld b e the o b je c t o f d is p e r s a l s tu d ie s . T h e r e le a s e -r e c a p t u r e tech n iq u e w ill be u se fu l in eva lu atin g sa m p lin g m eth od s n e c e s s a r y to d e te rm in e the p op u la tion le v e l in in fe ste d fa r m s . Such in v e s tig a t io n s a re o f the u tm ost v a lu e , s in ce c r o p dam age is v ir tu a lly in v is ib le in the f ie ld and m a n ife s ts it s e l f on ly at the pod b re a k in g sta g e .

T he r o le o f ants w ithin the e c o s y s te m o f the c o c o a fa r m has been the o b je c t o f a n u m ber o f stu d ies in the p a st, but it is not yet u n d erstood in quantitative t e r m s . L a r g e - s c a le e x p e r im e n ts on fo o d ch a in s shou ld g iv e va lu ab le in fo rm a tio n w ith r e g a rd to the a g r icu ltu r a l im p o rta n ce o f the s p e c ie s O e co p h y lla lon g in od a (L a t r e i l le ) , C r e m a to g a s te r a fr ica n a (M ayr) and d e p r e s s a (L a t r e i l le ) , and P la ty th y re a co n ra d t i E m e ry as p r e d a to rs on the c o c o a ca p s id s D ista n tie lla th e o b ro m a and S a h lb e rg e lla s in g u la r is (H aglund).

T h e study o f fo o d ch a in s w ill in c r e a s e u nderstan d in g o f in s e c t ic id e s id e e ffe c ts in h e a v ily sp ra y e d p lan tation s that r e su lt in ou tb rea k s o f le p id o p te ro u s p e s ts [13] such as the c o c o a trunk b o r e r (E u loph on otu s m y r m e le o n F ld r . ) and the pod husk m in e r (M a rm a ra s s p . ). T h is w ork c o u ld be in co r p o r a te d in the la r g e - s c a le 32P su p erp h osp h ate e x p e r im e n ts b e in g planned by the C o co a R e s e a r c h Institute in c o -o p e r a t io n w ith the IA E A . The w ide s p e c ie s d iv e r s ity in a t r o p ic a l en v iron m en t p re se n ts p r o b le m s w hich can be so lv e d on ly by a lo n g -t e r m a p p roa ch .

T h e u se o f la b e lle d b a its w ill p ro v id e in fo rm a tio n on the fo ra g in g ran ge o f an ts, w h ich is n eeded fo r p r e d a c io u s s p e c ie s and e s p e c ia lly fo r th o se attending the v e c t o r o f the Sw ollen Shoot v iru s .

R e la tiv e ly lit t le is known about p o llin a tion o f the c o c o a f lo w e r . The d is ta n ce o v e r w hich c r o s s p o llin a tion by the c e ra to p o g o n id m id g e s F o r c ip o m y ia a sh an tii (In gram and. M a c fie ) , F o r c ip o m y ia in g ra m i (C a rte r ) and L a s io h e le a lit o ra u r e a (Ingram and M a c fie ) takes p la ce is an im portan t fa c to r in p lantations o f s e l f - s t e r i l e c lo n e s , w h ere co m p a tib le p o llin a to r t r e e s have to be p ro v id e d . E x p e r im e n ts with r a d io a c t iv e ly la b e lle d p o llen have been su g g e s te d to c la r i fy th is qu estion .

T he study o f p la n t -in s e c t r e la t io n s h ip s by m ean s o f r a d io t r a c e r s cou ld y ie ld in form a tion on the p h y s io lo g ic a l a s p e c ts o f h op per bu rn , w h ich is ca u se d by E m p o a s ca s p e c ie s on young c o c o a [14 ]. E x p e r im e n ts on the feed in g b eh a v iou r o f P la n o c o c c u s c i t r i (R is s o ) in c o m p a r is o n w ith that o f P s e u d o c o c c u s n ja le n s is w ould be d e s ir a b le to obtain in fo rm a tio n on the re la t iv e fr e q u e n cy o f v iru s tr a n s m is s io n by th ese two s p e c ie s .

G e n e ra lly sp eak in g , w ith in se c t p r o b le m s the im p o rta n ce o f the m u lt ifa c to r a p p roa ch , i . e . o f an in teg ra ted v ie w , is now a p p re c ia te d to a g re a te r exten t. C on com ita n tly w ith th is tren d o f v iew in g the p est p ro b le m aga in st the b a ck g rou n d o f the c o c o a e c o s y s te m , a vast n u m ber o f qu estion s w ill a r is e w hich can be a n sw e re d on ly th rough r a d io is o to p e s tu d ie s . A l though it w ill be so m e tim e b e fo r e the m u lt i - fa c t o r sy s te m o f th is t r o p ic a l en v iron m en t can be u n d e rsto o d in quan tita tive te r m s , m any p r o b le m s h ith erto in a c c e s s ib le to e x p e rim e n ta tio n can be ta ck led by m ean s o f t r a c e r te ch n iq u es .

T h is b r ie f r e v ie w o f the s c o p e o f r a d io is o to p e s in the study o f c o c o a in se c t p r o b le m s is p r e se n te d to d e m o n stra te how m u ch t r a c e r m eth od s

14 MARCHART

can co n tr ib u te to p r o g r e s s in a g r icu ltu r a l r e s e a r c h , w h ich is o f v ita l im p o rta n ce fo r a d ev e lop in g cou n try lik e Ghana.

R E F E R E N C E S

LI] CORNWELL, P. B . , Trop. A g r ie . , Trin. 34 (1957) 117.[2] CORNWELL, P. B . , Bull. ent. Res. 49. (1958) 613.[3 ] CORNWELL, P. B . , Annual Report W . A. C .R . I. (1953 -54 ).[4 ] CORNWELL, P. B ., Bull. ent. Res. £7 (1956) 137.[5 ] ROI VAIN EN, О. H . , Rep. C o co a Res.Inst. Ghana, 1 9 6 6 /6 7 , in the press.[6 ] PICKETT, A. D . , MARCHART, H . , Rep. C ocoa lies. Inst. Ghana, 196 5 /66 , in the press.[7 ] MARCHART, H . , Q . Rep. Int. Capsid Res. T eam 4 (1966).[8 ]- MARCHART, H ., LESTON, D . , Rep. C ocoa Res. Inst. Ghana, 1 9 6 5 /6 6 , in the press.[9 ] MARCHART, H ., "R adiotracer study o f the predators on D istantiella theobrom a (Distant) (H em iptera:

M iridae)” , these Proceedings, S M -1 0 2 /1 .[10 ] LESTON, D . , Ghana J. A g r ie . , in the press.[11 ] MARCHART, I I . , unpublished.L12] LODOS, N .. Bull. ent. Res. 57 2 (1967) 289.[13] ENTWISTLE, P. F . , in Agriculture and Land Use in Ghana (WILLS, J. B., Ed. ) Oxford University Press,

London (1962) 342.[14 ] PICKETT, A. D . , Q . Rep. Int. Capsid Res. T eam 1 (1967) 19.

D I S C U S S I O N

W . J . K L O F T : You m en tion that D ista n tie lla is a p h loem fe e d e r .We know that so m e p h lo e m -fe e d in g in s e c ts , su ch as ap h id s, m e a ly b u gs and o th e r s , s e c r e te h oneydew . W e have a lso o c c a s io n a l ly found h on ey - dew s e c r e t io n in a H e te ro p te r , the s u g a r -b e e t feed in g s p e c ie s P ie s m a quadratum (T iu p id a e). R a d io a ctiv e honeydew s e c r e t io n , i f it o c c u r s , c o u ld be a p o s s ib le s o u r c e o f e r r o r in y o u r e x p e r im e n ts , as it w ould be taken up by the ants. I know that you re g a rd e d as p r e d a to rs on ly th ose ants w hich w ere o b s e r v e d d ir e c t ly attack in g the c a p s id s , but I sh ou ld n e v e r th e le s s be v e r y in te re s te d to lea rn i f any honeydew s e c r e t io n o c c u r s .

II. M A R C H A R T : D ista n tie lla th e o b ro m a d oes not s e c r e te h oneydew .I a g r e e , h o w ev er , that w ith o th er in se c ts su ch as P seu d ococc -u s n ja le n s is , f o r e x a m p le , t r a n s fe r o f the la b e l by honeydew s e c r e t io n cou ld in te r fe re w ith p r e d a t o r -p r e y e x p e r im e n ts . The tro p h ic p os ition o f ants is s o m e t im e s d ifficu lt to a s ce r ta in and has to be e s ta b lish e d by o b s e rv a t io n .

D. LINDQUIST: Y ou r e le a s e d 50 la b e lle d in se c ts p e r t r e e ; co u ldyou g iv e an e st im a te o f the 'n o r m a l' p opu la tion p e r t r e e ? A ls o , what p e rce n ta g e o f the la b e lle d in se c ts r e le a s e d w as d e s tro y e d by p re d a to rs du ring the 4 8 -h o u r te s t p e r io d ?

H. M A R C H A R T : D istan tie lla o c c u r s on ly in sm a ll n u m b ers ; b e ca u seo f th is it is a lw ays d iff icu lt to obtain s ta t is t ica lly s ig n ifica n t sa m p le s iz e s in s tu d ies o f b in o m ics and re la te d p r o b le m s . A n um ber as low as 100 in s e c t s /a c r e can ca u se a p p re c ia b le d am age, and 4 -5 in s e c t s /t r e e w ould be a fa ir ly high n atu ral popu la tion d en sity . T he p e rce n ta g e o f c a p s id s k illed by p re d a to rs in m y e x p e rim e n t w as about 2% in 48 h o u rs . T h is w ould in d ica te that p r e d a to rs a re an im portan t fa c to r in ca p s id m o r ta lity ; h o w e v e r , fo r the re a so n s m en tion ed in the p a p er , p red a tion in ten sity in m y e x p e r im e n ts w as p ro b a b ly h igh er than u nder natu ral co n d it io n s .

SM -102/1 15

W . J . LE QUESNE: I u nderstan d that a r t i f i c ia l feed in g is o ftend iff icu lt in the c a s e o f H o m o p te ra . I have seen a r e ce n t p a p er s u g g e s t ing the tech n ique o f u sin g a m em b ra n e with the flu id u nder s lig h t p r e s s u r e . W ould you c a r e to co m m e n t m o r e on y o u r tech n iq u e?

H. M A R C H A R T : T he d iff icu lty you m en tion w as an ticip a ted , but ittu rn ed out that the c o c o a ca p s id feed s on a so lu tion a p p lied to f i lt e r p aper to an extent s u ffic ie n t to obta in la b e lle d in s e c ts o f any d e s ir e d le v e l o f ra d io a ct iv ity . I have a ls o fed th em th rough P a r a f i lm © m e m b ra n e s to d e te rm in e the am ount o f liq u id diet taken up per- day.

E .M . SH U M AKO V: H ave r a d io is o to p e s been u sed in Ghana to studythe p o llin a tion o f the c o c o a tr e e by M e le id a e ?

II. M A R C H A R T : Not y et, but su ch an in vestig a tion is planned fo rthe fu tu re.

ETUDE, AU MOYEN DE L 1 OR RADIOACTIF 198Au, DU RAYON D' ACTION DE COLONIES DE BOURDONS (Bombus sp.) EN VUE DE LA POLLINISATION DES PLANTES CULTIVEES

J . LECOMTE ET A . POUVREAUSTATION DE RECHERCHES SUR L'ABEILLE ET LESINSECTES SO CIA U X,BU RES-SU R-YV ETTE, FRANCE

Abstract — Résumé

ETUDE, AU MOYEN DE L'OR RADIOACTIF 198A u , DU RAYON D ’ ACTION DE COLONIES DE BOURDONS ( Bombus sp .) EN VUE DE LA POLLINISATION DES PLANTES CULTIVEES. Il est généralem ent très fa c ile de procéder au m arquage d 'une co lo n ie au m oyen d 'un radioisotope en profitant des incessants échanges de nourriture entre les individus. Les bourdons, bien que vivant en c o lo n ie , ne procèdent pas à des échanges directs. C ependant, il a pu être m is en év id en ce q u 'il existait de continuels transferts par l'in term éd ia ire des ce llu les à m ie l (troph allaxie secon daire). Dans ces conditions le m arquage d 'une co lo n ie de bourdons est aussi fa c ile à réaliser que ce lu i d 'une ruche d 'a b e ille s . L’ o r - 198 a é té utilisé avec succès pour réaliser ces essais qui ont permis d 'ob ten ir des renseignem ents importants et inédits sur le com portem ent des butineuses des co lon ies de bourdons p lacées a proxim ité des cham ps de légum ineuses en vue de favoriser la production de graines. L 'accou tu m an ce h un nouveau site , la fixation sur une esp èce v ég é ta le , le rayon d 'a c tio n , le déterm inism e des lignes de v o l et des secteurs de butinage ont pu ainsi être étudiés.

USE OF RADIOACTIVE GOLD, 198Au, TO STUDY THE RADIUS OF ACTIO N OF COLONIES OF BUMBLE-BEES ( Bombus sp .) WITH A VIEW TO POLLINATION OF CULTIVATED PLANTS. It is generally very easy to label a co lon y with a rad io isotope, taking advantage o f the constant exch an ge o f food between individuals. B u m ble-bees, although liv in g in a c o lo n y , do not engage in direct exch a n g e , but it has been shown that continual transfer takes p lace through the honey ce lls (secondary trophallax ia ). In these conditions a b u m b le -b ee co lon y can be labe lled as easily as a h ive o f bees. G o ld -198 was successfully used for the tests, which enabled us to obtain important and unpublished inform ation on the behaviour o f workers in b u m b le -b ee co lon ies situated c lo se to fields o f legum inous crops, with a v iew to enhancing grain production. Questions that have been investigated by this m ethod includ e the tim e required to b ecom e fam iliarized to a new site , fixation on a particular species o f plant, the radius o f action and the determ inate character o f the flight paths and the honey-gathering sectors.

1. IN TRO D U CTIO N

D epuis q u e lq u es an n ées le s bou rd on s font l 'o b j e t de n o m b re u se s r e c h e r c h e s en vue de le u r u tilisa tion pou r a s s u r e r la p o llin isa tio n de ce r ta in e s p lantes c u lt iv é e s , en p a r t ic u lie r d es L é g u m in e u s e s . C ependant, nous m anquons sou vent d 'in fo r m a t io n s de ba se su r la b io lo g ie de c e s in s e c t e s , c e qui ne fa c ilite pas le u r u tilisa tio n à l 'é c h e l l e a g ro n o m iq u e .

On peut co n s ta te r , par e x e m p le , la r a r e té des d on n ées con cern a n t le co m p o rte m e n t de butinage qu i, pou rtan t, s e ra ie n t e s s e n t ie l le s .

P ou r ten ter de c o m m e n c e r à c o m b le r ce tte la cu ne nous avon s e n tre p r is une étude du ray on d 'a c t io n et des p a r t icu la r ité s du butinage en e ffectu an t

17

18 LECOMTE e t POUVREAU

un m arqu age des bu tin eu ses au m oyen d es r a d io is o to p e s en nous in sp ira n t de travau x an a logu es e ffe c tu é s su r l 1 a b e ille d om estiq u e par p lu s ie u rs au teu rs [ 1 ,2 ] .

2. M ETH O D ES DE T R A V A IL

2 .1 . Le m arquage

P o u r d iffé re n te s r a is o n s déjà so u lig n é e s lo r s d es travau x portant su r l 'a b e i l l e , nous avon s voulu u t i l is e r l ' o r r a d io a c t if , 198 Au, qu i, par sa p é r io d e su ffisan te pou r t r a v a i l le r e ff ic a c e m e n t m a is a s s e z c o u r te pou r ne pas co n ta m in e r le m ilie u , continue de nous a p p a ra ître c o m m e un tr a c e u r tr è s a p p ro p r ié à nos r e c h e r c h e s .

R a p p elon s que 19BAu e s t un é lém en t de 2 , 7 j de p é r io d e ém ettant en tre a u tres un ray on n em en t gam m a de 0 ,4 1 1 M eV .

C et é lém en t peut ê tre l iv r é sou s fo r m e de so lu tion c o llo ïd a le fa c i le m ent a b s o r b é e par le s a b e ille s ou le s b o u rd o n s , m é la n gée a v ec une so lu tion de s u c r e ou de m ie l.

De m ê m e qu ' en ce qu i c o n c e rn e le s a b e il le s , nous avon s e s t im é q u 'u n e a c tiv ité m oyen ne par in se c te d 'e n v ir o n 1 ¿¿Ci p e rm e tta it une d é te c t io n fa c i le et lim ita it l ' i r r a d ia t io n d es b o u rd o n s . L 'a c t iv i t é to ta le de la c o lo n ie de bou rd on s éta it a in s i au m a xim u m de 2 à 3 m C i dans le c a s de c o lo n ie s d 'im p o r ta n c e n o rm a le .

Un p ro b lè m e se p osa it cepen dan t, a p r io r i , en c e qu i c o n c e r n e la d is tr ib u tion du ra d io é lé m e n t tr a c e u r . L a q u e stio n e s t fa c ile m e n t r é s o lu e ch e z l 'a b e i l l e d om estiq u e , le s fo u r m is , le s gu êpes et le s te r m ite s , g râ ce à la p a rt icu la r ité p ré se n té e par c e s in s e c te s d 'é c h a n g e r con tin u e llem en t en tre eux de la n o u rr itu re . Il su ffit dans c e s con d ition s que q u e lq u es in d iv idu s re ço iv e n t le tr a c e u r pour que 1' e n se m b le de la c o lo n ie so it m a rq u ée au bout d 'u n la p s de tem p s tr è s co u r t , et c e c i de m a n iè re r e la t iv e m ent u n ifo rm e . C ette p a rt icu la r ité a d 'a i l le u r s fa c ilité un grand n om b re de travau x su r la s o c io lo g ie des in s e c te s .

M ais ch ez le s b ou rd on s , qui son t pourtant des in s e c te s s o c ia u x , ce phénom ène de tra n s fe r t de n o u rr itu re , g é n é ra le m e n t connu sou s le nom de tro p h a lla x ie ,se m b la it ne pas e x is te r .

Il e st en e ffe t im p o ss ib le de co n s ta te r 1' e x is te n ce d 'é c h a n g e s d ir e c ts en tre in div idu s appartenant à la m êm e c o lo n ie . P ou rtan t il a été p o s s ib le de d é m o n tre r [3] l 'e x i s t e n c e d 'u n e trop h a lla x ie in d ire c te d 'u n type nouveau .

Une seu le o u v r iè r e d 'u n e co lo n ie de B om bu s h ypnorum c o m p o s é e d 'e n v ir o n so ixa n te in div idu s re çu t à l 'a i d e d 'u n e p ipette une d iza in e de m ill im è tr e s cu b es de m ie l contenant qu e lq u es m ic r o c u r ie s de 198A u. C ette o u v r iè r e fut r e lâ ch é e et il fut p o s s ib le de co n s ta te r que la n o u rr itu re in g é ré e par un seu l individu se d iluait a v ec une tr è s grande ra p id ité dans la p op u la tion . Cinquante et un pour cen t des bou rdon s p ré se n ta ie n t des t r a c e s a isém en t d é c e la b le s de r a d io a ct iv ité 6 h seu lem en t a p rè s le re tou r de 1' o u v r iè r e .

D iffé re n te s o b s e rv a t io n s nous ont en su ite m on tré qu ' il e x is ta it bien une trop h a lla x ie in d ire c te du fa it de 1' habitude d es b ou rd on s de v e n ir à le u r re to u r d é g u rg ite r de la n o u rr itu re dans des «p o ts à m ie l» qu i se rv e n t à l 'a l im e n ta t io n de l 'e n s e m b le de la p op u la tion . C ette p a rt icu la r ité p e rm e t

S M - 102/3 19

donc d 'o p é r e r le m a rq u a ge par ra d io é lé m e n t d 'u n e c o lo n ie de bou rd on s a v ec autant de fa c i l i té que dans le ca s d es au tres in s e c te s s o c ia u x .

D eux tech n iq u es peuvent ê tre e n v is a g é e s : la p r e m iè r e c o n s is te à don ner la n o u rr itu re contenant le t r a c e u r à une ou p lu s ie u rs o u v r iè r e s au m oyen d 'u n e pip ette ou à l 'a id e d 'u n « n o u r r is s e u r e x t é r ie u r » , c 'e s t - à - d ire d 'u n p etit r é c ip ie n t p la cé aux en v iro n s im m é d ia ts de la c o lo n ie .

La se co n d e c o n s is te à in tro d u ire dans la c o lo n ie un ré c ip ie n t contenant la n o u rr itu re m a rq u é e . Ce r é c ip ie n t jou e le r ô le de pot à m ie l et s e r t à l 'a l im e n ta t io n d 'u n grand n om b re d 'in d iv id u s . La p r e m iè r e technique p e rm e t de m ieu x c o n t r ô le r le s quan tités de n o u rr itu re in g é r é e s , don c le s d o s e s r e çu e s par le s in s e c te s ; la se co n d e p e rm e t d 'e f f e c t u e r p lus r a p id e m ent un m arqu age plus g lo b a l. D 'a u t r e p a rt , dans c e d e r n ie r c a s , le pot à m ie l a r t i f ic ie l resta n t en p la ce e s t con tin u e llem en t v is it é e t , b ien que son a c tiv ité par unité de v o lu m e d é c r o is s e en fon ction de la p é r io d e du r a d io é lé m e n t , c e t ap p ort p è rm e t de c o m p e n s e r le s e ffe ts de la p é r io d e b io lo g iq u e ch e z le s in s e c te s et d 'e f f e c t u e r la d é tection dans de m e i l le u r e s con d ition s le s d eu xièm e et t r o is iè m e jo u r s de l 'e x p é r ie n c e .

2 .2 . L a d é tection

De m êm e que dans nos travau x a n té r ie u rs su r le s a b e ille s d o m e stiq u e s ,il nous a se m b lé p lus e f f ic a c e de ca p tu r e r tou s le s in d iv idu s re n c o n tré s dans la zon e étu d iée a v ec un f i le t à in se c te afin de m e s u r e r à l o i s i r le u r ra d io a ct iv ité à l 'a i d e d 'u n d é te cte u r p o r ta tif , c e c i étant l ié à n otre p r é o ccu p a tio n d 'u t i l i s e r le s d o s e s le s p lus m in im e s co m p a tib le s a v e c n otre ex p é rim e n ta tio n .

L e d é te cte u r u tilis é éta it un d é te cte u r p o r ta t if à s c in tilla t io n (S P P 2 ).L a d é tection éta it a s s u ré e su r le te r r a in p a r deux équ ip es de deux p e r so n n e s , l 'u n e capturan t le s b ou rd on s a v e c le f i le t , l 'a u t r e pratiquant le u r exam en au m oyen du s c in t il la te u r .

3. R E SU L TA T S

L e s e x p é rim e n ta tio n s e ffe c tu é e s en 1966 et 1967 ne peuvent e n co r e ê tre c o n s id é r é e s que co m m e des m is e s au point de la m éth od e ; pourtant e l le s nous ont d é jà ap porté d es re n se ig n e m e n ts in té re ssa n ts que nous a llon s c o n s id é r e r san s r e n t r e r dans le d éta il des o b s e rv a t io n s .

Nous avons a in s i pu co n s ta te r que le s b ou rd on s , de m êm e d 'a i l l e u r s que le s a b e ille s d o m e stiq u e s , a g ra n d issen t g ra d u e lle m e n t le u r ra y on d 'a c t io n a p rè s d ép la cem en t de la c o lo n ie dans un t e r r it o ir e inconnu. L e s bu tin eu ses n 'o n t g u ère ten dance à s 'é c a r t e r beau cou p durant le p r e m ie r jo u r et nous . n ' en v ou lon s pou r p reu ve que le s p ro p o rt io n s d 'in d iv id u s m a rq u é s r é c o lt é s dans une popu lation de bu tin eu ses dans un rayon d 'e n v ir o n deux cen ts m è tre s au tou r de la c o lo n ie .

L e 15 ju in 1966, jo u r d 'a r r i v é e de la c o lo n ie su r le te r ra in d 'e x p é r ie n c e , nous avons cap tu ré s ix o u v r iè r e s m a rq u é e s su r un tota l de 168, tand is que le len d em a in nous n 'e n tro u v io n s que une su r un tota l de 140.L e 22 ju in 1967, jo u r d 'a r r i v é e de la c o lo n ie , nous ca p tu r ion s s ix ind iv idus m a rq u és su r un tota l de 75; le len d em a in nous n 'e n tro u v io n s que un su r un tota l de 141. P ou rtan t des c o n t r ô le s nous ont m on tré q u e , dans le s deux

20 LECOMTE e t POUVREAU

c a s , plus de 95% des bu tin eu ses éta ien t m a rq u é e s durant toute la d u rée de l 'e x p é r ie n c e et q u 'e l l e s pouvaient ê tre fa c ile m e n t re co n n u e s .

Un d eu xièm e re n se ig n e m e n t e s t c e lu i r e la t if à l 1 e ffe t du m ic r o r e l ie f . P lus e n co r e que pour l 'a b e i l l e d om estiq u e le s o b s ta c le s p la cé s à p ro x im ité im m éd ia te de la ru ch ette contenant la c o lo n ie nous sem b len t jo u e r un r ô le su r la d ir e c t io n c h o is ie par la m a jo r ité des b u tin eu ses . C es deux don nées ont pu ê tre v é r i f ié e s a u ss i b ien en ce qui. c o n c e rn e B om bu s t e r r e s t r is que B . L a p id a r iu s .

On a pu ég a lem en t fa ire q u e lq u es o b s e rv a t io n s su r le s p r é fé r e n c e s a lim e n ta ire s des c o lo n ie s , la co n jo n ctio n des deux d e r n ie rs fa c te u rs entraînant une tr è s g ran d e h é té ro g é n é ité de la d is tr ib u tion des o u v r iè r e s de la m êm e co lo n ie dans 1' e sp a ce en viron nant.

4 . CONCLUSION

L a m éthode de m a rq u a ge par ra d io é lé m e n t se m b le don c p a r fa ite m ent a p p lica b le à l 'é tu d e du butinage d es b o u rd o n s . L a m éthod e p résen te d 1 in co n te s ta b le s avan tages par ra p p o rt à c e l le qu i c o n s is te r a it à u t ilis e r d es m a rq u e s c o lo r é e s , c a r e lle e st bien m oin s longue à m e ttre en oeuvre et ne p ertu rb e pas la c o lo n ie . 11 se m b le d 'a i l le u r s p a rt icu liè re m e n t avantageux de tr a v a i lle r en m êm e tem p s su r p lu s ie u rs c o lo n ie s d 'e s p è c e s d if fé r e n te s , ce qu i augm ente le n om b re d es r e p r is e s et des d on n ées u tile s pou r 1' in te rp ré ta tio n , sans su p p lém en t de tra v a il.

L e s p r e m ie r s ré su lta ts obtenus son t e n co u ra g e a n ts . Ils sem b len t m o n tre r un grand p a r a llé lis m e en tre ce que nous sa von s du butinage de l 'a b e i l l e d om estiq u e et c e lu i des b o u rd o n s . P ou rtan t, le s p r é c is io n s que l 'o n p ou rra ob ten ir se ro n t p r é c ie u s e s en vue de l 'u t i l i s a t io n de c e s in s e c te s pou r a s s u r e r la p o llin isa tio n de c e r ta in e s p lantes cu lt iv é e s .Il s e r a it sou h aitab le que c e s étu des so ie n t p o u rsu iv ie s su r un grand n om bre d 'e s p è c e s et dans des b io to p e s d iffé re n ts ; m a lh eu reu sem en t, la seu le autre tentative e ffe ctu é e à n otre co n n a issa n ce dans ce sen s s 'e s t s o ld é e par un é c h e c , le s au teu rs [4] n 'a y a n t pu r e tr o u v e r le s bou rdon s m a r q u é s .

R E F E R E N C E S

[ 1 ] COURTOIS, G . , LECOMTE, J . , Int. J. appl. Radiat. Isotopes 5 (1959) 265.[ 2 ] LECOMTE, J . , Annls A b eille 3 (1960) 311.1.3] LECOMTE, J . , C .r . hebd. Séan c. A cad . S c i. 251 (1963) 366-1.Г4 ] DIABOLA, J . , TAIMR, L. , Acta S oc . ent. Bohem. 62 (1965) 413.

D I S C U S S I O N

II. M A R C H A R T (C h a irm a n ): O f a ll f lo w e rs po llin a ted by in se c ts in y ou r p lo ts , what p ercen ta g e w ould you est im a te as po llin a ted by bum ble b e e s ?

J. L E C O M T E : T h is v a r ie s g re a tly depending on the c r o p in vo lved and a n um ber o f o th er fa c to r s , m a in ly c l im a t ic . F o r e x a m p le , in F ra n ce

SM~ 102/3 21

about 100% o f red c lo v e r p ollin a tion (T e tr a p lo id v a r ie t ie s ) takes p la ce via bum ble b e e s .

E . IiO K BEU : Have you co n tem p la ted , o r indeed t r ie d , la b e llin g p o lle n ?

J. L E C O M T E : A m ethod has been d e v e lop ed w hich c o n s is ts o f g row in g lu ce rn e in a nutrient so lu tion conta in in g 3i-P . The p o llen b e c o m e s la b e lle d , and can be d e tected in n eigh bou rin g flo w e rs by au tora d iogra p h y . The range o f d is p e r s a l o f the p o llen s e e m s to líe v e r y sh o r t , le s s than 1 m e tre .

C .A . P E L E R E N T S : How m any in s e c ts w ere th ere in y o u r bum ble bee c o lo n ie s , and w e re they re a re d as c o lo n ie s o r a s se m b le d fro m in d iv idu a ls ?

J. L E C O M T E : The co lo n y s iz e ran ged fro m 150 to 300. T h ey w ere p rod u ced by re a r in g fr o m a s in g le qu een .

M . COH EN : The la b e lle d honey w as in the c o lo n y fo r a p e r io d o f t im e , and th e r e fo re the la rv a e w ere both fed on it and e x p o se d to the rad ia tion . W as th ere any e ffe c t on the la r v a e , and w as the num ber o f adults that e m e rg e d fr o m them n o rm a l?

J . L E C O M T E : J m ade no s p e c ia l study o f the e ffe c t o f rad ia tion on the la rv a e , but d ev e lop m en t o f the c o lo n ie s se e m e d n o rm a l.

G . L E M ASN E: T o what extent w as d is p e r s a l o f the bum ble bees m o d ifie d by tra n sp o rt o f the c o lo n y f rom the la b o r a to r y to the ex p e rim e n ta l a r e a ?

J. L E C O M T E : H on ey -g a th er in g a c t iv ity w as m od ified bv tra n sp o rt , and in p a rt icu la r the range o f a ction w as re d u ced during the re o r ie n ta tio n p e r io d . T h is in te re s ts u s , b e ca u se it w ould be advantageous to red u ce the range o f the w o rk e r s and thus im p ro v e p o llin a tion . H ow ever , we a re a lso p e r fo rm in g e x p e r im e n ts w ith c o lo n ie s w hich have not been tra n sp o rte d .

RADIOISOTOPES IN THE INVESTIGATION OF INTERRELATIONSHIPS BETWEEN APHIDS AND HOST-PLANTS

W. KLOFT, P. EHRHARDT AND H. KUNKELIN STITU TE OF APPLIED ZOOLOGY,UNIVERSITY OF BONN,BONN, FEDERAL REPUBLIC OF GERMANY

Abstract

RADIOISOTOPES IN THE INVESTIGATION OF INTERRELATIONSHIPS BETWEEN APHIDS AND HOST-PLANTS. A ctiv ities o f the aphid on the host-plant, such as probing, starting o f feed in g and salivation, can be observed by la b e llin g either host-plant or insect with a rad ioactive isotope. T h e course o f plant sap uptake, assim ilation into the organism , haem olym ph circu la tion , and incorporation into different tissues, esp ec ia lly salivary glands and em bryos, w ere investigated with this m ethod. A rtific ia l feed in g o f aphids through m em branes enabled isotope studies to be perform ed independently o f the host-plant. Studies o f e x cretion v ia honeydew , saliva and n ew -b om larvae w ere also carried out. D eta iled results are presented on the processes o f saliva in jection and transport in the plant tissue, w hich are important from the point o f view o f phytopathology and virus transmission.


H om op tera as p la n t-su ck in g in s e c ts fe e d in d iffe re n t t is s u e s o f th eir h o s t -p la n ts . A c c o r d in g to K unkel [1 ], tw o m a in ty p es a re d is tin g u ish ed w ith r e g a rd to th e ir p h y s io lo g y o f n u trition . T h e f ir s t type tak es its fo o d out o f con d u ctin g t is s u e s and w as f o r m e r ly c a lle d 'P h lo e m s a u g e r ' o r 'X y le m s a u g e r ' [2 ]; K unkel in tro d u ce d the m ore , g e n e ra l te r m 's y s te m b i- b i t o r 1. T he se co n d ty p e fe e d s in n o n -co n d u ctin g t is s u e s and is c a lle d 'P a r e n c h y m s a u g e r '. F o r S tern orrh y n ch a the te r m 'l o c a lb ib i t o r ' is p r o p o s e d [1].

F o r a lon g t im e the p h y s io lo g y o f n u trition and v a r io u s a s p e c ts o f in s e c t -h o s t -p la n t r e la t io n s h ip s w e re in v es tig a ted by m ea n s o f r a d io i s o to p e s . T h is w as done m a in ly w ith p h lo e m fe e d e r s , e s p e c ia l ly aphids o f the fa m ily A p h id id a e . T h e A ph id idae a re m o s t im portan t a s plant p e s ts and v iru s v e c t o r s , and su ch e x p e r im e n ta l w ork co u ld e lu c id a te m o r e d e ta ils o f n utrition a l p h y s io lo g y in o th e r aphid fa m ilie s . S im ila r e x p e r im e n ts w e re s ta r te d w ith C h a etop h orid a e [3] and 'lo c a lb ib i t o r s ' o f d iffe re n t aphid fa m ilie s [4 ]. T h is w ork con tin u es in sp ite o f e x p e r im e n ta l d if f ic u lt ie s .

2. U P T A K E O F FO O D

Is o to p ic a l ly la b e lle d n atu ral o r a r t i f i c ia l fo o d can be fo llo w e d during the c o u r s e o f in g e stio n and d ig e s t io n by aphids by m e a su r in g th e ir r a d io a c t iv ity . A p a r t icu la r advantage o f th is m eth od is that the uptake o f ra d io a ct iv e su b s ta n ce s th rough the stin gin g and su ck in g m outh p a rts m in im iz e s o v e r a l l con tam in a tion [5 ], s in c e on ly th ese p a rts c o m e into con ta ct

23

24 KLOFT e : a l .

w ith the la b e lle d m a te r ia l . R a d io a ctiv e p h o sp h o ro u s w as the p r e fe r r e d is o to p e , b e ca u se ph osph ate is abundant in a ll t is s u e s , w e ll tr a n s lo ca te d in s ie v e -t u b e s , and p la ys an im portan t r o le in the m e ta b o lis m . M ost o f o u r e x p e r im e n ts w e re done w ith aphids su ck in g on h e rb a ce o u s p la n ts .

T o find th e ir fo o d , ap h ids m ust r e c o g n iz e th e ir h o s t -p la n ts and w ithin th e se the s ie v e -tu b e s . "Before pen tratin g d e e p e r a r e a s o f the plant t is s u e , ap h id s p ie r c e the e p id e rm is and one o r two c e l l la y e r s b ey on d , a b e h a v iou r w hich is ca lle d p ro b in g -p u n ctu r in g . N eith er m e a su re m e n t n or the s e n s it iv e a u to ra d io g ra p h ic te ch n iq u es d e te cte d r a d io a c t iv ity in sid e ap h ids a fte r such p ro b in g [ G - 81. M e a su re m e n ts sh ow ed that the in s e c ts take up fo o d on ly a fte r stin g in g when th ey re a ch the p h lo e m . T h e a c tiv ity then su dden ly in c r e a s e s . A ph id a c t iv ity m e a su re m e n ts and h is t o lo g ic a l ex a m in a tion s o f s ty le ts and sa liv a sh eaths in plant t is su e s d em on stra ted the fo llo w in g th ree p o in ts :

(1) M ea su rem en t o f the a c t iv ity o f in d iv idu a l aphids sh ow ed that the s h o r te s t p e r io d r e q u ire d to r e a c h a s ie v e -tu b e w as about sev en to ten m in u tes f 6, 9, 10].(2) G roup a c tiv ity m e a su re m e n t sh ow ed that on ly about 10% n eed ed th is m in im u m tim e . The sp eed o f t is su e p en etra tion up to the depth o f the s ie v e -tu b e s v a r ie d fr o m 5 to 30 m inu tes fo r s in g le aphids [4 , 10, 11].(3) A c e r ta in p e rce n ta g e o f aphids fa ile d to r e a c h a s ie v e -tu b e , a p h en o m en on w hich depen ds on the a n a to m ica l data o f a plant s p e c ie s and its d iffe re n t p a rts [6 , 7, 8, 10]. T h e fa ilu re to pu nctu re a s ie v e -tu b e w as c a lle d 'F e h ls t i c h 1. F o r in sta n ce , out o f a g rou p o f M egou ra v ic ia e su ck ing on .th e s tem o f V ic ia faba on ly 01% re a ch the p h loem in one hour and a ll aphids a fte r 5 h ou rs [7 ]. A m o r e d e ta iled a n a ly s is in clu d in g the in flu en ce o f te m p e ra tu re on th ese c o m p le x p r o b le m s is in p r o g r e s s [4].

A n oth er p r o b le m is the re co g n it io n o f su itab le h o st-p la n ts by aphids. S e v e r a l m e ch a n ism s s e e m to be in vo lved , fo r ex a m p le the g u s ta to ry d is c r im in a tio n by the ep ip h a ry n g ea l o rga n [7 ]. T h is w as show n by the fa ct that _iVL v ic ia e [8] and A ph is sa m b u c i [11] take up r a d io a c t iv ity out o f A lliu m sch o e n o p ra su m and o th er n o n -h o st -p la n ts at ra te s co r r e s p o n d in g w ith in itia l uptake in su itab le h o s t-p la n ts . A fte r th is in itia l p ro b e aphids try to le a v e the n o n -h o s t -p la n t . A fter pu n ctu rin g the s ie v e -tu b e , the aph id s r e g is te r e d a rap id in c r e a s e o f ra d io a ct iv ity . D uring the f ir s t th ree to fiv e h o u r s ', uptake (m e a su re d as c o u n ts /m in ) w as lin e a r w ith t im e , la te r on it b e ca m e ex p on en tia l. A fte r about 24 h ou rs the a c t iv ity d id not in c r e a s e , even when feed in g con tin u ed . A ph osph ate sa tu ra tion had a p p a re n tly been re a ch e d . Uptake and e x c r e t io n se e m e d to b e equ al [6 , 7].A s im ila r uptake w as found fo r the aes tiv a tin g la rv a e o f C h a etop h oria x an th om ela s K och on m a p le le a v e s . T h e la tte r re su lt , d e m o n stra te d with t r a c e r m eth od s, w as c o n tr a r y to the p re v a ilin g kn ow ledge that th ese la rv a e w e re not in a ctiv e but in gested fo o d [3 ].

In gestion o f a r t i f ic ia l d ie ts w as in ves tig a ted by m ea n s o f 32P , 1311 and SuRb w ith A ph is fabae [1 2 -1 4 ] . T h is m ethod p ro v id e d a known d iet by se p a ra tin g th ese p a r a s it ic in s e c ts fr o m th e ir natu ral h o s t -o r g a n is m w ith its u ndefined fo o d su p p ly . F u rth e r m o r e , it w as p o s s ib le to in trod u ce into aph id s a ll w ater so lu b le su b sta n ces w hich w e re not tra n s lo ca te d in p la n ts . O ne ap p lica tion o f th is m eth od is the fo llo w in g : it w as s tr ik in g that aphidsco u ld b e r e a r e d o v e r 11 g e n e ra tio n s on a to ta lly syn th etic diet con ta in in g

SM -102/4 25

no s t e r o ls . A fte r add ition o f s o d iu m - 14C -a c e ta te to the d ie t, la b e lle d c h o le s t e r o l c o u ld be d e te cte d in n o rm a l aphids w ith s y m b io te s , but not in aphids f r e e o f s y m b io te s [15 ]. F o llo w in g the m ethod in tro d u ce d by M itt le r , w e u sed the s o - c a l le d sa ch e ts : a nutrient so lu tio n is e n c lo s e dbetw een two s tre tch e d m e m b ra n e s o f P a r a f i l m ® and e v a p ora tion and con tam in a tion a re thus p rev en ted . T h e in flu en ce o f e c o lo g i c a l fa c to r s su ch as te m p e ra tu re , a ir h u m id ity , ligh t and day p e r io d on the fo o d uptake o f A p h is fabae w as in v estig a ted [16 ].

3. A SSIM ILA TIO N AN D D ISTR IB U TIO N IN M . v ic ia e '

T he f ir s t d e tection o f r a d io a c t iv ity in the h aem olym p h o f M . v ic ia e o c c u r r e d 30 m in u tes a fte r the beg in n in g o f fo o d uptake. T h e 32p le v e l in c r e a s e d l in e a r ly up to 10 h o u rs . An a n a ly s is o f the ph osph ate fra c tio n p r im a r i ly sh ow ed a high le v e l o f in o rg a n ic ph osp h ate . A fte r t r a n s fe r o f aphids to n o n -r a d io a c t iv e p la n ts , the o r g a n ic p a rt (A T P , A D P , G - l - P , G - 6 - P , F - 6 - P , F - l , 6 - P ) 1 in c r e a s e d [7 ]. Due to the ra p id p a ssa g e o f the t r a c e r into the h aem olym p h , the d iffe re n t t is s u e s and o rg a n s o f the aphids qu ick ly b e c a m e r a d io a c t iv e . T hus the e m b ry o s o f a r t i f ic ia l ly fed A . fabae had a h igh a c t iv ity on ly on e h our a fte r in g estion began [17 ]. The t r a c e r w as in co r p o r a te d into a lm o s t fu lly d ev e lo p e d e m b r y o s , e . g.M . v ic ia e fed on la b e lle d fa b a : the f ir s t ra d io a ct iv e la rv a e ap peared s ix h ou rs a fte r feed in g . The uptake o f the t r a c e r into the s a liv a r y glands w as o f s p e c ia l in te re s t w ith r e g a rd to sa liv a tio n and is d e s c r ib e d in a se p a ra te se c t io n . R a d io iod in e s e e m e d to have s p e c ia l a ffin ity fo r the c u t ic le [ 14].

4. E X C R E T IO N AND SA L IV A T IO N

E lim in ation o f r a d io is o to p e s b y the aphid is p o s s ib le th rough s a l iv a t io n , p rod u ction o f l a r v a e , m o u l t in g , and e x c r e t i o n o f h o n e y d e w . T h e b io lo g ic a l h a l f - l i f e is d e te rm in e d by the c o u r s e and v e lo c i ty o f a ll these p r o c e s s e s , and v a r ie s fr o m about 30 to 50 h ou rs depending on the stage [5 ]. T h ese data in d ica ted an e x tr e m e ly high m e ta b o lis m co m p a re d w ith r e s u lts fo r t e r r e s t r ia l H e te ro p te ra , w h ich sh ow ed h a lf - l iv e s o f betw een6 and 40 days [18].

T h e f ir s t r a d io a c t iv ity in the honeydew o f aphids su ck in g on la b e lle d p lants ap p ea red not e a r l ie r than 2.5 h ou rs a fte r the s ta rt o f uptake [5].T h e a c t iv ity o f the h oneydew re a ch e d a m a xim u m valu e a fte r about 20 h o u rs , r e f le c t in g a t im e la p se o f 20 to 24 h ou rs n eeded to r e a c h m a x im um a ctiv ity . T he count ra te o f honeydew p ro d u ce d by JVL v ic ia e on V . faba am ounted to about 3% and 1% p e r d rop le t o f the aphids a c t iv ity a fte r 4 and 24 h ou rs , r e s p e c t iv e ly . T he co r r e sp o n d in g data fo r a e s t i -

1 ATP = aden osine-tri-phosphate ADP = aden osine-d i-phosphate G - l -P = g lu c o s e -1 -phosphate G -u -P = g lu cose-6 -ph osp h ate F -6 -P = fructose-G -phosphate F - l , 6 -P = fru ctose -1 ,6 -p h osp h a te

26 KLOFT et a l .

vating la rv a e o f C h aetoph oria x an th om ela s w e re about 70 to 25%, su g gestin g that th ese la rv a e a s s im ila te le s s ph osph ate than o th e r p h lo e m -s u c k in g aphids w hich d ev e lop w ithout d iapau se [3].

A ph id sa liv a is im portan t in the tr a n s m is s io n o f c ir c u la t iv e plant v ir u s e s b e ca u se it m ight p r o v id e a m e ch a n ism fo r the in trod u ction o f the v iru s into the s ie v e -tu b e s . A c c o r d in g to stu d ies by K loft and K unkel [10] on M yzu s a s ca lo n ic u s and b y E h rh ardt [7] on M. v ic ia e , 32P w as f ir s t e x c r e te d in the sa liv a fiv e h ou rs a fte r the start o f feed in g . T he in se c ts w e re t r a n s fe r r e d to a n o n -r a d io a c t iv e tes t plant a fte r a r e la t iv e ly sh o rt feed in g t im e , and the v e r y low a c t iv it ie s in the in je c te d sa liv a w e re ch e ck e d b y a u tora d iogra p h y . T h e se e x p e r im e n ts m ight s e r v e as a m o d e l fo r d e te rm in in g c ir c u la t io n t im e s o f c e rta in plant v ir u s e s .

P h o sp h o ru s -3 2 is s e c r e te d a long w ith sa liv a on ly during p ie r c in g and r e tr a c t io n , as w as found in e x p e r im e n ts with M. a s ca lo n ic u s [9 ]. T h e in je c te d a c t iv ity did not depend on the du ration o f feed in g ; h o w e v e r , sbRb w as s e c r e te d con tin u ou sly by A . fabae feed in g on V . faba le a v e s [13 ]. T h is ph enom enon w as brou gh t about by the flu id phase o f sa liv a s e c r e t io n [19 ]. T h rou gh sa liv a tion , 1 .42% 0/h o u r o f the o r ig in a l a c t iv ity o f the aphid w as t r a n s fe r r e d into the le a f. If it w as a ssu m e d that the r a d io a c t iv ity w as u n ifo r m throughout an aphid w eigh in g 0 .5 m g , the r e su lts in d ica ted that sa liv a w as in je c te d into the plant at a ra te o f about 0.7 / jg /h o u r . T h is is equ iva len t to 0 .7 X 10"3 ^ i /h o u r . An a v e ra g e va lu e o f about 3.7 X 10 "4 jul w as found, su ggestin g that the ad d ition a l flu id sa liv a w as p ro b a b ly in je c te d m o r e o r le s s con tin u ou sly during feed in g [13 ]. L a b e llin g o f aphid sa liv a w as p o s s ib le by ap p lica tion o f 131I v ia the plant o r an a r t i f i c ia l d iet [14 ]. O th er ty p es o f p la n t-su ck in g a r th ro p o d s su ch as th rips [20] and sp id e r m ite s [21] a lso in je c te d sa liv a during feed in g , as w as d em on stra ted by the u se of. 32P .

5. D ISTRIBUTION AND E F F E C T S O F SA LIV A

W ith a u tora d iog ra p h ic m eth od s it w as show n that 32P - la b e l le d sa liv a in je c te d by M . a s ca lo n ic u s w as d is tr ib u ted in V io la t r i c o lo r le a v e s . The sp re a d in g o f sa liv a depended on the du ration o f su ck in g tim e and m a in ly fo llo w e d the v e in s [9]. T he p r e s e n c e o f ra d io a ct iv e m a te r ia l in le a f v e in s ad ja cen t to feed in g pu n ctu res m ade by 86R b -la b e lle d A . fabae w as d em on stra ted [13] (F ig . 1).

A phid feed in g d ir e c t ly a ffe c te d the p h y s io lo g ic a l p r o c e s s e s o f the h o s t-p la n t, i . e . p h o to sy n th e s is , re sp ira t io n and tra n sp ira tio n . S a liva se e m e d to be the m ost in flu e n t ia l 'fa c to r h e re [9]. A plant la b e lle d with tr it ia ted w ater [23] show ed changed tra n sp ira tio n a fte r be in g p ie r c e d by an aphid . T he phosphate m e ta b o lis m o f plant p a rts in flu en ced b y the s a liv a o f su ck in g in se c ts w as in v es tig a ted with t r a c e r m eth od s. U ptake, o f 32P in dam aged a r e a s o f P ic e a n e e d le s ca u sed by the s tro n g ly p h y to to x ic sa liv a o f the aphid , E latob iu m ab ietin u m , w as le s s than that in h ealthy p a rts [22 ]. T he d is tr ib u tio n o f 32P in a le a f w ith a pouch g a ll o f T e tra n e u ra u lm i d iffe r e d fr o m that in a n o rm a l le a f o f the sa m e sh oot.T h e g a ll it s e l f had a g ra d ie n t, with a high le v e l o f r a d io a ct iv ity a rou n d the la ce r a t io n . It w as co n c lu d e d that the a ccu m u la tion o r exch an ge o f p h o s phate v a r ie d with the p h yto tox a em ia in du ced by the sa liv a o f ap h id s. If the p a ra s ite in flu en ced a d iffe re n t ia te d t is s u e , the exch an ge o f ph osph ate

S M -102/4 27

(с)

FIG. 1. An autoradiographic presentation o f V ic ia faba leaves showing the distribution o f rad ioactiv ity in jected by Aphis fabae Scop , w hich had ingested w Rb: (a) after 10 m in (b) after 1 h, and (c ) after 24 h o f feeding.

FIG. 2. S ch em atic sum m ary o f radioisotope m ethods used:(a) Aphids (w hite), transferred to a rad ioactive plant (b la ck ), ingest the rad ioactive food and b e co m e rad ioactive (b lack ).(b) Feeding aphids w ith an artific ia l d ie t: the rad ioactive ly labelled d ie t (b lack ) is enclosed betw een two m em branes o f Parafilm ( r ) , w h ich are stretched over a glass ring to form an aphid ca g e .(c ) Labelled aphid (b lack ) is transferred to a n on -ra d ioa ctiv e plant (w hite).(d) R adioactive aphid in jects labe lled saliva w hich disperses in the le a f (dotted area) prim arily along the veins.

28 KLOFT et a l .

d e c r e a s e d . H ow ever , the ex ch an ge in cr e a s e d with e a r l ie r and s tro n g e r a tta ck s on n o rm a lly d iffe re n t ia te d plant tis su e s [24, 25].

6. CONCLUSIONS

R a d io is o to p e s have p ro v e d to be u se fu l to o ls fo r studying v a r io u s a s p e c ts o f the a p h id 's b e h a v io u r , its e c o lo g y , n u trition a l p h y s io lo g y and re la t io n sh ip with its h o s t -p la n ts . T h ey a re a lso valu ab le in in v e stig a tio n s o f p h y top a th o log ica l p r o b le m s and the p r o b le m o f v iru s tra n s m is s io n by ap h id s. F ig u re 2 su m m a r iz e s s c h e m a t ic a lly so m e o f the r a d io is o to p e m eth od s u sed .

R E F E R E N C E S

[1 ] KUNKEL, I I . , Z . angew. Z o o l . 54 (19C7) 37-68 .[21 KLOFT, W. , Entomophaga 5 (I9 60 ) 43 -54 .[3] EIIRHARDT, P . , Z . angew. Z o o l . 5 4 (1 9 6 7 )2 1 -3 6 ,[4] KUNKEL, II. , unpublished.[5 ] KLOFT, W ., EIIRHARDT, P ., in Radioisotopes and Radiation in E ntom ology, IAEA, Vienna

(1962) 181 -90 .[6] EIIRIIARDT, P ., Experientia Г7 (1061) 4ÍJ1.[7] EHRIIARDT, P . , Z . Morph. O kol. T iere 52 (1963) 597-G77.[8 ] EHRÍIARDT, P . , Experientia 19 (1963) 204.[9J KLOFT, W . , H abil. Arbeit, Würzburg 1056; Z . angew. Entom ol. 45 (I9 60 ) 337 -81 and 4o

(I9 6 0 ) 4 2 -7 0 .[10] KLOFT, W . , KUNKEL, I I ., Verhdl. 11. Int. Kongr. Entom ol. W ien 1960 1 (1901) 746 -49 .[11] KUNKEL, H . , D octora l thesis, Bonn (1966) 172.[12] DANNEEL, I . , Z . angew. Z o o l . 54 (1967) 181 -82 .L13] LAMB, К .Г . , E1IR11ARDT, P . , MOERICKE, V . , Nature, Loud. 2 1 4 (1 9 0 7 ) 002.[14] EIIRIIARDT, P . , LAMB, K. P . , Int.J . appl. Radiât. Isotopes 18 (1967) 543 -44 .[15] EHRHARDT, P . , Experientia, in the press.[16] DANNEEL, I . , unpublished.[17] DANNEEL, I . , Z . angew. Z o o l . , in the press.[18 ] SCHLAGBAUER, A . , Int. J. appl. Radiat. Isotopes 18 (1967) 505 -60 3 .[19] MILES, P. W. , Nature 183 (1959) 756.[20] KLOFT, W . , EHRHARDT, P . , Naturwissenschaften 46 (1959) 586 -87 .[21] LIESERING, R . , Z . Pflam enkrankheiten (Pflanzenpathol. ) und Pflanxenschutz 67 (I9 60 ) 524 -42 . L22] KLOFT, W . , EHRHARDT, P ., Phytopatliol. Z . 35 (1959) 401 -10 .[23] KLOFT, W . , SCHULZE, E. - F . , Atom praxis 13 (1967) 265 -69 .[24] KUNKEL, H . , Z . angew. Z o o l . , in the press.[25 ] KUNKEL, I I . , Phytopathol. Z . , in the press.

D I S C U S S I O N

R. C A VA L L O R O : W ere the A p h id idae c o lo n ie s you stud ied o f m ixedc o m p o s it io n , conta in in g c e r ta in p r o p o rt io n s o f sex u a l fo r m s , w in g le ss p a rth en og en etic fe m a le s and pupae, o r did they c o n s is t s o le ly o f in se c ts at on e stage o f d ev e lop m en t? T h is s e e m s to m e im portan t s in c e , fr o m a r a d io iso to p e study 011 trop h ic re la t io n sh ip s betw een M yzus p e r s ie a e S u lz e r and the h ost plant N icotian a tabac urn Sam s un w hich I have been con d u ctin g s in ce 19G1, I have found that the feed in g b eh a v iou r o f the in se c t v a r ie s depending on what stage it is in.

S M - 102/4 29

I sh ou ld a lso w e lco m e d eta ils oi' the a r t i f ic ia l n u tritiv e m ed iu m you u sed , in p a r t icu la r on its pH and its e f fe c t on en d osy m b ion ts o f the ap h id s.

F in a lly , I sh ou ld lik e to c o m m e n t on 131I d is tr ib u tion in in s e c ts . M y w ork at the E u ratom r e s e a r c h c e n tre at Isp ra has show n that th is is o to p e c o n c e n tra te s in c o n s id e r a b le qu an tities in the c u t ic le , as w e ll as in the fat b o d ie s . T h e se s tu d ies c o v e r e d a la rg e n um ber o f s p e c ie s , in clu d in g the O rth op tera C o n o ce p h a lid a e , C ry llid a e and G ry llo ta lp id a e , the L e p id o p te ra N octu id a e , T h a u m etop oe id a e , S a tu rn iid ae , L a s io ca m p id a e , P a p ilio m id a e and P ie r id a e and the H y m en op tera A rg id a e .

W. J . K L O F T : O u r n o rm a l b a s is fo r any c o m p a r is o n is the b e h a v io u r ,fo o d uptake, h oneydew s e c r e t io n , e tc . o f adult w in g le ss p a rth en og en etic fe m a le s ; up to now m o st o f o u r w ork has been w ith th is fo r m . In a b so lu te te r m s o f c o u r s e , the ra te o f fo o d uptake by la rv a e is lo w e r , but in r e la tion to bod y w eight it is h ig h er . T h is is a lso true o f h oneydew s e c r e t io n (se e M itt le r 's w ork on T u b e ro la ch m u s and P . F h rh a r d t 's on M egou ra v i c ia e ) . At p re se n t we have lit t le k n ow ledge o f the fo o d uptake o f w in ged fo r m s ; the on ly study we have m ade w ith A p h is fab ac w as to d e term in e the la st fo o d uptake o f nym phs b e fo r e , and the f ir s t o f the adult a fte r , m ou ltin g to the w in ged sta g e . T h is is im p ortan t in the con tex t o f v iru s v e c to r in g by the w inged fo r m .

T he pH v a lu es o f the m ed iu m w e re c lo s e to n eu tra l, but s lig h tly a lk a lin e (pH Т .2 -7 .4 ). I do not know about any e ffe c t o f pH v a r ia t io n s on e n d o sy m b io n ts ; fo r in fo rm a tio n on th is point you sh ou ld co n ta ct m y c o lle a g u e , P . E h rh ard t.

T o y ou r co m m en t on 131I co n ce n tra tio n , I w ould add that r e su lts have a lso b een obta in ed by m y c o lle a g u e , A . S clilag ba u er (P h . D. T h e s is ,Bonn, 19G7), on the s to ra g e o f 131I in the c u t ic le o f v a r io u s I le te ro p te ra .T he is o to p e is bound in the lip o p ro te in s o f the c u t ic le , and a fte r tanning th is b e c o m e s i r r e v e r s ib le (se e a ls o v a r io u s p a p e rs by M m e. F u zea u - B r a e s ch ).

R a ch e l G ALU N : M itt le r found that the ph ag ostim u lan ts o f aphidsa r e am in o a c id s and s u c r o s e , w hich a r e p r e se n t in a ll p la n ts . H ow, th e r e fo r e , do the ep h ip h a ryn g ea l r e c e p t o r s d iffe re n t ia te a c ce p ta b le and u n a ccep ta b le p la n ts?

W. J. K L O F T : A p a rt fr o m the su b s ta n ce s you m en tion , m in e ra l sa ltsa lso have a g u s ta to ry e f fe c t , as E h rh ardt has show n. H o w e v e r , a l l th ese su b s ta n ce s a re co m m o n p h loem sap com p ou n d s w hich can be found in p lants o f d iffe re n t s p e c ie s and even g e n e ra o r fa m ilie s . T h e m o s t im p ortant fa c to r in d is tin g u ish in g h ost-p lan ts fr o m n o n -h o st-p la n ts s e e m s to be the e x is te n c e o f s e c o n d a r y plant su b s ta n ce s , p r e se n t in v e r y sm a ll qu an tities and as yet v ir tu a lly unknown. E . W e n s le r has found, fo r e x a m p le , that the ca b b a g e aphid , B r e v ic o r y n e b r a s s ic a e , is a ttra c te d by m u stard o i l s , e s p e c ia l ly s in ig r in . In this c a s e , h o w e v e r , an oth er s e n s o r y o rga n is in vo lved ; th is is situ ated on the tip o f the la b iu m and a llo w s d is c r im in a tion fr o m ou ts id e .

R a ch e l G A LU N : How did you d iffe re n t ia te betw een plant and aphidtra n sp ira tio n ?

W. J . K L O F T : T he e f fe c t s o f feed in g on plant tra n sp ira tio n can bed e te cte d 5 -1 0 m in u tes a fte r pu n ctu rin g by the ap hids has begun. A s we m en tion in the p a p e r , uptake o f p h loem sap can b eg in at the e a r lie s t 8 -1 0 m inutes a fte r p u n ctu rin g ; h o w e v e r , fo r an aphid to in g est, a b so r b , and beg in tr a n sp ir in g tr it ia te d w a ter n eeds at le a s t 30 m in u tes . A s we

30 KLOFT et a l .

r e m o v e o u r aphids fr o m the plant a fte r a sh o rt t im e , no d if f icu lt ie s a r is e in d istin gu ish in g the tw o ty p es o f tra n sp ira tio n . A m uch m o r e im portan t s o u r c e o f e r r o r w ould be the s e c r e t io n o f tr it ia te d w ater with the h on ey dew , w hich b e g in s on ly 2 -2 \ h ou rs a fte r the s ta rt o f feed in g .

R a ch e l G ALU N : Is it not p o s s ib le that the in c r e a s e d tra n sp ira tio nra te o f the p lants is due s im p ly to w ounding, ra th e r than to any s p e c i f i c e f fe c t o f the ap h id s?

W . J . K L O F T : I have dea lt w ith th is a s p e c t in Z . an gew . E n tom ol. 45(1960) 3 3 7 -8 1 . The w ounding e f fe c t is in deed p re se n t, but m o r e im p ortan t a r e the fr e e am in o a c id s in the a p h id s ' sa liv a . T h e ir am ount and sp e c tru m a r e c lo s e ly re la te d to the p h yto tox ic e f fe c t o f aphids on tra n sp ira tio n , p h o to sy n th e s is and r e s p ir a t io n o f plant t is s u e s (K loft and E h rh a rd t, P h ytop ath o l. Z . ^5 (1959) 401).

W . J . L E QUESNE: Y ou m en tion in y o u r p a p er that sod iu m a ce ta te la b e lle d w ith c a r b o n - 14 is c o n v e r te d into c h o le s te r o l i f en d osy m b ion ts a r e p r e se n t . Is it a lso p o s s ib le that e ith e r the in se c t o r the en d osy m b ion t c o u ld c o n v e rt plant s t e r o id s , su ch a s s i t o s t e r o l o r s t ig m a s te r o l , into a n im a l s t e r o id s , such as c h o le s te r o l?

W . J . K L O F T : T h is m igh t be p o s s ib le , but we have not yet s ta rte d e x p e r im e n ts w ith p h y to s te ro ls . A s we have been ab le to sh ow , a r t i f ic ia l feed in g o f aphids op en s up a w ide ran ge o f e x p e rim e n ta l s tu d ies on b io sy n th e s is in in s e c ts .

K. N. M E H R O T R A : S in ce th is e f fe c t w as v e r y c le a r ly d em on stra ted in the c a s e o f phytophagous in s e c ts , e s p e c ia l ly lo c u s ts , I w ould think that s im ila r ph enom ena m u st a lso take p la ce in aphids.

F . T . P H IL L IP S : What ran ge o f w ind sp e e d s w as u sed in the t r a n s p ira tio n e x p e r im e n ts w ith tr it ia te d w a te r?

W. J . K L O F T : W e had w ind sp e e d s ran gin g fr o m 0 to a p p ro x im a te ly0 .5 m / s (1 m ph). T h e w ind sp e e d ca n .b e ch an ged , but is a lw a ys p r e c i s e ly c o n t r o lle d and m e a su re d w ith a s p e c ia l flow sp eed m e te r .

II. M A R C H A R T (C h a irm an ): I take it that the am ount o f Н гО vap ou rlo s t by e v a p o ra tio n fr o m the in s e c ts w hich you in trod u ced into the test c h a m b e r fo r the tra n sp ira tio n s tu d ies w as s m a ll c o m p a re d w ith the w ater lo s s fr o m the le a v e s , so that th e re w as no change in the ra te o f le a f tra n sp ira tio n ca u se d by a lte ra tio n o f the am bien t h um idity .

W . J . K L O F T : T h e w ater lo s s o f the aphids is in deed e x tr e m e lylow i f c o m p a re d with the l e a f 's tra n sp ira tio n .

II. M A R C H A R T : H ave you c o n s id e r e d the a p p lica tion o f y o u r m eth od sto the study o f s y s te m ic a n ti-fe e d in g co m p o u n d s?

W. J . K L O F T : Not s p e c i f i c a l ly . We a re at p re se n t p e r fo r m in g ad eta ile d a n a ly s is o f a ll o u r fo o d uptake data, w ith a v ie w to finding the a r t i f i c ia l nutrient m ed iu m w h ich is m o s t a ttra c tiv e to the in s e c ts . We have s ta r te d w o rk w ith s y s te m ic in s e c t ic id e s , and a re studying th e ir e f fe c t on fo o d uptake by m e a su rin g the a c t iv ity o f the ap h id s. T he s p e c if i c a c t iv ity o f the so lu tio n s u sed is a lw a ys e x a c t ly the sa m e . An advantage o f o u r tech n iqu e is that w e do not c o m e up aga in st the p r o b le m o f m e ta b o l is m in the plant - w e a re w ork in g w ith an a r t i f ic ia l sy s te m w h ich has no m e ta b o lis m , so that the in s e c t ic id e com p ou n d and the m e ta b o lite s fo r m e d can be ch e ck e d fo r both in s e c t ic id a l and a n ti-fe e d in g e f fe c t s .M iss D an neel, who is m en tion ed in the r e fe r e n c e s to o u r p a p e r , is w ork in g on th is .

RADIOISOTOPES IN STUDIES ON THE ECOLOGY OF TICK VECTORS OF DISEASE*

D .E . SONENSHINE OLD DOMINION COLLEGE,NORFOLK, Va. ,UNITED STATES OF AMERICA

Abstract

RADIOISOTOPES IN STUDIES ON THE ECOLOGY OF TICK VECTORS OF DISEASE. This paper dem onstrates the feas ib ility o f mass rearing o f ra d io isotope-tagged im m ature ticks by c o lle c t in g the progeny o f engorged fem a les o f three species inoculated with ca rb on -1 4 g lu cose or g ly c in e prior to ov iposition .The incorporation o f rad ioch em ica ls internally into ticks reduces the ch a n ce o f loss o f the radioactive label when m oulting occu rs, or under natural conditions.

Most treated ticks laid eggs. The amount o f rad ioactiv ity in the progeny cou ld be con trolled by con tro llin g the size o f the dose adm inistered to the parent ticks. H ow ever, d ifferen ces in radioactiv ity in the progeny o f treated ticks in relation to the day o f ov ip os ilion were noted . This a ctiv ity d eclin ed progressively through the ninth day . D ifferences were also noted in relation to the ra d ioch em ica l used.Most o f the carb on -1 4 g ly c in e (7 9 .0 °]o) rece ived by the engorged fem a les rem ained in the parents, whereas most o f the ca rbon -14 g lucose (7 7 . fpjo) rece ived was transferred to the progeny. H atching o f eggs labe lled by this method was less than in untreated oviposits . Radiosensitivity in the eggs was also noted and was related to the size o f the dose adm inistered to the parent t ick . No hatching occu rred when the average radioactiv ity o f the la b e lled eggs ex ceed ed 637 counts/m in per egg over background. N evertheless, m any h ighly radioactive eggs hatched , and larvae with counts as high as 510 counts/m in per larva over background were observed. The b io lo g ic a l characteristics o f the tagged larvae were apparently unaffected by in corp o ration o f rad iochem ica ls into these individuals. The proportion o f marked larvae w hich attached to hosts was sim ilar to the proportion o f unmarked larvae w hich attached. The duration o f survival o f fasting, rad io isotope-tagged larvae, under laboratory con d ition s , was sim ilar to the period o f survival o f nonradioactive larvae. N o apparent loss in rad ioactiv ity in fasting larvae held for up to 70 days under laboratory conditions was d e le cted .

Ib is dem onstration o f the fea s ib ility o f mass rearing and lon g -te rm survival o f rad io isotope-tagged im m ature ticks suggests that it is now possible to apply this ra d io e co lo g ica l techn ique to obtain important new knowledge, on the e co lo g y o f tick vectors o f disease.


T he ap p lica tion o f r a d io is o to p e la b e ll in g m eth od s to stu d ie s with t ick s w as c o n s id e r e d by s e v e r a l w o r k e r s , n otab ly Knapp et a l. [1 ], Quan et al.[2], Hyland and H am m er [3], B abenko [4] and S a m o s tr e ls k ii and D a iter [5]. M o re r e c e n t ly , Sonensh ine and Y un ker [6] d e m o n stra te d the fe a s ib i lity o f m a s s p rod u ction o f r a d io is o to p e -ta g g e d im m a tu re t ick s by in ocu la tion o f e n g o rg e d fe m a le s p r io r to eg g la y in g , w h ile Sonensh ine and C lark [7 ] sh ow ed that it w as p o s s ib le to r e ca p tu re th e se im m a tu re s .

T o r e a l iz e fu lly the p oten tia l u se fu ln e ss o f the r a d io is o to p e tagg in g m eth od , it is n e c e s s a r y to d e m o n stra te the fe a s ib ility o f m a ss p rod u ction o f adequ ately m a rk e d in d iv id u a ls o f the v e c t o r s p e c ie s , at low c o s t , and w ith a la b e l that m ay be r e g a rd e d as m o r e o r le s s p erm a n en t fo r that stad iu m . It is a lso e s s e n t ia l that the b io lo g ic a l c h a r a c t e r is t ic s o f la b e lle d

* This study was supported by the United States A tom ic Energy C om m ission under Contract No. A T (4 0 -] ) 3534.

31

32 SONENSUINE

in d iv id u a ls , e . g . h o s t -s e e k in g b e h a v io u r , s u r v iv a l in the n atu ral en v iron m en t and re la te d a ttr ib u tes , be s im ila r to th o se o f n o n -la b e lle d t ic k s . F in a lly , it m u st be d e m o n stra te d that the ta gged s p e c im e n s r e le a s e d in a n atu ral a re a m ay be re ca p tu re d in adequate n u m b ers and r e a d ily id e n tifie d .

T h is p a p er d e s c r ib e s the la b o r a to r y s tu d ies w hich w e re p e r fo r m e d to d e te rm in e the fe a s ib i l it y o f ap p lying r a d io is o to p e ta gg in g m eth od s to the study o f the e c o lo g y o f t ick s . M o re s p e c i f i c a l ly , it d e s c r ib e s the r e s u lt s o f la b o r a to r y stu d ies on the e f f ic ie n c y o f the la b e ll in g m eth od , ra d io s e n s it iv ity , h atch ing , tra n sta d ia l t r a n s m is s io n , and a co m p a r is o n o f the b io lo g ic a l c h a r a c t e r is t ic s o f m a rk ed and u nm ark ed p op u la tion s .F ie ld t r ia ls to d e te rm in e the u se fu ln e ss o f the tech n iqu e u nder n atu ral c o n d itio n s a re d e s c r ib e d e ls e w h e r e [7].

2. M A T E R IA L S AND M ETH O D S

T h re e s p e c ie s o f t ick s w e re u sed in th e se in v e s tig a t io n s , n am ely D e rm a c e n to r v a r ia b il is (Say), D e rm a ce n to r an d erson i S tile s , and A m b ly o m m a a m erica n u m (L in n a eu s). T ic k s w e re h e ld in an in cu b a torat 80 ± 2 ° F and 95% r . h. , du rin g eg g la y in g , h atch ing , o r m ou ltin g p e r io d s . R a d io a ctiv ity m e a su re m e n ts w e re done with a N u cle a r C h icag o M od el 470 gas flow d e te c to r and M od el 8166 d eca d e s c a le r .

In o cu la tion s w e re m ade a c c o rd in g to the m eth od d e s c r ib e d by Son en sh in e and Y u n k er [6 ]. E n g org ed fe m a le t ick s w e re p o s it io n e d on a b in o cu la r s t e r e o s c o p ic m ic r o s c o p e stag e with the aid o f p la s t ic in e , and th is a s s e m b ly w as p la ce d a d a jcen t to a m icr o m a n ip u la to r . A 50-jul H am ilton sy r in g e with a 3 0 -g a u g e n e e d le , con ta in in g the d e s ir e d v o lu m e o f so lu tion , w as fitted onto the m icro m a n ip u la to r and the m ic r o m e t e r c o n t r o ls w e re used to gu ide the n eed le in to the bod y o f the t ick . T h e so lu t io n s in ocu la ted in the e a r l ie r ph ase o f th is study w e re not o s m o t ic a l ly b a la n ce d ; th o se in o cu la te d la te r in the study w e re b a la n ce d by the ad d ition o f sa lin e to a p p rox im a te a co n ce n tra tio n o f 1. 0% N aC l.

E g g s c o l le c te d f o r r a d io a ct iv ity d e te rm in a tio n s w e re s e le c t e d at ran dom and se g re g a te d in to sa m p le s o f 25, and cou n ts fo r ea ch sa m p le w e re r e c o r d e d Cor 3 -m in u te p e r io d s . T h re e sa m p le s o f 25 e g g s each w e re draw n fr o m each o v ip o s it . O v ip o s its w e re c o l le c t e d fr o m e g g - la y in g t ick s on days 1, 5, 9 and 13 o f the o v ip o s it io n p e r io d and ch e ck e d fo r r a d io a c t iv ity as d e s c r ib e d ab ove .

T h e to ta l n u m ber o f e g g s in an o v ip o s it w as d e te rm in e d by o b s e rv in g the w eigh t o f a known n um ber o f ra n d om ly s e le c te d e g g s , to d e te rm in e the a v e ra g e w eigh t p e r eg g , and then w eigh in g the e n tire eg g m a s s . The to ta l n u m ber o f eg gs in the m a ss w as com p u ted fr o m th ese tw o f ig u r e s . W eight d e term in a tion s w e re m ade with a M e ttlcr m ic r o g r a m b a la n ce . The p r o p o r t io n o f e g g s w hich h atch ed w as d e te rm in e d by cou n tin g unhatched e g g s .

T he e f f ic ie n c y o f the r a d io la b e llin g p r o c e s s w as eva lu ated . T he a c c u r a c y o f the ra d io a c t iv ity m e a su re m e n ts r e p o r te d fo r the c o m m e r c ia l r a d io a c t iv e p re p a ra t io n s u sed in th is study w as d e te rm in e d by p re c ip ita tin g 100 la m b d a a liq u ots , a p p ro p r ia te ly d ilu ted on g re a s e d p la n ch ets , and fo llo w in g each tr a n s fe r w ith tw o r in s e s o f the la m bd a p ip e ttes with d is t il le d w a ter , and d ry in g s lo w ly u nder an in fr a -r e d la m p . T h re e sa m p le s

S M -102/ñ 33

w e re p r e p a re d fr o m each o f tw o c o m m e r c ia l p re p a ra t io n s r e p o r te d to con ta in 50 Ci o f 14C -la b e lle d g ly c in e . F o llo w in g the a ssa y o f the c o m m e r c ia l p re p a ra t io n s , 100 la m bd a a liqu ots (undiluted) o f the sa m e a s sa y e d so lu tio n s w e re w ithdraw n and added to the d is s o lv e d , n e u tra liz e d b od y con ten ts o f each o f th ree e n g o rg e d fe m a le t ick s w hich had not r e c e iv e d any r a d io is o to p e . S am ples w e re then w ithdraw n fr o m each o f th e se new p re p a ra t io n s , d ilu ted and ch e ck e d fo r r a d io a c t iv ity in the sa m e m an n er as ab ove . In add ition , t ick s r e c e iv in g in o cu la tio n s o f known co n ce n tra tio n s o f r a d io c h e m ic a ls w e re d is s o lv e d in a c id p r io r to o v ip o s it io n , d ilu ted and n e u tra liz e d , and a liqu ots w e re p re c ip ita te d onto p la n ch ets . F in a lly , the e n tire o v ip o s its o f in ocu la ted e g g -la y in g p a ren ts w e re c o lle c te d , d is s o lv e d and n e u tra liz e d , and a liqu ots w e re w ithdraw n fo r c o m p a r is o n o f th is m od e o f r a d io a ct iv ity m e a su re m e n t with the m e a su re m e n ts m ade u sin g e n tire e g g s .

C hanges in r a d io a ct iv ity in t ick m a te r ia l du rin g the p r o c e s s e s o f (1) hatch ing , and (2) m ou ltin g fr o m la rv a to nym ph w ere stud ied by d e te rm in in g the ra d io a c t iv ity o f each eg g , e n g o rg e d la rv a , subsequent stadium and shed m a te r ia l .

C o m p a r iso n o f b a s ic b io lo g ic a l c h a r a c t e r is t ic s o f ra d io la b e lle d t ick s w ith u n trea ted t ick s w as m ade with D. v a r ia b i l is . T he ph enom ena stud ied in clu d ed (1) w illin g n e ss to feed , (2) lo n g e v ity and (3) r e te n t io n o f r a d io a c t iv ity . F ou rteen g rou p s o f 50 unfed la rv a e , each sev en days o ld and h e ld at 95% r .h . and 80 ± 2°F , with in c r e a s in g da ily in cr e m e n ts o f in ca n d e sce n t illu m in a tion , w e re e x p o se d to a lb in o r a ts . One h a lf o f the sa m p le g rou p s w e re draw n fr o m the se co n d -th ro u g h -fo u r th day r a d io is o to p e -ta g g e d p ro g e n y o f a s in g le fe m a le . T he re m a in in g t ick s w e re n o n -r a d io a c t iv e in d iv id u a ls draw n at ran dom fr o m u n trea ted p a ren ts . A ll la rv a e w e re co n fin e d se p a ra te ly , 5 0 /r a t , by m ea n s o f m u slin clo th around each an im al. E ach an im al w as h e ld o v e r w ater in a sep a ra te tra y . T he c lo th w as r e m o v e d a fte r 24 h o u rs . E n g o rg e d la rv a e w e re c o l le c te d and the n u m ber o f t ick s r e c o v e r e d w as r e c o r d e d . L on gev ity w as stud ied by c o lle c t in g sa m p le s o f 100 la rv a e fr o m both la b e lle d and n o n -la b e lle d p opu la tion s im m ed ia te ly , a fte r h atch ing . T h e t ick s w ere o b s e r v e d e a ch day f o r e v id e n ce o f m o r ta lity until a ll w e re dead . R eten tion o f r a d io a ct iv ity w as stu d ied by h old in g s to ck pop u la tion s o f 100 la rv a e and w ith draw in g 10 in d iv id u a ls at 2 -w e e k in te r v a ls , k illin g them with c h lo r o fo r m and d e te rm in in g th e ir ra d io a c t iv ity . T h is p r o c e d u r e w as n e c e s s a r y s in c e a n aesth etic te ch n iq u es w e re u n re lia b le and c a r e had to b e taken to avo id con ta m in a tion o f the d e te c to r sy s te m by e sca p in g la rv a e . T h e se s tu d ies w e re con tin u ed fo r 70 days.

3. RE SU L T S

M ass p ro d u ctio n o f r a d io la b e lle d la rv a e and nym phs o f th ree s p e c ie s by in ocu la tion o f p r e o v ip o s it in g fe m a le t ick s w as a c c o m p lis h e d . In form ation w as obta in ed on the fo llo w in g a s p e c ts : the re la t io n s h ip betw een the a ctiv ity o f the d o s in g so lu tion and r a d io a ct iv ity in the p ro g e n y o f trea ted p a re n ts ; the d e c lin e in ra d io a ct iv a tio n o f p rogen y du rin g o v ip o s it io n ; r a d io s e n s it iv ity ; d if fe r e n c e s in r a d io a ct iv ity in im m a tu re s in re la t io n to the r a d io c h e m ic a l u sed ; the to ta l n u m ber o f eg g s p ro d u ce d and the

3

34 SONENSHINE

p e r cen t hatch ing ; lo s s o f a ctiv ity with h atch ing , fe e d in g and m ou ltin g ; b io lo g ic a l c h a r a c t e r is t ic s o f tagged t ick s .

In o cu la tion s . A ll but tw o o f the 32 e n g org ed fe m a le D. v a r ia b ilis in ocu la ted with b u ffe re d sa lin e so lu tion s con ta in in g fr o m 12. 5 to 25. O yC i 14C -g lu c o s e la id eg g s . A ll o v ip o s it in g tre a te d p a ren ts tr a n so v a r ia lly tra n sm itte d la rg e am ounts o f r a d io c h e m ic a l to th e ir p rog en y . The a v e ra g e ra d io a ct iv ity d e te cte d in day 1 o v ip o s its ran ged fr o m 55. 8 c o u n ts /m in p e r e g g to 976. 9 c o u n ts /m in p e r egg , w ith a m ean activ ity o f 3 9 3 .6 co u n ts /m in p er egg.

R a d io a ctiv ity o f p rogen y in re la t io n to d o s e . T he tr a n s fe r o f r a d io is o to p ic m a te r ia l to the p rogen y o f tre a te d t ick s w as stud ied by d e te rm in in g the r a d io a ct iv ity o f ra n d om ly s e le c te d sa m p le s o f th e ir e g g s and la rv a e .T he r e s u lts o f th e se d e term in a tion s fo r D. v a r ia b il is a re show n in F ig . 1.

10,000 9000 -■

8000 -

7000 -

6000|-

5000 '

1000 9 0 0 ,

800 -

700 :

6 0 0 -

5 0 0 1

RADIOACTIVITY IN D. V A R IA B IL IS EGGS AND LARVAE

IN RELATION TO DAY OF OVIPOSITION

Legend

= E g g s w ith g lu co se

= E g g s w ith g ly c in e

= L o rvo e w ith g ly c in e

J_____1DAY OF O V IP O S IT IO N

FIG. 1. Graphs showing changes in rad ioactiv ity in eggs and larvae from ca rb on -1 4 treated D . variabilis fem ales in relation to day o f oviposition . D ata for larvae from glucose 14C -treated parents was not ava ilab le . A ll estim ates corrected for d iffe ren ce in e f fic ie n cy betw een d irect counting (ra d ioa ctiv ity ) and counting from dissolved m aterial.

SM -102/5 35

T h e se v a lu es m u st be r e g a rd e d as r e la t iv e e s t im a te s , s in ce m e a su re m e n t o f r a d io a ct iv ity in th is m od e (w hole e g g s ) w as in e ffic ie n t as c o m p a re d with p re c ip ita te s fr o m d is s o lv e d tick m a te r ia l . T h e in co r p o r a t io n o f r a d io c h e m ica l in to e g g s and la rv a e sh ow ed a re la t io n s h ip (1) with the p e r io d o f o v ip o s it io n , (2) with the a ctiv ity o f the r a d io c h e m ic a l so lu tion in ocu la ted in to the p a ren t, and (3) with the r a d io c h e m ic a l u sed . The v a r ia t io n in a ctiv ity in the p rog en y in re la t io n to the p e r io d o f the o v ip o s it io n p r o c e s s in w hich the e g g s w e re d e p o s ite d w as d e te rm in e d by c o lle c t in g the o v ip o s its o f days 1, 5, 9, and 13. F ig u re 1 sh ow s the a ctiv ity p r e s e n t in e g g sa m p le s at th e se fo u r d iffe re n t t im e p e r io d s , based upon id e n tica l s iz e sa m p le s fr o m 33 D_. v a r ia b il is fe m a le s in ocu la ted with 14C -la b e l le d g ly c in e and 30 D. v a r ia b il is in ocu la ted with 14C -la b e l le d g lu c o s e . The r a d io a c t iv ity o f the g ly c in e in o cu la tio n s ra n ged fro m 10. 5 to 60. 0/uCi p e r t ick , and that o f the g lu c o se in o cu la tio n s fr o m 12. 5 to' 25. 0 n C i. In the g ly c in e tre a te d t ick s , the a v e ra g e a c t iv ity p e r eg g w as g re a te s t on the f i r s t day o f o v ip o s it io n , and th is a c t iv ity d e c lin e d r e g u la r ly until the ninth day. T h e re a fte r , the change in a ctiv ity w as v e ry s ligh t, and the a v e ra g e a c t iv ity p e r eg g on day 13 w as s lig h tly h igh er than on day 9 o f the o v ip o s it io n p e r io d . The sa m e tren d is apparent in the g lu c o se tre a te d t ick s , but the d e c lin e in a c tiv ity w as m o r e ra p id betw een the f i r s t and the fifth day than w as o b s e r v e d in the c a s e o f the g ly c in e in o cu la tio n s .

T o c o m p a re the re la t io n sh ip betw een the a v e ra g e a c t iv ity in co r p o r a te d in to the eg gs and the s iz e o f the d o se a d m in is te re d to each t ick , d o s e s o f 10. 5 /nCi, 25. 0 /uCi, 50 /uC i, and 60 ;uCi o f 14C g ly c in e , and 6. 5 /nCi,12. 5 jnCi and 25. 0 ^ C i o f 14C g lu c o s e w e re a d m in is te re d to the e n g org ed fe m a le s . E ach in o cu la tio n at a g iven d o s e le v e l w as r e p lic a te d fr o m six to sev en t im e s . T h e t r a n s fe r o f r a d io a c t iv ity fr o m p a ren t to e g g m a ss in re la t io n to th e se g ra d ed d o sa g e s is show n in P’ig s 2 and 3. E ggs obta in ed fr o m p a ren ts in ocu la ted with la rg e d o s e s g e n e ra lly had h igh er a ctiv ity m e a su re m e n ts than th o se obta in ed fr o m p a ren ts in ocu la ted with s m a lle r d o s e s . T h is is m o s t ev iden t in the c a s e o f the g lu c o s e tre a te d t ick s .

S im ila r s tu d ies w e re done w ith D. a n d e rso n i and A. a m e rica n u m ,M ost in ocu la ted fe m a le s o f both s p e c ie s la id eg gs and r a d io a c t iv ity w as r e a d ily d e te cte d in the p ro g e n y , at le a s t th rough the la r v a l stag e .

T o ta l n u m ber o f e g g s p r o d u c e d . T he m ean w eight o f 18 e n g o rg e d D. v a r ia b il is fe m a le s taken im m e d ia te ly a fte r d rop p in g fr o m th e ir h osts w as 639. 1 m g . T h e a v e ra g e w eigh t o f the eg g m a s s e s p ro d u ce d by 25 t ic k s , w h ere it w as p o s s ib le to c o l le c t and w eigh the e n tire o v ip o s it , w as 237. 3 m g . T he a v e ra g e n um ber o f e g g s p ro d u ce d b y 48 o v ip o s it in g in ocu la ted fe m a le s w as 4002 . T h u s , the p a ren t t ick c o n v e r ts a p p ro x im a te ly 37. 1% o f the e n g o rg e d bod y w eigh t in to e g g s . L a rg e e g g m a s s e s w ere a lso p ro d u ce d b y in o cu la te d D. a n d e rso n i and A. a m e rica n u m fe m a le s .T h e a v e ra g e n u m ber o f e g g s p ro d u ce d by the tre a te d D. a n d e rso n i w as 6632 and that p ro d u ce d by tre a te d A. a m erica n u m fe m a le s w as 4463 .

H atching (r a d io s e n s it iv ity ) . L a rv a e h atch ing fr o m r a d io la b e lle d o v ip o s its w e re le s s ra d io a c t iv e than the e g g s fr o m w hich th ey hatched (F ig . 1). T h e a v e ra g e re d u c t io n in r a d io a c t iv ity w as g re a te s t f o r the day 1 o v ip o s its , l e s s f o r the day 5 o v ip o s it s , and le a s t in the la te r day o v ip o s it io n p e r io d s . T he a v e ra g e d ro p in r a d io a ct iv ity r e p re s e n te d a l o s s o f 55% fr o m that p r e se n t in the e g g m a s s , F u rth e r , h igh activ ity , le v e ls w e re co m p le te ly le th a l to the e g g s . A lthough o v ip o s its with

36 S0NENS1 UNE

RADIOACTIVITY IN EGGS IN RELATION TO DOSE (GLYCINE C-14)

FIG. 2. Graphs showing re la tive d ifferences in rad ioactiv ity in eggs from g ly c in e 14 С -treated D. variabilis parents in relation to dose used. Each point represents average o f three sam ples from oviposits o f ea ch o f G to 7 fem a le ticks inoculated w ith the sam e dose. Л11 estim ates corrected for d iffe ren ce in e f f ic ie n cy betw een d irect counting (radioactiv ity ) and counting from dissolved m aterial.

a c t iv it ie s o f up to 976 c o u n ts /m in w ere ob ta in ed , no h atch ing w as o b s e r v e d . In the c a s e o f th ree fe m a le s in o cu la te d w ith 60 juCi o f 14C g ly c in e and th re e w ith 2 5 /líC í o f 14C g lu c o s e , day 1 o v ip o s its w ith a c t iv it ie s o f 7 5 7 .8 c o u n ts /m in p e r eg g , 636. 8 c o u n ts /m in p e r eg g , 686. 0 c o u n ts /m in p e r egg ,933. 0 c o u n ts /m in p e r eg g , 887. 8 c o u n ts /m in p e r eg g , and 976. 9 c o u n ts /m in p e r e g g , r e s p e c t iv e ly , w e re obta in ed . H ow ever , no hatch ing o c c u r r e d in any o f the eg gs p ro d u ce d by th e se fe m a le t ic k s . H atch ing w as c o n s id e r a b le at lo w e r a c t iv ity le v e ls , in m any c a s e s e x ce e d in g 50%.T he re la t io n s h ip betw een the m ean r a d io a c t iv ity o f the e n tire eg g m a s s and the p e r cen t h atch in g w hich o c c u r r e d in the p ro g e n y o f the 14C - tre a te d p a ren ts (g ly c in e and g lu c o s e ) is show n in T a b le I. N ote that the o v e r a l l p e r cen t o f h atch in g w as 50 .4% .

SM -102/5 37

10,0009 .0 0 0

8.0007 .0 0 0

6.000

5 ,0 0 '

4 .0 0 C

3 ,0 0 0

1.0009 0 0

8 0 0

7 0 0

6 0 0

5 0 0

RADIOACTIVITY IN EGGS IN RELATION TO DOSE (GLUCOSE C-14)

ADMIN ISTERED TO PARENT TICKS (D. variabilis)

;I ' - V

Legend

D ose a d m in is te re d

o m m w o = 2 5 m ic ro cu rie s/fem a le

• =12.5 m icrocunes/fem o le

-■•■■•••с = 6 0 m ic rocuries/fem ale

6 7 8 9

DAY OF O V IP O S IT IO N

FIG. 3. Graphs showing re la tive d ifferen ces in rad ioactiv ity in eggs from glucose 14C treated D . variabilis fem ales in relation to dose used. Each point represents average o f three sam ples from oviposits o f each o f ti to 7 fem a le ticks inocu la ted w ith the sam e dose. A ll estim ates corrected for d iffe ren ce in e f fic ie n cy betw een d irect counting (radioactiv ity ) and counting from dissolved m ateria l.

Although a la r g e p r o p o r t io n o f the e g g s d ied , ap paren tly as a r e s u lt o f the le th a l e f fe c t s o f the h igh r a d io a c t iv ity le v e ls u se d , m any in d iv idu a l la r v a l s p e c im e n s w hich h atch ed had high cou n t r a te s . L a r v a e w ith coun ts as h igh as 511 c o u n ts /m in p e r la rv a o v e r b a ck g rou n d w e re d e te cte d . In on e sa m p le , draw n fr o m the p ro g e n y o f a 14C -g lu c o s e tre a te d p a ren t,54% o f the la r v a e had cou n ts h ig h er than 100 c o u n ts /m in p e r la r v a o v e r ba ck g rou n d .

C o m p a r is o n s be tw een r a d io n u c lid e s . T he r a d io a c t iv ity o b s e r v e d in e g g s and la rv a e ob ta in ed fr o m D. v a r ia b il is fe m a le s tre a te d with sev en r a d io c h e m ic a l so lu t io n s , r e p re s e n t in g s ix r a d io n u c lid e s , is show n in T a b le II. In add ition to the high a c t iv it ie s obta in ed in e g g s and la rv a e fr o m p a ren ts tre a te d w ith 14C co m p o u n d s , c o n s id e r a b le a ctiv ity r e la t iv e

38 SONENSHINE

TABLE I

The re la t io n sh ip between hatching o f ra d io a ct iv e egg masses and the amount o f r a d io a c t iv ity adm inistered to engorged female

Dermacentor v a r ia b i l is

A. In ocu la tion s with Carbon-14 g lu cose (uniform ly la b e lle d )

R ep lica te No.

Percent hatching with in ocu la tion s o f :

12.5 ^C i 25 .0 MCi

1 49.8 0 .02 69.1 32.33 73.4 33.44 74.2 50.15 34.8 56.36 - - 13.8

Mean 60.3 31.0

Б. In ocu la tion s w ith Carbon-14 g ly c in e (uniform ly la b e lle d )

R e p lica te No.

Percent hatching with in ocu la tion s o f :

10 .5 jiiCi 2 5 .0 /jC i 50.0juCi 6 0 .0 ^ Ci

1 80.0 57.7 64.8 39.42 - - 58.0 35.6 2 .63 - - 90.9 0 .04 - - 82.0 0 .05 - - 73.0 - -

6 - - 59.8 - -7 54.0 “ “

Mean - - 67.9 50.2 10.5

Dash in d ica tes that the p rop ortion hatching was not determined.

to the sm a ll s iz e o f the d o s e w as d e te cte d in the p rogen y o f t ick s tre a te d w ith s tro n t iu m -9 0 . T h is w as a lso tru e o f t ick s in ocu la ted with p h o s p h o r u s -3 2 , but th o se fe m a le s d ied v e r y q u ick ly a fte r p ro d u c in g sm a ll o v ip o s i t s , and m o s t e g g s fa i le d to hatch . F u rth er stu d ies with 90Sr a re in p r o g r e s s .

E ff ic ie n c y of ra d io la b e llin g te ch n iq u e . K now ledge o f the e f f ic ie n c y o f th is m ethod o f m a ss p rod u ction o f tagged t ick s w as sought to fa c ilita te d e te rm in a tio n o f the d o sa g e fo r any g iven r a d io la b e llin g p r o b le m . T o

SM-102/5 39

TABLE II

Radioactivity in progeny (eggs and larvae) of D. variabilis females inoculated with

seven different radiochemicals

RadiochemicalNo.

Inoculations

Mean activity

Inoculation (pCi)

Mean radioactivity (all ticks)

in CPM over background

Meanall eggs all larvae

Carbon-14 Glycine 33 38.5 91.4 45.0

Carbon-14 Glucose 7 17.5 122.0 93.1

Cerium-144 1 0.04 2.5 *

Cesium-137a 3 0.09 8.5 *

Strontium-90b 4 0.05 18.5 9.5

Phosphorus-32 6 5.8 211.6C 26.0

Glycine 4 25.0 6.0 *

CPM = Counts p e r m inute a Activity determined with gas flow counter. Activity also determined

with solid scintillation detector, and with very similar results.

b Including 1 A. americanum

c Egg laying ceased shortly after day 1, parent ticks died.

* Not determined.

evalu ate th is e f f ic ie n c y , it w as n e c e s s a r y f i r s t (1) to d e te rm in e the a c c u r a c y o f the r a d io a c t iv ity v a lu es r e p o r te d by the m a n u fa c tu re rs o f c o m m e r c ia l p re p a ra t io n s u sed in th is r e s e a r c h , (2) to c h e c k the e f f ic ie n c y o f the sa m p le p re p a ra t io n tech n iq u e , and (3) to d e te rm in e the p r o p o rt io n o f the r a d io c h e m ic a l d o s e a ctu a lly r e c e iv e d by the r e p le te fe m a le t ick p r io r to o v ip o s it io n .

Tw o c o m m e r c ia l p re p a ra t io n s o f the sa m e r a d io c h e m ic a l ( 14 С g ly c in e ) and fro m the sa m e su p p lie r w e re te s te d . T he r e s u lts o f the te s ts in d ica ted a d ev ia tion o f 5. G% and 1. 8%, r e s p e c t iv e ly , fr o m the re p o rte d a ctiv ity p r e se n t in ea ch s o u r c e so lu tion . T h e e f f ic ie n c y o f the sa m p le p re p a ra tio n tech n iq u e w as ch e ck e d by u sin g 100 la m bd a a liq u ots o f the p r e v io u s ly a ssa y e d c o m m e r c ia l p r e p a r a t io n s , added to ea ch o f th ree n o n -r a d io a c t iv e fe m a le tick so lu t io n s . S ince no in o cu la tio n s w e re m ade

4 0 SONENSHINE

TABLE III

Efficiency of the preparation technique for determining the amount of radioactivity in engorged female Dermacentor variabilis*

Radioactivity in counts per minute over background

Sample No. Tick No. 1 Tick No. 2 Tick No. 3

1 3,990.3 4,365.4 4,379.8

2 2,938.0 4,536.9 4,907.5

3 9,644.7 4,878.3 4,530.3

Mean 4,605.8 4,593.5 4,605.8

Mean all 3 tick solutions - 4,614 CPM over background. 22,377.9 DHM.

True activity =

Expected activity (based on an assay value of 50.9 microcuries in original solution) = 6,650.9 + 67 counts per minute, or 0.0145 micro-cur íes.

Observed activity (in microcuries) = 0.0101 microcuries.

Efficiency of detection is 69.7%.

* 100 lambda aliquots of the radioactive glycine source solution were withdrawn and pipetted into each fully dissolved, neutralized solution, followed by 2 rinses. Samples were prepared after allowing for equilibrium to be reached.

D P M = Disintegrations per minute

in the c a s e o f th ese th re e in d iv idu a l t ic k s , it sh ou ld have been p o s s ib le to d e te ct a ll o f the added a c tiv ity fo llo w in g a p p ro p r ia te d ilu tion s to p e r m it p re p a ra t io n o f sa m p le s o f m in im a l th ick n e ss . In fa c t , on ly 69. 7% o f the p r e d ic te d a ctiv ity w as d e te cte d {T a b le III). The sa m p le th ick n e ss o f the p re p a ra t io n s stu d ied ra n ged fr o m 1. 3 to 2. 5 m g / c m 2. A c co r d in g to W ang and W illis [8 ], l o s s o f d e tection e f f ic ie n c y at su ch sa m p le th ic k n e s s e s is on ly about 2 -3 % . H en ce, s e l f -a b s o r p t io n is p ro b a b ly not s ig n ifica n tly in v o lv e d , and the e r r o r d e te rm in e d ab ove is a c ce p te d as in d ica t iv e o f the tru e e f f ic ie n c y e r r o r in ty p ic a l sa m p le s p r e p a re d fr o m a ll the r e p le te fe m a le t ic k s . T h e c o r r e c t io n fa c to r o f 1 .4 4 , com p u ted fr o m th e se data, e x p r e s s e s th is d ev ia tion betw een the a c c u r a c y o f the a ssa y o f p u re r a d io c h e m ic a l v e r s u s the m ix tu re o f r a d io c h e m ic a l and

SM-102/5 41

TABLE IV

Efficiency of technique of direct inoculation of Carbon-14 labelled compounds

into engorged female ticks

Radioactivity(Microcuries)reportedinoculated

Estimated activity in tick

(microcuries)

Percentefficiency(uncorrected)

Percentefficiency(corrected)

10.5 0.182 1.7 2.5

12.5 0.318 2.5 3.6

12.5 1.260 10.1 14.5

12.5 1.818 14.5 20.9

12.5 1.206 9.7 14.0

25.0 3.974 15.9 22.9

25.0 4.030 16.1 23.2

25.0 2.944 11.8 17.1

60.0 10.800 18.0 25.9

Mean 24.2 2.948 11.1 17.7

tick m a te r ia l , and th is c o r r e c t io n w as ap p lied to a ll the e f f ic ie n c y e s t im a te s g iven be low and in T a b le s IV and V.

T he r e s u lts o f the d e te rm in a tio n o f r a d io a c t iv ity p r e se n t in in ocu la ted e n g o rg e d fe m a le t ick s p r io r to o v ip o s it io n a re show n in T a b le IV . N ine t ick s w e re stu d ied . T he c o r r e c t e d e s t im a te s o f the e f f ic ie n c y o f th ese in o cu la tio n s v a r ie d fr o m 3. 6 to 25. 9%, w ith a m ean o f 17. 7%. In ten o th er t ick s in w hich the e f f ic ie n c y o f t r a n s m is s io n to e g g s w as d e te rm in e d , the cu m u la tiv e to ta l o f a c t iv ity in both e g g s and spent p a ren ts in d ica ted an in o cu la tio n e f f ic ie n c y o f betw een 15. 3% and 75. 6%. T h e o v e r a l l a v e ra g e e f f ic ie n c y o f in o cu la tio n , b a se d on a ll 19 s p e c im e n s , w as es t im a te d at 35. 1%.

Data on the r e la t iv e e f f ic ie n c y o f t r a n s o v a r ia l t r a n s m is s io n o f the tw o r a d io c h e m ic a ls u sed by in ocu la ted p a ren ts w e re obta in ed and are su m m a r iz e d in T a b le V . O nly t ick s in w hich the e n tire eg g m a ss cou ld b e c o l le c t e d and in w hich a ty p ic a l s iz e o v ip o s it o c c u r r e d w e re u sed . T h e e n tire eg g m a s s e s w e re d is s o lv e d p r io r to h atch ing . T he m ea n e f f ic ie n c y o f tr a n s o v a r ia l t r a n s m is s io n o f r a d io g lu c o s e and r a d io g ly c in e in the 10 t ick s stu d ied w as 60. 6%. T he m ean p r o p o r t io n o f the o r ig in a l in o cu la tio n

42 SONENSHINE

TABLE V

Transovarial transmission of radiochemicals (Carbon-14 labelled glycine and glucose) in

Dermacentor variabilis

Radioactivity(micro

Activity in microcuries* Efficiency (%)

Total3 Total Total Efficiency Efficiency b Efficiencyccuries) activity remain trans of inocu of trans of trans

inocu received ing in ferred lation mission ovariallated (parent) parent to eggs (parent) to eggs transmission

Glycine

60.060.025.0

31.59517.6769.006

27.76012.5487.197

3.8355.1281.809

52.729.536.0

6.48.5 7.2

14.029.020.1

Glucose

12.5 1.915 0.755 1.160 15.3 9.3 60.6

12.5 5.535 1.071 4.464 42.8 35.7 80.7

12.5 9.258 1.539 7.718 74.1 61.7 83.4

12.5 7.718 2.138 5.580 61.7 44.6 72.3

25.0 18.901 2.344 16.557 75.6 66.2 87.5

25.0 18.602 3.882 14.720 74.4 58.8 79.1

25.0 15.958 3.345 12.613 63.8 50.5 79.0

* All estimates corrected by a factor of 1.44 (see text).

a Cumulative total eggs plus spent parent.

k Estimate of proportion of original dose which was incorporated in the

eggs.

c Estimate of the proportion of the dose received by the preovipositing female tick which was transferred to the eggs.

d o s e w hich re a ch e d the e g g s w as 34. 9%. T he to ta l a c t iv ity p r e se n t in both sp en t paren t and to ta l eg g m a s s in d ica te s an a v e ra g e e f f ic ie n c y c o n s id e r a b ly g re a te r than the 17. 7% obta in ed fo r the nine n o n -o v ip o s it in g fe m a le s d e s c r ib e d a b ove . In the la te r g rou p (ten e g g -la y in g fe m a le s ) , the r e s u lt s in d ica ted an in o cu la tio n e f f ic ie n c y o f 52. 6%. It m ay a lso be n oted that in the th re e t ick s in o cu la te d w ith r a d io g ly c in e , the am ount o f r a d io c h e m ic a l r e m a in in g in the sp en t p a ren t w as 4. 6 t im e s the am ount t r a n s fe r r e d to the e g g s . H ow ever , in the t ick s in o cu la te d w ith r a d io g lu c o s e , 77. 5% o f the to ta l a ctiv ity e st im a ted to have been r e c e iv e d b y the p a ren t t ick w as tr a n s fe r r e d to the p rog en y .

F e e d in g . T h e p r o p o rt io n o f r a d io la b e lle d ^ v a r ia b il is la rv a e w hich fe d as co m p a re d with n o n -la b e lle d la rv a e fe e d in g on the sa m e h o st s p e c ie s w as stu d ied . The ra d io a ct iv e la rv a e had coun t r a te s a v e ra g in g

S M - 102/5 43

TABLE VI

Comparison of the Feeding of Labelled (Carbon-14) and Non-labelled D. variabilis Larvae

on Albino Rats

RadioactiveTicks

Non-labelled Ticks

No.exposed

No.recovered % Feeding

No.exposed

No.recovered % Feeding

50 8 50 15

50 18 50 2

50 12 50 11

50 12 50 9

50 14 50 23

50 16 50 7

50 5 50 12

Total 350 85 24.3% 350 79 22.6%

110 c o u n ts /m in p e r la rv a . T he m ean p r o p o r t io n o f r a d io a c t iv e la rv a e w hich fe d w as 24. 3%; that o f n o n -la b e lle d la rv a e w hich fe d w as 22. 6% (T a b le VI).

S u rv iv a l. Studies w e re p e r fo r m e d on s u rv iv a l o f D. v a r ia b ilis im m a tu re s u n der c o n tro lle d con d ition s o f 95% r .h . and 8 0 ± 2°F , with both r a d io la b e lle d and n o n -la b e lle d la rv a e (T a b le VII). S am p les o f la rv a e h eld u nder th e se co n d itio n s su rv iv e d fo r up to 70 d a ys . T he e la p se d t im e w hen 50% o f the n o n -la b e lle d t ick s d ied w as 62 d a ys . T h e e la p se d t im e when 50% o f the r a d io is o to p e -ta g g e d la rv a e d ied w as 63 da ys .E v id e n ce o f lo n g s u r v iv a l o f ra d io iso to p e -ta g g e d la rv a e w as a ls o obta in ed fr o m fie ld data. A to ta l o f 46 in d iv id u a ls r e le a s e d betw een 11 A ugust and 29 A ugust, 1966, w as re ca p tu re d on m ic e (w h ite -fo o te d m ic e and a m eadow v o le ) fr o m 14 M a rch th rough 11 A p r il , 1967, in d ica tin g su rv iv a l fo llo w e d by h o s t -s e e k in g b eh a v iou r fo r a p p ro x im a te ly 8 m on th s [7].

S u rv iva l o f D. v a r ia b il is im m a tu re s at h igh te m p e ra tu re (80°F ) is l im ite d . H ow ever , at lo w e r te m p e ra tu re s , e .g . 50°F , s u r v iv a l o f m o s t in d iv id u a ls in an o v ip o s it f o r up to 8 m on th s has b een o b s e r v e d .

B io e lim in a tio n . T he m ean a c t iv it ie s d e te cte d in la r v a l sa m p le s draw n fr o m a s to ck popu la tion at 2 -w e e k in te rv a ls is show n in T a b le VIII. L it tle change in r a d io a c t iv ity a p p ea rs to have o c c u r r e d d u rin g th is p e r io d . T he s u r v iv a l o f D. v a r ia b il is la rv a e at the te m p e ra tu re u sed is l im ite d , as noted a b ove , and the study cou ld not be con tin u ed beyon d the s ix t ie th day. A dd ition a l e v id e n ce o f lo n g -te rm re te n tio n o f ra d io a c t iv ity

44 SONENSHINE

TABLE VII

Survival of radioactive and non-radioactive D. variabilis larvae at 80 + 2°F and 95% r. h.

Number of survivors

Day ofObservation Radioactive Non-radioactive

1 100 10014 97 9935 96 8942 86 8845 80 8350 80 8052 80 7654 80 7055 67 7056 64 7060 64 6262 64 5063 38 4365 20 3169 5 1270 0 1272 - 175 . ” 0

w as obta in ed fr o m the f ie ld c o l le c t io n n oted ab ove . Indiv idual s p e c im e n s w ith coun t r a te s as high as 59 c o u n ts /m in w e re re ca p tu re d on the h ost a n im a ls trap p ed in the r e le a s e a re a , in d ica tin g re ten tion o f a ctiv ity up to 8 m on th s.

T ra n sta d ia l t r a n s m is s io n . S am ples o f new ly hatch ed la rv a e fr o m the s e c o n d -th ro u g h -fo u r th day o v ip o s it o f a s in g le D. v a r ia b il is fe m a le w e re te s te d fo r r a d io a c t iv ity . T h e a v e ra g e n u m ber o f coun ts p e r m inute o v e r b a ck g rou n d p e r la rv a w as 106. 1. T h e se la rv a e w e re a llow ed to fe e d on a lb in o r a ts . The a v e ra g e a c t iv ity in a sa m p le o f 50 e n g o rg e d la rv a e w as 5 1 .4 c o u n ts /m in p e r la r v a o v e r b a ck g rou n d , w ith a ran ge o f fr o m7 c o u n ts /m in p e r la rv a to 108 c o u n ts /m in p e r la rv a . A sa m p le o f25 nym phs w hich m ou lted fr o m th e se e n g o rg e d s p e c im e n s w as coun ted se p a r a te ly , as w e re the sh ed sk in s . T he a v e ra g e r a d io a c t iv ity in the n ym phs w as 3 8 .4 c o u n ts /m in p e r nym ph o v e r ba ck g rou n d . T he a v e ra g e a c t iv ity in the sh ed sk in s w as 62. 7 c o u n ts /m in p e r la r v a l sk in o v e r b a ck g rou n d (T a b le IX ).

4 . DISCUSSION

T h e in o cu la tio n s w ith r a d io g lu c o s e r e p o r te d in th is p a p er r e su lte d in o v ip o s it io n by 93. 3% o f the tre a te d t ic k s . H en ce, o v ip o s it io n by

S M - 102/5 45

TABLE VIII

Retention of radioactivity by fasting Dermacentor variabilis larvae

stored for long periods

Day Sample No.aAvg. СШ/larva over background

0 1 46.7

14 2 38.8

28 3 42.3

42 4 58.8

56 5 27.5

70 6 49.3

a Each sample contained 10 specimens. All were living specimens, except the sample taken on the 70th day. All remaining larvae died between the 56th and 70th day; these specimens were segregated into three groups of 10 each, counted, and the mean activity was computed

for the three samples.

a lm o s t a ll o f the t ick s r a d io la b e lle d by th is m eth od m ay b e e x p e cte d w hen the o s m o t ic p r e s s u r e o f the c a r r ie r so lu tio n is ad ju sted to a va lu e m o r e c lo s e ly r e s e m b lin g that o f the h a em olym p h . T he a b ility to lay e g g s d o e s not ap pear to be cu r ta ile d by the in -v iv o in co r p o r a t io n o f so ft b e ta -e m itt in g ra d ia tion .

T h is m ethod o f r a d io la b e llin g im m a tu re t ick s a p p ea rs to o f fe r m o r e advantages than o th er te ch n iq u es r e p o r te d in that (1) the ra d io a ct iv e m a te r ia l is in co r p o r a te d in to the b o d ie s o f the tagged s p e c im e n s and, h e n ce , it is not r e a d ily lo s t , (2) it is s im p le to apply, {3) it is not w astefu l o f m a te r ia l , and (4) the s iz e o f the d o se a d m in is te re d can be c o n tro lle d .

R a d io se n s it iv ity o f the e g g s r e p re s e n ts one o f the m a jo r lim itin g fa c to r s on the u se fu ln e ss o f the ra d io in o cu la tio n m ethod fo r ta gg in g im m a tu re t ick s . No hatch ing o c c u r r e d in o v ip o s its in w hich the a v e ra g e count ra te p e r e g g e x ce e d e d 637. H atching o c c u r r e d at lo w e r a c t iv ity le v e ls , but w as qu ite o b v io u s ly r e la te d to the r a d io c h e m ic a l and the d ose a d m in is te re d (T a b le I). G ro s ch [9, 10] a lso found re d u ce d e g g lay in g and d im in ish ed hatch ing in the w asp (H a b ro b ra co n sp . ) fe d on r a d io ph osp h oru s and z in c -6 5 . A n oth er im p orta n t lim ita tio n on the u se o f th is m eth od f o r m a ss r e a r in g o f ta gged t ick s is the p r o g r e s s iv e d e c lin e in r a d io a ct iv ity w ith the p e r io d o f o v ip o s it io n by the paren t t ick s (F ig . 1).T h e r e a s o n fo r th is is unknown. C on seq u en tly , p rogen y p ro d u ce d la te in the e g g -la y in g p e r io d w e re o ften found to have r a d io a c t iv ity le v e ls w h ich w e re u n s a t is fa c to r y fo r f ie ld s tu d ies .

46 SONENSHINE

TABLE IX

Radioactivity (Carbon 14) in engorged larvae, nymphs, and shed skins of D. variabilis3

Radioactivity in counts per minute over background

Nymphs molted from LarvalTick No. Engorged Larvae Engorged Larvae Skins

1 22 39 3082 12 49 653 37 33 1134 36 47 205 70 29 396 106 10 337 22 20 378 21 6 199 29 32 29

10 64 28 3711 23 3 1312 107 34 92

13 86 40 72

14 59 46 43

15 73 32 45

16 32 11 4317 8418 60 23 89

19 22 92 83

20 75 56 116

21 36 13 29

22 106 87 98

23 96 35 65

24 67 38 78

25- 63 33 39

Mean + 1 S. E 56.7 + 6.1 34.8 + 4.4 62.7 + 6.3

a Larval progeny of female No. 17, inoculated with approximately 50 aíCÍ Carbon-14 glycine. Larvae were collected from the oviposits of days2 through 4. The average number of counts per minute over background

per unfed larva was 106.1.

* Died without moulting.

T h e e n o rm o u s r e p ro d u c t iv e ca p a c ity o f ixod id t ick s co m p e n sa te s f o r the l o s s e s noted ab ove . The e st im a te d a v e ra g e n u m ber o f eg gs p ro d u ce d by 15 u n trea ted D. v a r ia b ilis fe m a le s w as 4156 . T he m axim u m n u m ber o b s e rv e d w as 6173. R a d io in ocu la ted fe m a le s p ro d u ce d on ly s lig h tly fe w e r e g g s . T h e a v e ra g e n u m ber fo r 48 t ick s w as 4002. A n oth er im p orta n t co n s id e r a t io n is the te m p o ra l d is tr ib u tio n o f the eg g la y in g p r o c e s s . F ig u re 4 sh ow s the a v e ra g e d a ily eg g p rod u ction o f 15 D. v a r ia b il is fe m a le s h e ld in the dark at 85% r .h . and 8 0 ± 2 ° F . By r e fe r r in g to F ig . 4, it m ay be found that 59. 2% o f the e g g s a re d e p o s ite d by the end o f the fifth day o f o v ip o s it io n , and a ll but 6% are la id b y the end o f the

S M - 102/5 47

AVERAGE DAILY EGG PRODUCTION

DAYS

FIG. 4. A verage da ily egg production o f 15 D . variabilis fem ales held at 80 ± 2° F. and 85% rela tive hum idity.

tenth day. T h is k n ow led ge o f the c h a r a c t e r is t ic s o f the e g g -la y in g p r o c e s s in t ick s m ay be u sed , a long w ith in fo rm a tio n on the e f f ic ie n c y o f in o cu la tio n , tr a n s o v a r ia l t r a n s m is s io n , and lo s s o f a c t iv ity w ith h atch ing , to d e te rm in e the b e s t m eth od o f m a ss r e a r in g o f t ick s f o r u se in f ie ld s tu d ies .

P re p a ra t io n o f the r e le a s e p op u la tion . An e x a m p le o f the a p p lica tion o f the m ethod fo r m a ss r e a r in g o f r a d io is o to p e -ta g g e d t ick p opu la tion s m ay be g iven by a ssu m in g the o b je c t iv e o f p ro d u cin g 100 000 D_. v a r ia b il is la rv a e with an a v e ra g e r a d io a ct iv ity o f not le s s than 50 c o u n ts /m in p e r la rv a . The r a d io c h e m ic a l s e le c te d is g lu c o s e 14C. S ince a p p ro x im a te ly h a lf o f the r a d io a ct iv ity in co r p o r a te d in the eg g s is lo s t du ring h atch ing , the in ocu la tin g d o se p e r p a ren t t ic k m u st b e ad ju sted to co m p e n sa te fo r it . By r e fe r r in g

48 SONENSHINE

TABLE X

Determ ination o f the dose ( in m icrocu ries) o f Carbon-14 g lu cose necessary to ra d io a ct iv a te the

maximum number o f D. v a r ia b i l is larvae w ith a minimum o f 100 CEM/larva over background

Day of o v ip o s it io n

Avg. N o.a Eggs

Avg. expected^ a c t iv it y (СШ)

A c tiv ity in to ta l ovipos i t

(m icrocu ries)A c t iv it y /

larvae (CPM)

1 483 260 0.274 1282 537 220 0.258 1083 552 180 0.218 884 467 140 0.143 695 452 100 0.099 496 392 90 0.077 D7 320 79 0.055 I8 266 68 0.039 S9 230 57 0.029 C

10 172 53 0.020 A11 107 50 0.012 R12 84 46 0.008 D13 42 43 0.004 E

D

a Data from Figure 4.

b Data from Figure 1.

to F ig . 1, it m ay b e o b s e r v e d that the p r o g r e s s iv e d e c lin e in r a d io a c t iv ity p e r e g g w ith tim e o f o v ip o s it io n is lin e a r , w ith a m idpoin t n ear the fifth day. H en ce , the a ctiv ity o f the fifth day o v ip o s it m ay be u sed as the b a se le v e l fo r d e term in in g the d o s e . E ggs p ro d u ce d a fte r th is day a re d is ca rd e d . T he d o se re q u ire d to a ctiva te the fifth -d a y o v ip o s it to a le v e l o f 100 c o u n ts /m in p e r e g g o v e r b a ck g rou n d is 0. 099 m ic r o c u r ie s (a v e ra g e o f 452 eg g s X 100 c o u n ts /m in X d e te c to r e f f ic ie n c y , o r 1 0 0 /2 0 . 6, d iv id ed by the n um ber o f d is in te g ra tio n s p e r m inute p e r m ic r o c u r ie , o r2 220 000). T h e a c t iv it ie s r e q u ire d fo r the o th er o v ip o s it io n p e r io d s can a lso be d e te rm in e d fr o m the data in F ig s 1 and 4. T h e se c a lcu la tio n s a re show n in T a b le X . W hen the a ctiv ity o f the r a d io c h e m ic a l w hich m ay be e x p e cte d to r e m a in in the spent p a ren t is in clu d ed (T a b le V ), the to ta l a ctiv ity w hich m u st be r e c e iv e d by the p r e o v ip o s it in g fe m a le is 1. 5 9 4 /jC i. S ince the e f f ic ie n c y o f in o cu la tio n has been est im a te d to be 35. 1%, the d o se r e q u ire d to obtain the d e s ir e d m in im a l le v e l o f la b e ll in g o f the m a xim u m n u m ber o f la rv a e is 4. 54/uCi. T h e fin a l e x p e cte d y ie ld o f la r v a e , tak in g in to accou n t the lo s s due to ra d io s e n s it iv ity (T a b le I), is 1 868. In ocu la tion o f a to ta l o f 57 e n g o rg e d fe m a le t ick s w ill be n e c e s s a r y to obta in the r e q u ire d n u m ber o f la rv a e .

In p r a c t ic e , the in v e s tig a to r m ay w ish to in c r e a s e the d o se to en su re adequate la b e llin g . F u rth e r , a m o n ito r in g ch e ck o f the hatch fr o m ea ch

SM - JO-2/5 49

o v ip o s it w hich is sch e d u le d fo r u se as a com p on en t o f the r e le a s e popu la tion sh ou ld be in clu d ed in the la b o r a to r y p r o to c o l to e n su re that a ll r e le a s e d cick s a re su itab ly la b e lle d .

B io lo g ic a l c h a r a c t e r is t ic s o f r a d io is o to p e -ta g g e d t i c k s . An im p orta n t co n s id e r a t io n w hich b e a rs upon the a p p lica tion o f ra d io la b e llin g m eth od s in e c o lo g ic a l s tu d ies is w hether the b io lo g ic a l c h a r a c t e r is t ic s o f r a d io is o to p e -ta g g e d in d iv id u a ls a re s im ila r to th o se o f u n trea ted sp e c im e n s . T h e data r e p o r te d in th is p a p er su g g e s t that they a re ve iy s im ila r . No apparent d e tr im e n ta l in flu en ce w as noted when fe e d in g o f ra d io a ct iv e la rv a e w as c o m p a re d w ith that o f n o n -r a d io a c t iv e la rv a e . T he p ro p o rt io n o f the t ick s (e x p o se d to a h ost, a lb in o ra t) w hich fe d w as a lm o s t id e n tica l in each c a s e , and it w as not s ig n ifica n tly d iffe re n t fr o m the p r o p o rt io n r e p o r te d by Sonensh ine and A tw ood [11 ] f o r th is sa m e h ost. S u rvival o f r a d io is o to p e -ta g g e d la rv a e a p p e a rs to b e s im ila r to s u r v iv a l o f n on - la b e lle d la rv a e . T he e la p se d tim e when 50% m o rta lity o c c u r r e d w as a lm o st id e n tica l in both c a s e s (62 and 63 d a ys , r e s p e c t iv e ly , at 80°F and 95% r . h. ). In ad d ition to la b o r a to r y e v id e n ce , r e c a p tu r e s o f o v e rw in te r s u r v iv o r s a fte r a p e r io d o f up to 8 m on th s w e re m a d e. L a rv a e r e le a s e d in A ugust 1966 at a study s ite n ear M o n tp e lie r , V irg in ia , w e re re ca p tu re d on sm a ll m a m m a ls in M a rch and A p r il 1967 [7]. T h u s, a ll the e v id e n ce obta in ed to date su g g e s ts that r a d io la b e l le d t ick s m ay be r e g a rd e d as s im ila r to n o n -la b e lle d la b o r a to r y r e a r e d t ick s . H ow ever , th is d o e s not im p ly that they a re s im ila r to w ild in d iv id u a ls liv in g in the n atural en v iron m en t. F u rth er study with la b o r a to r y r e a r e d p op u la tion s r e le a s e d in n atural and s im u la ted en v iron m en ts w ill be n eeded to d e te rm in e the d e g re e o f b e h a v io u ra l s im ila r ity betw een th e se t ick s and the n ative o n e s , as w e ll as to d e te rm in e the p r o p o r t io n o f the r e le a s e d p opu la tion w hich s u r v iv e s fr o m one y e a r to the next.

In addition to d e te rm in in g w hether ra d io a ct iv e t ick s w e re b io lo g ic a lly s im ila r to n o n -la b e lle d in d iv id u a ls , it is a lso o f in te r e s t to d e te rm in e how lo n g the r a d io a c t iv e la b e l r e m a in s in each sta g e . In f r e e - l iv in g in s e c ts , b io e lim in a t io n o ften a ccou n ts f o r m o r e lo s s o f r a d io is o to p ic m a te r ia l than n u c le a r d is in te g ra t io n s . T h e b io lo g ic a l h a l f - l i f e o f a ra d io n u clid e is v e r y d if fe r e n t fr o m the p h y s ic a l h a lf - l i fe [12]. In t ic k s , h o w e v e r , th ere is no d a ily ex ch an ge o f nutrien t and w aste m a te r ia l d u ring the n o n -p a ra s it ic p e r io d s . C on seq u en tly , lo n g -t e r m re te n tio n o f r a d io a ct iv ity m ay be e x p e cte d . T h e data in T a b le VIII le n d s su p p ort to th is h y p o th e s is . U n fortu n ately , fu rth e r study o f r e te n tio n o f ra d io a c t iv ity by fa s tin g la rv a e co u ld not be con tin u ed at the te m p e ra tu re u sed (80°F ) b e c a u se o f death o f a ll o f the s p e c im e n s . F u rth er in v e s tig a t io n o f this p ro b le m w ill b e done w ith t ick s h e ld at lo w e r te m p e ra tu re s . Data fr o m the f ie ld study a lso su p p ort the e x p e cta tio n o f lo n g -t e r m re te n tio n o f ra d io a c t iv ity . O v e rw in te r s u r v iv o r s w e re c o l le c t e d w hich had a c t iv it ie s as h igh as 59 c o u n ts /m in p e r la r v a o v e r ba ck g rou n d .

R a d io la b e llin g o f nym phs r e s u lts fr o m m ou ltin g o f fe d la b e lle d la rv a e (T a b le IX ). T he a c tiv ity le v e ls found in th e se nym phal t ick s w e re adequate fo r d e te ct io n o f m o s t in d iv id u a ls . T he data su g g e s ts that p opu la tion s o f ta gged nym phs su ita b le f o r u se in f ie ld s tu d ies m ay a lso be p rod u ced by th is m ethod .

4

50 SONENSHINE

5. SU M M ARY AND CONCLUSIONS

T h e fe a s ib i l it y o f m a ss r e a r in g r a d io is o to p e -ta g g e d im m a tu re t ick s by m ea n s o f in ocu la tin g e n g o rg e d fe m a le s w ith r a d io c h e m ic a ls is d e m o n stra te d in th re e s p e c ie s o f Ix od id a e . T h e p r e s e n c e o f r a d io a c t iv ity m ay be d e te cte d r e a d ily in in d iv id u a l la rv a e o r nym phs.

T h e in co r p o r a t io n o f r a d io c h e m ic a ls in te rn a lly in to t ick s r e d u c e s the ch a n ce o f lo s in g the r a d io a c t iv e la b e l w hen m ou ltin g o c c u r s , o r u n d er n atu ral co n d it io n s . F u rth e r , the am ount o f r a d io a c t iv ity in the s p e c im e n s can be c o n t r o lle d by c o n tro ll in g the s iz e o f the d o s e a d m in is te re d to the p a ren t t ic k s . H ow ever , d if fe r e n c e s in r a d io a ct iv ity in the p ro g e n y o f tre a te d fe m a le s in r e la t io n to the day o f o v ip o s it io n w e re noted . T h is a ctiv ity d e c lin e d p r o g r e s s iv e ly through the ninth day. D if fe r e n c e s w e re a lso n oted in re la t io n to the r a d io c h e m ic a l u sed . M ost o f the 14C g ly c in e (79 . 0%) r e c e iv e d by the e n g o rg e d fe m a le s re m a in e d in the p a ren ts , w h e re a s m o s t o f the 14C g lu c o s e (77 . 5%) r e c e iv e d w as tr a n s fe r r e d to the p ro g e n y . H atching o f e g g s la b e lle d by th is m eth od w as le s s than in u n trea ted o v ip o s it s . T h e d e g re e o f r a d io s e n s it iv ity w as r e la te d to the am ount o f r a d io a ct iv ity in the d o s e g iven to the p a ren ts . L a rg e d o s e s w e re le th a l to m any e g g s . No h atch ing o c c u r r e d when the a v e ra g e a c t iv ity o f the la b e lle d e g g s w as 637 c o u n ts /m in p e r eg g o v e r ba ck g rou n d , o r g r e a te r . N e v e r th e le s s , n u m e ro u s h igh ly ra d io a ct iv e la rv a e h atch ed , in c lu d in g in d iv id u a ls w ith cou n ts as h igh as 511 c o u n ts /m in p e r la r v a o v e r ba ck g rou n d .

T h e b io lo g ic a l c h a r a c t e r is t ic s o f the tagged la rv a e w hich h atch ed d id not ap p ear to have b een a ffe c te d by the p r e s e n c e o f ra d io a ct iv ity .T h e p r o p o r t io n o f m a rk e d la rv a e w hich attach ed to h o sts w as s im ila r to the p r o p o r t io n o f u nm ark ed la rv a e . T he tim e o f s u rv iv a l o f fa s tin g , r a d io is o to p e -ta g g e d la r v a e , u n der la b o r a to r y co n d it io n s , w as s im ila r to the p e r io d o f s u rv iv a l o f n o n -r a d io a c t iv e la rv a e . No apparent lo s s in r a d io a c t iv ity w as d e te cte d in fa s t in g la rv a e h eld f o r up to 70 days u n d er la b o r a to r y co n d itio n s .


T h e author w ish e s to e x p r e s s h is s in c e r e s t a p p re c ia tio n to M r. J. Stout f o r h is a s s is ta n c e in tra p p in g the w ild m a m m al h osts w hile he w as a s s o c ia te d with O ld D om in ion C o lle g e , N o r fo lk , V irg in ia , and to M rs . J . T ig n e r , a lso o f th is in stitu tion , f o r h e r a s s is ta n c e in the la b o r a to r y s tu d ies d e s c r ib e d in th is co m m u n ica tio n .

R E F E R E N C E S

[1] KNAPP, S. E., FARINACCI, C. J., HERBERT, С. M., SAENGER, E. L . ,A method of labelling the lone star tick (Amblyomma american»™) with a radioactive indicator (P^2). J. Econ. Entomol. 49 (1956) 393.

[2] QUAN, S. F., HARTWELL, W. V . , SCOTT, K. S., PENG, С. T., Cerium 144 as a tag for arthropods of medical importance. Trans. Royal Soc. Trop. Med. Hyg. 51 1 (1956) 87.

SM-102/5 51

[3] HYLAND, К. E., Jr., HAMMAR, J. L., Transovarial passage of radioactivity in ticks labelled with C*-4 glycine. J. Farasitol. 45 Suppl. 2 (1959) 24.

[4] BABENKO, L. V., The use of radioisotopes for labelling ticks.Med. Parazitol. i Parazitan. Bolezni. 29 (1960) 324.

[5] SAMOSTREL'SKII,. A. lu., DAITER, A. B., Experiment of the use of radioactive isotopes to mark Ornithodoros papillipes ticks. Tr. Inst. Epidem. Paster. ¿9 (1966) 86.

[6] SONENSHINE, D. E., YUNKER, С. E., Radiolabelling of ticks by inoculation. In preparation.

[7] SONENSHINE, D. E., CLARK, G. М., Field trials on radioisotope tagging of ticks. In preparation.

[8] WANG, С. H., WILLIS, D. L., Radiotracer Methodology in Biological Science (PRENTICE HALL, INC.), Englewood Cliffs, New Jersey (1965) 382.

[9] GROSCH, D. S., SULLIVAN, R. L., The effect of ingested radiophosphorus on egg production and embryo survival in the wasp Habrobracon. Biol. Bull. 102 (1952) 128.

[10] GROSCH, D. S., Distribution of Zinc-65 in the wasp, Habrobracon, and its effect on reproduction. Nature. 195 (1962) 356.

[11] SONENSHINE, D. E., ATWOOD, E. L., LAMB, J. T., Jr., The ecologyof ticks transmitting Rocky Mountain spotted fever in a study area in Virginia. Ann. Entomol. Soc. Amer. J>9 6 (1966) 1234.

[12] CROSSLEY, D. A., Jr., Consumption of vegetation by insects. Proc. First Natl. Symposium Radioecology. (1963) 427.

D I S C U S S I O N

H. M A R C H A R T (C h a irm an ): Have y ou e st im a ted the ap p rox im a te ra d ia tion d o se r e c e iv e d by the o v a r ie s o f the fe m a le t ic k s ?

D. E . SONENSHINE: No, we do not have the equ ipm ent fo r th is . H ow ever , I am c o n c e rn e d with r a d io is o to p e s in ocu la ted in to the fe m a le s and so fa r , with d o s e s o f up to 60juCi o f 14C p e r t ick , I have not seen any ev id e n ce o f ra d ia tio n dam age to the o v a r ie s . The a v e ra g e n u m ber o f e g g s d e p o s ite d by in ocu la ted fe m a le s w as a lm o st the sa m e as that p ro d u ce d by n o n -in o cu la te d fe m a le s .

D. S. GROSCH : I fe e l it sh ou ld be b o rn e in m ind that the en erg y o f the be ta ra y s and th e ir ra n ge in t is su e a re im p ortan t in in vestig a tin g the d o se le v e l to le ra te d . F o r in sta n ce 14C, in any fo r m , can be to le ra te d in m u ch la r g e r qu an tities than 32P .

I shou ld be in te r e s te d to h e a r w hether you have any l i fe -s p a n data on the o v ip o s it in g fe m a le s . T h e p a ttern on you s l id e s r e s e m b le d the se n ile d e c lin e seen with H a b ro b ra co n and D ro s o p h ila — though , o f c o u r s e , the t im e -s c a le w ould not be id e n tica l.

D. E . SONENSHINE: The fe m a le Ixod id t ick la y s a ll h e r e g g s in one b r ie f o v ip o s it io n p e r io d , and d ie s on , o r sh o r tly a fte r , co m p le t io n o f th is p r o c e s s , s o th e re is no q u estion o f se n ility h e re .

D. S. G ROSCH : T hen co u ld you te l l us w hether the m ea n day o f death is day 15? I am try in g to r e la te th is to som eth in g fu ndam en ta l in in s e c t p h y s io lo g y and I b e lie v e that the fat body is v e r y m u ch in vo lved in in s e c t o v ip o s it io n .

D. E . SONENSHINE: T he m ean day o f te rm in a tio n o f e g g la y in g w as not com p u ted e x a c t ly , but it is c e r ta in ly around 15 da ys . T h e actu al

52 SONENSHINF

l i fe -s p a n o f the fe m a le is l e s s s ig n ifica n t, s in ce she m ay w e ll su rv iv e fo r a sh o rt p e r io d a fte r o v ip o s itin g .

R a ch e l G ALU N : I w ould ju st lik e to point out that t ick s do not in fa c t have a fat body.

D. S. GROSCH : Then by what m ech a n ism do th ose p r o c e s s e s o c c u r in t ick s w hich a re p e r fo rm e d by the fat body in in s e c ts ?

R a ch e l G ALU N : T ic k s u tiliz e b lo o d v e r y s lo w ly and u se the gut as a s to r in g p la ce fo r food .

D. S. GROSCH : H ow ever , the in s e c t fa t bod y is m o r e than a p la ce f o r s to r in g sudan p o s it iv e m a te r ia l.

D. E . SONENSHINE: T h is is an in te re s t in g point.W. F . BA LD W IN : Y ou m en tion ed that eg g b a tch es la id b y fe m a le s

tre a te d with 14C in g lu c o s e sh ow ed a su bstan tia l and p r o g r e s s iv e d e c lin e in r a d io a ct iv ity through the f i r s t nine d a ys o f o v ip o s it io n . T h is d ro p is o f the o r d e r o f 20% and m igh t w ell in te r fe r e with the study. C ould you g iv e u s an in d ica tion o f the re a so n s fo r th is d im in ish in g d e g re e o f ra d io a c t iv ity ?

D. E . SONENSHINE: I can not g iv e a d e fin it iv e an sw er to th is . H ow ever , I cou ld sp e cu la te that it m ay be re la te d to the p o s itio n o f the e g g s in the r e p ro d u c t iv e tr a c t . P e rh a p s e g g s iii the o v id u cts and th ose in the o v a ry have d iffe re n t r a te s o f uptake o f m e ta b o lite s .

ETUDE ECOLOGIQUE DE DEUX PUCERONS VECTEURS DES VIROSES DE LA BETTERAVE

E. FRANÇOIS* A. R IG A * ET R. IM PEN S**CENTRE DE RECHERCHES AGRONOMIQUES DE L 'E T A T , .GEMBLOUX, BELGIQUE


ECOLOGICAL STUDY OF TWO APHID VECTORS OF THE SUGAR-BEET VIRUS. The dispersal o f Myzus persicae Sulz and Aphis fabae Scop, labelled with 32P was studied from 19fi3 to 1965 in sugar-beet plots.

The aphids were reared on beets in fected with a virus causing severe yellow in g and sprinkled with a solution containing 1 m C i o f sodium orthophosphate (32P). Aphids w hich rem ained for 24 hours on these plants were sign ificantly la b e lled , as were also their first-generation descendants. The a c t iv ity o f the insecls was recorded by a ga s-flow detector with a low background. The rad ioactive beets in fected with the virus and laden with aphids were p laced in the centre o f the plots. After successively increasing dispersal tim es, the largest possible num ber o f aphids was captured in each test, either m anually or in traps. The co-ord in ates and the precise iden tifica tion o f each la b e lled aphid w ere entered on charts, som e o f which are discussed by the authors.

The experim ents dem onstrated the m obility o f the ap lerae, w hich were found at average distances o f 6. 5 and 7 metres for Myzus persicae and Aphis fa b a e , respectively . H ow ever, som e apterae o f both species were found at distances o f over 15 metres after the sam e period.

The role o f the winged species as virus carries over short distances should not b e underestim ated.The a ctiv e character o f both apterae and winged species is em phasized.

ETUDE ECOLOGIQUE DE DEUX PUCERONS VECTEURS DES VIROSES DE LA BETTERAVE. La dispersion de Myzus persicae Sulz et d ' Aphis fabae Scop. marqués à Г aide de phosphore-32 a été su iv ie , au cours des années 1963 à 196Г>, dans des parcelles de betteraves sucrières.

Les pucerons sont é lev és sur des betteraves virosées ( in fectées par le virus de la jaunisse grave de la betterave) et arrosées d 'u n e solution contenant ] m C i d *orthophosphate de sodium ( 3ZP). Des pucerons ayant séjourné 24 heures sur ces plantes sont s ign ifica tivem en t marqués; leur descendan ce de la prem ière génération l 'e s t aussi. L 'a c t iv ité des insectes est d é c e lé e par un détecteur à courant d e g a z , de grande sensibilité et à fa ib le bruit de fond. Les betteraves radioactives virosées et chargées d e pucerons sont p lacées au centre des parcelles. Après des tem ps de dispersion croissants, on prélève, dans chaque parcelle le m axim um de pucerons, soit m anuellem en t, soit à l 'a i d e de pièges. Les coordonnées et l ’ identité précise de chaque puceron marqué sont reportées sur des plans. Certains de c e u x -c i sont com m en tés par les auteurs.

Les essais ont prouvé la m ob ilité des form es aptères des deux espèces. Les Myzus et Aphis aptères sont retrouvés, en m oyenne, respectivem ent à 6, 5 m ètres et à 7, 0 mètres de leur point d e départ, après 24h euresdedispersion . Certains individus des deux espèces sont capturés à plus de lb m ètres au bout du m êm e laps de temps.

La part d 'in terven tion des form es ailées dans la dissém ination des viroses à cou rte distance ne. sem ble pas n ég ligeab le .

Le caractère actif des déplacem ents des form es aptères et a ilées a pu être souligné.

IN TRO D U CTIO N

V e cte u rs p r in cip a u x de la ja u n is se g ra ve de la b e tte ra v e (V JG B ),M yzu s p e rs ica e Sulz et A ph is fabae Scop . son t t r e s com m u n s en B e lg iq u e .

* Station de ch im ie et de physique agricoles.* * Station d 'e n to m o lo g ie : actu ellem en t assistant à la Facu lté des S cien ces agronom iques de l 'E ta t,

G em bloux.

53

54 FRANÇOIS et al.

P lu s ie u r s c h e r ch e u rs ont d ém on tré 1' e f f ic a c ité su p é r ie u re du p r e m ie r v e c te u r : W atson et al. [1] au R oy a u m e-U n i, en com p a ra n t 1' in c id e n ce annuelle de la m a lad ie a 1' abondan ce re la t iv e des deux e s p è c e s , B jo r l in g [2 ] en tira n t p a rti du fa it que la v ir ó s e ne se p ré se n te pas en Suède ce n tra le et en com p a ra n t d ire c te m e n t sa d is p e r s io n par chacun d es deux v e c te u r s . B jo r l in g et O ss ia n n ilsso n [3 ] ont ég a lem en t pu m o n tre r au la b o r a to ir e que, m a lg ré d es d if fé r e n c e s dans l 'e f f i c a c i t é de t r a n sm iss io n d e s deux e s p è c e s , M yzus p e rs ica e d é te rm in e de t r o is a qu atre fo is plus d 1 in fe c t io n q u 1 A ph is fabae . L es travau x de H eathcotè et C ock ba in [4J c o n firm e n t c e s r é su lta ts . De p lu s, i ls m on tren t que le s fo r m e s a ilé e et ap tère d e s deux v e c te u r s tran sm etten t a u ss i a isém en t le VJGB.

L e s r ô le s r e s p e c t i fs d es fo r m e s a ilé e et ap tere dans la d is p e r s io n d es v iru s p e rs is ta n ts et s e m i-p e r s is ta n t s son t d i f f i c i l e s a d é te rm in e r . D av ies[5 ] in s is te su r 1' im p o rta n ce d e s a p tères dans c e d om ain e . B roa d b en t [6], B roa d b én t et G re g o ry [7], B road ben t [8, 9 ] ne p artagen t pas ce tte op in ion .D e n om breu x au teu rs, cepen dan t, m aintiennent q u 'u n e bonne p art de la d is p e r s io n d es v iru s p e rs is ta n ts et s e m i-p e r s is ta n t s au se in du cham p e s t due aux a p te re s . R ibbands [10] p a r e x e m p le , pou r s u iv re la d is p e r s io n d es ja u n is se s g ra v e et m o d é ré e , n ' é tu d ie que le c o m p o r te m e n t d es a p tères de M yzu s p e r s ic a e .

Une tech n ique perm ettan t de r e p é r e r le s aphides s 1 a v é ra it n é c e s s a ir e afin de su iv re le u r s d é p la ce m e n ts en cham p et de p r é c i s e r le r ô le jo u é p a r le s fo r m e s ap tère et a i lé e . B jo r l in g et a l . [11 ] u t ilis è r e n t le r a d io p h o s - p h o re 32 P p ou r m a rq u e r le s aphides et le s r e p é r è r e n t par a u to ra d io g ra p h ie . C ependant i ls ne r é u s s ir e n t a m a rq u e r d 'u n e fa çon d é c e la b le q u 'u n e fr a c t io n (év a lu ée a m o in s d 'u n t ie r s ) des in d iv idu s. Ils ob tin ren t n éan m oin s d e s re n se ig n e m e n ts t r è s in té re ss a n ts su r la d is p e r s io n d es deux v e c te u r s . C 'e s t en nous fondant su r le s travau x de c e s c h e r ch e u rs , m a is en u tilisa n t une m éthode de d é tection p lu s e f f ic a c e , que nous avons p o u rsu iv i c e s étu des au c o u r s d es années 1963 a 1965.

M ETH O D E S

L e s aphides des deux e s p è c e s son t é le v é s e n se m b le su r d e s b e t te ra v e s de la v a r ié té K le in w a n zleben E . c u lt iv é e s en pots de 30 cm de d ia m è tre .L e s p la n t e s -s o u r c e s son t, su ivan t le s an n ées, in o cu lé e s ou non de VJGB (so u ch e du L a b o ra to ire de p h y to v iro lo g ie de G em blou x ). Q u elqu es jo u r s avant le début de 1' e x p é r ie n c e , le s p a r c e l le s son t tr a ité e s au m oy en d 'u n in s e c t ic id e a c o u r te r é m a n e n ce , tandis que le s p ots son t m is dans des sea u x en p o ly e th y len e et a r r o s é s au m oy en d ’ une so lu tion d 'o r th o p h o s p h a te de sod iu m r a d io a c t if ( lm C i p a r pot et p a r p lan te). Le su r len d em a in , i i s son t r e c o u v e r ts d 'u n e c o i f fe de gaze em p êch an t toute fu ite d 'in s e c t e s .

B jo r l in g et al. [11 ] ont m on tré que le s aphides son t ra p id em en t m a rq u é s de c e tte fa ço n , qu ' i ls le re s te n t pendant au m oin s 2 a 3 se m a in e s et q u 'u n e p a rtie de la r a d io a ct iv ité p a sse a la d e sce n d a n ce . D epu is , d 'a u t r e s travau x su r 1 'a s s im ila tio n et l 'e x c r é t i o n du 32P ch e z le s aph id es [1 2 -1 6 ] ont fa it p r o g r e s s e r nos c o n n a is sa n c e s .

L e s b e tte ra v e s a in si c o i f fé e s sont tra n sp o rté e s av ec p ré ca u tio n deux a t r o is jo u r s p lus ta rd au m ilie u d es p a r c e lle s e x p é r im e n ta le s et e n fo u ie s a r a is o n de qu atre p lantes p a r p a r c e lle . L es c o i f fe s son t a lo rs e n le v é e s et in trod u ites su r p la ce dans de gran ds s a c s en po ly é th y lèn e , afin •

S M - 102/6 55

d 'e m p ê c h e r la d is sé m in a tio n a c c id e n te lle d e s in s e c te s m a rq u é s . L es p a r c e l le s ont é té in s ta llé e s en 1963, 1964 et 1965 dans un ch am p d ég ag é , h om og èn e , situ é a 100 m è tr e s de la sta tion c lim a to lo g iq u e de G em blou x .L e n o m b re et la d is p o s it io n d es p a r c e l le s ont v a r ié au c o u r s d e s t r o is an n ées. Cependant le s co n d itio n s du s e m is n 'o n t pas été m o d if ié e s :50 cm en tre le s l ig n e s et p lantes d is ta n tes de 33 cm dans le s lig n e s . L es lig n e s de b e t te ra v e s son t n u m é ro té e s et une b e tte ra v e su r 10, dans la lig n e , r e ç o it un n u m éro d 'o r d r e . L e s r é c o l t e s co m m e n ce n t g én éra lem en t le len d em a in du tra n sp o rt d es p la n t e s -s o u r c e s au cham p. E l le s sont fa ite s m a n u ellem en t. Une équ ipe de cap tu re co m p re n d un r é c o lt e u r et un a ide . L e p r e m ie r d é c o u v re , id e n tifie et p r é le v e le p u ce ro n au m oyen d 'u n p in ceau . L e se co n d p o rte la p la n ch ette où son t c o l lé e s le s cu p u les d e s tin é e s a le r e c e v o ir et note le s c o o rd o n n é e s de la p lante et le n u m éro d 'o r d r e de la cu pu le . L e s cu p u les son t ad aptées a l ' équ ipem en t de co m p ta g e et ont r e çu , au p r é a la b le , une goutte de g ly c é r in e et d 'in s e c t ic id e . E l le s sont d is p o s é e s dans le p a sse u r d 'é c h a n t i l lo n s san s au tres p r é p a r a t i fs . De n om b reu x p iè g e s ja u n es son t ég a lem en t d is p o s é s dans le s p a r c e l le s et le s aph id es a ilé s qu i y son t tro u v é s son t p la c é s dans le s m ê m e s cu p u les en vue du co m p ta g e .

L a ra d io a c t iv ité d es in s e c te s e s t é ta b lie au m oy en d 'u n d é te cte u r G e ig e r -M ü lle r a cou ran t de gaz , à fa ib le m ou vem en t p r o p r e et de grande s e n s ib il ité (fe n ê tre t r è s m in ce de qu elque 0, 125 m g p a r c m 2 ) co u p lé a une é c h e lle de co m p ta g e ad équ ate1 . L 'e m p lo i d 'u n p a sse u r autom atique (c a p a c ité : 50 u n ités) et d 'u n e c a lc u la tr ic e im p rim a n te nous a p e r m is de m e s u r e r 1' a c tiv ité de m i l l ie r s d 'in s e c t e s en qu e lq u es se m a in e s et d 'o b t e n ir a in s i ra p id em en t le s in fo rm a tio n s n é c e s s a ir e s tout en m enant le s r é c o lt e s su r une gran de é c h e lle . Suivant c e p r o c e s s u s , i l a été p o s s ib le de tr a ite r p r è s de 4 000 p u ce ro n s en 1963, qu elque 10 000 en 1964 et 11 000 en 1965.L e s éch a n tillon s son t c o m p té s pendant t r o is m in u tes . Seuls le s p u ce ro n s donnant, a p rè s t r o is c o m p ta g e s , une m e s u r e re p ro d u c t ib le su p é r ie u re a t r o is m ou v em en ts p r o p r e s son t c o n s id é r é s co m m e r a d io a c t ifs . A chaque début d 'e s s a i , on p r é lè v e un éch a n tillon d es aph id es se trouvant s u r le s p lan tes r a d io a c t iv e s . D ans tou s le s c a s , le s in s e c te s so u m is au com p ta g e son t s ig n ifica t iv e m e n t m a rq u é s . L e s r é su lta ts son t a lo rs r e p o r t é s su r le s p lans d e s p a r c e l le s .

ESSAIS DE 1963

C es e s s a is , d e s tin é s p r in c ip a le m e n t a la m is e au point d es m o d a lité s e x p é r im e n ta le s , ont n éan m oin s donné d e s in d ica tio n s in té r e ss a n te s su r la d is p e r s io n d es deux v e c te u r s .

Q uatre p a r c e l le s de b e t te ra v e s s u c r iè r e s (<5, y , a , /3, ) de 20 m X 20 m ont été é ta b lie s dans un ch a m p d 'a v o in e , la d is ta n ce en tre le s p a r c e l le s étant de 15 m . L e s p la n t e s -s o u r c e s , c h a rg é e s d es deux e s p è c e s aph id iennes et in o cu lé e s de VJG B , ont é té in trod u ites dans le s p a r c e l le s r e s p e c t iv e m e n t le s 9, 16, 29 ju ille t et 7 août 1963.

1 Tracerlab « O m n i-g u a rd » low -backgroun d counting system. M ouvem ent propre: 1 cpm ; diam ètre du détecteur: 25 m m ; rendem ent du détecteu r voisin de 25% dans les con d ition s d e nos com ptages.

56 FRANÇOIS e t al.

P a r m i le s 1084 M yzus p e rs ica e ca p tu ré s dans le s qu atre p a r c e l le s ,14, s o it 1, 3%, éta ien t m a rq u é s (6 a ilé s et 8 a p tè re s ). En ce qui c o n c e rn e A p h is fabae, 135 in d iv idu s m a rq u és (25 a ilé s , 86 a p tè re s et 24 fo r m e s « im m a t u r e s » ) ont été d é n o m b ré s dans un éch a n tillon n age de 271b in d iv id u s, c e qui r e p ré s e n te p r è s de 5% d es ca p tu res .

L e s don nées obtenu es su r la d is p e r s io n des in s e c te s sont e n co r e a s s e z l im ité e s . L es a p tè re s ont é té , p ou r la p lu part, r e tr o u v é s a p ro x im ité im m é d ia te d es p la n t e s -s o u r c e s , dans un rayon ne dépassan t pas l m , au c o u r s d e s t r o is p r e m ie r s jo u r s suivant le lâ ch e r . N otons cependant q u 'u n M yzu s p e rs ica e ap tère a été d é co u v e rt a 12 m d es p la n t e s -s o u r c e s , 2 jo u r s a p rè s le lâ ch e r , et q u 'u n A ph is fab æ ap tère a été cap tu ré a une d is ta n ce de 2, 3 m a p rè s 1 jo u r de d is p e r s io n .

La fig u re 1 donne, à t itr e in d ica tif, la d is p e r s io n de la ja u n isse o b s e r v é e 4 se m a in e s a p rè s le lâ ch e r des aphides dans une d es p a r c e lle s de 1' e s s a i (p a rc e lle a , lâ ch e r du 16 ju ille t 1963).

ОО о

о

®® a и ® ®

1 о<®¡ о

© о о

о

Légende .<§) plante * sourceО plante v.rosée ® plante virosée sur laquelle

des aphides marqués ont été trouvés

E chelle :

FIG. 1. Essais de 1963, p a rce lle a : distribution des plantes présentant les sym ptôm es du VJGB et lo ca lis a tion des aphides m arqués capturés.

C om m e on pouvait s 'y attendre [10], la d en sité d es p lantes v ir o s é e s e s t p lus é le v é e a p ro x im ité des p la n te s -s o u r c e s . De p lu s, le s p lan tes su r le s q u e lle s un aphide r a d io a c t if a été cap tu ré ont d év e lop p é le s sy m p tô m e s de la m a la d ie .

SM-102/6 57

En 1963 le s ca p tu re s , é ta lé e s dans le te m p s , avaient été e ffe c tu é e s su r la m ê m e p a r c e lle et avaient pu in flu e n ce r la d is p e r s io n u lté r ie u re des ap h id es. En ou tre ce tte fa ço n d 'o p é r e r augm entait le s r is q u e s d 'e n t r a in e m en t a c c id e n te l d es aphides m a rq u é s .

L e s e s s a is de 1964 ont é té co n çu s pou r v é r i f ie r le s o b s e rv a t io n s fa ite s 1' année p ré cé d e n te su r la m o b ilité d es deux e s p è c e s aph id ienn es c o n s id é r é e s , tout en évitant le s in con v én ien ts en qu estion .

Cinq p a r c e lle s r e c ta n g u la ire s id en tiqu es (40 m X 10 m ) dont le grand axe éta it o r ie n té su ivant la d ir e c t io n des vents d om in an ts, ont é té e n c la v é e s dans un cham p d 'a v o in e . Q uatre b e t t e r a v e s -s o u r c e s ont été p la c é e s au c e n tre de chaque p a r c e l le , au début de chaque e s s a i , et nous avons ad m is p a r h ypoth èse que la d is sé m in a tio n d es aphides se fa it de la m ê m e façon dans tou tes le s p a r c e l le s . L es p r é lè v e m e n ts d 'in s e c t e s ont eu lieu , dans une p r e m iè r e p a r c e lle , 24 h a p rès le lâ ch e r d es p u ce r o n s . L e s lig n es ce n tra le s ont été p r o s p e c té e s b e tte ra v e par b e tte ra v e , le s au tres par cou p s de son de en fon ction d e s d is p o n ib ilité s en p e rso n n e l. L e s o i r m ê m e , cette p r e m iè r e p a r c e lle a été tra ité e av ec une so lu tion a 0, 3% de P h o sd r in . L es au tres p a r c e l le s ont é té p r o s p e c té e s et tra ité e s de la m ê m e fa ço n r e s p e c t iv e m ent 2, 3, 4 et 6 jo u r s a p rè s le lâ c h e r . D es p iè g e s jau n es ont été d is p o s é s le lon g du cô té n o r d -e s t de chaque p a r c e lle et ont été d é p o u illé s chaque jo u r . Q uatre e s s a is s e son t su ccé d é su r c e s c in q p a r c e l le s . Ils ont débuté r e s p e c t iv e m e n t le s 1er, 15, 29 ju ille t et 5 août 1964.P o u r c e d e rn ie r e s s a i , le s ca p tu re s n 'o n t eu lieu que 1 jo u r a p rè s le lâ ch e r . L e s con d ition s c lim a tiq u e s ont é té , dans l 'e n s e m b le , fa v o ra b le s a la pu llu lation d es p u ce ro n s .

L a fig u re 2 donne la lo c a lis a t io n des a ilé s , d es a p tè re s et d es fo r m e s im m a tu re s d es deux e s p è c e s ca p tu ré e s m a n u ellem en t et dans le s p iè g e s jaun es a p rè s 1 jo u r de d is p e r s io n (2 e e ss a i) et 1' e f fe c t i f d é ta illé de 1' é ch a n tillon de s o u s -p a r c e l le s id é a le s de 2, 5 m de cô té .

L a fig u re 3 r a s s e m b le tou tes le s ca p tu re s e f fe c tu é e s a p rè s 1, 2, 3,4 et 6 jo u r s de d is p e r s io n au c o u r s de ce d e u x ièm e e s s a i (15 - 21 ju ille t ) . Enfin , dans le s tab leau x I et II son t cu m u lé e s le s ca p tu re s d 'in d iv id u s a p tè re s et a i lé s , r é a l is é e s au c o u r s d es qu atre e s s a is a p rè s 1 et 2 jo u r s de d is p e r s io n , le s aph id es étant c la s s é s en fo n ctio n de le u r é lo ig n em en t par ra p p o rt a le u r point de d épart.

A p tè re s

L e s a p tè re s , tant ceu x d ' A p h is fabae que ceu x de M yzu s p e r s ic a e , sont t r è s m o b ile s : n eu f A ph is fa b æ su r 17 ont é té r e tr o u v é s a p lu s de 5 m d e s p la n t e s -s o u r c e s a p rè s 2 jo u r s de d is p e r s io n , a lo r s que c e ra p p o rt a été de 33 in d iv idu s su r 100 ch e z M yzu s p e r s ic a e .

T r o is A phis et s ix M yzu s ont été r e tr o u v é s a p lu s de 15 m de le u r point de d ép art a p rè s 2 jo u r s de d is p e r s io n .

A ilé s

L e s a ilé s des deux e s p è c e s ont été r e tro u v é s dans le s p a r c e l le s à d es d is ta n ce s v a r ia b le s et, en c e qui c o n c e rn e le s ca p tu re s m a n u e lle s , dans

E SSA IS DE 1964

10 11 12 13

С E■э • в

2

• АО

(33),-ii6k

OU (29)-’/lCWAt f*ï114 15 16

со 2

(15)(в)(31 НЭОУмI (6)®

j25)_(7) (14)J(26);. (32)'- (24)....

A

..(13)2ЛД „(34,^ ^ « б Н 3)>:7ТЬб)

~ е з )сП*)(17) <1П ..(9>------ (35);

(21) ' (38)

(12):

J22;

<«Ч*2(п о и м )

Aphis Myzus fabae persicae

A a ilé Д ■ ap tè re °• im m ature О

Echantillonnage

Nombre aphides ( Af et Mp )

capturés

А

•

sur

bett

erav

esda

ns

pieg

es

(Лaicо

ОО

Оsu

rbe

tter

aves

idans

pi

eges

lia

unes

®

a..

betterave - source piège de la p a rce lle piège des autres

p a rce lle s t lignes de bette raves

E c h e lle j / en m /

0 2,5 /о■Q a 2 Д- i 3/ - _ - 1 - _ - i - 1 _ . _ - _ _ - 1 - _ 1 1.--- -3 - - 540-13

A u ■ 2c - _ - - - 3 _ _ w 7 - 1 . _ _ - _ 1 _ _ 1 1 --- 5 --- 1.---TJa* • -0- « 1 C - - - 1 - 2 - - - 1 - - - - - _ - 1 - - - ----- 1.--- 2- -6

A5 Д- i 6¿ 1 12 1 14 3 14 _ 9 1 6 _ 8 1 9 4 13 3 14 3 8 2 1 1 8 - 5.6 3.1 319- 2B -c ■ 1 a- ■ Ac - 6 - 8 - - 3 1 2 1 3 1 1 2 2 _ 3 _ 2 2 _ 3 - 3 --- --- 1.

.—, • - 0 - в -С - 5 - 6 1 3 1 2 1 - - 1 3 - 5 - 6 1 7 1 1 2 2 1 - --3 1 - - 1 -JC A5 ДЗ J 9/ 1 3 3 6 4 11 4 5 - 10 3 17 2 12 1 5 1 13 3 14 3 14 2 111 3- 6.411 3 -172 7

С E ■ 2o2 ■ 6с - ■- - 10 - 4 _ 3 - 5 i, 3 12 1 _ 6 - 2 2 _ _ 1 2 2 -- 1--- 1- 1 -cо *1 02 в 4С 3 3 2 1 3 1 2 - 1 1 1 3 9 6 2 9 - 4 4 2 1 -, 5 ----- 4.-3- 1 ---a. A3 Д- i 2 L - 10 4 _ 2 1 3 1 2 1 1 _ 4 7 _ 7 _ 7 _ 4 3 - 8.6.- 1 2 41-9

D ■- ■2 a- ■ 4с - 4 4 _ 3 _ 6 - 4 _ 2 _ 6 _ _ - 3 _ _ _ 3 _ 7 - 7.--- ---------i/i • -0- « -С - 2 - - - 1 - - 1 2 - - - 1 - - - 4 - - - - - 1 - 2.---1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

FIG. 2. Essais d e 1964: captures après 1 jour de dispersion (d eu x ièm e essai, 16 ju ille t).

-siv

EC H ELLE •n m

FIG. 3. Essais de 1964: captures après 1, 2 , 3, 4 et 6 jours d e dispersion (d eu x ièm e essai, 16 -21 ju il le t ) .Aphis Fabae: ▲ a ilé , ■ aptère, • im m atureM y2 us persicae: A a ilé , Q aptere, О im m atureX ; betterave-sou rceG : p iè g e jeuneI -V Î : jours après le lâcher.

SM

-102/6


T A B L E A U I. R E P A R T IT IO N D E S A P T E R E S C A P T U R E S A P R E S 1 E T 2 JO U R S D E D IS P E R S IO N E N F O N C T IO N D E L ' E L O IG N E M E N T P A R R A P P O R T A U X P L A N T E S -S O U R C E S E s s a is de 19 64 : c a p t u r e s c u m u lé e s d e s q u a tre e s s a i s

D istan ce (m )M yzus persicae

1 jour 2 jours

A phis fabae

1 jour 2 jours

0 - 5 21 46 4 4

5 - 1 0 8 8 2

10 - 15 8 3 2 2

15 - 20 4 2 1 _ 2_

41 59 7 10

A ptères rad ioactifs 100 17

T o ta l des captures 349 468

T A B L E A U II. R E P A R T IT IO N D E S A IL E S (C A P T U R E S M A N U E L L E S E T P IE G E S ) C A P T U R E S A P R E S 1 E T 2 JO U R S D E D IS P E R S IO N E N F O N C T IO N D E L 'E L O I G N E M E N T P A R R A P P O R T A U X P L A N T E S - S O U R C E S .E s s a is de 1 9 6 4 : c a p t u r e s c u m u lé e s d e s q u a tre e s s a i s

D istan ce (m )M yzus persicae

1 jour 2 jours 1 jour

A phis fabae

2 jours

0 - 5 4 2 1 7

5 - 1 0 - 1 ■ 1 -

10 - 15 - - - 2

15 - 20 2 - 1 -

P ièges à 2 0 m 5 - 3 5

11 3 6 14A ilé s ra d ioa ctifs 14 20T o ta l des captures 544 1476

d es d ir e c t io n s o p p o s é e s . E l le s r e p ré s e n te n t r e s p e c t iv e m e n t 14% et 118% d es cap tu re d 'a p t è r e s ch e z M yzu s p e r s ic æ et ch e z A ph is fabae .

F o r m e s im m a tu res

C es fo r m e s ont été r e tr o u v é e s dans le s p a r c e l le s dans tou tes le s d ir e c t io n s et a d es d is ta n ce s v a r ia b le s d e s p la n t e s -s o u r c e s . Il n 'e s t pas

SM-102/6 61

p o s s ib le de fa i r e la d is tin c tio n e n tre c e l le s e n g e n d ré e s p a r le s a p tè re s su r le s p lan tes r a d io a c t iv e s ou a p rè s d is p e r s io n et c e l le s d escen d an t d es alatae v ir g in ip a r e s . D e toute fa ço n , le s d is ta n ce s a u x q u e lles c e s fo r m e s im m a tu re s ont été r e tr o u v é e s a p rè s 24 h de d is p e r s io n sont du m êm e o r d r e de g ra n d eu r que c e l le s o b s e r v é e s p ou r le s fo r m e s a ilé e s et a p tè re s .

■ESSAIS DE 1965

L 'e x ig u ït é d es p a r c e l le s u t i l is é e s en 19 63 -64 im p liq u a it ce r ta in s in co n v é n ie n ts : le m ic r o c l im a t a in s i r é a l is é peut fa u s s e r p a rt ie lle m e n t la d is p e r s io n et m o d if ie r le s c o n d itio n s de v o l d es p u ce r o n s . P o u r y r e m é d ie r , nous avons é ta b li en 1965 un cham p de b e t te ra v e s d 'u n e s u p e r f ic ie de 1 ha 40 a au ce n tre duquel une p a r c e lle c a r r é e de 60 m de c ô té éta it p iqu etée (so it e n v iro n 130 lig n e s de b e t te r a v e s ) . D es p iè g e s ja u n es (144 au tota l) ont é té d is p o s é s s e lo n qu atre c e r c le s co n ce n tr iq u e s a 20, 28, 40 et 60 m d e s b e tte ra v e s r a d io a c t iv e s . C e l l e s - c i , c h a rg é e s de c o lo n ie s d e s deux e s p è c e s , ont été a m en ées au c e n tre de la p a r c e lle le 20 ju ille t et le lâ ch e r des p u ce ro n s a eu lieu a 11 h. L e r e le v é d es p iè g e s jau n es a été e ffe c tu é le jo u r m ê m e en tre 14 et 17 h a in s i que le s 22, 23, 26, 27 et 28 ju ille t en tre le s m ê m e s h e u re s . L e s ca p tu re s m a n u e lles ont débuté le21 ju ille t , le s p r é lè v e m e n ts se fa isan t su r le s 10e , 20e , 30e . . . b e tte ra v e s d es lig n e s p a ir e s de la m o it ié su p é r ie u re de la p a r c e l le (v o ir f ig .4 ) . L e22 ju ille t 1' au tre m o it ié de la p a r c e l le a été p r o s p e c té e de la m ê m e fa ço n .L e 3e jo u r (23 ju ille t ) , le s c a p tu re s ont été fa ite s su r le s 11e , 21e , 31e . . . p lantes d es lig n e s c e n tra le s (lig n e s 49 a 80). De fo r te s p lu ie s ont in te r rom pu le s ca p tu re s , qui ont été r e p r is e s le 26 ju ille t , s o it a p rè s 6 jo u r s de d is p e r s io n , su r le s 7 e , 1 7 e, 27e . . . p lantes d es lig n e s im p a ir e s de la p a rtie s u p é r ie u re ; e l le s ont été p o u rs u iv ie s le 27 ju ille t , s e lo n le m êm e sch é m a , dans la p a rtie in fé r ie u r e de la p a r c e l le . E n fin , le 8 e jo u r(28 ju ille t ) , le s ca p tu re s ont été e f fe c tu é e s su r tou tes le s p lantes d 'u n c a r r é centred de 10 m de cô té .

L e s con d ition s c lim a tiq u e s de 1 ' été 1 9 6 5 ont été p a rt icu liè re m e n t in c lé m e n te s . L e s o b s e rv a t io n s m é té o ro lo g iq u e s fa ite s a G em blou x au c o u r s de la t r o is iè m e d éca d e de ju ille t son t g ro u p é e s dans le tableau III.

L e s f ig u r e s 5 et 6 re p ro d u ise n t le s a n é m o g ra m m e s (v ite s s e et d ir e c t io n d es ven ts) con cern a n t la p é r io d e e n v isa g é e (2 0 -2 8 ju ille t 1965).L a fig u re 5 c o m p re n d , en ou tre , un p r o f i l d es v ite s s e s de vent é tab li su r le r a y -g r a s s avois in an t le cham p de b e tte ra v e s .

A p tè re s

L e tableau IV donne la r é p a r tit io n d es a p tè re s c a p tu ré s en fon ction d es d is ta n ce s a u x qu elles i ls ont été r e tr o u v é s p a r ra p p o r t aux p la n te s - s o u r c e s a p rè s 1, 2 et 3 jo u r s de d is p e r s io n .

Seuls t r o is M yzu s p e rs ica e ont é té r e tr o u v é s a d es d is ta n ce s in fé r ie u r e s à 5 m , 3 jo u r s a p rè s le lâ c h e r . En c e qui c o n c e rn e A ph is fa b æ , deux in d iv idu s ont été ca p tu ré s r e s p e c t iv e m e n t a 6 et 7 m de le u r point de d épart, 1 jo u r a p rè s le lâ c h e r , deux au tres ont é té p r is a 14 et 21 m a p rè s 2 jo u r s de d is p e r s io n , en fin t r o is au tres a p tè re s ont été lo c a l i s é s a 6, 13 et 14 m au bout de 3 jo u r s .


•t ‘ j*

•i v> •?

FIG. 4. Essais de 1965: schém a général d e dispersion des aphides m arqués capturés.

TABLEAU III. OBSERVATIONS METEOROLOGIQUES FAITES A GEMBLOUX (21-31 JUILLET 1965)

R a dia tion so la ire (h ) P récip ita tion s (m m )H u m id ité T em pératu re

m oy en n e sou s-abri a

( t )

H euresob servées

M oyenne 1 9 4 5 -5 9 (2 1 -3 1 j u i l . )

Hauteurob servée

M oyen n e 1 9 4 5 -5 9 ( 1 -3 1 j u i l . )

re la tiv e a

6 ° )

4 4 ,2 5 7 0 ,5 2 7 1 ,6 8 1 .0 7 7 ,9 1 5 ,7

a M oyen n e d e trois ob serva tion s fa ite s à 8 , 14 e t 17 h.

SM-102/6 63

Profil de vitesses de vent [calcu lées sur 10minutes)

1 600 h 1 <J)0 h 1 Q00 h

km h*lvitesse du vent

20 ; vu « ... г - ; I" ... Г ....Г Т . г п г ■'■■-f

- к ; 1 ... - f ...| — ___— —

:. г-г..л

r-Hк '-

! : . ч ; 1 1 * ' i ' i 1 il i

i h s l i ; ......... . S • • ?- j ? fih LSrÂï - ■ ;...... . _ J ...Q. .. I * i _j i i a iiLi

FIG. 5. Essais d e 1965 : aném ogram m es des 20, 21 et 22 ju ille t et profil des v itessesd ev en t du 20 ju ille t.

L e s co n d itio n s c lim a tiq u e s d é fa v o r a b le s dans le s q u e lle s ont été e ffe c tu é e s le s ca p tu re s n ' ont pas p e r m is de r e t r o u v e r un p lu s grand n om b re d 'in d iv id u s . L a ca p tu re de 12 a p te re s au c o u r s de c e t e s s a i


FTG. 6. Essais d e 19B5 : aném ogram m es des 2 3 -2 8 ju ille t.

S M - 102/6 65

T A B L E A U IV . R E P A R T IT IO N D E S A P T E R E S M A R Q U E S C A P T U R E S A P R E S 1, 2 E T 3 JO U R S D E D IS P E R S IO N E N F O N C T IO N DE L 'E L O I G N E M E N T P A R R A P P O R T A U X P L A N T E S -S O U R C E S E s s a is de 1965

D istance(m ) 1 jour

M yzus persicae 2 jours 3 jours 1 jour

A phis fa b æ 2 jours 3 jours

0 - 5 - - 3 - - -

5 - 10 - - - 2 - 1

10 - 15 - - - - 1 2

> 15 - - - - 1 -

T A B L E A U V . R E P A R T IT IO N D E S A IL E S M A R Q U E S E N F O N C T IO N D E L ’ E L O IG N E M E N T P A R R A P P O R T A U X P L A N T E S -S O U R C E S

D istance

(m )

M yzus persicae

Jour de captu re

A phis fa b æ

Jour de captu re

0 1 2 3 6 7 8 0 1 2 3 6 7 8

0 - 5 1 2 1

5 - 1 0 - - - 1 - - - - 1 - - 1

10 - 15 1 1 1

15 - 20 1(P) 2

2 0 - 25 3 1 - 4

2 5 - 30 - - 1(P) - - - 1(P) - - - - 1

30 - 35 ............................................. - - - - - 1 1

35 - 40 - - - 2 (P ) - - - - - - 1 - -

> 40 1

T ota l des a ilés m arqués

T ota l des captures

5

276

25

1017

P = captures dans un p iè g e jau n e .

(9 A ph is fa b æ et 3 M yzu s p e r s ic a e ) é ta ie cependan t le s o b s e rv a t io n s fa ite s le s deux années p ré cé d e n te s r e la t iv e s a la g ran de m o b ilité des deux e s p è c e s . En ou tre , 7 in d iv id u s (5 A ph is et 2 M y zu s ) ont été r e tr o u v é s en d e h o rs du s e c te u r , d e s vents ayant so u fflé au c o u r s de l 'e s s a i .

A ilé s

L e s ca p tu re s e f fe c tu é e s du 20 au 28 ju ille t fig u ren t dans le tableau V. T re n te aphides ont é té ca p tu ré s m a n u ellem en t, 5 M yzu s (dont 4 dans le s

S

6G FRANÇOIS et al.

p iè g e s ) et 25 A phis (dont 1 dans un p iè g e ). P a rm i le s 30 a ilé s ca p tu ré s ,10 (3 M yzu s et 7 A p h is ) 1' ont é té en d e h o rs du se c te u r , d es vents ayant so u ff lé au c o u r s de la p é r io d e e n v isa g é e (fig . 4 ). R a p p elon s que c e s vents ont so u fflé r é g u liè re m e n t du s u d -o u e s t , a v ec d es d ir e c t io n s e x tr ê m e s ne d ép a ssan t pas le n o r d -o u e s t et le s u d -s u d -e s t , s i nous ne ten on s pas co m p te du vent t r è s fa ib le qui a so u ff lé au c o u r s de la nuit du 20 au 21 ju ille t .

L e s a ilé s r e tr o u v é s dans d es d ir e c t io n s o p p o s é e s ont p ro b a b le m e n t v o lé a fa ib le hauteur, a u -d e s s u s des p la n tes , et pendant le s p é r io d e s où le vent éta it r e la t iv e m e n t c a lm e . L 1 in d iv idu cap tu ré le 20 ju ille t v e r s 15 h dans le p ièg e ü (A ph is fa b æ ) a été r e tr o u v é au s u d -e s t de la p a r c e l le , le vent ayant so u fflé du su d -o u e s t au s u d -e s t en tre le lâ ch e r et la cap tu re , a une v ite s s e o s c illa n t en tre 5 et 20 k m /h a 10 m de hauteur. (L e p r o f i l d e s v it e s s e s de vent de la f ig u r e 5 a é té é ta b li su r un gazon p r o ch e du ch a m p de b e t te r a v e s .)

DISCUSSION

M yzu s p e rs ica e a to u jo u rs été c o n s id é r é co m m e un v e c te u r t r è s m o b ile . C ependant, le s d is ta n ce s p a rco u ru e s in d iq u ées par le s au teu rs son t b ien in fé r ie u r e s a c e l le s que nous avon s e n r e g is t r é e s . R a p p e lon s que c ' e s t D av ies [5] qui a a ttiré 1' attention su r la m o b ilité des fo r m e s a p tè re s de M yzu s p e rs ica e dans un ch am p de p o m m e s de t e r r e et su r le u r im p o rta n ce dans la d is sé m in a tio n d es v ir o s e s . En m arquant le s in d iv idu s au bleu d 1 an iline il put m e ttre en é v id e n ce d es d é p la ce m e n ts de l 'o r d r e de 7 y a rd s en 24 h. L e fa it que B jo r l in g et al. [11 ] ne sign a le que d es d é p la ce m e n ts l im ité s (8 ,4 0 m en 8 jo u r s ) peut s 'e x p l iq u e r par un m anque de s e n s ib il ité de la m éth od e au torad iograp h iq u e de d é te ctio n . R ibbands [10], en partant d 'u n n o m b re connu d 'a p t è r e s de M yzu s p e rs ica e p la cé s au c e n tre d 'u n e p a r c e lle de b e tte ra v e s s u c r iè r e s non e n c o r e in fe s té e , n ' a pu m e ttre en é v id e n ce que d es d ép la cem en ts re la t iv e m e n t lim ité s par su ite , s e m b le - t - i l , de la m éth od e d 'é ch a n tillo n n a g e adoptée.

P o u r A ph is f a b æ , la se u le donnée c o n c r è te r é s u lte de travau x de B jo r l in g et al. [11] : 3, 25 m en 8 jo u r s .

N os e s s a is nous ont p e r m i de m e ttre en é v id e n ce une m o b ilité beau cou p p lu s g ran de, tant ch e z le s a p tè re s d ' A ph is fa b æ que ch e z ceu x de M yzu s p e r s ic a e (7 m en 24 h, en m oyen n e), la p o s s ib il it é d 'u n tra n sp o rt par le vent étant e x c lu e . Il se m b le donc que 1' e f f ic a c ité m o in d re d e s a p tè re s d ' A ph is fa b æ so it due p r in c ip a le m e n t au fa it qu ' i l n ' e s t pas un v e c te u r de la ja u n isse m o d é ré e de la b e tte ra v e et que son e f f ic a c ité de tr a n s m is s io n d e s au tres v ir o s e s e s t m o in d re .

L ' im p o rta n ce d es a ilé s dans la d is sé m in a tio n r a p p ro ch é e d e s v ir o s e s ne peut cependan t ê tr e n ié e . E l le a é té su p p o sé e , d ès 1946, p a r B roadb en t[6]. R ibbands [10] lu i-m ê m e le u r attribu e un r ô le dans la d is p e r s io n r a p p ro ch é e d es v ir o s e s de la b e tte ra v e , b ien que le u r part d 'in te r v e n t io n ne so it pas conn ue. Shaw [17], étudiant le v o l d es a ilé s d ' A ph is fa b æ , d is tin g u e p a rm i c e u x - c i , ou tre ceu x qui e ffe ctu en t un lon g vo l m ig r a to ir e avant de se f ix e r et de se r e p r o d u ir e , d es in d iv idu s qui ne p renn en t le u r v o l qu ' a p rè s a v o ir d é p o sé u n c e r ta in n o m b re de la r v e s et qui ont p erd u , de ce fa it, une p a rtie de le u r é n e rg ie m ig r a to ir e . C es d e r n ie r s in te r v ien d ra ien t, d 'u n e fa ço n s ig n if ic a t iv e , dans la d is p e r s io n d es v ir o s e s a c o u r te d is ta n ce .

S M - 102/6 67

Le c a r a c tè r e a c t if de l 'e n v o l a é té étudié p a r H aine [18], a lo r s que la ph ase a c t iv e du v o l de M yzu s p e r s ic æ et d 1 Aphis fa b æ a fa it 1' o b je t des r e c h e r c h e s de M u e lle r [19 ] et de M u e lle r et U nger [20, 21]. C es au teu rs ont o b s e r v é d e s v o ls a c t ifs , c o n tre d e s vents pouvant a tte in d re 2 m /s (7 ,8 k m /h ). M a lg ré la den sité r e la t iv e m e n t fa ib le de n os é ch a n tillon n ag es en 1965, nous avons pu ca p tu re r un a s s e z grand n o m b re d 'a i l é s dans le ch am p e x p é r im e n ta l, dont un t ie r s se sont d é p la cé s dans d e s d ir e c t io n s o p p o s é e s aux ven ts . N os ca p tu re s sem b len t donc so u lig n e r le r ô le des a ilé s dans la d is p e r s io n r a p p ro ch é e d es v ir o s e s et le c a r a c tè r e a ctif de le u r v o l.

R E M E R C I E M E N T S

N ous ten on s a r e m e r c ie r M e s s ie u r s L . L a llem a n d , J. N ico la s ,B . T am in iau x et F . Dupont sans le dévou em en t de qui n os e s s a is n ' au raientpu a v o ir lieu .

R E F E R E N C E S

[1 ] WATSON, M .A . , HULL, R. , BLENCOWE, J. W. , HAMLYN, B .M .G . , Ann. appl. B iol. 3 8 (1 9 5 1 ) 743.[2 ] BJORLING, K . , A cta A gríe, scand. 2 (1952 ) 2 58.[3 ] BJORLING, К . , OSSIANN ILS SON, F . , Socker L4 1 (1958 ) 1.[4 ] HEATHCOTE, G . D . , COCKBAIN, A. J . , Ann. appl. Biol. 53 (1964) 259.[5 ] DAVIES, W. M. , Bull. ent. Res. 2 3 (1 9 3 2 ) 535.[6 ] BROADBENT, L. , Ann. appl. B iol. 33 (1946) 360.[7 ] BROADBENT. L . , GREGORY, P .H ., Ann. appl. B iol. 35 (1948) 395.[8 ] BROADBENT, L . , Ann. appl. B iol. 37 (1950) 58.[9 ] BROADBENT, L . . Proc. C on f. Potato Virus Diseases, Lisse, 13 -17 Aug. 1951, Laboratorium voor

B loem bollen onderzoek te Lisse 94, H, Veenm an & Zonen , W ageningen (1951) 9.[10 ] RIBBANDS, C . R . , Bull. ent. Res. 54 (1953) 267.[11 ] BJORLING, K ., LIHNELL, D . , OSS1ANNILSSON, F . . Acta Agrie, scand. _1 (1951) 301.[12 ] WATSON, M .A . , NIXON. H. L . , Ann. appl. B iol. 40 (1953) 537.[13 ] MORTREUIL, М. , С . r. Sdanc. Soc. B iol. 5 (1959) 755.[14 ] BANKS, C .J . , NIXON, H. , E ntom ología exp . appl. 2 (1959) 77.[15 ] BANKS, C . J . , R adioisotopes and Radiation in E ntom ology (Proc. Sym. Bom bay, 1960), IAEA, Vienna

(1962) 175.[16 ] KLOFT, W . , EHRHARDT, P ., Radioisotopes and Radiation in E ntom ology (P roc. Sym. Bom bay, 1960),

IAEA, Vienna (1962) 181.[1 7 ] SHAW, M .J .P . , Rep. Rothamsted exp . Stn for 1966 (1967) 201.[18 ] HAINE. E . , Nature 175 (1955) 474.[19 ] MUELLER, H .J. , Z . PflKrankh. PflPath. PflSchutz 69 (1962) 385.[2 0 ] MUELLER, H.J. , UNGER, K . , ZUchter 22 (1952 ) 206.[21 ] MUELLER, H .J. , UNGER, K . , Forschn Fortschr. 29 (1955) 229.

D I S C U S S I O N

H. M A R C H A R T (C h a irm a n ): In d is p e r s a l stu d ies one frequ en tly fin ds a dep en den ce o f m ig r a to ry a ctiv ity on the popu lation den sity . D id you o b s e r v e an e ffe c t o f th is kind in y o u r e x p e r im e n ts?

A. R IG A : W e w e re not ab le to in vestig a te th is e ffe c t , s in c e w e knew n e ith er the popu la tion d en sity on the h o st-p la n ts at the ou tset o f the e x p e r i m ent n or the ra tio o f in d iv idu a ls o f the tw o s p e c ie s on th e se p lants.

G8 FRANÇOIS e t al.

W. J. K L O F T : O verp op u la tion o f h ost-p lan ts by aphids o ften re su lts in m ig ra t io n ; w e o b s e rv e d th is ca u sa l r e la t io n in the la b o ra to ry and in the fie ld . In such c a s e s w inged as w e ll as w in g le ss ap hids, and even la rv a e , m ay be o b s e rv e d 'w a lk in g ' out. Y o u r in te re st in g r e s u lts c le a r ly d e m o n stra te the g rea t s ig n ific a n c e o f m ig ra t io n in the d is p e r s a l o f v ir u s -c a r r y in g aphids. O ur ow n o b s e rv a t io n s a re in c lo s e a g reem en t.

A . R IG A : O c c u r r e n c e o f w in ged aphids in o u r e x p e r im e n ts w as p ro b a b ly a co n se q u e n ce o f what is ca lle d the ' g rou p e f fe c t ' , lea d in g to the m ig ra t io n w e o b s e rv e d in the f ie ld when the c lo th c o v e r in g the h osts w as r e m o v e d . W e did not note w hether the w inged aphids w alked o r flew .

EFFECTS OF IRRADIATION ON INSECT HOST-PARASITE RELATIONSHIP

G. W. RAHALKAR AND V. RAMA KRISHNAN BIOLOGY DIVISION,BHABHA ATOM IC RESEARCH CENTRE,TROMBAY, BOMBAY, INDIA

Abstract

EFFECTS OF IRRADIATION ON INSECT HOST-PARASITE RELATIONSHIP. E ffects o f host irradiation on the developm ent o f its parasite were investigated . Fem ales o f Bracon brev icom is readily accepted irradiated larvae o f tile wax moth ( G alleria m e llo n e lla ) and rice moth (C orcyra ce p h a lo n ica ) for ov iposition . H ow ever, irradiated wax moth larvae adversely in fluenced the v ia b ility o f eggs laid on them and also the survival o f the parasite grubs feed in g on their bodies. The fe m a le grubs were a ffected more than the m ales. R ice moth larvae, on the other hand, exerted no significant in flu en ce on the v ia b ility o f parasite eggs, but adversely a ffected the survival o f the grubs. H ie progeny o f parents that had been reared on irradiated larvae also exhibited som e developm en tal changes although grown on non-irradiated host larvae, and these changes were more pronounced when G. m ellon e lla was used as the host insect.


A c ce le ra te d m a ss p ro d u ctio n o f en tom op h agou s in s e c ts is o ften n e c e s s a r y f o r inundation r e le a s e s to c o r r e c t any u p set in the natural p a r a s it e -h o s t ba lance '. P ro p a g a tio n o f en tom op h agou s in s e c ts in v o lv e s th ree c lo s e ly in te r re la te d o r g a n is m s : the en tom op h agou s s p e c ie s , its h ost, and the h o s t -p la n t o r fo o d su b s tra te . T he s u c c e s s o f a b re e d in g p r o g r a m m e is th e r e fo r e a m a tter o f sy n ch ro n iz in g th re e l i fe c y c le s , i . e. the plant, the ph ytophagou s in s e c t and the en tom op h agou s s p e c ie s . In the p rop a g a tion o f an e c to p a ra s ite , a con tin u ou s su pp ly o f the p a rt icu la r d ev e lop m en ta l stag e o f the h ost in s e c t r e q u ire d fo r p a ra s ite d ev e lop m en t is s o m e tim e s d if f icu lt to m ain ta in b e ca u se o f the in h eren t b io lo g ic a l p e c u lia r it ie s o f the h ost in se c t .

R ad ia tion s w e re show n to p ro lo n g the d e v e lop m en t o f in s e c ts , to in h ib it pupation and to in c r e a s e adult lo n g e v ity [1 -4 ] . One cou ld take advantage o f th e se fa c ts to e n su re a con tin u ed su pp ly o f the h ost f o r m a ss m u lt ip lica t io n o f the p a ra s ite . H ow ever , it w as thought p ertin en t to a s ce r ta in the in flu e n ce o f the ir ra d ia te d h o st on the d e v e lop m en t o f its p a ra s ite . In vestig a tion s w e re th e r e fo r e c a r r ie d out on the. d ev e lop m en t o f a b ra co n id p a ra s ite on tw o d iffe re n t h o sts .

2. M A T E R IA L S AND M ETH O DS

A b ra co n id p a ra s ite , B ra co n b r e v ic o r n is (H ym e n o p le ra ), and its two le p id o p te ro u s la b o r a to r y h o s ts , the r ic e m oth ( C o r c y r a ce p h a lo n ica ) and the w ax m oth (G a lle r ia m e llo n e l la ) , w e re taken fr o m la b o r a to r y cu ltu re s and u tiliz e d in th e se in v e s tig a t io n s . T he la rv a e o f the r ic e m oths w e re r e a r e d on cru sh e d sorg h u m con ta in in g 5% y e a s t and th o se o f the wax

69

70 RAHALKAR and RAMAKRISHNAN

m oth s on a m ed ium con ta in in g fo u r p a rts each o f b rok en w heat and wheat bran and one p a rt each o f y e a s t , m ilk p ow d er , honey and g ly c e r in e .

T o study the d e v e lop m en t o f the p a ra s ite on ir ra d ia te d h o st la r v a e , ten p a irs o f fr e sh ly e m e r g e d m a le and fe m a le adult p a ra s ite s w ere ca g e d in a g la s s ja r and p ro v id e d with 30 wax m oth la rv a e w hich had p r e v io u s ly b een su b je cte d to a gam m a ir ra d ia t io n d o s e o f 40 krud. A se co n d grou p o f ten p a irs w as su pp lied with u n irra d ia ted h ost la rv a e fo r p a ra s it iz a t io n and s e r v e d as c o n tro l. A fter a la p se of 24 h o u rs , the p a ra s it iz e d la rv a e w e re re m o v e d and the p a ra s ite e g g s c o l le c te d , coun ted and r e p la c e d on the sa m e h ost la rv a e fo r fu rth e r d ev e lo p m e n t. T h e p a ra s ite gi-ubs led on the bod y flu id s o f the h o st la rv a e and, when fu lly g row n , pupated. The se x o f the adults w as d e te rm in e d on th e ir e m e r g e n c e . F ro m th e se adults ten p a irs w ere s e le c te d in ran dom fa sh io n and kept s in g ly in g la s s v ia ls , each o f w h ich con ta in ed tw o d ro p s o f 10% h o n e y -w a te r fo r u se as fo o d .E ach p a ir w as p rov id ed da ily with fou r u n irra d ia te d h ost la rv a e fo r o v ip o s it io n . O b se rv a tio n s on the d a ily eg g la y in g by each fe m a le and the su bsequ en t d e v e lop m en t o f the e g g s to the adult stage w e re r e c o r d e d .In an oth er e x p e r im e n t the sa m e p r o c e d u r e s w e re re p e a te d with the la rv a e o f C_. ce p h a lo n ica as the h ost in s e c t .

3. R E SU L TS

When the fe m a le s w e re a llow ed to p a ra s it iz e wax m oth la rv a e , no s ig n ifica n t d if fe r e n c e in th e ir a c ce p ta n ce o f ir ra d ia te d and n o n -ir r a d ia te d h o st la rv a e w as o b s e rv e d . H ow ever , the v ia b ility o f the eg gs la id on ir ra d ia te d h osts se e m e d a d v e r se ly a ffc c te d (T a b le I). T h e g ru bs fe e d in g on ir ra d ia te d h ost la rv a e w e re s im ila r ly in flu en ced , on ly 55. 3% s u c c e s s fu lly c o m p le t in g th e ir d e v e lop m en t up to the adult stage , in co n tra s t to 73. 03% o f the g ru bs grow n on n o rm a l h o s ts . T h is d if fe r e n c e in grow th c h a r a c t e r is t ic s on the tw o h ost m ed ia w as s ta t is t ica lly s ig n ifica n t. T he s e x ra tio (m a le : fe m a le ) o f adults w hich d ev e lo p e d fro m g ru bs fe e d in g on ir ra d ia te d la rv a e w as a lso s ig n ifica n tly re d u ce d as co m p a re d with that o f the c o n tro l in se c ts .

T A B L E I. D E V E L O P M E N T O F B. b r e v i c o r n i s W IT H G. m e l lo n e l la AS H O ST IN S E C T

Irradiated host N on -irra d ia ted host

T o ta l num ber o f eggs la id b y 10 parasite fe m a le s in 24 hours

974 , 935

Per cen t v ia b ility o f

eggs

. 6G .8 3 7 5 .4 3

Per cen t la rvae b e c o m in g adults

Г1Г1 .ЗО 7 3 .0 3

S e x -r a t io (m a le : fe m a le ) 1 :0 .6 9 1 :1 .1 0 6

SM- 102/7 71

T A B L E II. D E V E L O P M E N T A L C H A R A C T E R IS T IC S O F T H E P R O G E N Y O F P A R E N T S R E A R E D O N G. m e l lo n e l la L A R V A E

Parents reared on

Irradia ted host N o n -ir ra d ia te d host

F ecu n d ity 6 2 9 .9 6 9 8 .9

Per cen t su ccessfu l 6 3 .9 6 6 .0 6c o m p le t io n o f d e v e lo p m e n tfrom eg g to adult

S e x -ra t io ( m a le : fe m a le ) 1: 0 .2 9 9 1: 0 . 5665

T A B L E III. D E V E L O P M E N T O F B . b r e v i c o r n i s W IT HC . c e p h a lo n ic a AS H O ST IN S E C T

Irradiated host N on -ir ra d ia ted host

T ota l n u m ber o f eg g s la id b y 10 parasite fe m a le s in 24 hours

27 4 323

Per cen t eggs v ia b le 8 6 .1 2 8 4 .8 4

Per cen t larvae, b e c o m in g adults

7 7 .5 6 9 5 .6 1

S e x -r a t io (m a le : fe m a le ) 1: 1 .0 1 1 1: 1 .0 6 3

P ro g e n y o f p a ren ts r e a r e d on ir ra d ia te d la rv a e a lso exh ib ited so m e d e v e lop m en ta l ch a n g es although grow n on n o rm a l h o st la rv a e (T a b le II). T h is w as ev iden t fr o m the fa c t that on ly 63. 9% o f the e g g s la id by fe m a le s r e a r e d on ir ra d ia te d h ost la rv a e d e v e lo p e d in to adults, w h e re a s 66. 06% o f the eg g s fr o m fe m a le s r e a r e d on n o rm a l h ost la rv a e co m p le te d d ev e lop m en t in to the adult stag e . A p re p o n d e ra n ce o f m a le s in the p rogen y o f paren ts r e a r e d on ir ra d ia te d h o sts w as a lso o b s e rv e d .

U nlike G. m e llo n e l la , the ir ra d ia te d la rv a e o f c e p h a lo n ica e x e rte d no in flu en ce on the v ia b ility o f the e g g s la id on them by n o rm a l fe m a le s (T a b le III). H ow ever , the p a ra s ite g ru bs fe e d in g on the bod y flu id s o f ir ra d ia te d la rv a e sh ow ed c o n s id e r a b le m o r ta lity . The r e d u c tio n in the n um ber o f g ru bs s u c c e s s fu lly d e v e lo p in g on ir ra d ia te d h o sts w as s ta t is t ica lly s ig n ifica n t when c o m p a re d with the n um ber d e v e lo p in g on n o rm a l h ost la rv a e . T he p rogen y o f pa ren ts r e a r e d on ir ra d ia te d la rv a e o f C. c e p h a lo n ica sh ow ed no d e v e lop m en ta l a b n o rm a lit ie s (T a b le IV ).

4 . DISCUSSION

T he a d v e rse e f fe c t on the d ev e lop m en t o f the p a ra s ite r e a r e d on ir ra d ia te d w ax m oth la rv a e in d ica ted a change in the su ita b ility o f the


T A B L E IV . D E V E L O P M E N T A L C H A R A C T E R IS T IC S O F T H E P R O G E N Y O F P A R E N T S R E A R E D O N C . c e p h a lo n ic a L A R V A E

Parents reared on

Irradiated host N on -irra d ia ted host

F ecu n d ity 4 9 3 .f i 4 2 5 .1

Per c e n t successfu l c o m p le t io n o f d e v e lo p m e n t from e g g to adult

64 . 03 62 . 38

S e x -r a t io ( m a l e : fem a le .) 1 :0 .4 1 5 8 1 : 0 .3 9 2 3

h ost in s e c t . P h y s io lo g ic a l ch an ges in in s e c ts fo llo w in g ir ra d ia t io n w e re re p o r te d by m any w o rk e r s . D iffe re n c e s in the h aem olym ph p ro te in fr a c t io n betw een ir ra d ia te d and c o n tro l E p h estia la rv a e w e re o b s e rv e d[5]. K r itsk ii and C h iw ei [6] r e p o rte d a c o n s id e r a b le fa l l in ad en os in e tr ip h osp h a te content and a ctiva tion o f a u to ly s is im m e d ia te ly fo llo w in g ir ra d ia t io n o f the b ee m oth (G a llfr ia m e llo n e l la ) la rv a e with 2 k rad . A de la y in pupation o f X - ir r a d ia te d D ro sp h ila m e la n o g a ste r la rv a e w as n o t ic e d by B ou rg in et a l. [7 ], who su g gested that the ra d ia tion e f fe c t w as an a b n orm a l phase betw een n o rm a l and actu a l pupation , and du rin g th is ph ase a lo s s in the w eight o f the la rv a e with a g re a tly r e d u ce d grow th ra te o f fat b o d ie s took p la ce .

T h e e ffe c t o f ra d ia tion in du ced p h y s io lo g ic a l ch an ges in the h ost w as m o r e p ron ou n ced on the p o s t -e m b r y o n ic d ev e lop m en t o f the p a ra s ite . The s ig n ifica n t d e c r e a s e in the m a le - t o - fe m a ie ra tio am on gst adults d e v e lo p in g on ir ra d ia te d h ost la rv a e (G. m e llo n e lla ) in d ica ted that the fe m a le p a ra s ite g ru bs su ffe re d g re a te r m o rta lity w hile fe e d in g on body flu id s o f the ir ra d ia te d h ost la rv a e . T h is cou ld be the r e s u lt o f d iffe re n t ia l n utrition a l r e q u ire m e n ts , both qu a lita tive and qu an tita tive, o f the m a le and the fe m a le p a ra s ite g ru b s . No e x p e r im e n ta l ev id e n ce in su p p ort o f the e x is te n c e o f su ch a n utrition al d im o rp h ism w as found in the ra d ia tion lite ra tu re .H ow ever , the e x is te n ce o f su ch a ph enom enon w as d em on stra ted in p a ra s it ic H ym en op tera . In C occop h a gu s c o w p e r i the h o st s p e c ie s su pportin g d e v e lop m en t o f m a le and fe m a le g ru bs a re d iffe re n t [8]. In E n c a r s ia spp . , e g g s d e v e lo p in g into m a le s w e re d e p o s ite d e n d o p a ra s it ica lly in le p id o p te ro u s h o s ts , and th ose d e v e lo p in g in to fe m a le s in h om op terou s h o s ts [9].

T h e p h y s io lo g ica l ch a n ges in the h o st not on ly a ffe c t the d ev e lop m en to f the p a ra s ite , but a lso that o f the p rogen y , even when the la tte r a re a llow ed to d ev e lop on n o rm a l h o sts . T h is a d v e rse e ffe c t on the p rogen y cou ld be the r e su lt o f inadequate n utrition o f the p a ren ts . F la n d e rs [10], w h ile studying the e ffe c ts o f c o ld s to ra g e on the p a ra s it ic H y m en optera , o b s e r v e d that: "the m e ta b o lic activ ity du ring the m atu re la rv a l o r pupal p e r io d s is dependent on n u tritive m a te r ia ls accu m u la ted in the bod y du rin g the im m a tu re la rv a l s ta g e s . T he o rga n s m o s t lik e ly to be a ffe c te d by in su ffic ie n t n utrition a re th o se u sed in re p ro d u c t io n , with the r e su lt that the g erm c e l ls a re a d v e r se ly a ffe c te d , and the sp e rm a to z o a in

SM- 102/7 73

p a rt icu la r la ck v ita lity . " In b r e v ic o r n is u n fe r t il iz e d eg g s d e v e lo p in to m a le s and fe r t i l iz e d on es in to fe m a le s . T he p re p o n d e ra n ce o f m a le s in the p rog en y o f pa ren ts r e a r e d on ir ra d ia te d h ost la rv a e co u ld be due to the p o o r v ita lity o f s p e r m s , with the re su lt in g re d u ctio n in the n um ber o f f e r t i l iz e d e g g s la id .

The d ev e lop m en ta l ch a n ges su ffe re d by the p a ra s ite g ro w in g on ir ra d ia te d r ic e m oth la rv a e w ere le s s p ron ou n ced c o m p a re d with th ose o c c u r r in g when w ax m oth la rv a e w e re u sed as h o s ts . T h is cou ld be attribu ted to the d iffe r e n t ia l r a d io s e n s it iv ity o f th e se tw o h ost s p e c ie s .A d o s e o f 15 krad p rev en ted pupation o f the wax m oth la r v a e , w h e re a s a d o s e o f 40 krad w as r e q u ire d to p ro d u ce the sa m e d e g re e o f in h ib ition in the c a s e o f the r i c e m oth (R ah a lk ar, unpublished data). W hiting [11] o b e rv e d no m u ta gen ic o r le th a l e f fe c t s on the p a ra s ite H a b ro b ra co n r e a r e d on h ea v ily ir ra d ia te d E p h estia la r v a e . T h is cou ld be due to the h igh er to le ra n c e o f th is in s e c t to ra d ia tio n . When fu ll -g r o w n E . kuhniella la rv a e w e re X - ir r a d ia te d with d o s e s ran g in g fr o m 40 to 160 k rad , 23. 16% o f the la rv a e w e re a liv e 30 to 40 days a fte r ir ra d ia t io n [12]. It was co n c lu d ed that ir ra d ia t io n o f the h ost in s e c t g re a tly in flu e n ce s the d e v e lo p m ent o f the p a ra s ite and a lso that o f its p rogen y in su ch a m an n er that ir ra d ia tio n o f a h ost in s e c t is o f doubtfu l v a lu e in m o d ify in g the d ev e lop m en t o f the h o s t in s e c t to su it the re q u ire m e n ts o f the p a ra s ite .

R E F E R E N C E S

[1 ] ABDEL-MALEK, A . S. , Bull. en l. Res. 52 4 (1 9 6 1 ) 701.[2 ] ERDMAN, H .E ., Int. J. R adial. B iol. -5 4 (1962 ) 331.[3 ] D AY, M .F .. OSTLER, 1 .1 . , Insect Pathology - an advanced treatise, V o l. 1, ch . 2: Physical

injuries (Steinhaus, E. A. , Ed. ) A ca d em ic Press, New York (19G3).[4 ] BALOCK, J. W. . Jr., BURD1TT, A . K . , CHRISTENSON. L. D. . ] . e c o n . Ent. 56 1 (1963 ) 42.[5 ] ERDMAN, H .E . , Research and developm ent in progress, B io logy and M ed icin e , Issue No. 2,

T ID -4 201 , A EC (N ov. 1963) 127.[6 J KRITSKII, G . A . , CHIWEI, C . , B iochem istry USSR. 26 (1961) 224.[7 ] BOURG IN, R .C . , KRUMINS, R. , Ql'ASTLER, I I . , Radiat. Res. 5 6 (1956) 657.[8 ] FLANDERS, S. E . , Univ. C a lif. Pubis Ent. 6 (1937) 401.[9 ] FLANDERS, S. E . , Entom ología exp. appl. 2 (1959) 125.

[1 0 J FLANDERS, S .E . , ] . e con . Ent. 31_ 5 (1938) 633.[ l l j WHITING, A .R . , Anat. R ec. I l l (1951) 565.[12] WHITING, A .R . , Anat. R ec. 108 (1950) 609.

D I S C U S S I O N

V. L A B E Y R IE : Do not the d if fe r e n c e s in y ou r r e s u lts f o r the wax m oth (G. m e llo n e l la ) and the r i c e m oth ( С . c e p h a lo n ica ) a r is e fr o m the fa c t that in u n irra d ia ted m e llo n e lla the e m b ry o n ic m o rta lity is a lrea d y h igh er than in u n irra d ia te d ce p h a lo n ica , s o that it cou ld be a ssu m ed that w hile ce p h a lo n ica is a fa v o u ra b le h o st, m e llo n e lla is not, and any fu rth er d e te r io r a t io n in the co n d itio n s has fa ta l r e s u lt s ?

G. W. R A H A L K A R : T he w ax m oth and the r i c e m oth ap p ear to be equ a lly su itab le h o s ts f o r the d e v e lo p m e n t o f th is p a ra s ite . T he d if fe r e n c e in p e rce n ta g e v ia b ility o f eg gs la id on u n irra d ia ted la rv a e o f th ese two m oth s can b e a s c r ib e d to n atu ral v a r ia t io n in flu en ced by e n v iron m en ta l


fa c t o r s . T he tw o m ain e x p e r im e n ts w e re p e r fo rm e d at d iffe re n t t im e s and th e se tw o c o n tro ls can not th e r e fo r e be d ir e c t ly c o m p a re d .

V. L A B E Y R IE : Is the v a r ia t io n g re a te r fo r is o la te d fe m a le s on ir ra d ia te d la rv a e ?

G. W. ilA H A L K A R : T h is v a r ia t io n is not s ig n ifica n tly la rg e .P . P E L E G R IN : D oes the 4 0 -k ra d d o se g iven to the h ost ca u se its

death o r s im p ly a r r e s t its d ev e lo p m e n t?G. W. R A H A L K A R : In the c a s e o f the r i c e m oth , pupation w as

in h ib ited ab ove d o s e s o f 40 k rad , but ir ra d ia te d la rv a e su rv iv e d fo r a c o n s id e r a b le p e r io d . In the c a s e o f the w ax m oth the la r v a l m o r ta lity cu rv e c l im b s ra p id ly .

INVASION OF CONIFER PLANTATIONS BY RADIOACTIVELY LABELLED Hylobius abietis L.

H. H. F.IDMANN .ROYAL COLLECT OF FORESTRY,STOCKHOLM , SWEDEN

Abstract

INVASION’ OF CONNER PLANTATIONS BY RADIOACTIVELY LABELLED Hylobius abietis L,I ly lob h is abietis L. w eevils, rad ioactiv cly labelled by dipping into solutions contain in g ‘16Sc, were released around two m od el areas representing recently cleared cutting areas freshly planted with Norway spruce. H alf o f the w eevils released around one m odel area were also labelled with different co lou r marks. The invasion, o f both the natural llylobius population and the released labe lled w eevils, was studied by m aking repeated inspections o f attraetant pine billets evenly distributed over the experim ental areas. At certain intervals the spruce plants on the areas were inspected for feed in g marks to evaluate the progress o f the attack.

The greatest num ber o f labe lled w eevils, am ounting to 13ид> o f those released, was found in one experim ental area one day after release. In this area, successively dim inishing numbers o f w eevils found at the billets (both labelled w eevils and natural population) and an increasing degree o f feeding on the plants indicated that the w eevils did nol leave the plantation but changed their behaviour. The w eevils often dug into the so il, which was proved by measurements o f ground rad ioactiv ity at b illets and stumps. In the other area, which had richer vegetation , on ly sm all changes in numbers occurred .

In a ll inspections, most o f the labelled w eevils w ere found near the border o f the experim ental area, but the percentage o f w eevils in the m id dle o f the area increased slow ly . O ne day after release the greatest distance a ra d io a ctiv e ly -p lu s -co lo u r -la b e lle d b eetle had m oved into the area was 34 m , w hile the average distance was only 4 .1 5 m . There w ere no significant differences in the speed o f invasion from different directions into the plantation. From the results and observations it can be con clu ded that most or a ll released w eevils invaded the plantations by craw ling, and not by flying.

T h e natural populations invaded on e experim ental area slow ly from the borders, and the other area faster and m ore evenly distributed. A ccord in g ly , the distribution o f dam age, by feed in g was uneven in one area and com paratively even in the other.

One practica l con sequ en ce o f this study is the recom m endation that plant protection measures be concentrated on the borders o f con ifer plantations. Even plants in the central area o f plantations should be protected . Concentrations o f w eev il-a ttractin g m aterials, such as attractant in sectic id e traps, should be avoided in the centres o f plantations.


T he w e e v il, H y lob ius a b ie tis L ,., c a u se s c o n s id e r a b le dam age in E u ropean c o n ife r p lan tation s by feed in g on the young p la n ts . T h e re are v a r io u s m eth od s o f p reven tin g su ch d a m a g e : keeping the w e e v ils aw ayfro m the p lantations (r e fo r e s ta t io n s e v e r a l y e a r s a fte r c le a r cutting, d im in ution o f b e e t le popu la tion , tra p d itch e s ), p ro te c t in g the p lants by in s e c t ic id e s (trea tm en t b e fo r e p lanting o r in the p lan tation s) o r c o n tr o llin g the w e e v ils irrthe plantations (tra p s , in s e c t ic id e - t r e a te d b ille t s , to ta l in s e c t ic id e trea tm en t o f p la n ta tion ). F o r m o r e than 15 y e a r s syn th etic in s e c t ic id e s have been u sed s u c c e s s fu lly ; n e v e r th e le s s the p ro b le m o f H y lob iu s c o n t r o l is not c o m p le te ly so lv e d and r e q u ire s g r e a te r k n ow led ge o f the e c o lo g y and the b eh a v iou r o f the s p e c ie s .

75

76 EIDMANN

Young c o n ife r p lants on fr e s h c le a r -c u t t in g a r e a s w h ere th e re are stu m ps and o th e r re m a in s o f c o n ife r t r e e s a re p a r t icu la r ly su sc e p t ib le to attack by H y lob iu s . T h e se p lantations a re invaded in sp r in g by H y lo b ius w e e v ils from the v ic in ity , w hich feed on the plants and la y th e ir e g g s on the stu m p s. T h e in va sion o f w e e v ils into the p lan tation s and the d is tr ib u tion o f th e ir feed in g in the p lantation have h ith erto been stud ied s y s te m a tic a lly o n ly to a lim ite d d e g r e e . In vasion and fe e d in g , h o w e v e r , a re c lo s e ly co n n e cte d with the p ro b le m o f c o n tro l.

T e m p e ra tu re , hum id ity (o f a ir and s o i l) and v egeta tion in flu e n ce the a p p ea ra n ce o f the w e e v ils , th e ir a c t iv it ie s and feed in g [1 -3 ] . S ch m idt [3] stud ied the m ig ra t io n s in e x p e r im e n ts w ith fed , c o lo u r -m a r k e d w e e v ils . T h e b e e t le s can fly ra th e r lon g d is ta n ce s , but, on the o th e r hand, the tr a p ditch c o n tro l m ethod is based on the fa ct that the w e e v ils o fte n invade the p lantations by c ra w lin g on the grou n d . T h ey p r e fe r to stay u nder fr e s h c o n ife r bark , but la te r le a v e it [4] and en ter the s o i l f o r o v ip o s it io n .

In 1966 and 1967 w e con d u cted e x p e r im e n ts to c la r i fy the a n sw e rs to the fo llo w in g q u estion s :

In what way do w e e v ils en terin g a p lantation fr o m its b o r d e r s d is p e r s e o v e r the a re a , and what is the tim e se q u e n ce o f the in v a s io n ?

Do the w e e v ils p r e fe r c e rta in d ir e c t io n s o f in v a s ion ?A re th e re d if fe r e n c e s betw een the m a n n er o f in va sion o f b e e t le s

sta rtin g c lo s e to the a re a and that o f b e e t le s com in g fro m a g rea tevr d is ta n c e ?

W hen and to what extent do the w e e v ils en ter the s o i l?What is the tim e se q u e n ce and the sp a tia l d is tr ib u tio n o f fe e d in g on

the p la n ts?

T he b e e t le s w ere r e le a s e d at the b o r d e r s o f the e x p e r im e n ta l a re a s and w e re la b e lle d so that they cou ld be d is tin g u ish ed fro m the natural [ ly lo b iu s popu lation . F o r s e v e r a l r e a s o n s , la b e llin g with a y - ray em ittin g ra d io iso to p e w as ch o se n : fast and e a sy la b e llin g , no e ffe c t on the b eh a v iou r o f the in s e c ts , easy d e te ctio n , and d e tection o f w e e v ils u n d er the s o i l su r fa c e . The e x p e r im e n ta l a re a s w ere la id out as m o d e l a r e a s , b e ca u se con d ition s in p lantations on natural c le a r -c u tt in g a re a s u su a lly v a ry in the d is tr ib u tio n o f stum ps and v egeta tion in su ch a m a n n er that it is d iff icu lt to an a lyse and g e n e r a liz e the re su lts .

2. E X P E R IM E N T A L M ETHODS

2 .1 . E x p e r im e n ta l a rea s

T he e x p e r im e n ta l a re a s w ere r e c ta n g le s m e a su rin g 80 m X 96 m .T h ey w ere situated in fo r e s t d is t r ic t s in ce n tra l Sw eden and w e re s u r roun ded on th ree s id e s by u n iform fo r e s t s . No c o n ife r t r e e s had been fe lle d within a d is ta n ce o f at lea st 2 km fro m the a re a s fo r m o r e than on e y e a r b e fo r e the e x p e r im e n ts . T h is e lim in a ted o th e r s o u r c e s o f a ttra c tio n fo r the w e e v ils . The a re a s w e re fa rm land with u n iform w eed v e g e ta tio n . The e x p e r im e n ta l a re a used in 1966 (V ârm lan d) had m o is t c la y e y s o i l with th ick w eed v egeta tion ; the e x p e r im e n ta l a re a in .1.967 (O sterg ô tla n d ) had d ry sandy s o i l with p o o r vegeta tion . T he a re a s w ere

S M - 102/9 77

planted with N orw ay s p ru ce in s p a c e s o f 2 m X 2 m and 5 0 -c m fr e s h ly cut p ine b i lle t s w e re la id out on the ground in sp a c e s o f 4 m X 4 m . C uttings o f pine tru n k s, 50 cm lon g and 12 -15 cm in d ia m e te r , w e re put v e r t ic a lly into the ground in s p a c e s o f 8 m X 8 m . T h ey p ro tru d ed 15 cm a b ove the s u r fa c e and re p re s e n te d a r t i f ic ia l stu m ps.

In th is w ay e v e ry m o d e l a re a o f 0 .7 7 ha had 120 stu m p s, 480 pine b ille t s and 1920 s p ru ce plants ev en ly d is tr ib u ted o v e r the a re a .

2 .2 . L a b e llin g and r e le a s e o f w e e v ils

T he w e e v ils w ere c o lle c te d in sa w m ills at the beg in n ing o f June and kept c o o l and without food until la b e ll in g .

In p r e lim in a ry e x p e r im e n ts with 60Co so lu tion (2 .5 /u C i/m l) w e e v ils w ere la b e lle d by d ipping them into the so lu tio n and by feed in g them on p ine bark trea ted with ra d io a c t iv e so lu t io n . A fte r s e v e r a l w eek s in c a g e s , both g rou p s o f w e e v ils behaved n o rm a lly and w e re su ff ic ie n t ly r a d io a c t iv e . A fte r being r in se d th orou g h ly in w ater , the d ipped w e e v ils lo s t m o re r a d io a ct iv ity than th o se la b e lle d with ra d io a ct iv e fo o d . N e v e rth e le ss , the dipping m eth od w as c h o s e n f o r the fie ld e x p e r im e n ts , b e ca u se the r e le a s e d w e e v ils w ere to be hungry .

B e ca u se o f its s h o r te r h a l f - l i f e 46Sc w as used in stead o f 60C o . The d ipping so lu tion s con ta in ed 46S c C l3 , with H20 and 20% a lco h o l as a w etting agen t. The s p e c i f i c a c t iv ity w as 1 4 .4 /u C i/m l in the 19G6 e x p e r im e n t and 4 4 .8 ц C i /m l in 1967. D ipping w as p e r fo rm e d in the open a ir n ea r the e x p e r im e n ta l a r e a s . T he dipping so lu tion w as in a pit in the g rou n d . The o p e r a to r w as sh ie ld ed by a s o i l em ban k m en t and lea d b r ic k s , v iew ed the so lu tion in a m i r r o r and m anipu lated with ro d s and c la m p s . S m all, p e r fo ra te d P V C b o tt le s , each con ta in in g 88 w 'eev ils , w e re d ipped into the ra d io a ct iv e so lu tio n and then put a s id e to d ry . E ach b o ttle w as u sed on ly o n ce . On both 11 June 1966 and 13 June 1967, 3872 w e e v ils w ere la b e lle d ra d io a ct iv e ly and r e le a s e d at equ a l d is ta n ce s on the b o r d e r s o f the e x p e r im e n ta l a r e a s . D ipping and r e le a s e took le s s than fo u r h o u rs .

1760 o f the w e e v ils r e le a s e d in 1967 w e re la b e lle d with d iffe re n t c o lo u r s on the e ly tr a b e fo r e ra d io a ct iv e la b e ll in g . F o u r g rou p s o f 440 w e e v ils each w e re la b e lle d with fo u r d iffe re n t c o lo u r s and r e le a s e d on each o f the fo u r s id e s o f the e x p e r im e n ta l a re a . T h us each sid e had H y lob iu s with a c e r ta in c o lo u r . In th re e c a g e s n ear the e x p e rim e n ta l a re a u sed in 1967, u ntrea ted , ra d io a c t iv e ly la b e lle d and r a d io a c t iv e ly - p lu s -c o lo u r - la b e l le d w e e v ils w ere kept f o r c o m p a r is o n .

2 .3 . In sp ection s

The e x p e r im e n ts w e re in sp e cte d at in te rv a ls until A u gust, the p u rp ose o f the in sp e ctio n s b e in g to exa m in e the p r e s e n c e o f la b e lle d and u n labelled w e e v ils and o f r a d io a ct iv ity at the pine b ille t s and a r t i f ic ia l stu m p s. The ra d io a ct iv ity w as d e te rm in e d w ith a s c in t il la t io n co u n te r and GM co u n te r s . Random sa m p le s w e re taken in s id e and o u ts id e the a r e a to d e te rm in e the p r e s e n c e o f w e e v ils and r a d io a ct iv ity on the p la n ts . The extent o f feed in g o n e v e ry plant w as ra ted fr o m 0 to 5.

In la te r in sp e c tio n s the dam age by feed in g and the b re e d in g on the stum ps w e re stu d ied .

3. RESULTS

3 .1 . R a d ioa ctiv ity and su rv iv a l o f w e e v ils

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In the 1967 e x p e rim e n t the r a d io a ct iv ity o f on e w e e v il on e day a fte r la b e llin g w as, on the a v e ra g e , about 0 .1 y C i. It d e c r e a s e d ra th e r ra p id ly , and fiv e days a fte r la b e llin g am ounted to o n ly about 50%, and a fte r 2 m onths (ca g ed w e e v ils ) about 7%. In the 1966 e x p e r im e n t the r a d io a c t iv ity w as lo w e r than in 1.967.

T h e d e c r e a s e o f r a d io a ct iv ity was ca u sed by the ra d io a c t iv e d eca y o f 46Sc and by the ra d io a ct iv e la y e r b e in g rubbed o f f the b o d ie s o f the in s e c t s . T he im p o rta n ce o f rubbin g o f f w as p ro v e d by tw o r e su lts o f the e x p e r im e n ts :

(1) T h e s o i l u nder the pine b i lle t s and at the stum ps o ften sh ow ed c o n s id e r a b le ra d io a ct iv ity (s e e s e c t io n 3 .2 ) .(2) In the ca g e s 2 m on th s a fte r la b e llin g the dead w e e v ils had a s ig n if i can tly h igh er ra d io a ct iv ity than the l iv e I ly lo b iu s .

No o b v io u s d if fe r e n c e s betw een the d e g re e o f s u rv iv a l o f u ntreated , ra d io a c t iv e ly - la b e l le d w e e v ils and that o f r a d io a c t iv e ly -p lu s -c o lo u r - la b e lle d w e e v ils in the ca g e s w ere o b s e r v e d . In the e x p e r im e n ta l a re a , th e r e w as no g rea t d if fe r e n c e betw een the p r e s e n c e o f r a d io a c t iv e ly - la b e lle d w e e v ils and that o f r a d io a c t iv e ly -p lu s -c o lo u r - la b e l le d w e e v ils .

3 .2 . T im e seq u en ce o f w e e v il a p p e a ra n ce

In both e x p e r im e n ts r a d io a ct iv ity in the s o i l w as a lre a d y o b s e r v e d on e day a fte r r e le a s e o f the w e e v ils . It w as thus apparent that the w e e v ils so o n en tered the s o i l , a lso at the s tu m p s. The n u m ber o f p la ce s w ith r a d io a c t iv ity in the s o i l g ra d u a lly in c r e a s e d . R a d io a ctiv ity in the s o i l d o e s not im p ly the p r e s e n ce o f a la b e lle d w e e v il; in m any p la ce s exam in ed no b e e t le s w ere found. T he w e e v ils had ap paren tly le ft th ese p la c e s , but lo s t so m e o f the ra d io a ct iv e la y e r on th e ir b o d ie s by fr ic t io n w ith s o i l p a r t ic le s (s e e se c t io n 3 .1 ).

T h e n um ber o f la b e lle d w e e v ils found in on e in sp e ctio n in the 1966 e x p e r im e n t did not e x ce e d 0 .7 % o f the b e e t le s r e le a s e d . T h e r e fo r e , ' the t im e seq u en ce fo r the w eev il a p p earan ce cou ld not be a n a lysed . In the 1967 e x p e r im e n t, h o w ev er , 18% o f the la b e lle d w e e v ils w e re found in the e x p e r im e n ta l a re a one day a fte r th e ir r e le a s e . T h is w as the h igh est n u m ber o f la b e lle d w e e v ils found. T he n u m ber o f ra d io a ct iv e w e e v ils found in th is a rea la te r d im in ish ed s u c c e s s iv e ly (F ig . 1).

T h e u n la be lled w e e v ils o f the natural popu lation invading the e x p e r i m en ta l a re a in 1967 rea ch ed th e ir g re a te s t n u m ber at the b ille t s b e fo r e the r e le a s e o f la b e lle d w e e v ils . T h ey then d e c re a s e d at a rate re se m b lin g that o f the la b e lle d on es (F ig . 1 ) . ' It m ay thus be con clu d ed that la b e lle d and u n la be lled w e e v ils behaved in the sa m e w ay. The d e c r e a s e o f w e e v ils found at the b ille t s d o e s not m ean that the b e e t le s le ft the a re a . The continu ou s in c r e a s e o f feed in g in the a re a (s e e s e c t io n 3.4 and F ig . 2) r e fu te s th is ; a change in the b eh a v iou r o f the w e e v ils is m o r e p ro b a b le .

In the 1966 e x p e r im e n ts th e re w as no such change in b e h a v io u r . The n u m ber o f u n labelled w e e v ils v a r ie d , but ir r e g u la r ly and without the

S M - 102/9 79

FIG. 1. Experim ental area, 1967 : m ean num ber o f unlabelled (W ildpop) and labe lled (Rad) H ylobius w eevils per pine b ille t at d ifferent tim es during the season.

FIG. 2. Experim ental area, 1967: m ean d egree o f feed in g per spruce plant at d ifferen t 't im es during the season.

m a rk ed d e c r e a s e show n in F i g . l . T h is is se e n fr o m the fo llo w in g e x tra c t fr o m the p r o to c o ls :

D ate o f in sp e c tio n 1 C .6 .6 6 . 1 9 .6 .6 6 . 2 2 .6 .6 6 . 2 7 .7 .6 6

N u m ber I ly lo b iu s /b il le t 0 .2 9 0 .2 0 0 .4 1 0 .2 3

In 1966 the m a xim u m n u m ber o f u n la be lled w e e v ils found in on e in sp e ctio n w as c o n s id e r a b ly lo w e r than in 1967.

T he d iffe re n t b eh a v iou r o f the w e e v ils w as p ro b a b ly co n n ected with d if fe r e n c e s in s o i l , v egeta tion , and lo c a l and m ic r o c l im a te betw een the tw o e x p e r im e n ta l a r e a s .

3 .3 . Spatial d is tr ib u tio n o f w e e v ils

T he la b e lle d w e e v ils began to invade the e x p e r im e n ta l a re a s sh o rtly a fte r r e le a s e . S ing le w e e v ils so o n re a ch e d the m id d le o f the a re a , but

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the m a jo r ity advanced s lo w ly . In a ll in sp e c t io n s , c o n s id e r a b ly m o r e ra d io a c t iv e I ly lob iu s w ere found in the o u te r re g io n s o f the e x p e r im e n ta l a r e a s than in the ce n tre . L a te r , h o w e v e r , th ere w as an in c r e a s e in the p r o p o rt io n o f la b e lle d w e e v ils that advanced fu rth e r into the a r e a s . The ex a m p les in F ig s 3 -5 illu s tra te th is p r o g r e s s .

Mosstorp• ••• • «

• о .

•

oo •

• •

9О •

•

Ф •

e •

• •

• •

• • • • • ,

• .

•

16.6.66. 19.6.66.

FIG. 3. Experim ental area, I9 6 0 : spatial distribution o f la b e lled w eevils on 16 June (le ft) and 19 June (right).

N

Àlsebo,K.6.67. S 0 5 10 15 20 m

FIG. 4. Experim ental area, 1967: spatial distribution o f Labelled w eevils on 14 June.

N

S M - 102/9 81

E

Àlsebo, 17.6.67. s Q 5 10 15 20m

FIG. 5. Experim ental area, 19G7: spatial distribution o f labelled w eevils on 17 June.

T he r a d io a c t iv e ly -p lu s -c o lo u r - la b e l le d w e e v ils in the 1967 e x p e r i m ent gave m o r e e x a ct in fo rm a tio n on the sp eed o f in v a s io n . F o r e v e ry c o lo u r - la b e l le d H ylobiu s found in the a re a , the sh o r te s t d is ta n ce to the lin e o f r e le a s e fo r the r e s p e c t iv e c o lo u r w as d e te rm in e d . One day a fter r e le a s e on e w eev il had a lrea d y advanced at le a s t 34 m . The g re a te s t d is ta n ce w as 90 m (13 days a fte r r e le a s e ) . On the o th e r hand, the m ean m in im um d is ta n ce in c r e a s e d with t im e , but w as co n s id e r a b ly lo w e r (F ig . 6 ). T he sp eed and d is ta n ce o f in va sion o f c o lo u r - la b e l le d w e e v ils w as about the sa m e fo r the d iffe re n t d ir e c t io n s o f in v a s io n .

T he u n la be lled w e e v ils o f the tw o n atu ral p opu la tion s d if fe r e d , not on ly as r e g a rd s the tim e se q u e n ce (s e e s e c t io n 3. 2), but a lso in th e ir sp a tia l d is tr ib u tio n in the two e x p e r im e n ta l a r e a s . In the 1966 e x p e r i m en t, the in sp e c t io n s m ad e in June re v e a le d m any m o r e b e e t le s in the o u te r re g io n s than in the ce n tre o f the a re a . At the end o f July, h ow ev er , th e r e w e re no lo n g e r any d is tin c t d if fe r e n c e s betw een the o u te r and in n er re g io n s (F ig . 7 ). In the 1967 e x p e r im e n t, the u n la b e lled w e e v ils w ere re la t iv e ly ev en ly d is tr ib u ted o v e r the a re a .

3 .4 . P r o g r e s s and d is tr ib u tio n o f feed in g

In both e x p e r im e n ta l a r e a s , the m ea n d e g re e o f feed in g p e r plant in c r e a s e d a fte r the m id d le o f June and continu ed to in c r e a s e until the

6

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FIG. 6. Experim ental area, 1967: m ean m inim um distance (in m etres) covered by ra d ioa ctiv e ly -p lu s - co lo u r-la b e lle d w eevils at different tim es after release.

FIG. 7. Experim ental area, 1966: m ean number o f unlabelled w eev ils per b ille t at d ifferent distances from the border o f the area.4 inspection dates: 1 = Id June 3 = 22 June

2 = 19 June 4 = 27 July

beg in n in g o f A ugust (F ig . 2 ). Both the n u m ber o f attacked plants and the extent o f feed in g on the s in g le p lants in c r e a s e d .

A s a re su lt o f the d if fe r e n c e s in sp atia l d is tr ib u tio n o f the -w eevils (s e c t io n 3 .3 ) , . th e r e 'w e r e a lso d if fe r e n c e s in the sp a tia l d is tr ib u tio n o f fe e d in g betw een the tw o e x p e r im e n ta l a r e a s . In the 1966 e x p e r im e n ta l a r e a , the d e g re e o f feed in g d e c r e a s e d w ith in cr e a s in g d is ta n ce ( F i g .8 ). T hus the la te r , m o r e random d is tr ib u tion o f w e e v ils did not r e su lt in a ran dom d is tr ib u tio n o f feed in g dam age to w a rd s the end o f the se a so n .In the 1967 e x p e r im e n ta l a re a , h o w e v e r , the plants in the o u te r p a rts w e re som ew h at m o r e h ea v ily attacked than th o se in the ce n tre , but o n ly by the m id d le o f June. T he o u te r p a rts had a h igh er p e rce n ta g e o f a ttack ed p lants and a h ig h e r m ean d e g re e o f feed in g p er plant. L a te r in the se a so n , feed in g w as d is tr ib u ted at r a n d o m .

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FIG. S. Experim ental area, 19Gri: M ean degree o f feed in g per spruce plant at different distances from the border o f the area.5 inspection dates: 1 = 1 3 /1 4 June 4 = 22 June

2 = 17 June 5 = 2S July3 = 20 June

3 .5 . Spatial d is tr ib u tio n o f I ly lob iu s la rv a e

In the autum n o f 1.966 the m ean n u m ber o f Ily lob iu s la rv a e fo r the w hole a re a w as o b s e r v e d to be 20. 8 la rv a e p e r stum p. N eith er d if fe r e n c e s in the n u m ber o f la rv a e in d iffe re n t p a rts o f the a re a n or d if fe r e n c e s in the sp eed o f d ev e lop m en t w e re noted . Thus e ith e r the w e e v ils d is p e r s e d e v e n ly o v e r the a re a fo r o v ip o s it io n o r - m o r e p ro b a b ly - the lim ited n u m ber ol' b re e d in g p la c e s re su lte d in c o m p le te u tiliza tio n o f the stum ps i r r e s p e c t iv e o f d if fe r e n c e s in d en sity w ithin the w e e v il popu la tion . Thein sp e c tio n o f stu m ps in the 1967 e x p e r im e n ta l a re a has nt>t yet been c o n clu d ed , but it is not ex p e cte d that d if fe r e n c e s w ill o c c u r .

4 . DISCUSSION

T he c o u r s e o f H .ylobius in v a s io n s and the d is tr ib u tio n o f w e e v ils and fe e d in g d am age depend on lo c a l con d ition s su ch as the su rro u n d in g s o f the p lantation , s o i l , v eg e ta tion , and the lo c a l and m ic r o c l im a t e . T h is w as d em on stra ted by the e x p e r im e n ts , w hich a n sw ered the q u estion s lis te d in the in tro d u ctio n .

W e e v ils c lo s e to the b o r d e r s o f a p lantation invaded the a re a s lo w ly and w e re fo r so m e tim e uneven ly d is tr ib u ted in the p lantation . D uring th is t im e the d en sity o f the w e e v ils d e c r e a s e d fr o m the b o r d e r s to the c e n tre . A p p a ren tly the in s e c ts m o v e d m a in ly o r e x c lu s iv e ly by cra w lin g .

T h e se w e e v ils o b v io u s ly did not" p r e fe r c e r ta in d ir e c t io n s o f in va sion o c c a s io n e d by c o m p a s s d ir e c t io n , p re v a ilin g wind o r the s lo p e o f the g ro u n d .

84 EIDMANN

W eev ils a r r iv in g at a p lantation fr o m a g re a te r d is ta n ce m ight in vade the a re a s lo w ly in the sa m e way as w e e v ils sta rtin g c lo s e to the b o r d e r s . On the o th e r hand, they m ight invade the a re a ra p id ly and in a sh o rt t im e d is p e r s e at ra n d om . It is an op en qu estion w hether su ch ra p id ly invading w e e v ils a lso m ov ed by c ra w lin g o r w hether th ey flew into the a re a .

T h e w e e v ils en tered the s o i l at any t im e and cou ld le a v e the s o i l to m o v e to o th e r p la ce s .

F eed in g began (in ce n tra l Sw eden) in June and continued until A u gust. The a b se n ce o f w e e v ils at a ttractin g b i lle t s did not im p ly that th ey had le ft the a re a . T h ey w ere ca p a b le o f changin g th e ir b eh a v iou r and ch o o s in g o th e r p la c e s .

The sp a tia l d is tr ib u tio n o f feed in g depended on the d is tr ib u tio n o f w e e v ils . P lan tation s attacked by s lo w ly invading w e e v ils su ffe re d the h e a v ie s t feed in g dam age in the o u te r a r e a s . If the w e e v ils invaded ra p id ly , the feed in g w as m o r e ran d om ly d is tr ib u ted in the a re a .

The fo llo w in g p r a c t ic a l c o n c lu s io n s w ere draw n fro m the r e s u lts :

C o n tro l m e a su re s to p r o te c t c o n ife r p lantations against H ylobiu s attack should be co n cen tra ted in the o u te r a re a s o f the p la n tation s .

P lan ts in the ce n tre o f p lantations a lso need p ro te c t io n , b e ca u se w e e v ils m igh t invade the a re a ra p id ly and cannot be stopped n ear the b o r d e r .

C on cen tra tion s o f w e e v il-a tt ra c t in g m a te r ia l in the in n er re g io n s o f p lan tation s should be a v o id ed .

A ttractan t w e e v il tra p s in the in n er p a rts o f p lantations w ould be u n s a t is fa c to r y as the s o le m ea n s o f c o n tro l.

R E F E R E N C E S

L l] BUTOVITSCH, V. , Das Flugverm 8gen des grosseil brauneil RHsselkafers. Forstwiss. ZentlB l. 54(1932) 446-GO.

[2 ] FISCHER, K. R . , Beitr3ge zur Fm.’Ihrungsbiologie von Hylobius abietis b. und Untersuchungen iiberd ie O k olog ie und K lim a to log ie seines Nahriingsraumes. Z . angew . Ent. 3 9 (1 9 3 2 ) 25 0 -7 7 .

13] SCHM IDT, W. , RUsselkafer - Bilanz, Berlin (1934).[41 NOVÁK, V . , TEMMLOVA, B., P fem noZenf klikoroha borovélio ( Ilylobius abietis L .) v ¿SSR a

rozbor souïasnÿch obrannÿch a ochrannÿch opatfen f, l.esn. ¿ a s . 10 (1964) 6 5 9 -7 8 .

D I S C U S S I O N

W .F . BA LD W IN : I shou ld lik e to m en tion that in 1958 S u llivan et a l.in Canada r e p o rte d on a o n e -y e a r fie ld study o f w hite p ine w e e v ils la b e lle d with fi0C o. In co n tra s t to the p re se n t study, the in s e c ts w ere la b e lle d with 75 ц C i o f S0Co, and the au th ors found that su ch a d o s e had no s ig n ifica n t e ffe c t on the d e g re e o f s u r v iv a l .

И. II. EID M AN N : U nfortunately , s e c u r ity regu la tion s in Sw eden donot a llow us to use th ese high a c t iv it ie s in the f ie ld .

вRADIATION E FF EC T STUDIES: NON-GENETIC

(Sessions III and IV)

C h a irm a n : H . N O T H E L

l-’ E D E R A L R E P U B L IC O F G E R M A N Y

CORRELATIONS, INTERACTIONS AND DIFFERENCES BETWEEN RADIATION EFFECTS ON LONGEVITY AND NATURAL AGING .

H. N ÔT HE L

I N S T I T U T E OF G E N E T I C S ,

FREE UN I V E R S IT Y OP BERLIN,

BERLIN, FEDERAL REPUBLIC OF G E R M A NY

Abstract

CORRELATIONS, INTERACTIONS ANDi DIFFERENCES BETWEEN RADIATION EFFECTS ON LONGEVITY AND NATURAL AGING. T h e n on -g en etic overa ll radiation response o f insects is expressed by the fitness com ponents 'd ev e lop m en ta l rate’ and 'adu lt life sp a n ', and is hence related to natural aging.

Based on D rosoph ila , with add itional remarks on other insects, the paper dem onstrates that, in the d evelopm en ta l stages, ion iz in g radiation affects the d ifferentiation processes. D istinct sensitive phases are apparent for the induction o f d ifferent injuries, as are e f fe c t iv e phases during wnich the dam age exhibits its lethal a ction . With increasing d ifferen tiation , the radiation sensitivity decreases. The primary b io lo g ica l dam age is, at least in part, som e kind o f som atic m utation .

A review o f adult irradiation response is also based on D rosoph ila. At least two d ifferent types o f induced m orta lity are ev iden t. Type 1 occurs early after irradiation with high doses. It is probably due to central nervous injury and is a com m on e f fe c t in insects, as is indicated by a com parison o f various species. T ype 2 is a m ore d elayed death at m edian and low doses. It is o f d ifferent appearance in various insects, and this heterogeneity is even seen within one sp ec ies . Thus, steriliz ing Drosophila fem ales m ay result in a considerable prolongation or in a drastic reduction o f lifespan.

E xperim ental ev id en ce is found in D rosophila against the 'in d u ced aging’ hypothesis o f radiation death. T yp e 1 m orta lity increases with increasing age at exposure. The amount o f the increase in sensitivity depends indirectly on b io lo g ica l a ge . Both o f these parameters are fixed by gen otype, as is the m ode o f interaction betw een them . T yp e 2 m orta lity follow s an in itia l latent period within an ‘ induced mortality* period . The latter lasts always the sam e t im e , independently o f age at irradiation. The latent period, how ever, is shortened in general with increasing age at exposure. This e ffe c t seem s to depend on m etabolic properties rather than on aging itself, as is indicated esp ec ia lly by a prolongation in early adult l i fe .


T he b io lo g ic a l e f f ic ie n c y o f the s in g le o r g a n is m as w e ll as o f a popu lation is e x p r e s s e d by its f itn e s s . H en ce , the o v e r a ll ra d ia tion r e sp o n se o f l iv in g sy s te m s m ay be m e a su re d by m ean s o f f itn e s s co m p o n e n ts . The n on -gen etic ; ra d ia tion e f fe c t s a re r e s t r ic t e d , then, to th ose com p on en ts o f f itn e s s in v o lv e d in the l i fe c y c le o f a s in g le sp e c im e n . In in s e c ts , th ese are m a in ly re p ro d u c tiv e ca p a c ity , d e v e lop m en ta l ra te , and adult life s p a n . T he r e p ro d u c t iv e ca p a c ity is a ffe c te d m a in ly by dom inant le th a ls in du ced in the g erm c e l ls and a lso b y le th a l e f fe c t s on the tro p h o cy te s [ 1 - 6 ] . T he o th e r tw o f itn e s s com p on en ts a re re la te d to 'a g in g '. T h e d e v e lo p m e n ta l rate r e f le c t s the preadu lt d iffe re n t ia tio n o r ag in g p r o c e s s e s in a w id e r s e n s e . The adult life s p a n r e f le c t s natural aging sen su s t r ic t o . H en ce , the rad ia tion e f fe c t s on both o f th em , and thus the o v e r a l l n o n -g e n e t ic ra d ia tion r e s p o n s e s o f in s e c ts , a re c o v e r e d by the to p ic 'c o r r e la t io n s , in te ra c tio n s and - d if fe r e n c e s betw een rad ia tion e ffe c ts on lo n g e v ity and n atu ra l a g in g '.

87

88 NOTHEL

2. RA D IA TIO N E F F E C T S ON D E V E L O P M E N T A L STAG ES

The se n s it iv it ie s o f the v a r io u s d e v e lo p m e n ta l s ta g e s to io n iz in g ra d ia tio n s a re ra th er s im p ly m e a su re d by s u rv iv a l to adulthood . The L D 50 e m e r g e n c e in D ro s o p h ila m e la n o g a ste r c le a r ly d epen ds on the stage ir ra d ia te d (F ig . 1). It in c r e a s e s fr o m 400 R du rin g c le a v a g e to m o r e than 40 000 R a fte r e x p o su re o f pupae. T h e se d if fe r e n c e s up to a fa c t o r o f 100 w e re d e s c r ib e d in 1927 by M a v or [1 3 J. T h ey a re the sa m e in o th e r in s e c ts as in D ro s o p h ila . F o r e x a m p le , the sa m e se n s it iv ity pa ttern as d e s c r ib e d in F ig . 1 w as found in H a b ro b ra co n [1 4 -1 6 ] , in D acus and in C e r a t it is s p e c ie s [17 ] and in A n astreph a [18J. On the o th e r hand, the ra d ia tion se n s it iv ity in the v a r io u s d ev e lop m en ta l s ta g e s o f D ro s o p h ila is con stan t i f m e a su re d b y m ean s o f a s in g le type o f c e l l , the sp e rm a to g o n ia , and by one e f fe c t on ly , the in du ction o f s e x -lin k e d r e c e s s iv e le th a ls (F ig . 1). T h e r e fo r e , du rin g e m b ry o n ic , la rv a l and pupal d ev e lop m en t, it is not on ly the ra d ia tion se n s it iv ity o f the w hole o r g a n is m w hich d e c r e a s e s , but a lso that o f c e r ta in c e l l s o r c e l l g rou p s w hich , in addition , m a y change in th e ir im p o rta n ce fo r the o v e r a l l ra d ia tion r e s p o n s e . H en ce , th e re a re s e n s it iv e p h a se s fo r the in d u ction o f a s p e c ia l in su lt, and e f fe c t iv e p h ases d u rin g w hich the in ju ry b e c o m e s v is ib le when the a ffe c te d sy s te m is n eed ed to c o m p le te d e v e lop m en t.

HOURS

1 -*#>2CLEAVAGEDIVISIONS

EARLY

BLASTO- GASTRU- DERM L ATION

î _ j Î p — 1 —>*-2— -------3 -------PRE-| PF-2HATCHING

LARVAL INSTARSPUPA

EMERGENCE

% SEX-LINKED RECESSIVE А K A f î tРТН Д 1Я P F R 1 K R I I I I I ILETHALS PER 1 KR

INOUCED IN SPERMATOGONIA

FIG. 1. LDS0-em erg en ce after X -irrad ia tion o f d ifferent developm ental stages o f D rosophila m elanogaster ( a t 2 5 e Q . LD50 data after F ritz-N igg li (7 -9 ), W urgler (10), and own unpublished results; m utation rates after Khishin (11) and (adults) Auerbach (12).

S M - 102/10 89

T A B L E I . D IF F E R E N T T Y P E S O F M O R T A L I T Y A F T E R X -R A Y I N G 1. 75 H O U R S O L D E M

B R Y O S O F D R O S O P H IL A W IT H D IF F E R E N T D O S E S . - D a ta f r o m F R I T Z - N I G G L I ( 7 ) and

S T E E N B E C K (1 9 )

d o s e in R t o t a l m o r t a l i t y f r e q u e n c ie s o f th e d i f f e r e n t t y p e s w ith in t o ta l m o r t a l i t y

im m e d ia t e d e a th e a r l y d e a th la t e d ea th

8 ,0 0 0 100 % 5 0 % 50 %40 0 90 % - 95 % 5 %200 6 5 % - 6 0 % 4 0 %100 20 % - 20 % 80 %

D iffe ren t e ffe c t iv e o r le th a l p h a ses in d ica te d iffe re n t le th a l e f fe c t s du rin g d ev e lop m en t. T h e se e f fe c t s m a y change w ith in c r e a s in g d o s e .A le th a l in ju ry with a la te e ffe c t iv e ph ase in du ced at low d o s e s w ill be su rp a sse d by an oth er one w ith an e a r ly e ffe c t iv e phase in du ced at the sa m e d ev e lop m en ta l stage at a h ig h er d ose le v e l . T h u s, X - ir r a d ia t in g e a r ly b la s to d e r m sta g e s o f D ro s o p h ila re su lte d in at le a s t th ree d iffe re n t le th a l p h ases depen d in g on d ose (T a b le I). O f th ese , the 'im m e d ia te death 1 ju st a fte r e x p o su re s e e m e d to in d ica te the co m p le te break dow n o f d iv id in g c e l l s . The 'e a r ly death ' o c c u r r e d until g a stru la tion . The in terp re ta tion that it w as due to the im p o s s ib i l ity to in itia te g a stru la tion w as co n firm e d by F r it z -N ig g l i [8 ]. E ven at the LDgo h atch ing sh e found no e a r ly death a fter the beg in n in g o f p r e g a s tru la r c e l l m o v e m e n ts at an age o f th ree h o u rs : e a r ly type m o r ta lity a fte r e x p o su re o f 2 .5 , 3, and o v e r 3 -h o u r -o ld e m b ry o s w as 90%, 5% and 0%, r e s p e c t iv e ly . H en ce , the s e n s it iv e phase f o r the in d u ction o f dam age w ith an e ffe c t iv e le th a l phase du rin g the e a r ly g a stru la tion en ds with the beg in n in g o f the la tte r . In o th e r c a s e s , som e tim e e la p se d betw een the tw o p h a ses . T h us, the 'la te death ' w as due to dam age in the d e v e lo p in g la r v a l o rg a n s , w hich p red om in a n tly in vo lved the h y p o d e rm is , e s p e c ia lly that o f the m outh re g io n , and the m u scu la tu re [8, 9 ] . A fte r the d iffe re n tia tio n o f la rv a l o rg a n s w as co m p le te d , the se n s it iv e phase fo r the in d u ction o f the above dam age ended at 7 h o u rs (F ig . 1). K illin g o ld e r e m b ry o s b e fo r e h atch in g re q u ire d d o s e s o f s e v e r a l thousand R , but the e f fe c t iv e le th a l phase fo r the dam age in du ced b e fo r e an age o f 7 h ou rs w as not re a ch e d until h atch ing , w hich n o rm a lly o c c u r r e d at 22 h o u rs . O nce the d iffe re n t ia tio n o f the la rv a l orga n s w as co m p le te d , e m b ry o n ic d ev e lop m en t and la r v a l life se e m e d no lo n g e r a ffe c te d at d o s e s b e lo w the L D 100 e m e r g e n c e ra n g e . Instead, in th ese sta g es d iffe re n t ia tio n and d e v e lop m en t o f the im a g in a i d is c s w as a ffe c te d . A gain , d is t in c t s e n s it iv e and e ffe c t iv e p h ases w e re found.

T h e s e n s it iv e p h ases w e re in te rp re te d as s ta g e s o f high gene action [9 ] . T h is poin t o f v iew w as c o n fir m e d b y ra d ia tio n -in d u ce d p h e n o co p ie s — m o d ifica t io n s co p y in g the ph enotypes o f m u tants. T h ey w e re in d u cib le at s e n s it iv e p h a ses thought to r e p re s e n t th ose d e v e lo p m en ta l sta g es at w hich the g en es in qu estion n o rm a lly b e c o m e a c tiv e .F o r ex a m p le , th e 'm u tan t D ich a ete o f D. m e la n o g a s te r had d o r s o c e n t r a l b r is t le s re d u ce d in n u m ber and the w in gs exten ded to 45° fr o m the bod y a x is . F r it z -N ig g l i [20] saw the sa m e m a lfo rm a t io n s in up to 100% o f the su rv iv in g f l ie s a fte r ir ra d ia t io n o f 5 -h o u r o ld p rep u p ae . T h e y w ere not found a fte r e x p o su re o f la rv a e o r o f o ld e r pupae. The qu estion a r is e s w hether one o f the p r im a r y ra d ia tion e ffe c ts is so m e kind o f so m a tic

90 NÔTHEL

m u tation . T h ese w ere in v estig a ted by P a tte rso n [23] in 1929, and r e ce n tly in s e v e r a l g en etic [24] and cy to g e n e tic [25] s tu d ie s . The id ea o f th e ir le th a l a ction on deve lopm en ta l s ta g e s w as stren gth en ed by so m e fin d in gs in D r o s o p h ila . A c co r d in g to U lr ich [2 6 ], in the youn g e m b ry o the n u clea ted p art o f the eg g is 182 tim e s as s e n s it iv e as the cy to p la s m . W ü rg le r [10] found that d u rin g c le a v a g e the r e la t iv e ly lo w e st se n s it iv ity a p p eared in the in terp h a se s ta g e s (F ig . 1). S e v e ra l authors sh ow ed by m ea n s o f s p e c ia l g en etic s to ck s that the le th a l rad ia tion e f fe c t s in du ced in la rv a e w e re : "a lm o s t e x c lu s iv e ly due to c h r o m o s o m e b rea k a ge fo llo w e d b y c h r o m o s o m e lo s s , c e l lu la r dam age o r death re su lt in g w h en ever a p o rtio n o f the gen om e w as no lo n g e r re p re s e n te d in at le a s t h ap lo id c o n d it io n " (O ste r ta g [27 J).

H en ce , in the d ev e lop m en ta l s ta g e s o f in s e c ts , so m a tic m u tation s, ra d ia tio n -in d u ce d p red om in a n tly du ring m ito tic d iv is io n s , r e su lt in death until e m e r g e n c e o r in m o d ific a t io n s v is ib le in the ad u lts. O ne ra th er u n s p e c if ic grou p o f such m o d ific a t io n s is the sh orten in g o f adult life sp a n (T a b le II), w hich depends on the d ev e lop m en ta l stage e x p o se d and on the d o s e . S ince in the adult in se c t d iffe re n t ia tio n is co m p le te d and m ito tic a c t iv ity has c e a se d in the so m a tic t is s u e s , a quite d iffe re n t rad ia tion r e s p o n s e shou ld be e x p e cte d when the 'a g in g ' adult in s e c t is e x p o se d .

T A B L E II . E F F E C T S O F X -I R R A D I A T I N G D E V E L O P M E N T A L S T A G E S O F H A B R O B R A C O N

J U G L A N D IS W IT H D IF F E R E N T D O S E S O N M E A N A D U L T L IF E S P A N (M ). - D a ta f r o m

C L A R K (2 1 ) and E R D M A N (2 2 )

E m b r y o s ( m a le s ) L a r v a e ( fe m a l e s ) w h ite P u p a e ( f e m a l e s )

d o s e in R . M d o s e in R M d o s e in R M

6 0 0 23 d a y s 0 28 d a y s 0 . 29 d a y s900 16 d a y s 1, 0 0 0 24 d a y s 5 , 00 0 22 d a y s

1, 200 14 d a y s 2 ,0 0 0 11 d a y s 1 0 ,0 0 0 12 d a y s1 ,5 0 0 8 d a y s 3 ,0 0 0 6 d a y s 1 5 ,0 0 0 10 d a y s

3. RA D IA TIO N E F F E C T S ON YOUNG AD U LTS

T h e in du ced red u ction o f m ean life sp a n a fte r ir ra d ia tio n o f adult D. m e la n o g a ste r m a le s and fe m a le s with v a r iou s d o s e s is show n in F ig . 2. T h e d o s e -a c t io n re la t io n sh ip s in d ica te a m uch lo w e r ra d io s e n s it iv ity o f adults c o m p a re d with that o f the d ev e lop m en ta l s ta g e s . T h e se re la t io n sh ip s w e re lin e a r , bul c o n s is te d o f tw o p a rts , sep a ra ted by a steep en in g . E a ch part w as b e lie v e d to in d ica te at le a st one type o f m o r ta lity . The e a r ly death, found at high d o s e s , is ca lle d 'type .1 m o r ta lity ' in th is p a p e r . The m o r e d e la yed death is c a lle d 'ty p e 2 m o r ta lit y '. The d o s e -a c t io n p a rt o f the la tte r w as in terru p ted in fe m a le s by a c o n s id e r a b le p ro lon ga tion o f life sp a n .

3. 1. T ype 1 m o rta lity

A change in the type o f m o r ta lity at h igh d o s e s w as c o n fir m e d ('F ig . 3). The a ccu m u la ted m o r ta lity ra te s a fte r ir ra d ia t io n o f D ro so p h ila fe m a le s with d iffe re n t d o s e s a re h e re g iv e n on a p ro b a b ility s c a le . A G auss

SM» 102/10 91

DArS40ртз30

II

20

FEMALES

MALES

0 10 20 40 60 80 100 120

DOSE IN KR

FIG. 2. M ean survival tim es in days after X -irrad ia tion o f D. m elanogaster m ales and fem ales w ith d ifferent doses. After N othel (28).

T Y PE 1 MORTALITY

T Y P E 2 - M ORTALITY

FIG .3. A ccum ulated m orta lity rates as a function o f tim e after X -irrad ia tion o f D. m elanogaster fem ales w ith high doses. Data after N othel (28).

d is tr ib u tion o f p e rce n ta g e a ccu m u la ted m o rta lity r e su lte d in a l in e a r r e g r e s s io n lin e . T h is lin e a r ity w as ap parent in the u n irra d ia ted c o n tro ls (0 kR ). It p e r s is te d at 67 kR, but had an in c r e a s e d r e g r e s s io n c o e ffic ie n t a fte r an in itia l latent p e r io d . H o w ev er , beg in n in g at 90 kR the d is tr ib u tion b e ca m e m o r e and m o r e b im od a l, an e a r ly type o f in du ced death o v e r lapp in g the 'n o r m a l ' on e.

F lie s sh ow in g type 1 m o r ta lity w e re c h a r a c te r iz e d by a s p e c ia l ra d ia tion sy n d ro m e : th ey w e re v e r y qu iet, unable to f ly one day a fte r

92 NÓTHEL

e x p o su re , and m o s t o f them sh ow ed u n co -o rd in a te d m ov em en ts o f lo c o m o t io n o rg a n s . T h ese c h a r a c t e r is t ic s in D ro so p h ila w e re s im ila r to th ose o f a c o m a in du ced at high d o s e s in H a b ro b ra co n [29 ] and D ah lbom in us [3 0 ] . It w as co n c lu d ed that type 1 m o r ta lity w as due to dam age in the ce n tra l n e rv o u s s y s te m . T h is is c o n s id e r e d co m m o n in a ll in s e c ts ir ra d ia te d w ith high d o s e s , s in ce at a d ose le v e l o f 150 to 200 kR, the T50 did not e x ce e d a few days in a ll in s e c ts tes ted (T a b le III). T h e T 50 is the tim e at w h ich 50% o f the an im als in v estig a ted d ied . By m ea n s o f th is T 50, the rad ia tion r e s p o n s e s o f v a r io u s s p e c ie s are co m p a re d at d iffe re n t d o se le v e ls in T a b le III.

T A B L E III. T 50 IN D A Y S A F T E R IR R A D IA T IO N O F N E W L Y E M E R G E D A D U L T S O F

V A R IO U S IN S E C T S P E C IE S A T D IF F E R E N T D O S E L E V E L S (IN k R )

I n s e c t 1 d o s e in k R : 0 1 0 -2 0 5 0 -7 0 1 5 0 -2 0 0 R e f e r e n c e s

O R T H O P T E R AP e r ip la n e t a a m e r ic a n a (m a le s ) 200 10 4 - W H A R T O N (3 1 )

C O L E O P T E R AT r ib o l iu m c o n fu s u m 290 20 - - C O R K (3 2 )

- 12 9 3 H A S S E T T (3 3 )

R h y z o p e r ta d o m in ic a 100 23 14 3 H A S S E T T (3 3 )

E p ila c h n a v a r i f e s t i s ( m a le s ) 34 16 - - H E N N E B E R R Y ( 34 )

P j s s o b e s s t r o b i ( m a le s ) 34 16 - J A Y N E S (3 5 )

A n th o n o m u s g r a n d is 30 10 - - D A V IC H (3 6 )H Y M E N O P T E R A

H a b r o b r a c o n ju g la n d is ( f e m a le s ) 25 13 12 6 C L A R K (2 1 )

H a b r o b r a c o n s e r in o p a e ( m a le s ) 62 36 18 _ C L A R K (3 7 )D IP T E R A

A e d e s a e g y p t i ( f e m a l e s ) 45 35 - - S T A H L E R (3 8 )

D r o s o p h i l a s u b o b s c u r a ( m a le s ) 4 7 34 14 - L A M B (3 9 )

D r o s o p h i l a m e la n o g a s t e r (m a le s ) 44 38 13 5 B A X T E R (4 0 )37 26 11 2 n O t h e l (2 8 )

3. 2. T yp e 2 m o rta lity

A c c o r d in g to T a b le III, w ithin the d o se range o f type 2 m o rta lity , d if fe r e n c e s betw een the v a r io u s in se c ts a re apparent, e s p e c ia l ly at low d o s e s (1 0 -2 0 kR ). T h ese d if fe r e n c e s b e co m e c le a r e r by c o n s id e ra t io n o f tw o e x a m p le s : (a) D o s e -a c t io n cu r v e s in D ro s o p h ila and P e r ip la n e ta m a le s a re co m p a re d in F ig . 4 . In co n tra s t to the fin d in gs in D ro s o p h ila , in P e r ip la n e ta the d e c r e a s e o f T 50 with in c r e a s in g d o se w as b im o d a l even at low d o s e s ; it w as c o n s id e r a b le betw een 2.5 and 10 krad and le v e lle d o f f at h ig h e r d o s e s , (b) A ccu m u la ted m o r ta lity r a te s (on a p ro b a b ility s c a le ) o f ir ra d ia te d and c o n tro l m a le s o f D ro s o p h ila and T r ib o l iu m are co m p a re d in F ig . 5. In the c o n t r o ls the r e g r e s s io n lin e s a re stra ig h t in both s p e c ie s . A fte r ir ra d ia t io n , th is type o f r e g r e s s io n p e r s is te d in D ro s o p h ila , but in T r ib o l iu m the p o s t - ir r a d ia t io n m o r ta lity d is tr ib u tio n w as b im o d a l. A sh arp in itia l in c r e a s e w as fo llo w e d by a re tu rn to the c o n tr o l r a te s . A s to o th e r d o s e s , in T r ib o liu m the in itia l in c r e a s e g oes up to 45% a ccu m u la ted m o r ta lity at 11 kR and to 100% at 20 kR . In

S M - 102/10

40 j -

DROSOPHILA 10 20 40 X-RAYS 60 KR 80

PERIPLANETA 5 10 20 /3-RAYS 30 К RAO 40

FIG. 4. T50 as a function o f dose after irradiation o f D rosophila and Periplaneta m ales. Periplaneta data after Wharton (31), D rosophila data after N óthel (28).

DAYS AFTER IRRADIATION, TR1BOUUM SCALE

25 50 7 Ю0 200 300 400 500

FIG. 5. A ccum ulated m orta lity rates as a function o f tim e in D rosophila and T ribolium m ales w ith and w ithout irradiation. T riboliu m data after Cork (32), D rosophila data after N othel (28).

94 NÔTHEL

FIG. 6. Fecundity (after exposition o f oogon ia ) and m ean survival tim es o f D . m elanogaster fem ales after X -irrad ia tion w ith different doses in per cent o f unirradiated controls (0 kR). A fter N othel (28).

D ro s o p h ila a c o r r e s p o n d in g d is tr ib u tion w as not found at low o r m ed ian d o s e s . H en ce it w as a ssu m ed that d iffe re n t in ju r ie s o c c u r r e d w ithin type2 m o r ta lity in the v a r io u s in s e c t s p e c ie s .

In D ro s o p h ila th e re e x is te d som e h e te ro g e n e ity w ith in type 2 m o rta lity , as b e c a m e apparent in tw o e x a m p le s a s s o c ia te d with ra d ia tio n -in d u ce d s te r i liz a t io n o f fe m a le s : (a) A p ro lon g a tion o f fe m a le life sp a n (F ig . 2) is show n in F ig . 6 to be c o r r e la t e d with s te r i liz a t io n . A c c o r d in g to N othel [28, 41, 4 2 ] , it w as due to the s te r i liz a t io n , s in ce s t e r i l iz a t io n w ith TTCM r e su lte d in the sa m e p ro lo n g a tio n e f fe c t as ir ra d ia t io n w ith11 k R . L am b [39 ] found a co r r e s p o n d in g p ro lo n g a tio n in n o rm a l fe m a le s , but not in th ose w hich g e n e t ica lly la ck the o v a r ie s , (b) An o p p o s ite e f fe c t o f fe m a le s te r i liz a t io n on life sp a n w as found to be due to a s p e c ia l h e re d ita r y fa c to r (N othel, u npu blish ed ). Its a ction is show n in T a b le IV. N o rm a l fe m a le s w e re perm a n en tly s t e r i l iz e d by dom inant le th a ls in du ced in the oo g o n ia . K illin g one o o g o n ia l c e l l r e su lte d in the r e s o r p t io n o f one o v a r io le [4 3 ] . 100% s t e r i l iz a t io n was in d u ced at 11 kR [5 ] , at w h ich d ose the o v a r io le s d im in ish ed so m e tim e a fte r e x p o s u re . In the c a r r ie r s o f the fa c to r in qu estion , perm a n en t s te r i liz a t io n w as in du ced at 4 kR, but in th e se fe m a le s o o g e n e s is w as not a ffe c te d and no o v a r io le s w e re r e s o r b e d . Instead , o v ip o s it io n w as b lo ck e d , the o v a r ie s b e c a m e e n la rg e d and the f ly d ied v e r y soon b e ca u se it cou ld not get r id o f the e g g m a s s e s .

S e v e ra l a ttem pts w e re m ade with v a r io u s in s e c ts to u n derstan d the n ature o f type 2 m o r ta lity . E x p e r im e n ts w ith sp lit d o s e s , sta rv a tion , n itro g e n o r p h osp h oru s tu rn o v e r , o r o th e r m e ta b o lic p a ra m e te r s did not y ie ld s u ffic ie n t in fo rm a tio n . Studies w ith d iffe re n t p lo id y le v e ls fa v o u re d the v ie w that even in adult in s e c ts s o m a t ic m u tation s w e re a p r im a ry e f fe c t o f ra d ia tion in ju ry . C la rk [21, 37] u sed h a p lo id s and d ip lo id s o f H a b r o b r a c o n . He found that the ra d ia tio n -in d u ce d d e c r e a s e in life sp a n w as m a rk e d ly in flu en ced by gen om e n u m ber (F ig . 7) and co n c lu d e d : "that

S M - 102/10 95

T A B L E IV . T H E B E A R IN G O N T 5Q O F

A S P E C IA L G E N E -C O N T R O L L E D T Y P E

O F R A D IA T IO N -IN D U C E D S T E R IL IT Y

IN D . M E L A N O G A S T E R F E M A L E S

e g g s / f e m a l eT 50 1П

d a y s 1 + 2 d a y s 8 -1 1 d a y s

n o r m a l f e m a le s :

0 k R 174 284 k R 122 154 27

f e m a le s w ith th e f a c t o rf o r " in d u c e d s t e r i l i t y " :

0 kR 174 284 k R 129 0 11

95%90

vmV - i - /I. ( h a p lo id J /

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5 50 !-

/ +/vltfV * --- « ----/

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■__j. 1 ___ ! I _!_20 30 40 50 60

D A YS A F T E R E X P O S IT IO N

70 80

FIG. 7. A ccum ulated m orta lity rales as a function o f tim e in haploid (+ or v l) and d ip lo id (+ / v l j m ales o f H abrobracon serinopae w ith and without X -irrad ia tion . A fter Clark and Rubin (37).

the d e c r e a s e in life s p a n b y X - ir r a d ia t io n is due to dam age o f a g en etic n a tu re " [3 7 ] . C la rk found c o n s id e r a b le d if fe r e n c e s betw een r a d ia t io n - in du ced m o r ta lity and n atu ra l death , s in c e , a c c o r d in g to the la tte r , h a p lo id s and d ip lo id s beh a v ed s im ila r ly (F ig . 7 ). On the o th e r hand, at le a s t in D ro s o p h ila , the shape o f the d o s e -a c t io n cu r v e s as w e ll as that o f the a ccu m u la ted m o r ta lity r a te s fa v o u re d the id ea o f 1 ra d ia tio n -in d u ce d ag in g1 w ith r e g a rd to type 2 m o r ta lity .

9G NÔTHEL

4. R A D IA T IO N E F F E C T S ON AGING A D U LT S

W ith in cr e a s in g age at ir ra d ia t io n the adult in s e c t d ied w ith d e c r e a s in g d o s e s [3 1 ] . S om e r e ce n t p a p e rs o f B a x te r and B la ir [4 0 ], L am b [4 4 ], and N othel [45] dea lt with th is ph enom enon in D r o s o p h ila . S in ce th ere w ere s e v e r a l d is c r e p a n c ie s in the in te rp re ta t io n s g iven by the au th ors c ited , we p e r fo rm e d so m e ad d ition a l e x p e r im e n ts with D. m e la n o g a s te r .The r e s u lts a re d e s c r ib e d h e r e , but the m a te r ia l and m eth od s u sed , as w e ll as d e ta iled s ta t is t ic s , w ill be pu b lish ed e ls e w h e r e .

4 .1 . T ype 1 m o rta lity

In ou r p re v io u s in v e s tig a t io n s [4 5 ], the L D 50 type 1 m o r ta lity d e c r e a s e d ra th e r ra p id ly w ith in cr e a s in g age at ir ra d ia t io n (F ig . 8 ). T he d e c r e a s e w as s im ila r in both s e x e s , w h eréa s natu ral ag in g depen ded on se x , as in d ica ted by m ean l i fe t im e s o f 41 .8 and 29.9 days in m a le s and fe m a le s , r e s p e c t iv e ly . H en ce it w as co n c lu d ed that the in c r e a s e in ra d ia tion se n s it iv ity w as due to aging o f the c e n tra l n erv ou s sy s te m (r e s p o n s ib le fo r type 1 m o r ta lity but not f o r n atural death) ra th e r than to that o f the w hole o rg a n ism [4 5 ] . The e x p e r im e n ta l data o f B a xter and B la ir [40] w e re in good a g re e m e n t w ith o u r fin d in gs , e x ce p t f o r one ra th e r im p ortan t poin t: th ese au th ors found the ra te s o f ag in g as in d ica ted by the ra d ia tion r e sp o n se to be in v e r s e ly p ro p o rt io n a l to n o rm a l life s p a n s o f m a le s and fe m a le s .

3 0

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5 0 6 0 7 0 8 0 9 0 10 0 110 12 0 130

LD 50 IN KR

FIG. 8. L D so-type 1 m orta lity as. a function o f age at X -irradia tion in D . m elanogaster m ales and fem ales. After N othel (45).

Both in te rp re ta t io n s w e re tes ted w ith the age depen den ce o f type1 m o r ta lity in m a le s and fe m a le s o f fo u r ad d ition a l and d iffe re n tD. m e la n o g a s te r s to ck s . T h ese ex h ib ited c o n s id e r a b ly d iffe re n t life s p a n s

S M - 102/10 97

¿ • 1:( L D j q IN HRÍ2» Ю6

FIG. 9. LD50-ty p e 1 m orta lity as a function o f age at X -irra d ia tion in D. m elanogaster m ales o f d ifferent strains. M ean lifespans o f unirradiated m ales in days:E/E 25 . 9 ± .8 , F /F 18. 3 = . 9, D /F 43. 4 ± 1 .3 , E /F 31. 8 * 1 .3 .

when u n irra d ia ted (F ig . 9) and w e re th e r e fo r e b e lie v e d to have d iffe re n t ra te s o f n atural aging. T o e s ta b lis h p r a c t ic a b le a g e - t o -L D 50 r e l a t i o n sh ip s , the r e c ip r o c a ls o f the sq u a re s o f LD 50 v a lu es w e re u sed ;1 0e : (L D 50 in kR )2 w as s y m b o liz e d by d. In F ig . 9, d p r o v e s to be a l in e a r fu n ction o f age at e x p o su re , d is tin c t in e v e r y genotype and se x . N atural aging w as m e a su re d by the a ccu m u la ted m o r ta lity r a te s , g iven in p ro b it units Z . Z w as a lin e a r fu n ction o f age in e v e r y genotype and sex (s e e F ig s 4 and 12, f o r e x a m p le ). S ince the age is a lin e a r fu nction o f d as w e ll as o f Z , Z is g iven in F ig . 10 as a fu n ction o f d fo r e v e r y genotype and se x te s te d . If, in d eed , the rad ia tion s u s c e p t ib il ity (in d ica ted by d) depen ded on agin g as an o v e r a ll e f fe c t (in d ica ted b y Z ), the fu nction Z = f (d) sh ou ld be equ a l in e v e r y genotype and se x . A c c o r d in g to F ig . 10, th is h o lds tru e on ly in so m e c a s e s . T h e r e fo r e , both n a tu ra l ag in g as in d ica ted b y life s p a n and agin g o f the ce n tra l n e rv o u s s y s te m as in d ica ted b y ra d ia tio n -in d u ce d type 1 m o r ta lity w e re fix e d by g en otype , as w as the m od e o f c o r r e la t io n betw een th em .

4 .2 . T y p e 2 m o r ta lity

In D ro so p h ila , type 2 m o r ta lity lo o k e d lik e 'ra d ia t io n -in d u c e d a g in g '. L am b and M aynard Sm ith [4 6 j found the p e rce n ta g e re d u ctio n in life sp a n

98 NOTHEL

П С. 10. A ccum ulated natural m ortality rates (Z ) as a function o f radiation sensitivity to type 1 m ortality (d) in D. m elanogaster m ales and fem ales o f d ifferent strains.

Z = probit units o f percentage accum ulated m ortality = linear function o f age in unirradiated controls,

d = 10 6 : (LD -0-ty p e 1 m orta lity in kR)2 = linear function o f age at irradiation.

FIG. 11. Relations in D . m elanogaster m ales betw een the age (A) at X -irrad ia tion w ith 42 kR, mean postirradiation survival tim e (M ), and m ean control survival tim e (C) at the sam e age.

S M - 102/10 99

by a g iven d ose to be a lw a ys the sa m e w h a tev er the c o n tr o l life s p a n (v a r ie d by te m p e ra tu re ) w a s . L am b [44] found the sa m e con stan t re la t io n a fte r ir ra d ia t io n at d iffe re n t a g e s . It w as, h o w e v e r , not c le a r w h eth er type 2 m o r ta lity depen ded on the agin g p r o c e s s e s in v o lv e d in the sh orten in g o f n atu ra l l i fe e x p e cta n cy o r w h eth er it r e f le c t e d a lte ra tio n s o f so m e o th er m e ta b o lic p r o p e r t ie s with a g e . T o tes t th is , w e paid s p e c ia l attention to the e a r ly days o f adult l i fe , s in ce du rin g th is p e r io d o f 'm e ta c h c m o g e n e s is ' th ere is an in c r e a s e in v a r io u s p h y s io lo g ic a l p r o p e r t ie s and a d e c r e a s e in n atu ra l l i fe e x p e cta n cy [4 7 ] .

A c c o r d in g to F ig . 11, the m ean su r v iv a l t im e s a fte r ir ra d ia t io n at d iffe re n t ages w e re in a n e a r ly con stan t re la t io n to th ose o f c o n t r o ls at the sa m e age. The sum o f p r e - plus p o s t - ir r a d ia t io n l i fe t im e s p roved to be fa r fr o m co n s ta n cy . T h ese re su lts a re in a g re e m e n t w ith th ose of L am b [44 ] and c o n tra ry to th ose o f B a x te r and B la ir [4 0 ] .

H o w ev er , in u sin g the m ean su rv iv a l t im e s on ly , so m e s im p lifica t io n s w e re m a d e . It se e m e d m o r e a p p ro p r ia te w ith ou r e x p e r im e n ta l m a te r ia l to u se the accu m u la ted m o rta lity r a te s . S om e e x a m p le s o f th is fo r m o f c o n s id e ra tio n are g iven in F ig . 12. T he ba ck b on e is the r e g r e s s io n line o f u n irra d ia ted c o n t r o ls . The lin e s o f the ir ra d ia te d sa m p le s s ta r t fr o m it, and to obtain th em the su m o f p r c -a n d p o s t - ir r a d ia t io n deaths w as se t at 100%. The p o s t - ir r a d ia t io n m o r ta lity c u r v e s co n s is te d o f two d is tin ct l in e a r co m p o n e n ts . The f i r s t w as equ a l to the n atu ra l m o rta lity and re p re s e n te d a la ten t p e r io d . T he se co n d exh ib ited a m u ch s tro n g e r in c r e a s e and r e p re s e n te d the 'in d u ced m o r ta lity 1. L atent p e r io d and m ed ian su rv iv a l tim e w ithin the in du ced m o r ta lity w e re eva lu ated f o r e v e r y age c la s s b y m ea n s o f p ro b it equ ation s . T h ey are co m p a re d in F ig . 13.It is ev iden t that, in dep en den tly o f age at ir ra d ia tio n , the sa m e tim e is a lw ays r e q u ire d until 50% o f the an im als have d ied w ithin the in du ced m o r ta lity p e r io d . The la te n cy , h o w e v e r , in c r e a s e s w ith in c r e a s in g age at e x p o su re up to a m a xim u m at about the th ird day, and then d e c r e a s e s . T he in itia l in c r e a s e in la te n cy w as s im ila r to the m e ta ch e m o g e n e s is o f so m e en zym e a c t iv it ie s in M u sca [47] and to the in c r e a s e in r e sp ir a t io n rate [48] o r o v i p o s i t i o n r a t e [5] in D r o s o p h i l a . H e n c e it i s p r o b a b l e that the a g e -d ep en d en t a lte ra tio n s in the se n s it iv ity to type 2 m o rta lity w ere due to s p e c i f i c m e ta b o lic ch a n g es ra th e r than to n atural ag in g it s e lf , and .that th is m o r ta lity w as not an 'in d u ce d ag in g1.

5. SU M M AR Y AND CONCLUSIONS

The n o n -g e n e t ic o v e r a l l rad ia tion r e s p o n s e o f in s e c ts is e x p r e s s e d by the f itn e s s com p on en ts 'd e v e lo p m e n ta l r a te ' and 'adu lt life s p a n ' and is h en ce re la te d to n atu ral ag in g. In the d e v e lop m en ta l s ta g e s , io n iz in g ra d ia tion a ffe c ts the d iffe re n t ia tio n p r o c e s s e s , at le a s t in part, by m eans o f s o m a t ic m u ta tion s. W ith in c r e a s in g d iffe re n t ia tio n , the ra d ia tion in ju ry d e c r e a s e s until a m a xim u m r e s is ta n c e is a ch iev ed about e m e r g e n c e .In the adu lts, the se n s it iv ity again in c r e a s e s w ith in c r e a s in g age at e x p o s u re . H ow ever , th is c o r r e la t io n d o e s not r e f le c t true in te ra c tio n s betw een n atu ra l ag in g and ra d ia tio n e f fe c t s , such as 'in d u ce d a ccu m u la ted a g in g '. D if fe r e n c e s betw een both p a ra m e te rs in d ica te that the se n s itiza tio n is a s e c o n d a r y e f fe c t o f m e ta b o lic a lte ra tio n s . T h e r e fo r e , the rad ia tion

ACCUMULATED

MORT

ALITY

IN

100 NÓTHEL

FIG. 12. A ccum ulated m ortality rates after X -irradia tion o f D. m elanogaster m ales at various ages with 42 kR.

H IN DAYS

FIG. 13. Latent periods (L) and m edian survival tim es within the 'induced m orta lity ' periods (IM) in days after X -irra d ia tion o f D. m elanogaster m ales at various ages w ith 42 kR.

S M - 102/10 101

r e sp o n se o f in s e c ts is not such a good to o l to e s ta b lish a m o d e l o f n atu ral ag in g as h as o ften been p r e d ic te d .

T he r a d io b io lo g ic a l in v e s tig a t io n s o f d e v e lop m en ta l s ta g e s turned out to be s tu d ies in d e v e lo p m e n ta l g e n e t ic s . In the adults, the in te r s p e c i f ic v a r ia t io n s in the ra d ia tion re sp o n se w ill not be ex p la in ed until th ere is ad d ition a l k n ow led ge on co m p a ra tiv e in s e c t p h y s io lo g y . The in tr a s p e c i f ic v a r ia b ility , h o w e v e r , p r o m is e s g ood r e s u lts . T h is is se e n in stu d ies w ith d iffe re n t p lo id y le v e ls in H a b ro b ra co n o r even in th o se on se c o n d a r y e f fe c t s on lo n g e v ity by s t e r i l iz in g D ro s o p h ila fe m a le s . H en ce the study o f n o n -g e n e t ic ra d ia tion e f fe c t s by m ea n s o f w e ll known g en etic devian ts sh ou ld be p rop ag ated .

R E F E R E N С. E S

(1 ) . D E M E R E C , M . , F A N O , U . , F r e q u e n c y o f d o m in a n t l e th a ls in d u c e d b y r a d ia t io n ins p e r m s o f D r o s o p h i la m e la n o g a s t e r . G e n e t ic s £ 9 (1 9 4 4 ) 3 4 8 -3 6 0 .

(2 ) V o n B O R S T E L , R . C . , R E K E M E Y E R , M . L . , R a d ia t io n - in d u c e d a n d g e n e t i c a l l y c o n t r iv e d d o m in a n t l e th a l i t y in H a b r o b r a c o n a n d D r o s o p h i la . G e n e t ic s 44 (1 9 5 9 ) 1 0 5 3 -7 4 ,

(3 ) L A C H A N C E , L . E . , R IE M A N N , J . G . , C y to g e n e t ic in v e s t ig a t io n s o n r a d ia t io n and c h e m ic a l l y in d u c e d d o m in a n t le th a l m u ta t io n s in o o c y t e s a n d s p e r m o f th e s c r e w - w o r m f ly . M u ta t io n R e s . J. (1 9 6 4 ) 3 1 8 -3 3 3 .

(4 ) E R D M A N , H . E . , M o d i f i c a t io n s o f p r o d u c t iv i t y in f lo u r b e e t l e s , T r i b o l iu m c a s ta n e u m H e r b s t , du e to X - r a y d o s e s , h y p o t h e r m ia , a n d th e s e x e x p o s e d . R a d ia t .R e s . 25 (1 9 6 5 ) 3 4 1 -3 5 1 .

(5 ) N Ô T H E L , H . , P e r E in f lu fi v o n R ô n tg e n s t r a h le n a u f V i t a l i t â t s m e r k m a le v o n D r o s o p h ila m e la n o g a s t e r . I I . U n te r s u c h u n g e n ü b e r d ie F e k u n d itá t . S t r a h le n t h e r a p ie 134 (1 9 6 7 )in p r e s s .

(6 ) N Ô T H E L , H . , ----- IV . U n te rs u ch u n g e n ü b e r d ie F e r t i l i t á t . S t r a h le n t h e r a p ie , in p r e s s .(7 ) F R I T Z -N T G G L I , H . , B i o l o g i s c h e A n a ly s e d e r S t r a h le n s c h a d ig u n g v o n D r o s o p h i la - E i -

e r n d u r c h 180 k V R ô n tg e n s tr a h le n und u l t r a h a r t e 31 M e V -S t r a h le n . N a t u r w is s e n - s c h a ft e n 39 (1 9 5 2 ) 4 8 5 -4 8 6 .

(8 ) F R I T Z -N I G G L I , H . , V e r g l e i c h e n d e A n a ly s e d e r S t r a h le n s c h â d ig u n g v o n D r o s o p h i l a - E ie r n m it 180 k e V und 31 M e V . F o r t s c h r . R Ô n tg e n s tr . 83 (1 9 5 5 ) 1 7 8 -2 0 0 .

(9 ) F R I T Z - N I G G L I , H . , " S t r a h l e n b i o lo g i e " , T h ie m e , S tu ttg a rt (1 9 5 9 ) .(1 0 ) W Ü R G L E R , F . E . , U L R IC H , H . , S C H N E ID E R -M IN D E R , A . , V a r ia t io n o f r a d i a s e n s i -

t iv it y d u r in g m e io s i s and e a r l y c le a v a g e in n e w ly la id e g g s o f D r o s o p h i la m e la n o g a s t e r . " R e p a i r f r o m G e n e t ic R a d ia t io n D a m a g e an d D i f f e r e n t i a l R a d ia s e n s i t iv i t y in G e r m C e l l s (S O B E L S , F . H . E d . ) , P e r g a m o n P r e s s , O x fo r d (1 9 6 3 ) 1 0 1 -1 0 6 .

(1 1 ) K H IS H IN , A . F . E . , T h e r e s p o n s e o f th e im m a t u r e t e s t i s o f D r o s o p h i la to th e m u ta g e n ic a c t io n o f X - r a y s . Z . V e r e r b u n g s le h r e 87 (1 9 5 5 ) 9 7 -1 1 2 .

(1 2 ) A U E R B A C H , C h . , S e n s i t iv it y o f th e D r o s o p h i l a t e s t i s t o th e m u t a g e n ic a c t io n o f X - r a y s . Z . V e r e r b u n g s le h r e JÏ6 ( 1 9 5 4 ) 1 1 3 -1 2 5 .

(1 3 ) M A V O R , J . W . , A c o m p a r is o n o f th e s u s c e p t ib i l i t y t o X - r a y s o f D r o s o p h i la m e la n o - g a s t e r at v a r i o u s s t a g e s o f i t s l i f e - c y c l e . J . e x p . Z o o l . 4 7 (1 9 2 7 ) 6 3 - 8 3 .

(1 4 ) A M Y , R . L . , A c o m p a r a t iv e s tu d y o f th e e f f e c t s o f f i - r a y s , - jp -r a y s , and X - r a y s on d e v e lo p m e n t in H a b r o b r a c o n . R a d ia t .R e s . 3 (1 9 5 5 ) 1 6 6 -1 8 1 .

(1 5 ) C L A R K , A . M . , T h e r e la t io n o f g e n o m e n u m b e r t o r a d i o s e n s i t i v i t y in H a b r o b r a c o n . A m e r . N a t u r a lis t 91 (1 9 5 7 ) 1 1 1 -1 1 9 .

(1 6 ) E R D M A N , H . E . , A n a ly s e s o f th e d i f f e r e n t ia l r a d i o s e n s i t i v i t y o f d e v e lo p in g r e p r o d u c t iv e t i s s u e s in H a b r o b r a c o n ju g la n d is (A s h m e a d ) t o io n iz in g r a d ia t io n . In t. J . R a d . B i o l . 3 (1 9 6 1 ) 1 8 3 -2 0 4 .

(1 7 ) B A L O C K , J . W . , B U R D I T T , A . K . j r . , C H R IS T E N S O N , L . D . , E f f e c t s o f g a m m a r a d i a t io n on v a r io u s s t a g e s o f t h r e e f r u it f l y s p e c i e s . J . E c o n , E n t o m o l . 5 £ (1 9 6 3 ) 4 2 - 4 6 ,

(1 8 ) B E N S C H O T E R , C . A . , T E L I C H , J . , E f f e c t o f g a m m a r a y s on im m a t u r e s t a g e s o f the m e x ic a n f r u it f l y . J . E c o n . E n t o m o l . 57 (1 9 6 4 ) 6 9 0 -6 9 1 .

(1 9 ) S T E E N B E C K , L . , H is t o l o g is c h e U n te r s u c h u n g e n z u m " F r ü h t o d " r o n t g e n b e s t r a h l t e r D r o s o p h i l a - E i e r . N a t u r w is s e n s c h a f t e n 4 8 (1 9 6 1 ) 310 .

(2 0 ) F R I T Z - N I G G L I , H . , E r s t e b i o l o g i s c h e V e r s u c h e m it d e m 3 1 - M e V - B e t a t r o n .A r c h . K la u s - S t i f t . V e r e r b . F o r s c h . 24 (1 9 5 1 ) 4 5 4 -4 5 9 .

102 NÔTHEL

(2 1 ) C L A R K , A . M . , S o m e e f f e c t s o f X - i r r a d i a t i o n on l o n g e v i ty in H a b r o b r a c o n f e m a le s . R a d ia t .H e s . 15_ ( 1 9 6 1 ) 5 1 5 - 5 1 9 .

(2 2 ) E R D M A N , H . E . , A d u lt l o n g e v i t y a s a s e n s i t i v e c r i t e r i o n o f r a d ia t io n - in d u c e d d a m a g e w h en 2 4 - h o u r H a b r o b r a c o n (H y m e n o p te r a ) e m b r y o s a r e X - r a y e d . J . E c o n . E n t o m o l ,53 (1 9 6 0 ) 9 7 1 -9 7 2 .

(2 3 ) P A T T E R S O N , J . T . , T h e p r o d u c t io n o f m u ta t io n s in s o m a t ic c e l l s o f D r o s o p h i la m e la n o g a s t e r b y m e a n s o f X - r a y s . J . e x p . Z o o l . 5 3 (1 9 2 9 ) 3 2 7 -3 7 2 .

(2 4 ) T O K U N A G A , C h . , A R N H E IM , N . j r . , A g e d e p e n d e n c e o f th e l o c a t i o n s o f X - r a y i n d u c e d s o m a t ic c r o s s i n g o v e r in D r o s o p h i l a . G e n e t ic s 54 (1 9 6 6 ) 2 6 7 -2 7 6 .

(2 5 ) F O X , D . P . , T h e e f f e c t o f X - r a y s on th e c h r o m o s o m e s o f l o c u s t e m b r y o s .I . T h e e a r l y r e s p o n s e s . C h r o m o s o m a ( B e r l . ) J_9 (1 9 6 6 ) 3 0 0 -3 1 6 .

(2 6 ) U L R IC H , H . , E in V e r g l e i c h d e r R o n tg e n s tr a h le n w ir k u n g a u f K e r n und P la s m a d e s D r o s o p h i l a - E i e s . -B io l . Z e n t r a lb la t t 74 (1 9 5 5 ) 4 9 8 -5 1 5 .

(2 7 ) O S T E R T A G , W . , T h e g e n e t i c b a s i s o f s o m a t ic d a m a g e p r o d u c e d b y r a d ia t io n in th ir d in s t a r l a r v a e o f D r o s o p h i la m e la n o g a s t e r . Z . V e r e r b u n g s le h r e £ 4 (1 9 6 3 ) 1 4 3 -1 6 2 .

(2 8 ) N Ô T H E L , H . , D e r E in flu fl v o n R o n tg e n s tr a h le n a u f V i t a l i t a t s m e r k m a le v on D r o s o p h i la m e la n o g a s t e r . X. U n te rs u ch u n g e n i ib e r d ie L e b e n s d a u e r . S t r a h le n th e r a p ie 126 (1 9 6 5 ) 2 6 9 -2 8 2 .

(2 9 ) H E ID E N T H A L , G . , T h e o c c u r e n c e o f X - r a y in d u c e d d o m in a n t le th a l m u ta t io n s in H a b r o b r a c o n . G e n e t ic s _ 3 0 (1 9 4 5 ) 1 9 7 -2 0 5 .

(3 0 ) B A L D W IN , W . F . , S im i la r i t i e s in k i l l in g b y h e a t and b y X - i r r a d ia t i o n in th e i n s e c t D a h lb o m in u s fu s c ip e n n is ( Z e t t . ). R a d ia t . R e s . 5 (1 9 5 6 ) 4 6 - 5 1 .

(3 1 ) W H A R T O N , D . R . A . , W H A R T O N , M . L . , T h e e f f e c t o f r a d ia t io n on the lo n g e v i t y o f th e c o c k r o a c h , P e r ip la n e t a a m e r i c a n a , a s a f f e c t e d by d o s e , a g e , s e x and f o o d in ta k e . R a d ia t . R e s . П (1 9 5 9 ) 6 0 0 -6 1 5 .

(3 2 ) C O R K , J . M . , G a m m a -r a d ia t io n an d l o n g e v i ty o f the f lo u r b e e t l e . R a d ia t . R e s . 7 (1 9 5 7 ) 5 5 1 -5 5 7 .

(3 3 ) H A S S E T T , C . C . , JE N K IN S , D . W . , U se o f f i s s i o n p r o d u c t s f o r in s e c t c o n t r o l . N u c le o n i c s H) (1 9 5 2 ) V¿_ 4 2 - 4 6 .

(3 4 ) H E N N E B E R R Y , T . J . , S M IT H , F . F . , M u C O V E R N , W .L . , S o m e e f f e c t s o f g a m m ar a d ia t io n and a c h e m o s t e r i la n t on the m e x ic a n b ea n b e e t le . J . E c o n . FJntom ol. 57 (1 9 6 4 ) 8 1 3 -8 1 5 . ~

(3 5 ) J A Y N E S , H . A . , G O D W IN , P . A . , S t e r i l i z a t io n o f the w h it e -p in e w e e v i l w ith g a m m a r a d ia t io n . J . E c o n . E n to m o l . 50 ( 1 9 5 7 ) 3 9 3 - 3 9 5 .

(3 6 ) D A V IC H , T . B . , L IN D Q U IS T , D . A . , E x p lo r a t o r y s t u d ie s on g a m m a r a d ia t io n f o r the s te r il iz a t io n o f the bo ll w e e v il. J . E con . E n tom ol. ^5 (1962) 164 -167 .

(3 7 ) C L A R K , A . M . , R U B IN , M . A . , T h e m o d i f i c a t io n b y X - i r r a d ia t i o n o f th e l i f e sp a no f h a p lo id s and d ip lo id s o f th e w a s p , H a b r o b r a c o n s p . R a d ia t . R e s . 15 (196 1) 2 4 4 -2 5 3 .

(3 8 ) S T A H L E R , N . , T E R Z I A N , L . A . , T h e r e s p o n s e o f b l o o d - f e d A e d e s a e g y p t i tog a m m a r a d ia t io n . J . E c o n . E n to m o l . 56 (1 9 6 3 ) 4 1 6 -4 1 7 .

(3 9 ) L A M B , M . J , , T h e e f f e c t s o f r a d ia t io n on th e lo n g e v ity o f fe m a le D r o s o p h ilas u b o b s c u r a .. J . I n s . P h y s io l . 10 (1 9 6 4 ) 4 8 7 -4 9 7 .

(4 0 ) B A X T E R , R . C . , B L A I R , H. A . , K in e t ic s o f a g in g a s r e v e a le d b y X - r a y - d o s e - l e t h a l i - ty r e la t i o n s in D r o s o p h i l a . R a d ia t . R e s . 30 (1 9 6 7 ) 4 8 - 7 0 .

(4 1 ) N Ô T H E L , H . , T h e in f lu e n c e o f X - r a y s on l o n g e v i t y , fe c u n d ity and f e r t i l i t y o f D r o s o p h i l a m e la n o g a s t e r . In t. J . R a d . B i o l . 6 (1 9 6 3 ) 3 8 1 -3 8 2 .

(4 2 ) N Ô T H E L , H . , D if fe r e n t t y p e s o f m o r t a l i t y in c lu d in g p r o lo n g a t io n o f f e m a le l i f e t im e a f t e r X - r a y i n g D r o s o p h i la m e la n o g a s t e r im a g in e s . " G e n e t i c s T o d a y " , P r o c . X I Int. C o n g r . G e n e t ic s , T h e H a g u e , 1 (1 9 6 3 ) 7 2 -7 3 .

(4 3 ) N Ô T H E L , H . , A n e s t im a t io n o f th e n u m b e r o f -a c t i v e o o g o n ia p e r o v a r i o le in D . m e la n o g a s t e r f e m a le s . D IS 4 2 (1 9 6 7 ) 55 .

(4 4 ) L A M B , M . J . , T h e r e la t io n s h ip b e tw e e n a g e at i r r a d ia t io n and l i f e - s h o r t e n i n g in ad u lt D r o s o p h i l a . MR a d ia t io n & A g e in g " (L IN D O P , P . J . , S A C H E R , G . A . , E d s . ) , T a y lo r & F r a n c i s , L o n d o n (1 9 6 6 ) 1 6 3 -1 7 4 .

(4 5 ) N Ô T H E L , H . , D e r E in flu fi v o n R o n tg e n s tr a h le n a u f V it a l i t a t s m e r k m a le v o n D r o s o p h i l a m e la n o g a s t e r . III. U n te rs u ch u n g e n U ber d en E in flu G d e s A l t e r s a u f d ie S t r a h le n e m p f in d l ic h k e it . S t r a h le n th e r a p ie 135 (1 9 6 8 ) in p r e s s .

(4 6 ) L A M B , M . J . , M A Y N A R D S M IT H , J . , R a d ia t io n and a g e in g in i n s e c t s . E x p .G e r o n t o l . _1 (1 9 6 4 ) 1 1 -2 0 .

(4 7 ) C L A R K , A . M . , R O C K S T E IN , M , , A g in g in i n s e c t s . " T h e P h y s io lo g y o f I n s e c t a " (R O C K S T E IN , M . , E d . ) , A c a d e m ic p r e s s . N ew Y o r k , 1 (1 9 6 4 ) 2 2 7 -2 8 1 .

(4 8 ) K E R R , R . W . , V a r ia t io n w ith a g e in the s u s c e p t ib i l i t y to D D T and the r e s p i r a t i o nr a t e o f m a le and fe m a le D r o s o p h i la m e la n o g a s t e r M g . B u ll . e n t . R e s . 45 (1 9 5 4 ) 3 2 3 -3 2 8 .

DESTRUCTION DES C E LLU LE S POLAIRES DE L 1 Œ U F DU DORYPHORE PAR IRRADIATION

R. BLUZATFACULTE DES SCIENCES D 'O R SA Y ,

UNIVERSITE DE PARIS,FRANCE


DESTRUCTION DES CELLULES POLAIRES DE L 'Œ U F DU DORYPHORE PAR IRRADIATION. L'irradiation lo ca lisée , au m oyen de rayons X d e fa ib le én erg ie , du p ô le postérieur de la blastula de Leptinotarsa decem lin eata Say (C h rysom elidæ ) est réalisée sans entraver la poursuite du dévelop p em en t em bryonnaire. L ’ exam en histologique des gonades des larves de prem ier stade m ontre qu*elles sont tota lem ent dépourvues de gonies lorsque la dose est suffisam m ent é lè v é e (10 000 R) pour entrafher la destruction certa in e des ce llu les polaires postérieures. L 'ex isten ce de gonades agam étiques, d ifféren ciées à Г im age des tém oins, montre que l 'é d i f ic a t io n de la partie m ésoderm ique de la gonade est indépendante de la présence des ce llu les germ inales in itia les. Les larves agam étiques ne sont pas v iab les, sauf ex ce p tio n , et deux hypothèses sont ém ises pour tenter d 'e x p liq u e r c e fa it. Il sem ble q u 'u n phénom ène de régulation intervienne au cours du développem ent larvaire ou nym phal des gonades dont le nom bre de gonies est inférieur à c e lu i des tém oins (ca s des œ ufs irradiés à 3000 R).

DESTRUCTION OF THE POLAR CELLS OF COLORADO BEETLE EGGS BY IRRADIATION. The rear pole o f the blastoderm o f Leptinotarsa d ecem lin eata Say (C hrysom elidae) is subjected to lo ca l irradiation with low -en ergy X -rays without disruption o f em bryon ic developm ent. H isto log ica l study o f the gonads o f grow ing larvae shows total absence o f gonia after a su fficien tly high dose (10 000 R) to cause certain destruction o f the rear polar ce lls . The existen ce o f agam ic gonads d iffering in appearance from those in the control ce lls ind icates that developm ent o f the m esoderm ic part o f the gonad is independent o f the presence o f in itia l germ inal ce lls . Two hypotheses are put forward to explain the fact that agam ic larvae are almost always n on -v ia b le . It would appear that in cases where the number o f gon ia is less than in the control ce lls ( e .g . eggs irradiated at 3000 R), a regulation phenom enon has c o m e into play in the course o f gonial developm ent in the larva or pupa.

1. ETU D E B IB LIO G R A PH IQ U E E T BU T DU T R A V A IL

E n tre 1908 et 1911, H egner [1, 2] m etta it en é v id e n ce l 'e x i s t e n c e de deux t is s u s , b ien d is t in c ts par le u r o r ig in e , dans la gonade du d o ry p h o re ( L e p t in o ta rsa d e ce m lin e a ta Say, C h ry s o m é lid e , C o lé o p tè re ).Au m oyen d 'u n e a ig u ille chaude, i l d é tru isa it le p ô le p o s té r ie u r de l 'œ u f , au stade b la s tu la , et con sta ta it 1' a b se n ce de c e llu le s g e rm in a le s dans le s gonades d es e m b ry o n s is s u s d es œ u fs o p é r é s . G eigy [3], puis A boim [4], r é a lis è r e n t le m ê m e type d 'e x p é r ie n c e ch e z D ro so p h ila au m oy en d 'u n e b rû lu re par rayon n em en t UV et c o n fir m è r e n t l 'e x i s t e n c e de gonades a g a m étiq u es ; i l s m o n tr è r e n t qu e le s gon a d es de ce d ip tère son t co n s titu é e s de deux m a s s e s m é s o d e r m iq u e s où v ienn en t se lo g e r , a p rè s m ig ra t io n , le s c e l lu le s p o la ir e s , in it ia le s d e s c e llu le s g e rm in a le s . Selon c e s au teu rs, l 'é d i f i c a t io n de c e s deux m a s s e s m é s o d e r m iq u e s est indépendante de la p r é s e n c e d es c e llu le s g e rm in a le s . D ep u is, d iffé re n ts au teu rs ont obtenu d es ré su lta ts s im ila ir e s : H aget [5 ] ch e z L e p tin o ta rsa ,

103

104 BLUZAT

A lleau m e et H aget [6] ch e z C a llip h o ra p a r m ic r o c a u té r is a t io n , Hataway et Seim an [7] ch ez D ro so p h ila , O elhafen [8] ch ez C u lex , Ju ra [9] ch ez D ro so p h ila par ir ra d ia t io n UV lo c a l i s é e , Id r is [10] ch ez C u lex par lig a tu re .

A l 'o p p o s é , C ou n ce et Seim an [11], sou m ettant des oeufs de D ro s o p h ila a un tra item en t par le s u lt r a -s o n s , ont obtenu deux e m b ry o n s ayant d es gonades s itu é e s dans la r é g io n a n té r ie u re , a p ro x im ité de l 'œ s o p h a g e : pou r c e s au teu rs , le s c e l lu le s p o la ir e s ont atteint une m a s s e m é s o d e rm iq u e an tér ieu re et induit a c e t e n d ro it la fo r m a t io n d es gon a d es. L e s o b s e rv a t io n s de C ou n ce et Ede [12 ] su r un m utant de D ro s o p h ila , et le s ré su lta ts de P o u lso n et W aterhou se [13] v iennent à 1' appui de ce tte h yp oth èse . C es d e r n ie r s au teu rs, a p rè s b rû lu re d es c e llu le s p o la ir e s de l 'œ u f de D ro so p h ila par rayon n em en t UV, co n s ta tè re n t, s o it la p r é s e n c e de qu e lq u es gon ies dans le s gonades des e m b ry o n s ob ten u s, so it l 'a b s e n c e to ta le de gon a d es. Ils co n c lu re n t que la p r é s e n ce de c e l lu le s p o la ir e s est n é c e s s a ir e à l 'é d i f i c a t io n de la p a rtie m é s o d e rm iq u e de la gonade. A n d erson [14 ], s ' appuyant su r le s t r o is travau x c ité s c i -d e s s u s et su r ce lu i de D avis (non pu b lié ) ch e z C ulex et L u c il ia , co n c lu t à l 'e x i s t e n c e in d iscu ta b le de l 'in d u c t io n de la p a rtie m é s o d e rm iq u e de la gonade par le s c e l lu le s p o la ir e s . Il c o n s id è r e en e ffe t que le s gon a d es a g am étiq u es , m is e s en é v id en ce par de n om breu x au teu rs, ne son t pas in té g ra le m e n t m é s o d e r m iq u e s .

Ce tra v a il a eu p ou r but de fo u rn ir d es in d ica tion s su r le r ô le des c e l lu le s p o la ir e s , en lo s détru isa n t au m oyen d 'u n e n ou v e lle tech n iq u e : l 'i r r a d ia t io n lo c a l i s é e du p ô le p o s té r ie u r de la b la s tu la par d e s ra y o n s X de fa ib le é n e rg ie . C ette étude a été p o u rsu iv ie p r in c ip a le m e n t dans deux d ir e c t io n s :- e x is te n c e e t s tru ctu re d e s gon ades,- p o s s ib il it é s de d év e lop p em en t a p rè s l 'i r r a d ia t io n .

2. M A T E R IE L E T TECH NIQUES

L e s œ u fs , c o l lé s par la fe m e lle su r une fe u ille de p om m e de t e r r e , son t r é c o l t é s a u ss itô t pon du s. Ils son t p la cé s a 16°C dans une en ce in te h u m ide. Dans c e s con d ition s de d év e lop p em en t, le stade b la s tu la jeu n e (1000 à 2000 noyaux) e st atteint en 24 h, le stade b la s tu la âgé (16 000 noyaux) en 48 h [15]. Ils sont p la cé s su r une plaque de p lom b de 5 m m d 'é p a is s e u r , enduite d 'u n e m in ce co u ch e de g r a is s e à v id e . C ette plaque e s t p e r c é e de can au x ayant 0, 5 m m de d ia m è tre . L e s œ u fs son t d is p o s é s de fa ço n te l le que se u le la p a rtie p o la ir e p o s té r ie u r e (1 /8 de la lon gu eu r to ta le ) s o it a ttein te p a r le s ra y on s X lo r s q u e la p laqu e, r e to u rn é e , e s t p la cé e su r une en ce in te en p lom b.

L e s ir ra d ia t io n s son t e ffe c tu é e s au m oyen d 'u n tube H olw eck m uni d 'u n e an ticath ode en m olyb d èn e et d 'u n e fe n ê tre en alum in ium (0, 05 m m ), ém ettan t un rayon n em en t X peu pén étran t: la cou ch e de d e m i-a b s o r p t io n e s t ég a le a 1 m m d ’ eau. L 1 a p p a re il fon ction n e sou s une ten sion de 37, 5 kV et av ec une in ten sité de 15 m A , L a d o s im é tr ie , e ffe c tu é e d 'u n e p a rt p a r la m éthode ch im iqu e au su lfa te fe r r e u x et d 'a u t r e part a v ec un d o s im è tr e P h illip s , indique un déb it de 3 000 R /m in à 118 m m de la fe n ê tre . L e s d o s e s u t i l is é e s , 1 000 R à 30 000 R , son t d é liv r é e s à déb it con stan t, le s .d u r é e s d 'ir r a d ia t io n v a r ia n t de 20 s a 10 m in .

SM- 102 /11 105

A p rè s ir ra d ia t io n le s œ u fs son t in cu bés à 25°C dans une en ce in te hu m ide. L e s la r v e s son t tu ées à la n a is sa n ce au m oyen de v a p e u rs de c h lo r o fo r m e puis f ix é e s par le m élan ge de C arn oy durant 1 h (a c id e a cé tiq u e : 1 v o lu m e ; c h lo r o fo r m e : 3 v o lu m e s ; a lc o o l a b so lu : 6 v o lu m e s ) . L e s la r v e s son t en su ite in c lu s e s dans la p a ra ffin e (5 4 -5 6 °C ) a p rè s désh y d ra ta tion p a r l 1 a lc o o l bu ty liq u e . L e s co u p e s s é r ié e s (6 ц ш ) sont c o lo r é e s p a r un m éla n g e d 1 é r y th r o s in e -a u r a n t ia -o ra n g é G (5 m in) puis par le b leu de to lu id in e (1 m in 30 s ) , d é sh y d ra té e s p a r le b en zèn e et m on tées dans le bea u m e du Canada.

3. ETU D E DES GONADES

T é m o in s

L e s gon ad es de la la r v e n a issa n te du d o ry p h o re se p résen ten t sou s deux a s p e c ts se lo n le s e x e : deux m a s s e s o v o ïd e s ch ez la fe m e l le , deux h a ltè re s ch e z le m â le . E l le s son t s itu é e s dans la p a rtie p o s té r ie u r e de l 'a b d o m e n , de chaque c ô té de l ' in t e s t in m oy en . E lle s son t e n to u ré e s d 'u n e gaine de g r o s s e s c e l lu le s a d ip e u se s . A l 'in t é r ie u r de chaque gonade on d istin gu e deux c a té g o r ie s c e l lu la ir e s :- de p e tites c e l lu le s b a n a le s , d 'o r ig in e m é s o d e rm iq ü e ,- de g r o s s e s c e l lu le s c a r a c t é r is é e s par un noyau sp h é r iq u e , tr è s vo lu m in eu x (d ia m è tre > 1 0 /um), fa ib lem en t c o lo r a b le et contenant un g ro s n u c lé o le ; une m in ce co u ch e de cy to p la s m e p é r ip h é r iq u e t r è s b a so p h ile (c o lo ra t io n in ten se par le b leu de to lu id in e ).

C es c e l lu le s , g o n ie s p r im o r d ia le s , son t p lus n o m b re u se s dans la gonade m â le , où e l le s son t r é p a r t ie s de fa ço n é g a le dans le s deux re n fle m e n ts .

L a r v e s is s u e s d 'œ u fs ir r a d ié s

Irra d ia t io n au stade b lá s tu la jeu n e

1000 R (22 c a s ) : 40% de gon ades d ép ou rv u es de gon ies40% de gon ades ayant un n om b re v a r ia b le de g on ies 20% de gon ades n o rm a le s .

3000 R (28 c a s ) : 70% de gon ad es d ép ou rv u es de g on ies30% de gon ades ayant un n o m b re v a r ia b le de g o n ie s .

10 000 R (36 c a s ) et 30 000 R (20 c a s ) : tou tes le s g on a d es son td ép ou rv u es de g o n ie s , a 1' e x ce p t io n de t r o is c a s , p ro b a b le m e n t dus à une m is e en p la c e d e s œ u fs d é fe c tu e u se au m om en t de l ' i r r a d ia t io n .

Irra d ia tio n au stade b la s tu la âgée

10 000 R (24 c a s ) : 60% de gon a des d ép ou rv u es de g on ies40% de gon a d es ayant un n o m b re v a r ia b le de g o n ie s .

106 BLUZAT

A s p e c t d es gonades

D ans tou s le s ca s e x a m in é s , deux gon ades b ien d is t in c te s e x is te n t ; E l le s son t to u jo u rs s itu é e s a le u r e m p la ce m e n t anatom iqu e n o rm a l, m a is , le p lu s sou vent, le s g o n ie s p r im o r d ia le s m anquent to ta lem en t ou son t peu n o m b r e u s e s . L e s g on a d es , d ép ou rv u es de g o n ie s , ont une ta ille n ettem ent in fé r ie u r e a c e l le d e s gon a des té m o in s , m a is c o n s e rv e n t p a rfa ite m e n t le u r f o r m e : o v o ïd e ou en h a ltè re .

D is c u s s io n

C e s e x p é r ie n c e s m on tren t c la ir e m e n t que la p a rtie m é s o d e r m iq u e de la gonade peut s 'é d i f i e r et se d i f fé r e n c ie r se lo n le s e x e , m a lg ré l 'a b s e n c e d es c e llu le s g e rm in a le s in it ia le s . C ette c o n c lu s io n va a 1 'e n co n tre de c e l le de ce r ta in s au teu rs [13, 14] qui, a p rè s d e s tr u c t io n d es c e llu le s p o la ir e s , n 'o n t pas co n s ta té la p r é s e n c e de gon ad es a g a m é tiq u e s . Il e s t ra iso n n a b le de p e n se r que c e s au teu rs ont peu t- ê tr e d é tru it , ou tre le s c e l lu le s p o la ir e s , le s c e l lu le s b la s to d e r m iq u e s qui fo u rn is s e n t n o rm a le m e n t le m é s o d e r m e de la r é g io n m oyen n e a b d om in a le [5, 6],

4 . PO SSIB IL IT E S DE D E V E L O P P E M E N T A P R E S IR R A D IA T IO N

A p rè s ir ra d ia t io n du p ô le p o s té r ie u r de la b la stu la , jeu n e ou âg ée , le d év e lo p p e m e n t e m b ry o n n a ire se p ou rsu it ju s q u 'à 1 'é c lo s io n (90% d e s c a s ) san s que l 'o n p u isse o b s e r v e r un re ta rd .

L 'é le v a g e de 150 an im aux is s u s d 'œ u fs ir r a d ié s par 10 000 R au stade b la s tu la jeu n e m o n tre que la p lu pa rt d 'e n t r e eux m eu ren t d ès le p r e m ie r stade la r v a ir e , san s s 'ê t r e n o u r r is . Aucun adulte n 'a pu ê tr e obtenu.

L ' é le v a g e de 200 anim aux ir r a d ié s p a r 3 000 R a fo u rn i 60 adultes ayant d e s gonades ty p iq u es , à 1' e x ce p t io n de qu atre d 'e n t r e eux. C e u x -c i , p ou rv u s d 'u n Ira ctu s gén ita l n o rm a l, p ré se n ta ie n t d es te s t ic u le s de fo r m e typ ique (h a ltè re ): le u r d ia m è tre é ta it iden tiqu e à la la rg e u r du sp e rm id u c te , so it 0, 1 m m .

L e s la r v e s ayant à la n a is sa n ce un p etit n om b re de g on ies évo lu en t en ad u ltes n orm a u x , p a r fa ite m e n t fé c o n d s : i l s e m b le é ta b li q u ’ il s e p rod u it une ré g u la t io n au c o u r s de la c r o is s a n c e la r v a ir e .

D is c u s s io n

D eux h yp oth èses p erm etten t d 'e x p l iq u e r le s p o s s ib il it é s de s u r v ie e x trê m e m e n t fa ib le s d es la r v e s a g a m é tiq u e s :

a) L e s c e llu le s p o la ir e s de L e p t in o ta rsa au raien t d es p o ten tia lités a u ss i éten du es que ch e z le s D ip tè re s s u p é r ie u r s : e l le s d on n era ien t n a is sa n ce , d 'u n e part, aux g o n ie s et, d 'a u t r e part, à c e r ta in e s c e llu le s de l 'in t e s t in m oyen , à l ' im a g e de c e qui e s t connu ch e z D ro s o p h ila [13].

b) L a d e s tru ctio n de la to ta lité d es c e l lu le s p o la ir e s par le s ra y o n s X ne p o u rra it s 'e f f e c t u e r sans e n tra în e r la d e s tru c tio n de c e llu le s b la s to d e rm iq u e s v o is in e s . C ette se co n d e h yp oth èse e st d 'a u ta n t p lus v r a is e m b la b le q u 'i l e s t n é c e s s a ir e d 'i r r a d i e r toute la r é g io n p o la ir e

S M -1 0 2 A 1 107

p o s té r ie u r e : le s c e l lu le s p o la ir e s ne se détach en t pas en une m a s s e , e x te rn e à 1' œ u f, c o m p a ra b le à c e l le o b s e r v é e ch e z D ro s o p h ila .

R E F E R E N C E S

[1 ] HEGNER, R .W . , B iol. Bull. 1 6 (1 9 0 8 ) 19.[2 ] HEGNER, R .W . . B iol. Bull. 20 (1911 ) 237.[3 ] GE1GY, R ., Revue Suisse Z ool. 38 (1 9 3 1 ) 187.[4 J ABOIM, A. N .. Revue Suisse Z o o l . 52 (1 9 4 4 ) 53.[5 ] HAGET, A . . C . r. Séan c. S oc . B iol. 147 (1953 ) 673.[6 ] ALLEAUME, N .. HAGET, Л .. P. V. S oc. S ci. Phys. Nat. de Bordeaux (1961 ) 92.[7 ] H ATA W AY , D. S . . SELMAN, G .G . , J. Em bryol. exp . Morph. 9 (1 9 6 1 )3 1 0 .[8 ] OEI.HAFEN, F . . A rch . EntwMech. Org. 153 (1 961 ) 120.[9 J JURA, С . , A cta b io l. cra cov . 7 (1965) 89.

[1 0 ] IDRIS, Arch. EntwMech. Org. 152 (1 960 ) 230 .[11] COUNCE, S.J. , SELMAN, G .G . , J. Em bryol. exp . Morph. 3 (1955) 121.[121 COUNCE, S.J. . EDE, D. A. , J. Em bryol. exp . Morph. 5 (1957) 404.[13 ] POULSON, D .F . , WATERHOUSE, D. F . , Ausl. J. b io l. S ci. 13 (1960 ) 541.[14 ] ANDERSON, D. T . , A . Rev. Ent. П (1966 ) 23.[15 ] SCIINETTER, W . , Arch. EntwMech. Org. 155 (1965) 637.

D I S C U S S I O N

R . C A V A L L O R O : D o you have any data on the ra d ia tio n se n s it iv ity o f c o lo r a d o b e e t le e g g s ir ra d ia te d ' i n to to ' ?

R. B L U Z A T : Y e s , I have r e s u lts f o r the beg in n in g o f e m b ry o g e n e s is .R. C A V A L L O R O : I sh ou ld lik e to m en tion , i f I m a y , that I have

r e ce n t ly co m p le te d s o m e w ork on the sa m e s p e c ie s u s in g X - r a y equ ipm ent at 200 kV , 10 m A cu rre n t, with a d o se ra te o f 650 R /m in , and found that o f e g g s g iven a d o s e o f 1000 R at 25°C tw e n ty -fo u r h ou rs a fte r o v ip o s it io n on ly 5. 81% h atch ed , and that a d o se o f 2000 R ca u sed to ta l e m b ry o m o rta lity .

R . B L U Z A T : R e su lts obta in ed u n der m y co n d itio n s o f w ork a re v e r y c lo s e to y o u r s , i . e . L D 99 is betw een 600 and 800 R. On the o th er hand, the e a r l ie r d e v e lo p m e n ta l s ta g e s ( fro m o n se t o f in tra v ite llin e m u ltip lica tio n ) a re m o r e re s is ta n t to ra d ia tio n , i . e . LE^9 > 1000 R . M o r e o v e r , in the la tte r c a s e the la rv a e obta in ed a re v ia b le and n orm a l.

G. L E M ASNE : I sh ou ld lik e to h ear a lit t le m o r e on the ph enom enon o f la rv a e w ith a re d u c e d n um ber o f gon ia a fte r ir ra d ia t io n trea tm en t d e v e lo p in g in to p e r fe c t ly fe r t i le adu lts, as d e s c r ib e d in se c t io n 4 . 4 o f y o u r p a p er . F ir s t ly , what do the gonads o f the youn g la rv a e lo o k lik e ?S econ d ly , what s o r t o f r e g u la to ry m e ch a n ism had you in m in d ? Is g on ia l m u lt ip lica t io n r e su m e d , and i f s o what fa c t o r s c o m e in to p la y ? It s e e m s that th is r e g u la to ry p r o c e s s w ould be w orth studying.

R . B L U Z A T : The gonads we saw in th e se ir ra d ia te d la rv a e lo o k e d the sa m e as th ose o f the c o n tro l in s e c ts . It w ould a p p ear that regu la tion is not due to fu rth e r m u lt ip lica t io n o f the gon ia , but the p r o c e s s e s in vo lved have s t i l l not b een e lu cid a ted .

V. L A B E Y R IE : Is it not p o s s ib le that the m e s o d e r m ic m a ss o f the gonad is in stru m en ta l in in du cin g m u lt ip lica t io n o f gon ia r e d u ce d a p p re c ia b ly in n u m ber by ir ra d ia t io n , s in ce the n u m ber o f gon ia is n o rm a lly a

108 BLUZAT

fu n ction o f a m a ss ra t io o f m e s o d e r m ic c e l ls to g on ia l c e l ls in the e a r ly s ta g e s o f d e v e lop m en t b e fo r e d iffe re n t ia tio n in to oo g o n ia o r sp e rm a to g o n ia ?

R. B L U Z A T : Indeed , it is h igh ly lik e ly that th is ra t io p la y s a part in gon ia m u ltip lica tio n . W e cannot ru le out the p o s s ib ility that the m e s o d e r m ic t is su e c o n tro ls gon ia m u lt ip lica t io n m o r e o r le s s d ir e c t ly .

QUELQUES EFFETS DE L'IRRADIATION AUX RAYONS GAMMA SUR Spodoptera (Laphygma) exigua Hb. (LEPIDOPTERA: NOCTUIDAE)*

M. AN WAR**INRA, STATIO N DE ZOOLOGIE AGRICOLE,CENTRE DE RECHERCHES AGRONOMIQUES DU SU D -EST,M O N TFAVET, FRANCE


SOME EFFECTS OF G AM M A IRRADIATION ON Spodoptera (Laphygm a) Exigua Hb. (LEPIDOPTERA: NOCTUIDAE). A study has been m ade o f the e ffe c t o f gam m a rays from a co b a lt -6 0 source on the eggs, larvae, pupae and adults o f Spodoptera exigua H b ., a lepidopteron which is harm ful to crops in many clim ates . The insect was reared on an artific ia l diet based on pow dered cabba ge and w hear-germ . The irradiation o f o n e - and fo u r -d a y -o ld eggs with 3 ,6 and 9 krad showed that none o f the o n e -d a y -o ld eggs hatched, w hile the fo u r -d a y -o ld ones gave 52, 18 and 0% o f adults respectively-. The larvae hatched from irradiated eggs showed retarded developm ent, and the butterflies which deve lop ed w ere listless and o f low fertility.” Mature larvae irradiated at 3 and 5 krad g iv e 100 and 22. 5% o f adults respectively ; irradiation at 7 krad or m ore com p le te ly stops the production o f adults. Irradiation o f young pupae either kills them or leads to the hatching o f m alform ed adults. Irradiation at 50 krad o f pupae at the end o f their developm ent has no e ffe c t on hatching. The adults from nymphs irradiated at 50 krad show a fertility reduced to 0 .5 % , w hile the adults w hich have just hatched show total sterility after irradiation at the sam e dose. Higher doses reduce the frequency o f m ating, w hile fem ales irradiated at 30 krad and m ated with norm al m ales lay in fertile eggs.

QUELQUES EFFETS DE Ll IRRADIATION AUX RAYONS G AM M A SUR Spodoptera (Laphygm a) exigua Hb. (LEPIDOPTERA: NOCTUIDAE). L 'e ffe t des rayons gam m a d'une source au co b a lt -6 0 a été étudié sur les œ ufs, larves, pupes et adultes de Spodoptera exigua H b ., Lépidoptère nuisible aux cultures sous de n om breux c lim ats. L 'in secte était é le v é sur m ilieu a rt ific ie l à base de poudre de chou et de germ e de b lé . L 'irradiation d 'œ ufs de 1 e i 4 jours à 3, 6 e t9 k ra d s m ontre q u 'il n 'y a aucune é c lo s ion pour ceux de 1 jour et que ceux de 4 jours donnent 52, 18 et 0% d'adultes. Les larves issues d’ œ ufs irradiés montrent un retard de développem ent et les papillons éc los sont sans vigueur et peu fécon ds. Les larves âgées irradiées à 3 et5 krads donnent 100 et 2 2 ,5 % d 'adultes; l'irradia tion à 7 krads et plus ne donne pas d 'adultes. L 'irradiation des pupes jeunes provoque une m orta lité des pupes ou des éclosion s d 'adultes m al form és. L1 irradiation à 50 krads de pupes en fin de développem ent n 'a ffe c te pas les éclosion s. Les adultes issus de nym phes irradiées à 50 krads montrent une fertilité réduite à 0 ,5 % . Les adultes venant d 'é c lo re irradiés à 50 krads montrent une stérilité totale. Des doses plus é levées dim inuent la fréquence des accouplem ents. Des fem elles irradiées à 30 krads et a ccou p lées à des m aies normaux pondent des ceufs infertiles.

1. INTRODUCTION

Sp odoptera ex igua est une N o c tu e l le répandue dans le m on d e en t ie r , surtout dans le s r é g io n s t r o p i c a l e s , su b t r o p ic a le s et m é d ite r ra n é e n n e s . Sa ch e n il le ca u se des d é g â ts p a r fo is im p ortan ts à des cu ltu re s t r è s

. * Trava il e ffe ctu é en 1966-1967 pendant un stage en France organisé par 1‘ ASTEF (A ssociation pour l'organ isation des stages en France).

* * Adresse perm anente: Pakistan A tom ic Energy Com m ission.

109

110 ANWAR

v a r ié e s , co ton , tabac , v igne , lu ze rn e , p lantes m a r a î c h è r e s , etc .L 'a d u lte est un m ig r a te u r pouvant e f fe c tu e r des d é p la c e m e n ts im p o rta n ts .

Une seu le p u b lica tion , à n otre c o n n a is sa n c e , donne l e s ré su lta ts d 'é tu d e s d ' i r r a d ia t io n s , qui ont été p o u rs u iv ie s en Union sov ié t iqu e [1]. Etant donné l ' im p o r t a n c e éco n o m iqu e de l ' i n s e c t e , il nous a paru in té re ss a n t de r e p r e n d r e ce tte étude de fa ço n plus c o m p lè te ; nous v e r r o n s d 'a i l l e u r s que nos résu lta ts ne c o n c o rd e n t souvent pas a v e c ce u x de R a su lo v .

2. M ETH O D E D 'E L E V A G E

L 'é l e v a g e de S. ex igua sur m i l ie u a r t i f i c i e l est a s s e z f a c i l e . D if fé re n te s , f o r m u le s de m i l ie u ont été m i s e s au point [2, 3]; nous avons adopté une fo r m u le a s s e z v o is in e , m is e au point à la Station de z o o lo g ie de M ontfavet par G .G u e n n e lo n et S .P o it o u t (T ra v a u x non p u b lié s ) , c o m p o s é e e s s e n t ie l le m e n t de p o u d re de chou et de g e r m e de b lé , auxquels sont a jou tés l e s in gréd ien ts qui c o m p o s e n t habitue llem ent un m i l ie u s e m i - synthétique.

L 'é l e v a g e des la r v e s es t r é a l i s é su r ce m il ie u , qui e st d é co u p é en p et its cu b e s et d is p o s é dans des b o îte s en m a t iè r e p last iqu e (de 17X 1 0 , 5 X 2 , 5 c m ) . Chaque boîte cont ient en v iron une centa ine de l a r v e s , qui sont é l e v é e s dans une p iè c e c l im a t i s é e (2 5 ° C , 80% HR et é c la i r a g e de 18 h par tu bes f lu o r e s c e n ts ) . D ans c e s con d it ion s d 'é l e v a g e , la p r é ch ry s a l id a t io n débute de 11e jo u r . D ès le 10e jo u r le s b o î te s c o n t e nant l e s la r v e s sont p la c é e s dans un ré c ip ie n t p lus vaste r e m p l i au t i e r s de sa b le lé g è r e m e n t h u m id if ié . T ou s le s jo u r s , des la r v e s qui ne sont pas e n tr é e s dans le sa b le sont p la c é e s dans un autre ré c ip ie n t av e c du sa b le .L a ch ry s a l id a t io n c o m m e n c e le 12e jo u r et la sép a ra t io n d e s s e x e s avant l ' i r r a d ia t i o n a été e f fe c tu é e à c e stade. L a du rée de d é v e lo p p e m e n t des n ym p h es est en v iron de 7 jo u r s pou r le s f e m e l le s et de 8 jo u r s pou r l e s m â le s .

3. CONDITIONS E X P E R IM E N T A L E S E T M ETH O DES

L e s ir ra d ia t io n s ont été e f fe c tu é e s av e c un ir ra d ia te u r de 60C o de 1400 C i au S e r v ic e de r a d io - a g r o n o m ie du C e n tre d 'é tu d e s n u c lé a i r e s de C a d a r a c h e . L e débit de d o s e était d 'e n v ir o n 1040 r a d /m in . L a d o s i - m é t r i e a été e f fe c tu é e pou r chaque e x p os it ion .

P o u r l 'a c c o u p le m e n t d e s adultes, nous avons u ti l isé d e s b o î te s en p last iqu e de 8 c m de d ia m è tre et 5 c m de hauteur, av e c d e s c o u v e r c l e s a é r é s p a r un trou c i r c u la i r e de 4 cm de d ia m è tre et g r i l la g é . P o u r l e s c o n t r ô le s de fé co n d ité des adultes et de v iab il ité d e s œ u fs , on ta p is se l e s p a r o is de la ca g e a v e c du pap ier s u l fu r isé , su r leq ue l le s pa p il lon s d ép osen t l e u r s œ u fs . T o u s l e s t r o i s j o u r s l e s adultes sont ch a n gés de c a g e s , le s pontes sont c o m p té e s . Un c e r ta in n o m b r e de pontes sont g a r d é e s p ou r c o n t r ô le r la v iab il ité des œ u fs , et l e s la r v e s é c l o s e s sont c o m p té e s chaque jo u r .

On p la ce un seu l c ou p le p a r cage , a v e c d i f fé r e n te s co m b in a is o n s (m â le i r r a d ié X f e m e l le non i r r a d ié e ; f e m e l le i r r a d ié e X m â le non i r r a d ié ; té m o in , m â le et f e m e l le non i r r a d ié s ) . On in sta lle 20 c a g e s p a r c o m -

S M - 102/12 111

b ina ison . L e s f e m e l l e s sont d is s é q u é e s a p r è s le u r m o r t p ou r c o m p te r l e s s p e r m a to p h o r e s .

C e s c a g e s sont p la c é e s dans une p iè c e c l im a t is é e (2 2 ° C , 80% HR) à la lu m iè r e n atu re l le . On u t i l ise seu lem ent l e s adultes qui ont é c l o s dans l e s 24 h e u re s suivant l ' i r r a d ia t io n . L e s adultes sont n o u r r i s av e c une so lu t ion d 'eau et de m ie l à 10%.

4. R E S U L T A T S

4 . 1 . I r ra d ia t io n d e s œ u fs

L e s d o s e s d ' i r r a d ia t io n c h o i s i e s ont été de 3, 6 et 9 krad , ap p liquées à d e s œ u fs de 1 et 4 j o u r s (d u rée d 'in cu ba tion 4, 5 jo u r s à 22 °C ) .

L e s .œufs de 1 jo u r i r r a d ié s ne donnent aucune é c lo s io n .L 'e f f e t d es r a y o n s g a m m a sur le s œ u fs de 4 jo u r s est p or té dans le

tableau I. Nous v o y o n s que, s i l e s d o s e s de 3 et 6 krad ne diminuent que peu le p ou rce n ta g e d 'é c l o s i o n s , par co n t re l 'e f f e t de l ' i r r a d ia t i o n se fait sen t ir fo r te m e n t su r le d év e lop p em en t u lté r ie u r , la r v a i r e et nym phal, et l ' é c l o s i o n d 'a d u lte s . L e s la r v e s i s s u e s d 'œ u fs i r r a d ié s m ontrent un re ta rd de d é v e lop p em en t ; l ' i r r a d ia t i o n des œ u fs à 9 krad ne p e rm e t aucune fo r m a t io n d 'ad u ltes .

P r o v e r b s et Newton [4] et E lb a d ry [5] a ttira ient d é jà l 'a ttention sur ce tte p ro lon ga t ion d 'a c t io n de l ' i r r a d ia t io n des œ u fs , r e s p e c t iv e m e n t su r C a r p o c a p s a p o m o n e l la et G n o r im o s c h e m a o p e r c u le l la .

N ous ayons é g a lem en t e x am in é la fé co n d ité et la fe r t i l i t é des adultes is s u s d 'œ u fs i r r a d i é s . Il est r e m a r q u a b le de c o n s ta te r (tableau II) que ce tte act ion est t r è s m a rq u é e ; en p a r t i c u l ie r , l e s adultes i s s u s d 'œ u fs i r r a d ié s à 6 krad p résen ten t une v iab il ité d 'œ u fs nulle , q u ' i l s 'a g i s s e des m â le s ou des f e m e l l e s . A ce tte d o s e , la lo n gé v ité est a s s e z co u r te et l e s adultes sont sans v igu eu r et peu fé c o n d s .

4 . 2 . I r ra d ia t io n d e s la r v e s

L ' i r r a d ia t i o n d e s la r v e s du d e r n ie r âge à d i f fé r e n te s d o s e s de 3 et 5 krad p e r m e t une évo lu t ion ju sq u 'a u x adultes (tableau III).

L e s adultes i s s u s de la r v e s i r r a d ié e s à 3 et 5 krad m ontrent (tableau IV) une fe r t i l i t é d e s œ u fs d im in uée p a r ra p p o r t aux té m o in s , et une fé co n d ité rédu ite .

4 . 3 . I r ra d ia t io n d e s nym phes

4 . 3 . 1 . E c lo s io n

L e s n ym phes de 1 jo u r sont tu é e s par des d o s e s de 10 krad . L e s n ym phes de 3 jo u r s sont m o in s s e n s ib le s ; une d o s e de 20 krad ne cau se que 25% de m o r ta l i t é d e s nym phes .

D es n ym phes en fin de d év e lop p em en t (7 jo u r s p ou r l e s nym phes f e m e l l e s et 8 j o u r s p ou r l e s n ym phes m â le s ) supportent d e s d o s e s bea ucou p plus f o r t e s . Il faut a t te in dre 100 krad p o u r c o m m e n c e r à p r o v o q u e r une m o r ta l i t é s e n s ib le , et 120 krad pour e m p ê c h e r toute fo r m a t io n d 'a d u lte s n o rm au x .

T A B L E A U I. E F F E T S DU R A Y O N N E M E N T G A M M A SUR L E S Œ U F S A G E S DE4 JO U E S D E S p o d o p t e r a e x ig u a H b.

(2 0 0 œ u f s p a r d o s e )

D ose(krad)

Ceufsé c lo s

№ )

Larves parvenu es au dern ier stade

(%)

Larves m ortes au ■

dern ier stade

m

N ym phesm ortes

(%)

Ceufs donnant des adu ltes (% )

D urée m o y e n n e de d é v e lo p p e m e n t de

l ' é c lo s io n des œ u fs jusqu à 1* é c lo s io n des adu ltes

( j)T o ta l M â le F e m e lle

0 95 96 5 2 84 4 7 ,6 5 2 ,4 2 0 ,9

( té m o in )

3 87 76 8 15 52 4 7 , 7 5 2 ,3 2 4 ,1

6 86 63 18 59 18 6 3 ,6 3 6 ,4 2 7 ,5

9 65 13 92 1 0 0 0 0 , 0 0 , 0 0 , 0

ANVVAR

T A B L E A U I I . N O M B R E D ' A C C O U P L E M E N T S , F E C O N D I T E , F E R T I L I T E E T L O N G E V I T ED E S A D U L T E S D E S p o do pt e ra ex i g ua ISSUS DU C R O I S E M E N T D ' I N S E C T E S N O R M A U XA V E C D E S I N S E C T E S ISSUS D ' Œ U F S I R R A D I E S

D ose(krad )

N ature des cro isem en ts

P ou rcen tage des fe m e lle s

fé co n d é e s

N om b red 'a c co q p le m e n ts

parm â le

N om b red 'œ u fs

parf e m e l le

Cteufsé c lo s

(%)

L o n g é v ité des adu ltes

( j )

M a ie F e m e lle M â le F e m e lle

0 N N 1 0 0 2 ,6 0 6 1 1 ,7 8 7 ,5 1 0 ,3 9 ,6

3 I N 95 1 ,4 5 3 9 5 ,9 5 1 .8 9 ,7 9 , 4

3 N I 80 2 ,6 0 3 1 3 ,7 2 8 ,4 1 0 ,3 9 ,9

G I N 5 0 ,0 5 2 3 2 , 0 0 , 0 5 , 8 1 0 ,7

6 N I 45 0 ,4 5 1 1 6 ,4 0 , 0 1 0 ,5 5 ,0

N = non irra d ié , I = irra d ié .

SM

-102/12 113

T A B L E A U III. E F F E T S DU R A Y O N N E M E N T G A M M A SUR L E S L A R V E S DU D E R N I E R A G E D E S p o d o p t e r a e x ig u a H b .

(4 0 l a r v e s p a r d o s e )

D ose(krad)

N om b re d e larves m ortes

N om b re d e nym phes

m ortes

N om b re d 'a d u ltes

. m a lfo rm ês

N om b red 'a d u lte sn orm au x

0 0 0 0 40

3 0 0 0 40

5 0 4 27 9

7 9 9 2 2 0

1 0 28 1 2 0 0

15 33 7 0 0

114 A

NW

AR

T A B L E A U I V . N O M B R E D ' A C C O U P L E M E N T S , F E C O N D I T E , F E R T I L I T E E T L O N G E V I T ED E S A D U L T E S D E S p o d o p te ra ex ig ua Hb. I SS US DU C R O I S E M E N T D ' I N S E C T E S N O R M A U XA V E C D E S I N S E C T E S ISSUS D E L A R V E S I R R A D I E E S

D ose(krad )

N ature des c ro isem en ts

P ou rcen tagedes

fe m e lle sfé co n d é e s

N om b re d 'a c co u p le m e n ts

par m â le

N om b red 'œ ufs

parfe m e l le

ceu fs é c lo s

(% ) ■


( j )

M â le F e m e lle M â le F e m e lle

0 N N 90 2 .1 5 6 4 7 ,9 9 0 ,1 1 3 ,7 1 2 ,4

3 I . N 80 1 ,3 0 6 4 5 ,6 6 0 ,5 1 4 ,5 1 6 ,3

3 N I 80 1 ,3 5 1 8 7 ,8 3 3 ,9 1 7 ,0 1 3 ,3

5 I N 30 0 ,4 5 . 5 8 4 ,3 2 4 ,1 1 1 ,7 1 5 ,9

5 N I 40 0 ,5 0 1 1 0 ,5 1 3 ,6 1 7 ,8 8 ,9

N = non ir ra d ié , I = irra d ié .

SM

-102/12 115

116 ANWAR

4 . 3 . 2 . S tér i l i té obtenue

Une d o s e de 5 krad ap p liquée à d e s n ym phes de 1 jo u r donne d e s f e m e l l e s à fé co n d ité rédu ite et f e r t i l i t é nulle ; l e s m â le s m on tren t une fe r t i l i t é d 'e n v ir o n 42%.

Sur d es n ym phes de 3 jo u r s la d o s e de 10 krad donne une fé co n d i té et une f e r t i l i t é n u lles p ou r l e s deux s e x e s . D ans l e c a s des m â le s i r r a d ié s à 10 krad , i l n 'y a d 'a i l l e u r s pas d 'a c c o u p le m e n t s .

D e s d o s e s de 15 à 70 krad ont été ap p liq uées à d e s n ym ph es de 7 jo u r s ( f e m e l le s ) et de 8 jo u r s (m â le s ) . L a fé c o n d i té d e s f e m e l l e s .diminue av e c la d o s e m a is r e s te e n c o r e de 25% p a r r a p p o r t au té m o in à la d o s e de7 0 krad ( f ig . 1). L a fé co n d i té d es f e m e l l e s n o r m a le s a c c o u p lé e s à d e s m â le s i r r a d ié s ne p r é se n te une l é g è r e d im inution que pou r 70 krad.

L a fe r t i l i t é des f e m e l l e s i r r a d ié e s d é c r o î t t r è s v ite en fo n ct io n de la d o s e d ' i r r a d ia t io n pou r d e v e n ir nulle à 30 krad (f ig . 2). L a f e r t i l i t é des m â le s d é c r o î t p lus p r o g r e s s iv e m e n t , et dev ient p rat iqu em en t nulle à p a r t i r de 50 krad.

R a su lo v [1] indique a v o ir obtenu la s t é r i l i t é de m â le s i s s u s de n ym phes i r r a d i é e s à 9 et 11 krad , et d e s f e m e l l e s à 5 krad . C e s r é su lta ts sont é v id e m m e n t t r è s d i f fé re n ts d e s n ô t r e s . N os ré su lta ts p a ra is s e n t pourtant p lus c o n f o r m e s à ce qui a été obtenu su r d 'a u tr e s L é p id o p tè r e s de ta i l le c o m p a r a b le , par e x e m p le O str in ia n ub ila lis Ilb . [6], P e c t in o p h o ra g oss .yp ie l la [7] et su r d e s L é p id o p tè r e s de plus petite ta i l l e , p a r e x e m p le C a r p o c a p s a p o m o n e l la L . [8].

D e s o b s e rv a t io n s p r é c i s e s su r la v igu eu r se x u e l le ont été e f fe c t u é e s en utilisant c o m m e tes t le n o m b r e d 'a c c o u p le m e n t s s u c c e s s i f s . L e s i r ra d ia t io n s ju sq u 'à 50 krad ne dim inuent pas la v igu eu r s e x u e l le des m â le s ; la r é ce p t iv i té d es f e m e l l e s i r r a d i é e s p a ra ît s u p é r ie u r e à c e l l e d e s t é m o in s .

4 . 3 . 3 . A c t i o n r é s id u e l le de d o s e s s u b s té r i l i sa n te s

L a d o s e de 45 krad appliquée à des n ym ph es m â le s de 8 jo u r s l a i s s e une fe r t i l i t é de 10% ch e z le s m â le s é c l o s . Il est in téressan t d 'e x a m in e r l 'é v o lu t io n et la d e s ce n d a n ce de c e s œ u fs f e r t i l e s .

L e tableau V m on tre que l e s la r v e s d escen dan t de m â le s i r r a d ié s (à l 'é ta t de nym phe) à 45 krad présen ten t une fo r te m o r ta l i t é du p r e m ie r et d e u x iè m e stade, à la qu e l le s 'a jo u te une m o r ta l i té au m o m e n t de la n y m p h o s e . Seulem ent 10% de c e s la r v e s donneront d e s ad u ltes . C e s adultes e u x - m ê m e s , qu i constituent une g én éra t ion F l , m on tren t une fe r t i l i t é nulle (tableau VI).

Il y a donc là une act ion r é s id u e l le t r è s re m a r q u a b le de l ' i r r a d ia t io n .Si l ' o n r e g a r d e le résu lta t d 'e f f i c a c i t é to ta le , on voit que la d o s e de 45 krad appliquée à des m â le s p ro v o q u e une extinction c o m p lè te de la d e s ce n d a n c e m a lg r é une fe r t i l i t é de 10% d e s p r e m ie r s œ u fs obtenus. Il se r a i t in té re ssa n t de r e p r e n d r e l e s m ê m e s o b s e rv a t io n s aux d o s e s in fé r i e u r e s .

Une action identique a été s ign a lée sur d 'a u tr e s L é p id o p tè r e s . P a r e x e m p le С . p o m o n e lla [81 et P a r a m y e l o i s t ra n s i te l la [91.

S M - 102/12 117

Fécondité 6 0 0

5 0 0

4 0 0

3 0 0 ,

200

100

15 2 0 3 0 4 0 5 0 6 0 . 7 0Dose (k rad )

FIG. 1. Fécond ité résultant de croisem ents insectes normaux x insectes issus d e nym phes d e 7 j (fem elles ) et 8 j (m âles) irradiées aux doses de 15 à 70 krad (la fécon d ité des tém oins non irradiés était de 598 œufs).

% fertilité

Dose (krad )

FIG. 2. Fertilité résultant d e croisem ents insectes normaux x insectes issus de nym phes de 7 j (fem elles) et 8 j (m âles) irradiées aux doses de 15 à 70 krad (la fertilité des tém oins non irradiés était d e 9Г7<>).

4 . 4 . I r ra d ia t ion d e s adultes

L ’ ir ra d ia t io n d1 adultes dans la jo u r n é e suivant le u r é c l o s i o n a donné d e s r é su lta ts tout à fa it c o m p a r a b le s à ce u x des i r ra d ia t io n s de nym phes juste avant l ' é c l o s io n . L a d o s e de 40 krad p ro v o q u e la s t é r i l i t é des f e m e l le s et la i s s e une fe r t i l i t é de 11% c h e z l e s m â le s . L a d o s e de 50 krad est to ta lem ent s t é r i l is a n te p ou r le s deux s e x e s .

T A B L E A U V . D E V E L O P P E M E N T D E L A D E S C E N D A N C E D E N Y M P H E S I R R A D I E E S D E S p o d o p t e r a e x ig u a H b .( M A L E I R R A D I E X F E M E L L E NON IR R A D I E E ) A L A D OSE S U B S T E R I L I S A N T E

N ature des c ro isem en tsCeufs

aé c lo s

m

Larves m ortes aux p rem ier et

d e u x ië m e stades

№

Larves m ortes

au dernier stade

m

N ym ph esm ortes

m

Larves donnant des adu ltes F I

№ )

T o ta l M â le F e m e lle

M â le non irra d ié x

f e m e l le non irra d iée 9 1 ,0 4 .0 5 ,0 2 , 0 8 9 ,0 4 7 ,6 5 2 ,4

M â le irra d ié x

f e m e l l e n on irra d iée 9 ,7 7 0 ,5 1 1 , 0 8 ,5 1 0 , 0 5 9 ,1 4 0 ,9

a400 larves F I par c o m b in a is o n g a rd ées pour l 'é l e v a g e .

118 A

NW

AR

T A B L E A U V I . N O M B R E D ' A C C O U P L E M E N T S , F E C O N D I T E , F E R T I L I T E E T L O N G E V I T ED ' A D U L T E S F I I SSU S DU C R O I S E M E N T M A L E I R R A D I E A 4 5 k r a d X F E M E L L E NON I R R A D I E E

N atu re des c ro isem en ts

P ou rcen tagedes

fe m e lle sfé co n d é e s

N om b red 'a c co u p le m e n ts

parm â le

N om b re

d 'oeu fspar

f e m e l le

ceu fsé c lo s


( j)

№ )M â le F e m e lle

M â le n on irra d ié x

f e m e l l e non irra d ié e 1 0 0 2 ,6 0 6 1 1 ,7 8 7 ,5 1 0 ,3 ■ 9 ,6

M â le F I x

fe m e l l e n on irra d ié e 3 8 , 9 a 0 ,5 4 3 7 5 .6 0 , 0 Î . 8 1 3 ,8

M â le non irra d ié x

fe m e l l e F I 6 6 , I a 0 ,8 9 2 5 6 ,7 0 , 0 12 , 7 7 ,6

18 c a g e s par c o m b in a is o n .

SM

-102/12 119

120 ANWAR

R E M E R C I E M K N T S

L 'a u te u r r e m e r c i e M . F é r o n d 'a v o ir fa c i l i t é la r é a l i s a t io n et la r é d a c t io n de c e trav a i l dans son la b o r a to i r e . Il r e m e r c i e le G o u v e r n e m ent pakistanais et le G ou v ern em en t f r a n ç a is d 'a v o i r p e r m is l ' o r g a n i sation et le bon d é ro u le m e n t de ce stage .

R E F E R E N C E S

[ 1 ] RASULOV, F .K . , K hlopkovodstvo 7 (1963) 41 -42 .[ 2 ] ВОТ, I . , J. ent. S oc. sth. Afr. 29 (1967) 206 -09 .[ 3 ] SHOREY, H .H . , HALE, R .L . , J. e con . Em. 58 (1965) 5 2 2 -2 4 .[ 4 ] PROVERBS, M .D . , NEWTON, J. R . . Can. J. Z o o l . 4 0 (1 9 6 2 ) 401 -20 .[ 5 ] ELBADRY, E ., Ann. ent. S oc. A m . 58 (1965) 2 0 6 -0 9 .[ 6 ] ANWAR, M . , « Stérilisation de la pyrale du mais ( Ostrinia nubilalis H b .) par l'irradia tion aux

rayons g a m m a » , ces com ptes rendus, S M -102 /15 .[ 7 ] OUYE, M .T . et a l . , J. e con . Ent. 57 (1964) 38 7 -9 0 .[ 8 ] PROVERBS, M .D . , NEWTON, J. R . , Can. Ent. 94 (1962) 116 2-7 0 .[ 9 ] HUSSEINY, M .M ., MADSEN, H. F . , H ilgaidia 36 (1964) 113 -37 .

D I S C U S S I O N

C . F . CURTIS: I w as in te re s te d to note the high d o s e s o f ion iz ing rad ia tion r e q u ire d to s t e r i l i z e these L e p id o p te r a , in c o m p a r is o n with the d o s e s re q u ire d f o r D ip tera . Could you su ggest any explanation f o r th is g rea t d i f fe r e n c e ?

M . AN W AR: Nothing is r e a l ly known about why s o m e in s e c t s p e c i e s a r e m o r e r a d io s e n s i t iv e than o th e rs . P r o b a b ly the s i z e o f the. o r g a n is m , its m e t a b o l i s m , and the n u m ber and s i z e o f c h r o m o s o m e s a r e all c o n t r i buting fa c t o r s .

H. ER D M AN : Can S p odop tera ex igua be m a s s - r e a r e d ? With what n u m b e rs w e re you w orking?

M . A N W A R : I did not need many in s e c t s f o r m y p r e l im in a r y s tud ies .I a lw ays r e a r e d them in g ro u p s o f about 100 la r v a e p e r r e a r in g c a g e , on an a r t i f i c i a l m e d iu m , and u sed to obtain between 80 and 90% adults, which sh ow s that it is quite fe a s ib le to m a s s - r e a r them .

M . COHEN: S ince S . ex igua s e e m s to be e a s i ly r e a r e d , do you think it would be f e a s ib le to apply the ir ra d ia t ion technique to c o n t r o l o f th is in s e c t in the f ie ld and in v iew o f yo u r la b o r a to r y r e su lt s would s t e r i l i za tion o f f e m a le s be p r e fe r a b le to s t e r i l iza t io n o f m a le s?

M . A N W AR: W e did e x p e r im e n ts under la b o r a to r y cond it ion s with popu la tion s o f (a) pupae ir ra d ia te d at a late stage in th e ir d e v e lo p m e n t with a d o s e of 50 krad and (b) n o rm a l pupae, m ix e d in the fo l low in g p r o p o r t io n s : 1 n o rm a l p a ir f o r e v e r y 5 s t e r i l e p a ir s ; 1 n o rm a l p a ir f o r e v e r y 5 s t e r i le m a le s ; 1 n o rm a l pa ir f o r e v e r y 5 s t e r i le f e m a le s . F r o m the r e s u l t s o f th ese e x p e r im e n ts we found v e r y g rea t v a r ia t io n s in the v iab i l i ty o f e g gs la id by the f e m a le s . T h is show ed that in trodu ct ion o f s t e r i l e m a le s a p p re c ia b ly r e d u c e d egg v iab i l i ty , but an even g r e a te r d e c r e a s e was noted on s im u ltaneou s in trod u ct ion o f s t e r i le m a le s and fe m a le s ; it would thus a p pear that s t e r i le f e m a le s cou ld be used in the sa m e way as the m a le s to in c r e a s e the c h a n c e s o f s u c c e s s in an e r a d ica t io n p r o g r a m m e .

S M -102/12 121

R . B L U Z A T : D o you know the n um ber o f c h r o m o s o m e s S. ex igua has and have you c o m p a r e d th is with the n u m ber o f c h r o m o s o m e s p o s s e s s e d by c r e a t u r e s exhib iting a rad iation r e s is ta n c e o f the sa m e o r d e r ?

M . A N W A R : I 'm a fra id the an sw er on both s c o r e s is 'n o 1.J . E . SIMON: W as th e re any can n ib a l ism am o n g st S p odop tera la rv a e

during m a s s r e a r in g ?M. A N W A R : I have n e v e r o b s e r v e d any.J . E . SIMON: W e r e the la rv a e r e a r e d tog e th e r o r s e p a ra te ly ?M .A N W A R : T h e y w e re a lw ays r e a r e d in g ro u p s .

SENSIBILITE DES DIVERS STADES DE D E V E L O P P E M E N T DE Sitophilus zeam ais MOTS (S. oryzæ L. )A U X RADIATIONS IONISANTESEtude des stades endogés par radiographie etenregistrem ent actographique

P. PESSON ET G. K. GIRISH*

IN ST IT U T NATIONAL AGRONOMIQUE,PARIS, FRANCE

( P r é s e n t é p a r P . P é l e g r i n )


RADIOSENSITIVITY OF Sitophilus zeam ais MOTS (S . oryzae L. ) A T VARIOUS STAGES OF DEVELOPMENT: A STUDY OF THE LARVAL STAGES BY MEANS OF RADIOGRAPHY AND 'ACTOGRAPH IC' RECOR- DING. The paper describes an orig inal m ethod o f study, based on the em ploym ent o f an 'actograph ' w hich records the vita lity o f the larval stages and makes it possible to study the larva popu lation inside grains (actograph for e le ctro -a co u s tic detection deve lop ed by the INRA A coustic Physiology Laboratory, Jouy-en-Josas). The contam in ation rate and the death rate o f the larvae are observed by radiography.The e ffects o f irradiation at doses o f *2000, 4000, 8000 and 16000 rad are studied in the egg stage, on larvae aged 5 -7 days, 1 3 -15 days and 19-21 days and on adults, whose fertility is measured 0 -5 days, 5 -1 0 days and 10-15 days after irradiation. The e ffects o f irradiation on the second generation bred from the various stages irradiated are noted.

SENSIBILITE DES DIVERS STADES DE DEVELOPPEMENT DE Sitophilus zeam ais MOTS (S . oryzæ L. ) AUX.RADIATIONS IONISANTES: ETUDE DES STADES ENDOGES PAR RADIOGRAPHIE ET ENREGISTREMENT ACTOGRAPHIQUE. Les auteurs présentent une m éthode d 'étu de orig ina le , fon d ée sur l 'e m p lo i d 'un appareillage actographique, qui enregistre et traduit la v ita lité des stades endogés et perm et d 'apprécier la population larvaire à l 'in térieur des grains (actogtaphe à détection é lectro -a cou stiq u e m is au point par le Laboratoire de ph ysio log ie acoustique de l'IN RA, Jouy-en-Josas). Le contrôle du taux de con tam ination et de la m orta lité larvaire se fa it par radiographie. Les auteursont étudié les effets de l'irradiation (doses de 2000, 4000, 8000 et 16 000 rad)sur le stade œ uf, sur les larves âgées respectivem ent de 5 à 7 jours, 13 à 15 jours, 19 à 21 jours et sur les adultes (fécon d ité 0 à 5 jours, 5 à 10 jours, 10 à 15 jours après l'irradia tion ); ils ont éga lem ent fa it des observations sur les conséquences de l 'irradia tion sur la seconde génération issue des divers stades irradiés.

D iv e r s auteurs [1 -4 ] ont d é jà u t i l isé d e s tech n iques de d é te c t io n a c o u s tique ou le s o b s e r v a t io n s r a d io g ra p h iq u e s pour é tu d ier le dév e lo p p e m e n t à l ' in té r ie u r d e s g ra in s de c é r é a l e s de d iv e r s in s e c te s d e s d e n r é e s . Nous u t i l iso n s con jo in te m e n t c e s deux tech n iques en vue de p r é c i s e r la s e n s i b i l ité aux rad ia t ion s io n isan tes d e s stades e n d ogés du ch a ra n ço n des g ra in s .

L 'e n r e g i s t r e m e n t de l 'a c t iv i t é de c e s d iv e r s s tad es , avant et a p rè s i r ra d ia t io n , e s t obtenu g r â c e à un d é te c te u r é l e c t r o - a c o u s t iq u e a s s o c i é à un a m p l i f i ca te u r t r è s puissant, a p p a r e i l m is au point par R . G . B u s n e l1.

* Boursier du G ouvernem ent indien auprès de l'Institut national agronom ique (Laboratoire de z o o lo g ie ) , Paris.

1 Laboratoire de p h ysio log ie acoustique, Institut national de la recherch e agronom ique, 78 Jou y-en - Josas, France.

124 PESSON et GIRISH

Nous avons l im ité ce tte étude à S itophilus z e a m a is M ots ; L 'e s s e n t ie l de nos ré su lta ts est en a c c o r d av e c le s travaux a n té r ie u re m e n t publiés par d iv e r s c h e r c h e u r s et par n o u s -m ê m e s [5 -8 ] .

1. M A T E R IE L E T M ETH ODE

L 'é le v a g e de S. z e a m a is a été r é a l i s é su r d e s g ra in s de b lé , dans une étuve, av e c une hum idité re la t iv e de 7 5% ± 5% et une te m p é ra tu re con s tan te de 21° С ± Io С .

Cinquante g ra in s de blé , p ré a la b le m e n t bien con d it ion n és (HR et te m p é ra tu re in d iquées c i - d e s s u s ) sont a l o r s m is dans d e s petits tubes de m a t iè r e p lastique , en p r é s e n c e de 10 co u p le s de c h a ra n ç o n s , â g é s de 1 S 3 j o u r s . P o u r chaque e x p é r ie n c e on p ré p a re c inq ré p é t it io n s d 'un t e l éch a n t i l lon . L es g ra in s a ins i s o u m is à l 'o v ip o s i t io n furent c o n s e r v é s dans le s con d it ion s o p t im a le s p r é c i t é e s , en assu ra n t en p a r t i cu l ie r une h um idité re la t iv e fa v o ra b le en stockant le s tubes de ponte dans un e x s i c c a - te u r dont l 'hu m id ité était maintenue constan te par une so lut ion de p o ta ss e .Le te m p s de ponte des ad ultes était l im ité à 24 h ou 48 h, afin d 'a v o i r en su ite une population la r v a i r e d 'â g e u n i fo r m e . Con n aissant bien, dans c e s co n d it ion s , la d u ré e re la t ive des d iv e r s stades (œ u fs , l a r v e s et n ym p h e s ) , i l nous était f a c i le de d i s p o s e r au m om en t voulu de lo t s h o m o g è n e s pour le s i r r a d ie r .

La s o u r c e d ' i r r a d ia t io n u t i l i sé e était un ir ra d ia te u r y de 3000 C i de 60Co, dans leq ue l la s o u r c e était d is p o s é e se lo n le s g é n é r a t r i c e s d 'un c y l in d r e . La te m p é ra tu re dans l 'e n c e in te d ' i r r a d ia t io n était de 27° С ± 0 , 5° С. P o u r le s d iv e r s stades e x p é r im e n té s , quatre d o s e s d i f fé re n te s ont été u t i l i s é e s : 2000, 4000, 8000 et 16 000 rad, a d m in is t ré e s r e s p e c t iv e m e n t en des te m p s de 1 min 43 s e c , 3 min 26 .sec, Ci min 52 s e c et 13 min 44 s e c , en r a p p o r t a v ec l ' in ten s ité de rayonn em en t de la s o u r c e , qui est de 70 k ra d /h .

L 'a c t iv i t é des la r v e s dans les g ra in s était e n r e g is t r é e pour l 'e n s e m b le d e s 50 g ra in s de chaque lot, d 'une part avant l ' i r ra d ia t io n , d 'a u tre part à d e s in te r v a l le s de tem ps r é g u l i e r s a p rè s l ' i r ra d ia t io n . La date d 'a p p a r i t ion d e s adultes et leu r n o m b r e furent n o tés . L e s adultes is s u s des lots i r r a d ié s furent a lo r s m is à nouveau dans des con d it ion s fa v o r a b le s à la ponte sur de nouveaux g ra in s pendant 22 j o u r s . L 'a c t iv i té de leu r p ro gé n itu re fut évaluée par ac to g ra p h ie et le n o m b re d 'ad u ltes de d eu x iè m e g én éra t ion is su s de c e s g ra in s fut c o m p té . L e s lots de g ra in s i r r a d ié s ou ceux servant au d é v e lo p p e m e n t de la deu x ièm e g énérat ion furent, en fin d 'e x p é r ie n c e , e x a m in é s par ra d io g ra p h ie , pour a p p r é c ie r le taux de m o r ta l it é des stades e n d o gé s ou le p ou rcen tage de g ra in s attaqués.

2. R E S U L T A T S E T DISCUSSION

2 . 1 . Sen s ib il ité des œ u fs

La p r o c é d u r e e x p é r im e n ta le fut la suivante: 10 co u p le s de Sitophilus z e a m a is (âgés de 3 jo u r s ) furent m is , pour la ponte, en p r é s e n c e de 50 g ra in s de blé le 21 m ai 1966 (5 lo ts ) ; le s ad ultes furent r e t i r é s du blé le 23 mai et quatre lo ts furent i r r a d ié s à 2000, 4000, 8000 et 16 000 rad (le 23 m ai ég a le m e n t ) , l 'â g e des œ u fs au m o m e n t de l ' i r ra d ia t io n étant de 2 jo u r s

S M - 102/14 125

* Lf jí З/Ь/ 6 b. oí»*» О < ? -

T E M O IN 2 0 0 0 г . 4 О 0 Or . 8 0 0 0 г . 1 6 0 0 0 г

'I Awltiirtu

j d jÀJi:J .U É L _____ ............ ....................... .... ....... ..... ■_ ■ "

FIG. 1. A ctographies d e lots de 50 grains de b lé ayant reçu la ponte d e 10 couples d e S. zem ais pendant 48 h. Irradiation des grains a v ec les œufs inclus, ces derniers étant au plus âgés d e 2 jours. Actographies enregistrées 16, 21, 24 et 28 jours après l'irradia tion .

au m a x im u m ; le s a c t iv i t é s la r v a i r e s dans chacun des lo ts i r r a d ié s et dans le lot tém oin furent e n r e g i s t r é e s le s 8, 13, W i et 20 juin ( le s la r v e s étant donc â g é e s de 16 à 30 j o u r s ) .

La f igure 1, qui rep ro d u it l e s p o r t io n s des a c t o g r a m m e s obtenus dans c e s c ond it ion s , m on tre qu 'aucun d es lots i r r a d ié s n 'h é b e r g e a it de la rv e s a c t iv e s . Le lot tém oin , par c o n t re , m ontra it une ac t iv ité la r v a ir e qui a l la it en s 'a m p li f ia n t a v e c l 'â g e . Une d o s e d ' i r r a d ia t io n de 2000 rad suffit donc pour e m p ê c h e r le d év e lop p em en t de la ponte de Sitophilus z e a m a i s .

L 'e x a m e n d es r a d io g r a p h ie s r é v è le le s p iq û res de ponte, av e c un petit n o m b r e de débuts de g a le r ie s a l im e n ta ir e s , té m o ig n a g e s d 'é c l o s i o n s , „ dans le s lo ts i r r a d ié s à 2000, 4000 et 8000 rad . A 16 000 rad aucune g a le r ie n 'e s t d é c e la b le , i l n 'y a donc eu aucune é c l o s i o n des oeufs.

2 . 2 . Sens ib il ité d e s la r v e s

a) L a r v e s â g é e s de 5 k 7 jo u r s

La p r o c é d u r e e x p é r im e n ta le fut la su ivante; ponte ( c o m m e c i - d e s s u s ) du 9 au 11 m a i , incubation , puis i r ra d ia t io n le 23 mai; a c to g ra p h ie avant ir ra d ia t io n le 23 m ai, a p r è s ir ra d ia t io n du 24 m ai au 10 ju in ; contam ination pendant 22 jo u r s de nouveau blé par le s adultes i s s u s du lot tém oin et des lo t s i r r a d ié s .à 2000 et 4000 rad; a c to g ra p h ie des la r v e s de d e u x ièm e g én éra t ion du 8 au 13 août.

La f igure 2 donne l e s résu lta ts de ce tte e x p é r ie n c e . On n otera que l 'a c to g ra p h ie de c o n t r ô le , sur le s d iv e r s lots avant l ' i r r a d ia t io n , r é v è le une ac t iv ité fa ib le en r a is o n du t r è s jeu n e âge des la r v e s . Cette a ct iv ité va s ' a c c r o î t r e r é g u l iè r e m e n t a v e c l 'â g e dans le lot té m o in et le s lo ts i r r a d i é s à 2000 et 4000 rad. M ais pour le s lo ts i r r a d ié s à 8 0 0 0 e t l 6 000 rad, i l e s t évident que le d év e lop p em en t la rv a ir e est d é f in it ivem ent stoppé v e r s l 'â g e de 20 à 22 j o u r s . Il suffit donc d 'une do se de 8000 rad pour tuer dans un d é la i m a x im a l de 15 j o u r s d e s la r v e s â g é e s de ô à 7 j o u r s .

126

TEMOIN

PESSON et GIRISH

20 0 0 r. 4 0 0 0 r . 8 0 Ó Or. 1 6 0 0 0 г.

FIG. 2. A ctographies d e populations larvaires dans 50 grains de b lé , contam inés par 10 couples de Calandres, pendant 48 h. Irradiation des larves à l 'â g e d e 5 à 7 jours. A , actographies enregistrées avant l 'irra d ia tion ; B, enregistrem ents effectués 1, 3, 7, 11, 18 et 21 jours après l 'irra d ia tion ;C, actogram m es d e la deu xièm e génération larvaire provenant des adultes éc los du lo t tém oin et des lots irradiés à 2000 et 4000 rads.

La suite de l 'e x p é r ie n c e ( f ig .2 C ) portant su r le s adultes i s s u s des lo t s i r r a d i é s à 2000 et 4000 rad m ontre que c e s d e r n ie r s sont fé c o n d s et donnent une progén itu re la r v a i r e act ive .

A p r è s ra d iog rap h ie d e s d iv e r s lo ts en fin d 'e x p é r ie n c e , on peut d é n o m b r e r le s g ra in s attaqués, c e qui d ev ra it nous p e r m e tt re d 'é v a lu e r la m o r ta l i t é la r v a ir e , en tenant com p te du n o m b r e d 'ad u ltes é c l o s . T o u t e f o i s , nous con sta ton s que, dans le lot tém oin , pour 44 g ra in s attaqués (su r 50), on n 'a que 21 adultes, c e qui r e p ré s e n te dé jà une m orta l ité i m p ortante . E l le e st due en pa rt ie au fait que le m ê m e gra in h é b e rg e souvent deux la r v e s , p a r fo is t r o i s . En ou tre , nous avon s d é c e lé quelques a c a r ie n s p r é d a te u r s (P e d ic u lo id e s v e n t r i c o s u s ) . Il se r a i t sans doute p o s s ib le de

S M - 102/14 127

d é c o m p t e r le s m o r ta l i t é s dues à c e préd ateu r , m a is i l nous parâ ît s u f fisant de c o m p a r e r le s r a d io g r a p h ie s , qui m ontrent m a n ifestem en t que dans le s lo ts i r r a d ié s à 4000, 8000 et 16 000 rad le s g a le r ie s la r v a i r e s sont m o in s étendues que dans le lot tém oin , s igne d 'une m o r ta l i té d 'autant p lus p r é c o c e que la d o s e r e ç u e est plus f o r t e . Cette m o r ta l i té es t s i m plem ent e s t im é e de fa çon v a la b le en indiquant le n o m b r e d 'ad u ltes é c l o s en r e g a rd du n o m b r e de g ra in s attaqués. E l le est m a n i fes tem en t en rap p ort a v e c l ' i r r a d ia t io n d è s une do se de 4000 rad.

T é m o in 2000 4000 8000 16 000 radG ra in s attaqués 44 35 35 41 40A dultes é c l o s 21. 20 2 0 0

De m ê m e , le s r a d io g r a p h ie s de la d e u x ièm e g én éra t ion la rv a ir e m on tren t que le s adultes i s s u s de la r v e s i r r a d ié e s d e m eu ren t fé co n d s m ê m e pour le lot i r r a d ié â 4000 rad . M ais le s d on nées de l ' e x p é r ie n c e , oïi l 'o n c o m p a r e la d e s ce n d a n c e d 'un n o m b r e v a r ia b le de c o u p le s , ne p e r mettent pas de p r é c i s e r s i ce tte fé con d ité a subi une dim inution ch e z les ad ultes i s s u s d e s lo t s i r r a d ié s . T o u te fo is le s c h i f f r e s obtenus sou lignent su f f isam m en t la fe r t i l i t é de c e s in s e c te s :

T é m o in 2000 4000 radG ra in s attaqués 49 49 40Adultes é c l o s 52 46 30

L e s dates d 'é m e r g e n c e d e s p r e m ie r s adultes dans le s lo ts i r r a d ié s c o n c o r d e n t av e c c e l l e s d e s lo ts té m o in s , m a is le s d e r n iè r e s é m e r g e n c e s sont p lus ta r d iv e s . Dans la p r e m iè r e généra t ion , on r e lè v e le s dates du15 au 22 ju in pou r le lot tém oin , du 15 au 29 ju in pour le lot i r r a d ié à 2000 rad . Dans la d e u x iè m e g én éra t ion , on note r e s p e c t iv e m e n t : du 18 ju i l l e t au 9 août pou r le lot tém oin , du 18 ju i l l e t au 22 août pour le lot à 2000 rad, du 18 ju i l l e t au 30 août p ou r le lot à 4000 rad . Un r e ta rd du d év e lop p em en t est donc plus m a n ifeste dans la d e u x ièm e gén éra t ion .

b) L a r v e s â g é e s de 13 à 15 jo u r s

La p r o c é d u r e fut la m ê m e que p r é cé d e m m e n t : ponte du 9 au 11 mai; a c to g ra p h ie avant i r ra d ia t io n le s 25 et 31 m ai; ir ra d ia t io n le 31 mai ( la r v e s â g é e s de 13 à 15 j o u r s ) ; a c to g ra p h ie a p rè s i r ra d ia t io n du 1er au 8 ju in ; contam ination de nouveau b lé p a r le s adultes i s s u s d e s d iv e r s lo t s pendant 22 j o u r s ; a c to g r a p h ie d e s la r v e s de d e u x iè m e g é n é ra t io n du8 au 13 ju i l l e t . -

L 'a c t iv i té d e s la r v e s avant i r ra d ia t io n (f ig . 3) e s t b ea u cou p plus grande que dans l ' e x p é r i e n c e p r é cé d e n te , en r a is o n de le u r d é v e lo p p e m e n t plus a v a n cé . L 'a c to g r a p h ie a p rè s i r ra d ia t io n r é v è le que la d o s e de 16 000 rad stoppe p re sq u e im m é d ia te m e n t l 'a c t iv i t é la r v a i r e . P a r c o n t r e , le d é v e lo p p em ent s 'e f f e c t u e ju s q u 'à l ' é c l o s i o n d ' im a g o s dans le s lo t s i r r a d ié s à 2000, 4000 et 8000 rad . T o u te fo is , la c o m p a r a is o n d e s a c t o g r a m m e s r é v è le une ac t iv ité un peu m oin s fo r te que c h e z le s té m o in s , c o n s é q u e n c e d 'une ce r ta in e m o r ta l i t é la r v a ir e et d 'une p er te de v ita l i té . M ais on ne d é c è le un r e ta r d dans l ' é c l o s i o n d e s im a g o s que dans le lot i r r a d ié à 2000 rad pour leq ue l l e s ap p a r it io n s d ' im a g o s s 'é ta le n t du 15 ju in au

128 PESSON et G IRISH

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FIG. 3 . Irradiation d e larves âgées d e 13 à 15 jouis. A , actographie des populations larvaires avant irradiation; B, enregistrements e ffectués 1, 2 , 4 et 8 jours après l 'irra d ia tion ; C, actogram m es de la d eu xièm e génération larvaire engendrée par les adultes issus du lot tém oin et des lots irradiés à 2 0 0 0 , 4000 et 8000 rads. Noter que, dans ces derniers lots, l 'a c t iv ité des larves est moins forte: retard du développem ent et d e l 'ém erg en ce des adultes, associé à une m orta lité plus forte.

7 ju i l l e t , a l o r s que pour tous le s au tres lo ts ( tém oin s et i r r a d ié s ) ce tte p é r io d e va du 15 au 22 ju in .

L 'e x a m e n d e s r a d io g r a p h ie s a p rè s la fin d e s é c lo s io n s im a g in a le s r é v è le le s p r o p o r t io n s su ivan tes entre le n o m b r e de g ra in s attaqués et le n o m b r e d ' im a g o s s o r t i s :

T é m o in 2000 4000 8000 16 000 radG ra in s attaqués 26 34 36 30 28Adultes é c l o s 26 16 6 2 0

C e s r a d io g r a p h ie s m on tren t que, dans le s lo ts i r r a d ié s , p lu s ie u r s im a g o s sont m o r t s à l ' in t é r ie u r des g ra in s . En p a r t i cu l ie r , on peut en d é n o m b r e r huit dans le lot à 2000 rad. Ce fait p o u rra it t ra d u ire une

S M - 102/14 129

c e r t a in e d é f i c i e n c e d e s in s e c t e s et e x p l iq u e r par a i l le u r s le r e ta r d des é c l o s i o n s . P a r c o n t r e , dans le s lo t s i r r a d ié s à 4000 et 8000 rad , on d é c è le surtout une m o r ta l i t é l a r v a i r e , le s g a l e r i e s étant in a c h e v é e s et m o in s d é v e lo p p é e s , surtout dans le lot à 8000 rad .

L e s ad ultes i s s u s du lot t é m o in et des lo t s i r r a d ié s furent t r a n s f é r é s su r du g ra in sain , pendant 22 j o u r s . La s e co n d e g é n é ra t io n d e s d iv e r s lo t s donne r e s p e c t iv e m e n t 47 (lot té m o in ) , 45 (lot à 2000 rad ) , 27 (lot à 4000 rad) et 20 im a g o s (lot à 8000 ra d ) . L e s a c to g r a p h ie s d e s popu lations l a r v a i r e s c o r r e s p o n d a n te s r é v è le n t une ac t iv ité p lus ou m o in s fo r te , qui té m o ig n e d 'un r e ta r d dans l 'é v o lu t io n d e s la r v e s i s s u e s d e s lo t s i r r a d ié s . En e ffe t , a l o r s que l e s é c l o s i o n s d ' im a g o s dans le lot t é m o in et dans le lo t à 2000 rad s 'é ta la ie n t du 18 ju i l l e t au 22 août, e l l e s ont été n o té e s du 18 ju i l l e t au 31 août p ou r le lo t à 4000 rad, et du 9 au 30 août pour ce lu i à 8000 rad.

En ou tre , la r a d io g r a p h ie d e s popu lations la r v a i r e s de d e u x iè m e g é n é ra t io n r é v è le une m o r ta l i t é c r o i s s a n t e a v e c le s d o s e s d ' i r r a d ia t io n r e ç u e s par la g é n é ra t io n la r v a i r e p a ren ta le . On note en e f fe t :

T é m o in 2000 4000 8000 radG ra in s attaqués 48 50 39 42A du ltes é c l o s 47 45 27 20

On peut donc c o n c lu r e que le s d o s e s d ' i r r a d ia t io n in te r m é d ia ir e s en tre 8000 et 16 000 rad doivent en tra în e r 100% de m o r ta l i t é dans une population de la r v e s â g é e s de 13 à 15 j o u r s . L e s ad ultes i s s u s d e s la r v e s i r r a d i é e s à 2000, 4000 et 8000 rad d e m e u re n t fé c o n d s , m a is l eu r d e s c e n da nce subit un r e ta r d im p ortan t dans son d é v e lo p p e m e n t et p ré se n te une m o r ta l i té p lus grande que l e s té m o in s .

c) L a r v e s â g é e s de 19 à 21 jo u r s

L a p r o c é d u r e e x p é r im e n ta le fut la m ê m e que dans le s e x p é r i e n c e s p r é c é d e n te s . La f ig u re 4A m o n tre que l 'a c t iv i t é la r v a i r e avant i r r a d i a t ion était tout à fa it c o m p a r a b le dans le s d iv e r s lo t s . A p r è s ir ra d ia t io n ( f ig .4 B ) , ce tte a c t iv ité r e s t e a p p a re m m e n t n o r m a le dans l e s d iv e r s lo ts , m a is aucun adulte n 'é c l o t du lot i r r a d ié à 16 000 rad . L e n o m b r e d 'adu ltes i s s u s d e s au tre s lo t s e st r e s p e c t iv e m e n t de 28 (lot tém o in ) , 24 (2000 rad), 21 (4000 rad) et 13 (8000 rad ) . M ais nous n 'a vo n s pas obtenu pour c e s lo t s de docu m e n ts r a d io g ra p h iq u e s c o r r e c t s , par suite d 'un incident t e c h nique, et n 'a vo n s pu, en c o n s é q u e n c e , r a p p o r te r le n o m b r e d 'é c l o s i o n s au n o m b r e de g ra in s attaqués.

L e s dates d 'é m e r g e n c e d e s im a g o s s 'é ta le n t du 15 au 22 ju in pour tous le s lo ts , sau f pou r le lot à 2000 rad, pou r leq u e l on note , c o m m e dans le s e x p é r i e n c e s p r é cé d e n te s , c e r t a in e s s o r t i e s ta r d iv e s du 15 au 29 juin.

C e s ad ultes , m i s à co n ta m in e r du blé sa in pendant 22 j o u r s , ont donné d e s popu lations la r v a i r e s de d e u x ièm e g én éra t ion dont nous r e p r o d u ison s le s a c t o g r a m m e s r e s p e c t i f s (f ig . 4C ). L 'a c t iv i t é d e m e u r e fa ib le pou r le s l ig n é e s i s s u e s des lo t s i r r a d ié s à 4000 et 8000 rad , et p résen te un net r e ta rd par ra p p o r t au lot té m o in . L e n o m b r e d 'a d u lte s i s s u s de c e s d iv e r s lo t s a été r e s p e c t iv e m e n t de 50 (lot tém o in ) , 30 (2000 rad) et 12 (4000 rad ) ; le lo t à 8000 rad n 'a donné aucun adulte . L e s dates

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FIG. 4. Irradiation d e larves âgées de 19 à 21 jours, A , actogram m es avant l 'irrad ia tion ; B, après l 'irra d ia tion ; C f actogram m es des populations larvaires d e deu x ièm e génération engendrées par les adultes issus des lots irradiés et du lot tém oin . C om parer la dim inution croissante d 'a c t iv ité dans les lots à 2000, 4000 et 8000 rads avec les données fournies par la radiographie.

d 'é m e r g e n c e furent r e s p e c t iv e m e n t du 18 ju i l l e t au 22 août (lot tém oin ) , du 1er août au 3 se p te m b re (2000 rad) et du 9 août au 3 s e p te m b r e .(4000 rad ) .

L 'e x a m e n des r a d io g r a p h ie s de ce t te d e u x iè m e g én éra t ion la r v a ir e m o n tr e que le n o m b r e de g ra in s p résen tant d e s g a le r ie s la r v a i r e s e s t t r è s c o m p a r a b le ch e z le s té m o in s et c h e z le lot à 2000 rad (r e s p e c t iv e m e n t 42 et 45 g ra in s attaqués) . M ais dans le lot à 4000 rad, i l n 'y a que14 g ra in s présentant d e s g a l e r i e s a v a n c é e s , et deux seu lem en t dans le lot à 8000 rad . P a r co n tre , on o b s e r v e un grand n o m b r e de p iq û re s de ponte, non a s s o c i é e s à d es g a le r ie s , dans le lot à 4000 rad, et peu de p iq û r e s de ponte dans le lot à 8000 rad .

Il e s t év ident que le s ad ultes i s s u s d e s la r v e s i r r a d i é e s à 4000 et 8000 rad sont peu fé c o n d s ou peu f e r t i l e s . En p a r t icu l ie r , l e s adultes du lot à 4000 rad, s ' i l s la isse n t d 'a s s e z n o m b r e u s e s p iq û re s de ponte, d o iven t d é p o s e r d es œ u fs en m a je u re pa rt ie s t é r i l e s . On peut p e n s e r q u ' i l s 'a g i t là d 'e f f e t s de m utations lé ta le s dom inantes , p ro d u ite s par l ' i r r a d i a t ion .

2 . 3 . Sen s ib il ité d e s ad ultes - E f fe t s de l ' i r r a d ia t io n su r la fé co n d i té

S M - 102/14 131

A p r è s a v o ir s o u m is d e s ad ultes à d e s d o s e s d ' i r r a d ia t io n de 2000,4000, 8000 et 16 000 rad , nous le s avon s m is pon dre dans d e s lo t s de g ra in sa in . Chaque e x p é r i e n c e p orta it su r 10 c o u p le s d isp osa n t de 50 g ra in s de b lé . T o u s l e s c in q j o u r s , c e s in s e c t e s étaient t r a n s f é r é s dans un n o u veau m i l ie u , a fin d 'a p p r é c i e r le u r f e r t i l i t é 0 à 5 j o u r s , 5 à 10 j o u r s ,10 à 15 j o u r s et 15 à 20 j o u r s a p r è s l ' i r r a d ia t io n .

L 'a c t iv i t é d e s popu la tion s la r v a i r e s obtenu es dans le s d iv e r s lo t s fut e n r e g i s t r é e à p lu s ie u r s r e p r i s e s au c o u r s de leu r d é v e lo p p e m e n t (tous l e s 3 ou 4 jo u r s ) ; le n o m b r e d 'ad u ltes é c l o s et l e s da tes de le u r é m e r g e n c e ont été n o té s ; enfin, le s lo ts de g ra in s furent r a d io g r a p h ié s a p r è s la s o r t ie d e s d e r n ie r s im a g o s .

L e s adultes i s s u s de ce t te p r e m iè r e g én éra t ion furent m is pour la ponte dans de nouveaux m il ie u x , pou r c o m p a r e r le u r fe r t i l i t é .

a) D e sc e n d a n c e is su e d e s p on tes d é p o s é e s 0 à 5 jo u r s a p rè s l ' i r ra d ia t io n

L e s a c t o g r a m m e s ( f i g .5 ) r é v è le n t l 'a b s e n c e d 'a c t iv i té la r v a ir e dans le s lo t s d 'ad u ltes i r r a d i é s à 8000 et 16 000 rad . Le n o m b r e d ' im a g o s is su s d e s a u tres lo ts sont r e s p e c t iv e m e n t : 16 (lot tém oin ) , 14 (2000 rad) et9 (4000 rad ) . L e s da tes d 'é m e r g e n c e s 'é ta le n t du 1er au 7 ju i l l e t pour le s té m o in s , et du 1er au 12 ju i l l e t pou r le s lots i r r a d i é s . L e s r a d io g r a p h ies m ontrent une su rpopu lat ion d e s g ra in s dans le lot té m o in , c e qui exp lique une m o r ta l i té notab le (50 g ra in s attaqués ne donnent que 16 adultes) , m o r ta l i t é im putab le a u s s i p a r t ie l le m e n t à quelques a c a r ie n s p ré d a te u rs (P e d ic u lo id e s v e n t r i c o s u s ). C e p r é d a te u r se r e t r o u v e a u s s i dans le s lotsi r r a d i é s , m a is i l d e m e u r e év ident que le n o m b r e de g ra in s ne présentant pas de g a le r ie s la r v a i r e s s 'a c r o î t a v e c la d o s e d ' i r ra d ia t io n . Le n o m b r e de g ra in s attaqués p a r l e s d i f f é r e n ts lo t s fut de 38 (2000 rad ) , 19 (4000 rad) et 0 (8000 et 16 000 rad ) . Cependant, le n o m b r e de g ra in s présentant des p iq û r e s de ponte non a s s o c i é e s à d e s g a l e r i e s la r v a i r e s e s t t r è s é lev é dans c e s lo ts i r r a d ié s ; i l s sont p a r t icu l iè r e m e n t ap p arents dans le lot à 4000 rad et dans le lot à 16 000 .rad .

On peut en c o n c lu r e que, d è s 4000 rad, un c e r ta in n o m b r e d 'œ u fs ne sont pas fé co n d s , et que tous sont s t é r i l e s a p rè s ir ra d ia t io n à 8000 et16 000 rad . L e s adultes i s s u s d es lo t s à. 2000 et 4000 rad purent donner une d e s ce n d a n c e la r v a i r e (d eu x ièm e gén é ra t io n ) , c o m m e en tém oignen t les a c t o g r a m m e s (fig . 5C). Le n o m b r e d 'ad u ltes de d e u x ièm e g én éra t ion fut r e s p e c t iv e m e n t de 44 ( té m o in s ) , 40 (2000 rad) et 30 (4000 ra d ) . A nouveau, l e s dates d 'é m e r g e n c e , du 9 août au 2 se p te m b re dans le s lo ts i r r a d ié s , p résen ten t un c e r ta in re ta r d par ra p p o r t au tém oin (du 9 au 28 a o û t ) .L e s ra d io g r a p h ie s d e s g ra in s ayant h é b e rg é ce tte d e u x iè m e g énérat ion m on tren t dans le s t r o i s lo t s de t r è s n o m b r e u s e s g a le r ie s , l a r v a i r e s . Mais on note d e s g a l e r i e s la r v a i r e s qui s 'a r r ê t e n t e n .c o u r s de d é v e lopp em en t , t é m o ig n a g e d 'une c e r ta in e m o r ta l i té .

b) D e sce n d a n ce i s s u e d e s pontes d é p o s é e s 5 à 10 j o u r s a p r è s l ' i r ra d ia t io n

A nouveau, dans ce t te e x p é r ie n c e , se u ls le s lo t s à 2000 et 4000 rad donnent n a is sa n ce à ,d e s adultes, en petit n o m b r e d 'a i l l e u r s : 2 (4000 rad) et 6 (2000 rad ) , c o n t re 44 (tém oin ) . L e lot à 8Ó00 rad p ré se n te un a c t o -

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FIG. 5. Irradiation d 'adultes: ponte 0 à 5 jours après l'irradia tion , A , actogram m es des populations larvaires enregistrés à diverses périodes du dévelop p em en t; B, ém ergen ce des adultes d e d eu xièm e génération ; C, actogram m es des populations larvaires de d eu xièm e génération.

g r a m m e lé g è re m e n t p o s i t i f ( f ig . 6A) et la ra d io g ra p h ie u lté r ie u re d e s g ra in s r é v è l e au plus deux g a l e r i e s la r v a i r e s in a c h e v é e s . L 'é m e r g e n c e des ad u ltes dans le lot té m o in s 'é ta le du 1er au 12 ju i l le t , a l o r s q u 'e l le se p r o lo n g e ju sq u 'a u 3 août dans le lot à 2000 rad . L e s r a d io g r a p h ie s m o n trent un grand n o m b r e de p iq û r e s de ponte dans le lot à 4000 rad, c e l l e s - c i étant m o in s n o m b r e u s e s dans le s lo t s à 8000 et 16 000 rad . C e s p iq û re s non a c c o m p a g n é e s de g a l e r i e s la r v a i r e s tém oignen t se lo n toute v r a i s e m b la n ce d 'une m o r ta l i té im p o rta n te au stade « œ u f » (œ u fs in fé co n d s ) .

La d e u x ièm e g én éra t ion la r v a ir e e s t peu abondante dans le s lo t s à 2000 et 4000 rad, c o m m e le r é v è le n t le s a c t o g r a m m e s (f ig . 6C) et c o m m e le c o n f ir m e n t le s r a d io g r a p h ie s . L e s é m e r g e n c e s d 'ad u ltes ont été r e s p e c t iv e m e n t de 59 (tém oin ) , 15 (2000 rad) et 1 (4000 rad ) . Dans le lot à 2000 rad, l e s ra d io g r a p h ie s font a p p a ra ît re une c e r ta in e m orta l ité ta r d iv e : 4 im a g o s m o r ts dans le g ra in . P a r con tre , i l ne se m b le pas y a v o i r de m o r ta l i té au stade « œ u f » ou « j e u n e l a r v e » (19 g ra in s attaqués:15 im a g o s v ivants + 4 im a g o s m o r t s ) .

c ) D e sce n d a n ce is su e d e s pontes d é p o s é e s 10 à 15 j o u r s a p r è s l ' i r r a d ia t io n

L e s a c to g r a m m e s (f ig . 7) r é v è le n t une ac t iv ité la r v a ir e t r è s fa ib le et t e m p o r a i r e (début du d é v e lop p em en t ) dans le s lo ts i r r a d ié s à 2000,

S M - 102/14 133

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FIG. 7. Irradiation d ’ adultes: ponte 10 à 15 jours après l'irradia tion . A ctogram m e des populations larvaires. Une très fa ib le a ctiv ité tem poraire se m anifeste dans les lots à 2000, 4000 et 8000 rads. Aucun des lots-irradiés n 'a donné de descen dan ce v ia b le .


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FIG. S. Irradiation d ’ adultes: ponte 15 à 20 jours après l ’ irradiation. A , actogram m es des populations larvaires; В, ém ergence des adultes d e d eu xièm e génération pour le lot tém oin et le lo t à 2000 rads;C, actogram m es des populations larvaires de d eu xièm e génération.

4000 et 8000 rad. En fait, aucun adulte n 'e s t s o r t i de c e s lo t s , b e s r a d io g r a p h ie s m ontrent de r a r e s g a le r ie s la r v a i r e s , ja m a is a c h e v é e s , m a is par co n t re de n o m b re u x g ra in s av e c d e s p iq û re s de ponte, en p a r t i c u l i e r dans le s lo ts à 2000 et 4000 rad, 10 à 15 jo u r s a p r è s l ' i r r a d ia t io n , le taux de fe r t i l i t é des œ u fs est donc e n c o r e p lus fa ib le que dans l ' e x p é r i e n c e p ré cé d e n te .

d) D e sce n d a n ce la rv a ir e i s s u e des pontes d é p o s é e s 15 à 20 j o u r s a p rè s l ' i r ra d ia t io n

L e s a c to g r a m m e s se r é v è le n t à nouveau p os it i fs pour c e r ta in s lots i r r a d i é s , t r è s nettem ent pour le lot à 2000 rad, fa ib lem ent pou r c e lu i à 4000 rad (fig . 8). Ils d e m eu ren t nuls pour 8000 et 16 000 rad . L e s r a d io g r a p h ie s m on tren t d 'a s s e z n o m b r e u s e s g a le r ie s la r v a i r e s c o m p lè t e s dans le lot à 2000 rad : 22 g ra in s attaqués plus un c e r ta in n o m b r e de g ra in s p iqués , donnant 14 im a g o s . Dans le lot à 4000 rad, on ne d é c è le que deux g ra in s nettem ent m in és , m a is de n o m b re u x g ra in s p iqués ; aucun im a g o n 'e s t s o r t i de ce lot . Dans le s lots à 8000 et 16 000 rad, aucune g a le r ie l a r v a i r e n 'e s t d é c e la b le et on ne vo it que t r è s peu de p iq û res de ponte.

S M - 102/14 135

L e s im a g o s i s s u s du lo t à 2000 rad donnent, en d e u x iè m e g énérat ion , une population la r v a ir e a c t ive (f ig . 8C) et i l en so r t i t 42 im a g o s pour 44 g a l e r i e s la r v a i r e s r é v é l é e s par r a d io g ra p h ie .

I l s e m b le donc ra iso n n a b le de c o n c lu r e de c e s d iv e r s e s e x p é r i e n c e s portant su r d e s adultes i r r a d i é s que leu r fé con d ité d im inue gra d u e l le m e n t ju s q u 'à d e v e n ir nulle , du 1er j o u r au 1 5 e j o u r a p rè s l ' i r r a d ia t io n . M ais p a s s é c e d é la i , un r e g a in de fé co n d i té se m a n ifeste , au m o in s p ou r le s a d u ltes i r r a d ié s à 2000 rad . C e la p o u rra i t c o r r e s p o n d r e à une n ouvelle p o u s s é e de sp e r m a to g é n è s e c h e z les m â le s i r r a d ié s , c o m m e l 'av a ien t l a i s s é p r é v o i r le s o b s e r v a t io n s a n té r ie u r e s de P e s s o n et V e r n ie r [8].

e) I r ra d ia t io n d 'ad u ltes - E f f e t s g én érau x su r la d e s ce n d a n ce

Une autre e x p é r i e n c e a é té e n t r e p r i s e su r d e s lo t s de 100 in s e c te s ad ultes (50 m â le s + 50 f e m e l le s ) i r r a d i é s de 2000 à 16 000 rad , dont on a su iv i la d e s ce n d a n ce pendant deux g é n é r a t io n s s u c c e s s iv e s . On a noté le n o m b r e d ' im a g o s i s s u s de chaque g én éra t ion et le p ou rcen tag e de g ra in s attaqués dans chaque étape de l ’e x p é r i e n c e , étant entendu que le s g é n i t e u r s d isp o sa ie n t i c i d 'une m a s s e de g ra in im p ortan te pou r d é p o s e r leu r ponte. L e s r é su lta ts sont donnés à la f ig u re 9.

On con sta te qu 'une d o s e de 8000 rad est su ff isante , à l ' é g a r d des ad ultes , pour an n ih iler tout d é v e lo p p e m e n t la r v a i r e . On r e m a r q u e é g a l e m ent que d e s d o s e s d ' i r r a d ia t io n de 2000 à 4000 rad rédu isen t la d e s c e n dance r e s p e c t iv e m e n t de p r è s de 60% et de 85%.

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FIG. 9. D iagram m e soulignant l 'in flu e n ce d e l 'irra d ia tion sur des adultes de Sitophilus zem ais (50 couples . par ex p ér ien ce ), en com parant le nom bre d e leurs descendants et le nom bre de grains attaqués. G l:

descen dan ce d e prem ière génération. G2 descen dan ce de d eu x ièm e génération.

Cependant, à la d e u x iè m e g én éra t ion , le s ad ultes d e s lo ts i r r a d ié s à 2000 et 4000 rad sont c a p a b le s de re d o n n e r des popu lations la r v a i r e s a s s e z abondantes, qui attaquent le s g ra in s dans la p r o p o r t io n de 80% (2000 rad) et 40 à 45% (4000 rad ) .


CONCLUSION

P a r l ' e m p lo i c o m b in é de la ra d io g r a p h ie d e s g ra in s et de l ' e n r e g i s t r e m ent de l 'a c t iv i t é d es s tad es en d o gé s de Sitophilus z e a m a is à l 'a id e d 'un d é te c te u r é le c t r o - a c o u s t iq u e t r è s se n s ib le a v e c une t r è s fo r te a m p l i f i ca t io n , i l a été p o s s ib le de p r é c i s e r l 'e f f e t des rad ia t ion s io n isan tes su r le s d iv e r s stad es de d é v e lo p p e m e n t de ce t in s e c te . La m éthod e p e r m e t en p a r t i c u l i e r de d é c e l e r le s r é d u c t io n s de f e r t i l i t é en mettant en é v id e n ce d e s p iq û re s de pontes non a c c o m p a g n é e s de g a le r ie s la r v a i r e s et de s tad es e n d o g é s a c t i f s (œ u fs s t é r i l e s ) . L e s p r in c ip au x ré su lta ts o b s e r v é s sont le s su ivants :

- L a se n s ib i l i té de Sitophilus z e a m a is aux rad ia t ion s io n is a n te s d i f fè r e se lo n le stade de d é v e lop p em en t .

- L e stade « œ u f » e s t le plus s e n s ib le , le stade « l a r v e â g é e » (19 à 21 j o u r s ) le plus ré s is ta n t . T an dis qu 'une d o s e de 2000 rad suffit pou r e m p ê c h e r toute é c l o s i o n d e s œ u fs , i l faut 8000 rad pour a r r ê t e r c o m p l è t e m ent l ' é v o lu t io n d e s la r v e s â g é e s . Cette m ê m e d o s e de 8000 rad , ap p liquée à d e s ad ultes , annihile ég a le m e n t leu r d e s ce n d a n c e la r v a i r e .

-U ne d o s e de 16 000 rad es t n é c e s s a i r e pou r o b ten ir la d e s tr u c t io n c o m p lè t e d 'une population m ix te de la r v e s et d 'ad u ltes dans un lo t de g ra in s .

- L e s e f fe ts d e s rad ia t ion s à fa ib le d o s e (2000, 4000 et 8000 rad) s e font e n c o r e sen t ir su r la d e u x iè m e g én éra t ion i s s u e de la r v e s ou d 'ad u ltes

i r r a d i é s . I l s 'a g i t en p a r t i c u l ie r d 'une in fe r t i l i t é plus ou m o in s gra nd e d e s œ u fs , c o n s é q u e n c e v r a i s e m b la b le de m utations lé ta le s dom ina ntes , surtout fr é q u e n te dans le c a s d ' i r r a d ia t io n de la r v e s â g é e s (19 à 21 j o u r s ) .

- L e s adultes i r r a d i é s présen ten t, m ê m e a p rè s d e s d o s e s fa ib le s d ' i r r a d ia t io n (2000 rad) , une dim inution de leu r fé con d ité qui va en s ' a g g r a vant du l et au 1 5 e j o u r a p r è s l ' i r r a d ia t io n , et peut à c e m om en t about ir à une s t é r i l i t é de fa it . M ais , p a ssé c e d é la i (du 15e au 20 e j o u r a p r è s l ' i r r a d ia t io n ) , l e s in s e c t e s r e trou v en t une c e r ta in e fé co n d ité , c o n s é q u e n c e v r a i s e m b la b le d 'une r e p r i s e de la sp e rm a to g e n è s e ch e z le s m â le s .

R E M E R C IE M E N T S

C e t r a v a i l a été e f fe c tu é g r â c e à un co n tra t de r e c h e r c h e s p a ssé a v e c 1UNSERM (Institut nationa l de la santé et de la r e c h e r c h e m é d ic a le ) dans le c a d r e d e s a c t io n s c o n c e r t é e s « N u t r i t i o n » . I l nous e s t a g r é a b le d 'e x p r i m e r i c i aux r e s p o n s a b le s de la C o m m is s io n INSERM toute n o tre gra t itu d e pou r l 'a id e a p p o rté e à notre la b o r a to i r e et pou r a v o i r a in s i fa c i l i t é le d é v e lo p p e m e n t de n os r e c h e r c h e s su r l ' i r r a d ia t io n d e s in s e c t e s d e s d e n r é e s .

N ous a d r e s s o n s é g a le m e n t n os v i f s r e m e r c i e m e n t s à M. S im on , du C e n tre d 'é tu d e s n u c lé a i r e s de Saclay , à M. V a llé e , te ch n ic ie n photographe , et à M lle Joa n n es , a ide technique du L a b o r a to i r e de z o o lo g ie de l 'Institu t n a tiona l a g ro n o m iq u e , qui ont c o l l a b o r é à d iv e r s t i t r e s à c e t r a v a i l .

R E F E R E N C E S

[1]- ADAMS, R .E ., WOLFF, J .E ., MILNER, М ., SHELLENBERGER, I. A . , D etection o f internal insect infestation by sound am p lifica tion , C ereal Chem . 3 (1954 ) 2 7 1 -7 6 .

[2] BAILEY, S .W ., McCABE, J .B ., The detection o f im m ature stages o f insects within grains o f wheat,J. stored Prod. Res. 1 (1965 ) 20 1 -0 2 .

S M - 102/14 137

[3] KIRKPATRICK, R. L., WILBUR, D .A . , The developm ent and habits o f the granary w eev il, Sitophilus granarius, w ithin the kernel o f wheat, J. e co n . Ent. 58 3 (1965 ) 9 1 1 -8 5 .

14] MILLS, R. B ., WILBUR, D. A . , Radiography studies o f Angoum ois grain m oth developm en t in wheat, .corn and sorghum kernels, J. e con . Entom . 60 3 (1967) 6 7 1 -7 7 .

[5 ] CORNWELL, P. B ., The E n tom ology o f Radiation Disinfestation o f Grains, Pergam on Press, London(1966) 236 pp.

[6] PESSON, P ., U tilisation des radiations ionisantes ( 60C o ) pour la protection des denrées contre les insectes nuisibles. Recherches relatives à la déterm ination des doses m in im a utiles pour assurer la stérilité des insectes, Inds a grie , a lim en t. 3 (1 9 6 3 )2 1 1 -2 5 .

L7] PESSON, P ., L 'énergie nucléaire e s t -e lle u tilisable pour la protection des stocks de céréa les contre les in sectesî, Inform ation et D ocum entation A g rico les n °2 , C oopératives La Fayette, Paris (15 janvier 1965).

[8 ] PESSON, P ., VERNIER, J .M ., La protection des denrées contre les insectes ravageurs par l 'e m p lo i des radiations ionisantes en vue d 'ob ten ir la stérilité des insectes adultes. Etude particu lière de la réaction des gonades de Sitophilus granarius, Annls Nutr. et A lim en t. XVII 6 (1 9 6 3 ) 4 8 7 -9 7 .

D I S C U S S I O N

G. LE M ASNE: It would be in te re s t in g to find out f r o m P r o f e s s o r P e s s o n o r M r. G ir ish w hether they think the sounds r e c o r d e d by the e l e c t r o a c o u s t i c d e t e c to r a re em itted by the la rv a e during feed in g (by rubbing the ja w s against the gra in ) o r by m ov in g about in side the ca v ity they have dug f o r th e m s e lv e s in the gra in .

P . PESSON* : T he v ib ra t io n s r e c o r d e d by the e l e c t r o - a c o u s t i c d e t e c to r m ay be due to e ith e r type o f a c t iv ity . The a c to g r a p h ic r e c o r d in g o f the en tire d e v e lop m en t o f a g r a in - w e e v i l la rva f r o m hatching to e m e r ge n c e o f the im ag o (P . P e s s o n and M. O z e r , Ann. d es E p iphyties (1968), in the p r e s s ) show s that the feed in g a c t iv ity o f the la rv a is continuous, be ing in terru pted on ly in the p r o c e s s o f shedding o f the la rv a l skin. The p e r io d during w hich the pupal c a s e is p r e p a r e d and thè pupal p er iod i t s e l f a r e c h a r a c t e r i z e d by p e c u l ia r n o is e s . But these p e c u l ia r i t i e s can only be detected by using the a c tog rap h on a s ing le gra in o f a f fe c te d c o rn .

W . J . K L O F T : An e l e c t r o - a c o u s t i c a c tog rap h u sed during the i r r a d ia tion p r o c e s s would p o s s ib ly a l low r e g is t r a t io n o f d i r e c t r e s p o n s e s o f the in s e c t s . Do you know w hether B u sn e l 's apparatus w il l function when e x p o s e d to ion iz in g rad ia t ion ? I am su re that th is would p ro v id e an in te r e s t in g line o f e x p e r im e n ta l study.

P . P E L E G R IN : I am not su f f ic ie n t ly w e l l acquainted m y s e l f with M r . B u sn e l 's a c to g ra p h to be ab le to r e p ly . H ow ever , I b e l ie v e it should be p o s s ib le to in vest igate the in s e c t s during ir ra d ia t io n as you suggest , s in c e the rad ia t ion d o s e s in vo lved a r e re la t iv e ly weak.

Written reply received fo llow in g the Sym posium .

STERILISATION DE LA P Y R A L E DU MAIS (O strinia nubilalis Hb. )PAR L 'IR R AD IATIO N A U X RAYONS G AM M A*

M . ANWARINRA, STATION DE ZOOLOGIE AGRICOLE,

CENTRE DE RECHERCHES AGRONOMIQUES DU SU D -E ST ,MON T F A VET, FRANCE


STERILIZATION OF THE EUROPEAN CORN BORER Ostrinia nubilalis Hb. BY G AM M A IRRADIATION. Pupae and larvae o f Ostrinia nubilalis Hb. were irradiated with a co b a lt -6 0 source. The dose rate was about 1040 rad/m in. The e ff e c ts o f different doses on the survival o f the pupae, the length o f l i fe o f the adults, sexual behaviour, fecundity and fertility were studied. Larvae at the end o f rheir developm ent exposed to 5, 10, 15, 20, 25 and 30 krad gave respectively 90, 77, 23, 16, 13 and 10% o f pupae; the non-pupared larvae often rem ained a liv e in their cocoon s for four months or m ore . At 10 krad and above , no adults hatched. At 5 krad, 63% o f adults were obtained; the m ales and fem ales w hich hatched showed a fertility o f only about 12%, their sexual behaviour, fecundity and length o f l i fe being reduced. The sensitivity o f the pupae decreased with age; 6 - and 1 2 -d a y -o ld specim ens irradiated at 10 krad gave a hatching rate o f 42 and 100% respectively . A dose o f 20 krad applied to fem a le 1 2 -d a y -o ld pupae was sufficient to m ake the eggs in fertile , w hile a dose o f 40 krad was needed to ensure that irradiated m ales cou p led with norm al fem a les gave rise to totally sterile batches o f eggs.- When groups o f sterile butterflies (25 m ales, 25 fem ales or 25 individuals o f both sexes) were introduced in to,a ca g e containing f iv e norm al butterflies o f each sex, the number o f v ia b le eggs la id was reduced, the m axim um reduction being for the proportion 2 5 :2 5 : 5 : 5 . It would therefore seem advantageous to release both irradiated m ales and fem ales together.

STERILISATION DE LA PYRALE DU MAIS (Ostrinia nubilalis Hb) PAR L'IRRADIATION AUX RAYONS G AM M A. Les pupes et les larves de Ostrinia nubilalis Hb. ont éré^irradiées avec une source au co b a lt -6 0 . Le débit de dose était environ de 1040 rads/m in. Les effets de doses variées sur la survie des pupes, la lon gév ité des adultes, le com portem ent sexuel, la fécon d ité et la fertilité ont é té exam inés. Des larves en fin de développem ent exposées à 5, 10, 15, 20, 25 et 30 krads donnent respectivem ent 90, 77, 23,16, 13 et 10% de pupes; les larves non pupéfiées restent souvent vivantes dans leurs co con s pendant 4 m ois ou plus. A 10 krads et plus, aucun adulte n* é c lo t . A 5 krads, on obtient 63% d'adultes; les m ile s et fem elles é c lo s montrent une fertilité de seulem ent 1 2 % environ; m ais le com portem ent sexuel, la fécon d ité et la durée de v ie sont réduits. La sensibilité des pupes dim inue avec l ‘ âge; des pupes de6 et 12 jours irradiées.à 10 krads donnent 42 et 100% d*éclosions. La dose de 20 krads appliquée à des pupes de 12 jours suffit ch e z la fe m e lle à rendre les œ ufs infertiles. Il faut une dose de 40 krads pour que les m âles irradiés, a ccouplés à des fem elles norm ales, provoquent des pontes totalem ent stériles. Lorsque des groupes de papillons stériles (25 m aies, 25 fem e lle s ou 25 individus de chaque sexe) sont introduits dans une ca g e contenant 5 papillons normaux de chaque sexe, les nombres d*œufs viables pondus sont réduits; la réduction m axim ale intervient pour le m élange 2 5 :2 5 : 5 : 5 . Il paraît donc plus intéressant de lâcher à la fois les m âles et les fem elles irradiés.

* T ravail e ffectu é en 1966-1967 pendant un stage en France organisé par l'ASTEF (A ssociation pour l ’ organisation des stages en France).

**Adresse perm anente: Pakistan A tom ic Energy Com m ission.

139

140 ANWAR

1. INTRODUCTION

L 'a c t i o n d e s ray o n s X su r la P y r a le du m a is , O str in ia n ub ila lis Hb. (L e p id o p te ra : Pyra lidae ) a été étudiée par W a lk er et B r in d le y [1].L 'a c t i o n h istopatho log ique d e s ray on s g a m m a su r le s t i s s u s o v a r ie n s du m ê m e in se c te a été étudiée pa r Sun et L e e [2].

Il nous a paru in té re ssa n t d’ e x a m in e r l 'a c t i o n de r a y o n s g a m m a d’ une s o u r c e de 60C o su r le s la r v e s et l e s nym phes de la P y r a le du m a ïs .

2. M A T E R IE L E T TECHNIQUES D 'E T U D E

L e s in s e c te s u t i l i s é s proviennent d 'un é lev ag e perm anen t d 'O . n ub ila lis au la b o r a to i r e .

L e s la r v e s sont é l e v é e s su r un m il ie u a r t i f i c i e l à b a se de g e r m e de blé .suivant la m éthod e m i s e au point pa r M m e G uennelon, à la Station de z o o lo g ie a g r i c o l e de M ontfavet (travaux non p u b l i é s ) . L e s la r v e s sont a l im e n té e s in d iv idu e llem ent dans d e s tubes en m a t iè r e p last ique de5 c m de lon gu eu r et 1 c m de d ia m è tre , r e m p l i s aux deux t i e r s de m il ie u . L 'é l e v a g e est e f fe ctué dans une p iè c e c l im a t i s é e à une te m p é ra tu re de 25°C et une hum idité re la t iv e de 80%, é c la i r é e pendant 18 h e u re s par jou r pa r d e s tu bes f lu o r e s c e n ts . Dans c e s c on d it ion s , la d iapause de la la r v e d 'O . n ub ila lis e st é v itée . L a n ym ph ose s 'e f f e c t u e à p a r t i r du 16e jo u r de fa ç o n g é n é r a le ; la la rv e du d e r n ie r stade t i s s e un c o c o n de s o ie c o n t re la p a r o i du tube avant de se c h r y s a l id e r . Chaque jo u r l e s tubes contenant des n ym phes sont p r é le v é s et p la c é s dans une autre p iè c e c l im a t i s é e à une te m p é ra tu re de 22°C et une hum idité r e la t iv e de 80%. Avant l ' i r r a d i ation , l e s nym phes sont e x tr a it e s des tu bes et sont s é p a r é e s pa r se x e .L a d u ré e de la n ym phose est d 'e n v ir o n 12 jo u r s .

P o u r l 'a c c o u p le m e n t d e s adultes nous avons u t i l isé d e s c a g e s c y l in d r iq u e s en m a t iè r e p last ique de 20 c m de d ia m è tre et 25 c m de hauteur. L e s c o u v e r c l e s en g r i l la g e p last iqu e sont f ix é s a v e c une bande a d h é s iv e . P o u r le s c o n t r ô le s de fé cond ité des adultes et de v iab il ité des œ u fs , on ta p is s e l e s p a r o is de la cage a v e c du p a p ier s u l fu r isé , sur le q u e l l e s p a p il lon s d éposent l e u r s œ u fs .

Une fo is pa r sem a in e l e s adultes sont changés de c a g e s , l e s pontes sont c o m p té e s . Un c e r ta in n o m b r e de pontes sont g a r d é e s pou r c o n t r ô le r la v iab i l i té d e s œ u fs , et l e s la r v e s é c l o s e s sont c o m p té e s chaque jo u r .

On p la ce c inq c o u p le s pa r cag e , suivant d i f fé r e n te s c o m b in a is o n s (m â le i r r a d ié X f e m e l le non i r r a d ié e ; f e m e l le i r r a d i é e X m â l e non i r r a d ié ; ■ té m o in s , m a ie et f e m e l le non i r r a d ié s ) . On in sta lle c inq c a g e s pa r c o m b ina ison . L e s f e m e l le s sont d i s s é q u é e s a p r è s le u r m o r t p ou r c o m p te r l e s s p e r m a to p h o r e s .

Dans d 'a u tr e s e x p é r i e n c e s c o m p o r ta n t des m é la n g e s de popu la tion s , d e s c a g e s g r i l la g é e s sont u t i l i s é e s . C e s c a g e s sont an a logu es à c e l l e s q u 'u t i l is en t G uthrie et al. 13]. L a technique d 'obtent ion d e s pontes est la m ê m e que c e l l e adoptée pa r c e s au teu rs . L e s pontes sont r é c o l t é e s a p r è s 7 jo u r s et sont g a r d é e s pou r le c o n t r ô le de la v iab i l i té d e s œ u fs .

C e s c a g e s sont p la c é e s dans une p iè c e c l im a t is é e à une te m p é r a tu r e de 2 2 "C et une hum idité r e la t iv e de 80%, à la lu m iè r e n a tu re l le . On u t i l ise seu lem en t le s adultes qui ont é c l o s dans l e s 24 h e u r e s suivant l ' i r r a d ia t io n . L e s adultes sont n o u r r i s a v e c de l 'e a u s u c r é e à 5%.

SM -102/15 141

L e s i r ra d ia t io n s ont été e f fe c t u é e s a v e c un ir ra d ia te u r de 60С о de 1400 C i au S e r v i c e de r a d io - a g r o n o m ie du C e n tre d 'é tu d e s n u c lé a i r e s de C a d a r a c h e . L e débit de d o s e était d 'e n v ir o n 1040 r a d /m in . L a d o s i - m é t r ie a été e f fe c tu é e p ou r chaque ex p o s it io n .

3. R E SU L T A T S

3 .1 . I r ra d ia t io n d e s la r v e s

L e s la r v e s d u 'd e r n ie r âge (15 j) ont été i r r a d ié e s à d e s d o s e s de 5 à 30 krad . A p r è s l ' i r r a d ia t i o n l e s la r v e s sont r e t r a n s f é r é e s , in d iv id u e l le m ent, dans l e s tubes a v e c le m i l ie u f r a i s p ou r la n ym p h ose .

3. 1. 1. N y m p h o se s et é c l o s io n s

L e tableau I m o n tr e que, m ê m e à la p lus fa ib le d o s e de 5 krad , il y a d e s la r v e s qui ne se n ym phosent pas ; e l l e s resten t v iva n tes pendant p lus de 4 m o is dans une attitude c a r a c t é r i s t iq u e d e s l a r v e s en état de d iapause (a l o r s que l e s t é m o in s dans l e s m ê m e s c o n d it io n s se n ym phosent t r è s rap id em en t) . Nous n 'a v o n s cependant pas pu o b ten ir de r e p r i s e de d é v e lop p em en t , bien que le s la r v e s aient été p la c é e s dans d i f fé r e n te s con d it ion s de te m p é ra tu re . P lu s i e u r s au teu rs [4, 5] ont d é jà s ign a lé , c h e z d 'a u tr e s in s e c te s , que l ' i r r a d ia t i o n d e s la r v e s p r o v o q u e un a r r ê t

T A B L E A U I. E F F E T S DU R A Y O N N E M E N T G A M M A SUR L E S L A R V E S DU D E R N IE R A G E D ' O s t r in ia n u b i la l i s H b.(3 0 l a r v e s p a r d o s e )

D ose(krad)

N om b re d e larves m ortes avant la

n y m p h ose 3

N om b re de nym phes m ortes

N om b re d 'a d u ltes é c lo s

0 0 2 28

5 3 « 19

1 0 7 23 0

15 23 7 0

2 0 25 5 0

25 26 4 0

30 27 3 0

ay co m p r is les la rves dont l e d é v e lo p p e m e n t s' est a rrêté .

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de dév e lo p p e m e n t de type d iapause et V a s i ly a n (6] aurait m o n tré que , ch e z P e c t in o p h o ra m a lv e l la , l ' i r r a d ia t io n d e s l a r v e s du d e r n ie r âge c a u se un v é r i ta b le état de d iapause .

L e n o m b r e de n ym phes f o r m é e s dev ient t r è s fa ib le p ou r le s d o s e s s u p é r ie u r e s à 10 krad . M a is aucune é c lo s io n de n ym phes n 'e s t c o n s ta té e p ou r d e s d o s e s de plus de 5 krad ap p liquées aux la r v e s du d e r n ie r â g e .

3 . 1 . 2 . S tér i l i té obtenue

L e s adultes is s u s de la r v e s i r r a d ié e s à 5 krad m on tren t (tab leau II) une fé c o n d i té , une fe r t i l i t é et une lon gév ité a s s e z ré d u i te s . L a fe r t i l i t é est réd u ite à 12% en v iron , m a is la v igueur s e x u e l le s e m b le d im in u ée ; on con s ta te en p a r t i c u l ie r que de n om breu x c o u p le s d ' in s e c t e s ne peuvent pas se s é p a r e r a p r è s l 'a c c o u p le m e n t (28% d e s c o u p le s m â le i r r a d i é X fe m e l le non i r r a d ié e et 16% d e s c o u p le s m â le non i r r a d ié X f e m e l le i r r a d ié e ) .

T A B L E A U II. F E C O N D I T E , F E R T I L I T E E T L O N G E V I T E D E S A D U L T E S D ' O s t r in ia n u b i la l i s H b. ISSUS D E C R O I S E M E N T S D 'I N S E C T E S NON IR R A D I E S A V E C D E S IN S E C T E S ISSUS D E L A R V E S IR R A D I E E S A 5 k ra d

D ose(krad)

N aturedes

cro isem en tsN om bred 'œ u fs

parfe m e lle

N om b red 'œ u fs

L on g é v ité des adu ltes

(3 )

M â le F e m e lleé c lo s

M â le F e m e lle

0 N N 510 .60 ,8 2 0 , 8 2 0 , 6

I N 376 1 1 . 6 1 3 ,3 2 0 ,25

N I 76 1 3 .7 2 7 ,3 1 1 ,6

N = non irradié, I = irradié.

3 . 2 . I r ra d ia t ion d e s nym phes

3 . 2 . 1 . E c lo s io n s

D e s d o s e s de 10 et 15 krad ont été ap p liquées à des n ym phes d 'â g e s d i f f é r e n ts . On voit dans le tableau III que c e s deux d o s e s ont à peu p r è s la m ê m e a c t io n , l ' i r r a d ia t io n à 12 jo u r s ne diminuant pas le s é c l o s io n s p a r ra p p o r t aux té m o in s .

L e s d o s e s de 20 à 50 krad ont été ap p liq u ées à d e s n ym phes de 12 jo u r s . Seule la d o s e de 50 krad p r o v o q u e une l é g è r e dim inution d e s é c lo s io n s par ra p p o r t aux té m o in s .

S M -102A 5 143

T A B L E A U III. E F F E T S DU R A Y O N N E M E N T G AM M A SUR DES N Y M PH E S D 'O str in ia nub ila lis Hb. A D IF F E R E N T S AGES (60 n ym phes p a r c a s )

Age

(j)

Pourcentage d 'é c lo s ion des adultes par rapport aux tém oins

1 0 krad 15 krad

Tém oins 1 0 0 , 0 1 0 0 , 0

2 0 , 0 0 , 0

4 4 ,0 4 ,0

6 4 2 ,3 3 4 ,6

8 7 3 ,2 57 ,3

10 8 6 , 6 8 2 ,0

1 2 1 0 0 , 0 9 5 ,4

3. 2. 2. S tér i l i té obtenue

L a s té r i l i té a été e x a m in é e en mettant chaque f o i s en p r é s e n c e dans la m ê m e ca g e c inq m â le s et c inq f e m e l l e s i r r a d ié e s ou non. L e s d o s e s u t i l i s é e s a l l a i e n t d e 10 à 50 k r a d .

Un e s s a i su r d e s n ym phes â g é e s de 6 jo u r s à la d o s e de 10 krad a m o n tré une f e r t i l i t é c h e z les . m â le s i r r a d ié s réduite à 2 2 ,7 % ( tém oin 77, 6%).

L ' e s s a i c o m p le t a p o r té su r d e s n ym phes â g é e s de 12 j o u r s . L e n o m b r e m o y e n d 'œ u fs pondus par d e s f e m e l l e s n o r m a le s n 'e s t que fa ib lem en t in f lu encé p a r l 'a c c o u p le m e n t a v e c des m â le s i r r a d i é s , qu e lle que so it la d o s e . P a r co n tre , c h e z le s f e m e l le s i r r a d i é e s , le n o m b r e m oy en d 'œ u fs pondus d im inue p r o g r e s s iv e m e n t ( f ig . 1).

L a fe r t i l i t é d e s œ u fs pondus par des f e m e l le s i r r a d ié e s c r o i s é e s a v e c des m â le s non i r r a d ié s est p r e sq u e nulle d è s la d o s e de 10 krad et dev ient nulle à p a rt ir de 20 krad . L a fe r t i l i t é d e s œ u fs résu ltant du c r o i s e m e n t m â le i r r a d ié X f e m e l le non i r r a d i é e d im in ue p r o g r e s s iv e m e n t p ou r dev en ir nulle à 40 krad ( f ig . 2).

3 . 2 . 3 . V igu e u r se x u e l le

L a v igu eu r d es m â le s i s s u s de n ym phes i r r a d ié e s à d i f fé r e n te s d o s e s a été e x a m in é e . L e c r i t è r e était le n o m b r e de s p e r m a to p h o r e s p r é se n ts dans le s f e m e l le s a p rè s le u r m o r t . P o u r d e s d o s e s de 10 à 40 krad , le

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Fécondité • 6 0 0

5 0 0

4 0 0

3 0 0

200

100

c? irradiés X о normales

10 3 0 4 0 50Dose (krad)

FIG. 1. F écond ité résultant d e croisem ents insectes normaux x insectes issus d e nym phes de 12 j irradiées aux doses de 10 à 50 krad (la fécon d ité des tém oins non irradiés était d e 554 œ ufs).

% fertilité

Dose (krad)

FIG. 2 . Fertilité résultant de croisem ents insectes norm aux x insectes issus d e nym phes de 12 j irradiées aux doses de 10 à 50 krad (la fertilité des tém oins non irradiés était de 78<T/o).

n o m b r e m oyen d 'a c c o u p le m e n t s par m â le re s ta it c o m p a r a b le à c e lu i du té m o in (1 ,4 5 dans le s con d it ion s de nos e x p é r i e n c e s ) ; il était p lus réduit (0, 95) pour 50 krad. La v igu eu r se x u e l le d e s m â le s ne para ît donc pas d im in uée aux d o s e s s t é r i l i s a n te s . L o r s q u e des f e m e l le s i r r a d ié e s sont m i s e s en p r é s e n c e de m â le s n o rm au x , l e s n o m b r e s d 'a c c o u p le m e n t s p a r m â le ne su b issen t pas de d im inution , c e qui m o n tre que c e s f e m e l le s re s ten t a u ss i r é c e p t iv e s que d e s f e m e l l e s non i r r a d ié e s .

SM '102/15 145

T A B L E A U IV . V I A B I L I T E D E S Œ U F S D ' O s t r in ia n u b i la l i s H b. R E S U L T A N T DU M E L A N G E D 'I N S E C T E S S T E R I L E S E T D 'I N S E C T E S N O R M A U X EN D I F F E R E N T E S P R O P O R T I O N S

N om b re d 'a d u lte s par c a g eN om b re d ’ oeufs

fe rt ile s par f e m e l le n orm a le (p re m iè re se m a in e de p on te)

S tériles N orm au x

M â le F e m e lle M â le F e m e lle

0 0 5 5 183

0 0 5 5 2 0 4

0 0 5 5 230

0 0 5 5 218

25 25 5 5 0

25 25 5 5 0

25 25 . 5 5 0

25 25 5 5 52

25 0 5 5 1 1 1

25 0 5 5 14

25 0 5 5 1 0 1

25 0 5 5 54

0 25 5 5 2 0 1

0 25 5 5 1 0 2

0 25 5 5 150

0 25 5 5 26

10

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3 . 3 . M é la n g e s de popu lations

Q u e lqu es e s s a i s de m é la n g e s d ' in s e c t e s i r r a d ié s (à la d o s e de 40 krad ap p liquée à des nym phes de 12 jo u r s ) ou non en p r o p o r t io n s v a r ia b le s ont été r é a l i s é s . L e s r é su lta ts sont ind iqués dans le tableau IV. L e résu lta t le p lus r e m a r q u a b le r e s s o r t de la c o m p a r a is o n d es m é la n g e s , m â le s s t é r i l e s , f e m e l le s s t é r i l e s , m â le s n o rm a u x et f e m e l le s n o r m a le s , dans le s p r o p o r t io n s : 25 :0 :5 :5 et 25 :25 :5 :5 . L e fait de m é la n g e r c e s popu lations c o m p lè t e s , n o r m a le s et i r r a d ié e s , dans la p r o p o rt io n de 5 à 25 p ro v o q u e une s t é r i l i t é à peu p r è s a b so lu e . P a r c o n t r e , s i ce tte p r o p o r t io n de5 à 25 est l im ité e aux m â le s , la s t é r i l i t é p r o v o q u é e r e s te rédu ite . C e c i m o n tr e que, dans le s con d it io n s de nos e s s a i s , lè fait d 'a j o u t e r des f e m e l l e s s t é r i l e s joue un r ô le im portan t dans la dim inution de la f e r t i l i t é . L e m ê m e phénom ène a été m is en é v id e n ce par H u sse iny et M ad sen [7] su r P a r a m y e l o i s t r a n s i t e l la . P a r c o n t r e . P r o v e r b s et Newton [8], sur C a r p o c a p s a p o m o n e l la , indiquent q u ' i l para ît p r é fé r a b le de ne pas lâ c h e r de f e m e l l e s s t é r i l e s . D 'a u tre part , dans n otre e s s a i le fait de lâ c h e r seu lem en t des f e m e l le s s t é r i l e s (0 :25 :5 :5 ) ne p ro vo q u e pas r é g u l iè r e m e n t la s t é r i l i t é , c o n t ra ire m e n t aux r é su lta ts obtenus par E lb a d ry [9] sur G n o r im o s c h e m a o p e r cu le l la .

C e c i m on tre que le s r é su lta ts de m é la n g e s de populations s t é r i l e s et n o r m a le s peuvent ê tre t r è s d i f fé re n ts d 'un in se c te à l 'a u tr e . C e s d i f f é r e n c e s peuvent ê tre l i é e s à d ' im p o r ta n te s var ia t io n s dans le c o m p o r te m e n t , surtout dans l e s con d it ion s t r è s a r t i f i c i e l l e s du l a b o r a t o i r e . E l l e s ne constituent que des in d ica t ion s et doivent ê tre r e p r i s e s dans d e s cond it ion s au ss i p r o c h e s que p o s s ib le des c o n d it ion s n a tu re l le s .

R E M E R C I E M E N T S

L 'a u te u r r e m e r c i e M o n s ie u r M .F é r o n , D ir e c te u r de la Station, qui lui a o f fe r t dans s e s la b o r a t o i r e s de r e c h e r c h e s tou tes l e s fa c i l i t é s d é s i r a b le s pou r m e n e r à bien un tra v a i l s c ien t i f iqu e , et qui a bien voulu c o n s a c r e r une part ie de son te m p s à l 'é tu d e c r it iq u e de c e tra v a i l .

R E F E R E N C E S

[ 1 ] WALKER, J .R ., BRINDLEY. A . T . , J. e con . Ent. 56 (1963) 52 2 -2 5 .[ 2 ] SUN, S . , LEE, W ., A cta ent. sln. 14 (1965) 4 2 3 -3 1 .[ 3 ] GUTHRIE, W .D . , RAUN, E .S ., DICKE, F. F . , PESHO, G .R ., CARTER, S .W ., Iowa St. J. S c i. 40

(1965) 6 5 -8 3 ,[ 4 ] BAROUGHI-BONAB, H ., Etude du développem ent post-em bryonriaiie de l1 ova ire ch e z Ephestia

kühniella Z . (Lépidoptère Pyralidae), Effets des radiations ionisantes, Thèse de D octeur - ingénieur, U niversité de Paris (1965) 65p.

[ 5 ] PROVERBS, M .D . , NEWTON, J. R . , Can. J. Z o o l . 40 (1962) 40 1 -2 0 .[ 6 ] VASILY AN, V. V . , Ent. O bozr. 39 (1960) 5 99 -60 4 .[ 7 ] HUSSEINY, M .M ., MADSEN, H .F ., H ilgardia 36 (1964) 113 -37 .[ 8 ] PROVERBS, M .D . , NEWTON, J .R . , Can. Em. 9 4 (1 9 6 2 ) 116 2-7 0 .[ 9 ] ELBADRY, E ., J. e con . Ent. 57 (1964 ) 4 1 4 -1 5 .

S M -102/15 147

D I S C U S S I O N

R . C A V A L L O R O : Could you p le a se g ive us the r e c ip e f o r the sy n thetic m e d iu m on which you r e a r e d y o u r О . nub ila lis la rv a e ?

M . A N W A R : C e r ta in ly ; the m e d iu m used c o n s is t e d o f the fo l low in g :

D is t i l le d w ater 170 c m 3 C o r n oil 30. 4 cm

A g a r agar 5 g W e s so n m in e r a l sa lts 2 gC e l lu l o s e 6 g B r e w e r 1 s yeast 6 gWheat g e r m 10 g A s c o r b i c ac id 1 gC a se in p r o te o ly s a te 7 g B a lan ced v itam ins 4 gG lu c o s e 3 .5 g B e n z o ic ac id 0. 25 gC h o le s te r o l 0 .6 g Nipagin 0 .2 0 g

R . C . VON B O R S T E L : Is the s p e c i e s you studied h e re id en tica l to that studied by W a lk e r ( R e f e r e n c e [1] in yo u r p aper) and do you get the sa m e d o s e - e f f e c t c u r v e s f o r s p e r m ir ra d ia t ion ?

M. A N W A R : W e used the s a m e s p e c i e s (O str in ia n ub ila lis Hb. ) but W a lk e r 's s t e r i l i z in g d o s e was 32 krad o f X - r a y s , a d m in is te re d to the adult in se c t , w h e r e a s o u r s was 40 krad o f g a m m a rad ia t ion , a d m in is te re d to the m a tu re pupae.

R . C . VON B O R S T E L : I understand you used f iv e p a ir s o f m oths in the b reed in g tubes in each c a s e . It would a p p e a r that the p a ir m atings w e r e not w holly s u c c e s s f u l . I w o n d e r w hether one o f the f e m a le s in your m a s s m atin gs did not m a te . If such a v irg in fe m a le then la id h er eggs th is would giv.e an egg hatchability o f 80% in the c o n t r o l e x p e r im e n t . C o r r e s p o n d in g r e d u c t io n s would a l s o o c c u r in the ir ra d ia te d g ro u p s .

M. A N W A R : W e a lw ays obtained o v e r 90% mating , in c o n t ro l as well as in- s t e r i le in s e c t s .

I. A . KANSU: In y o u r w ork on O. n ub ila lis and S_. ex igua why did you i l lum in e the la b o r a to r y f o r 18 h ours a day? I a lw ays thought that these two s p e c i e s w e re l e s s a c t ive in the light.

M . A N W A R : W e u s e d f l u o r e s c e n t t u b e s f o r 18 h o u r s a d a y s i m p l y t op r e v e n t d i a p a u s e o f t h e l a r v a e .

I. A . KANSU: Y ou found a l i f e - s p a n o f 2 0 .8 days f o r the m a le and20. 6 days f o r the fe m a le o f the s p e c i e s in th is w ork . I w as under the i m p r e s s i o n that the f e m a le l iv ed the lo n g e r . I n o t ice that T a b le II a lso sh ow s a l i f e - s p a n o f 27. 3 days f o r the m a le . Cou ld you p le a s e e la bora te on th ese r e s u l t s ? ,

M. A N W A R : M y e x p e r im e n ta l w ork has n e v e r r e v e a le d a s ign if icant d i f f e r e n c e in lo n g e v ity o f the m a le and o f the f e m a le , but I did o b s e r v e that those m a le s w hich m ated on ly a few t im e s l iv e d lo n g e r than those which m ated m o r e often.

K .K . NAIR: Is th e r e any e v id e n ce f r o m y o u r stud ies to support the v iew that a f i r s t m ating is m o r e im portan t than subsequent m atin gs? I r a i s e th is point b e c a u s e it has been shown in s o m e s p e c ie s that if a n o rm a l f e m a le is m ated to an ir ra d ia te d m a le and then m ated to a n o rm a l m a le m o s t o f the e g gs a r e found to be s t e r i le .

M . A N W A R : I am a fra id I have not studied this point.B . R . L E R O I: F o r y o u r e x p e r im e n t on m ix in g tog e th e r n o rm a l and

s t e r i le m a le s and f e m a le s (T a b le IV) have you any r e s u l t s f o r a fte r the f i r s t w eek o f o v ip o s i t io n ? I am try in g to a s c e r ta in w hether when n o rm a l

148 ANWAR

f e m a le s show no in d icat ion o f fe r t i l i ty th is is an inherent in capab il i ty o r whether it is s im p ly due to a t im e - la g in the p h y s io lo g ic a l p r o c e s s in vo lved .

¡VI. A N W AR: No, I o b s e r v e d this in se c t f o r a p e r io d o f one w eek only. T he fe m a le m oths la id m o s t o f th e ir e g gs within the f i r s t w eek a fter being put in the ca g e s .

C . A . P E L E R E N T S : Do the v irg in f e m a le s o v ip o s i t and a re th e re any f e a tu re s w hereby it is p o s s ib le to d is t inguish between the s e x e s at the la r v a l and pupal stages?

M. A N W AR: Our r e s u lt s ind icated that v irg in f e m a le s cou ld lay up to 429 e g g s . We did not d if ferent ia te the s e x e s at the la rv a l stage , b e fo r e ir ra d ia t ion , but this was done on the pupae by exam ining the u n d e r s id e o f the abdom en with a m agn ify ing g la s s . In this c a s e , as f o r many s p e c i e s o f L e p id o p te r a , the p os it ion and a p p e a ra n ce o f the genital o r i f i c e is d i f fe ren t fo r the m ale and f e m a le .

B. NAGY: Could you g ive any m o r e d e ta i ls as to the stage o f d e v e lo p m ent o f th ose e m b r y o s which died in the egg? I should be in te re s te d to le a rn whether they died in an ear ly o r late stage o f th e ir e m b r y o n ic l i f e .

M. A N W A R : We ir ra d ia te d the m a le m oths at the pupal stage with s t e r i l i z in g d o s e s o f up to 40 krad , and found that th ere w as e m b ry o n ic de v e lo p m e n t up to the 'b la c k h ead1 stage in about 90% of the e g gs la id by the f e m a le s m ated with them . H o w e v e r , these la rv a e cou ld not hatch out of the she ll and eventually died .

K. NAGY: Do you think it will be f e a s ib le to c o n t ro l the E u ro p e a n c o rn b o r e r by m eans o f the s t e r i le -m a le technique? One has to bea r in mind that this s p e c ie s is v e ry w ide ly d is tr ibu ted and the adults can fly many m i le s in one night during the flight stage , e s p e c ia l ly in s t o r m y , windy w eather and this m ay contr ib u te s ign if ican t ly to d i s p e r s a l o f the adults .

M . A N W A R ; M y l a b o r a t o r y r e s u l t s , s e t out in T a b l e I V , showed' c l e a r ly that s t e r i le in s e c ts a r e c o m p e t i t iv e with n o rm a l in s e c ts . It is qu ite p ro b a b le that they w ill c o m p e te in the f ie ld as w ell .

V . L A B R Y R IE : T he se co n d set o f r e s u lt s in T a b le IV o f y o u r pa p ersh ow s only one g rou p o f f e m a le s out o f the fou r as lay ing e g g s . D o e s this m ean that the f ive f e m a le s o f each g rou p re m a in e d v irg in s and hence that the n o r m a l m a le s m ust have s e le c t e d the s t e r i le f e m a le s , o r that the fe m a le s m ated with s t e r i le m a le s ? Did you in vestigate f o r s p e r m a to p h o r e s in s id e the fe m a le s ?

M . A N W A R : T he adults o f O. n ub ila lis a r e capab le o f mating f r o m the f i r s t two days a fter hatching. I studied th ese in s e c t s f o r sev en days and I do not think that the f e m a le s rem a in e d unmated f o r as long as that. I did not, h o w e v e r , d i s s e c t the f e m a le s to look f o r s p e r m a to p h o r e s .

IN FLUEN CE OF DIAPAUSE ON THE RADIOSENSITIVITY OF KHAPRA B E E T L E LAR VAE

G .W . RAHALKAR AND K .K . N A IR*

BIOLOGY DIVISION,BHABHA ATOM IC CENTRE,

TROMBAY, BOMBAY, INDIA

Abstract

INFLUENCE OF DIAPAUSE ON THE RADIOSENSITIVITY OF KHAPRA BEETLE LARVAE. Studies to determ ine the effects o f varying periods o f diapause on the radiosensitivity o f the larvae o f TroftQderma granariuin showed p ractica lly no m orta lity in the irradiated larvae during diapause, but only after the diapause was broken at 3 8 °C . As the ca lcu la ted LD50S for 10, 20 and 30 days o f post-irradiation diapause were not sign ificantly d ifferent, it was also evident that increases in the length o f diapause tim e had no significant e ffe c t on post-diapause survival t im e . On the other hand, pupation seem ed to be in fluenced by tile duration o f diapause in the irradiated larvae . This e f fe c t was best d iscern ib le ar low doses. The s ign ifican ce o f these findings is discussed.


In a p r e v io u s c o m m u n ica t io n [1 ] , the authors r e p o r t e d that when the la rv a e o f the K hapra be e t le , T r o g o d e r m a g ra n a r iu m Piverts, w e re ir ra d ia te d with 5 and 8 krad and m aintained in a d iapau sing state, m o r ta l i ty w as r e m a r k a b ly low', and that it m a n ife s te d i t s e l f a fte r the d iapause was brok en by r a is in g the am bient te m p e ra tu re to 38°C. F u r th e r stud ies w e re cond u cted with the sa m e s p e c ie s to d e te rm in e w h eth er i n c r e a s e s in d iapause t im e at a m uch l o w e r te m p e ra tu re (20°C), a fte r i r ra d ia t io n with v a r io u s d o s e s o f g a m m a rad ia tion , would in f lu ence its radiation s e n s i t iv i ty with r e s p e c t to la r v a l m o r ta l i t y and pupation.

2. M A T E R IA L S AMD M ETHODS

The la rv a e o f the K hapra bee t le used in th ese stud ies w e re c o l le c t e d at ran dom f r o m a la b o r a t o r y cu ltu re o f 5th and 6th in s ta r la rv a e m aintained at 30° ± 2 d eg C . T h e y w o re ir ra d ia te d with d o s e s ranging f r o m 5 to 1 8 krad in a c o b a l t -6 0 s o u r c e at a d o s e rate o f 1.5 X .10° k r a d /h . A f t e r ir ra d ia t io n the la r v a e w e r e se p a r a te d into three g ro u p s and kept at 20° ± 0.5 degC with c ru sh e d wheat as fo o d . A fter 1.0, 20 and 30 days at this te m p e ra tu re the d iapau se was bro k e n by t r a n s fe r r in g the la rv ae to 38° ± 1 degC and they w e r e m ainta ined at this te m p e ra tu re t i l l the end o f the e x p e r im e n t . F ou r r e p l i c a t e s o f 50 la rv a e each w e r e used f o r each i r r a d ia t i o n d o s e le v e l . The sa m e n u m b e r o f r e p l i c a t e s o f 50 la rv a e each w e re s im u lta n e o u s ly kept as c o n t r o l . L a r v a l m o r ta l i t y and pupation in

* Present address: Pestology C enter, D ept, o f B io log ica l .Sciences, S im on Fraser University, Burnaby, B .C . , Canada.

149

150 RAHALKAR and NAIR

the v a r io u s g rou ps w e re r e c o r d e d at da i ly in te r v a ls . T he data on the cum ula t ive m o r ta l i t y a fte r 10 days o f post d iapau se w e re su b je c te d to p ro b it an a lys is f o r the ca lcu la t io n o f L D 50. The la r v a l m o r ta l i t y that o c c u r r e d during d iapause was not c o n s id e r e d in ca lcu la t in g L D 50.

3. R E SU L T S AND DISCUSSION

It is ev ident f r o m the r e su lts that p r a c t i c a l ly no m o r ta l i t y o c c u r r e d in the ir ra d ia te d la rv a e du ring d iapause at 20°C (F ig s 1 -3 ) . Radiation d am age m a n ifested i t s e l f on ly a fter the d iapause was brok en at 38°C.The ca lcu la te d L D 50 value's f o r 10, 20 and 30 days o f d iapause w e re 16.22, 16.03 and 16.9 krad, r e s p e c t iv e ly . As th ese values did not d i f fe r s ign if ican t ly , it was a lso ev ident that i n c r e a s e s in the length o f d iapause t im e had no s ign if ican t e f f e c t on p o s t -d ia p a u se su rv iv a l t im e (T a b le I).

t im e С d!)FIG. 1. Effect o f 10 days' post-irradiation diapause on larval m ortality ■ = 5 krad; Л = tí krad; * = 8 krad; □ = 10 krad; A - 14 krad; 0 = 18 krad

TIME Cd )FIG. 2 . Effect o f 20 days ’ post-irradiation diapause on larval m ortality

S M - 1 0 2/ 16 151

TiMECd)FIG. 3. Effect o f 30 days' post-irradiation diapause on larval m orta lity

T A B L E I. E F F E C T O F D U R A T I O N O F P O S T - I R R A D I A T I O N D IA P A U S E O N T H E L D 50(10 D A Y S ) F O R T H E L A R V A E O F T r o g o d e r m a g r a n a r i u m E V E R T S

P ost-irrad ia tion d iapau se in days

H eterog en e ity R egression eq u a tion LD5û ( krad) ± S .E .

F id u cia l lim its o f

LDjo w ith 95% c o n f i d e n ce

1 0 X2(5) = 28.1 Y = 2 .0 1 9 + 2 .4 6 4 X , 1 6 .2 2 ± 0 .0 6 .1 5 .0 8 0

1 7 .3 6 0

2 0 X2( 5 ) = 7 .2 Y = 1 .4 6 9 + 2 .9 3 X . 1 6 .0 3 ± 0 .0 2 1 4 .9 3 5

1 7 .1 2 5

30 " X * ( 5 ) = 1 5 .0 Y = 0 .4 8 9 + 2 .6 7 6 X 1 6 .9 ± 0 .0 1 7 . 1 5 .8 9 6

1 7 .0 0 4

Y = Probit k ill ; X = Lg d ose ; LD50 = R a d ia tion d ose c a lcu la te d to g iv e 50% k i l l .

B u rg e s [2 J studied the b e h a v io u r o f d iapau sing and n o n -d ia p a u s in g la rv a e o f T r o g o d e r m a g r a n a r iu m . He o b s e r v e d that a d o rm a n t la rv a spent lon g p e r io d s without fe e d in g o r m o v in g and that its rate o f r e sp ir a t io n w as p ro b a b ly at a b a sa l l e v e l . H o w e v e r , when thè te m p e ra tu re b e c a m e favo u ra b le , the la rv a e m e r g e d f r o m the d orm a n t state, with a c onsequ ent in c r e a s e in its ra te o f r e s p ir a t io n , which w as about two to s ix t im e s that o f a dorm a nt la rv a . T he in v e r s e re la t io n sh ip betw een su r v iv a l o f an i r ra d ia te d o r g a n is m and its m e ta b o l i c rate is w e l l known [ 3 ] . In the d iapau sing state, rad iation da m ag e r e m a in s latent and e x p r e s s e s i t s e l f on ly when the m e ta b o l ic ra te is a c c e le r a t e d b y b re a k in g the d iapause .T h is exp la ins the a b se n ce o f any p r o te c t iv e e f f e c t b y the in terven in g


diapause . A s im i la r e f f e c t was a lso o b s e r v e d in h ibernating s q u i r r e l s [4] and m a r m o ts [ 5 ] .

Pupation o c c u r r e d on ly a fte r the d iapause was brok en and o c c u r r e d m o s t ly within the f i r s t 10 days o f p o s t -d ia p a u se p e r io d . Although it was o b s e r v e d at d o s e s o f 10 krad and be low , adult e m e r g e n c e was seen on ly up to 6 krad, in d icat ing that at 8 and 10 krad death o c c u r r e d in the pupal stage (T a b le II).

T A B L E II. E F F E C T O F P O S T - I R R A D I A T I O N D IA P A U S E ON P E R C E N T A G E S O F A D U L T E M E R G E N C E ( T r o g o d e r m a g r a n a r i u m ) F R O M P U P A E F O R M E D

D ose (krad) Post- irrad iation d iapau se (days)

1 0 20 30

0 96 . 3 9 8 .9 9 9 .5

5 8 3 .7 8 5 .5 8 8 .3

6 56 . 6 6 3 .3 5 8 .2

8 0 . 0 0 . 0 0 . 0

1 0 0 . 0 0 .Û 0 . 0

The duration o f d iapause a lone had no s ign if ican t e f f e c t on the n u m b e r pupated in the n o n - i r r a d ia te d c o n t ro l . Pupation was 94.5, 94 .0 and 92.0%, r e s p e c t iv e ly , when the d iapau se la sted f o r 10, 20 and 30 days . H o w e ve r , pupation in the ir ra d ia te d g ro u p s was in f lu en ced , not on ly by the rad iation d ose , but a lso by the duration o f p o s t - i r r a d ia t i o n d iapause (F ig . 4).A n a ly s is o f v a r ia n ce o f the data on pupation in the ir ra d ia te d g ro u p s su b je c te d to d i f fe ren t p o s t - i r r a d ia t io n d iapause p e r io d s show ed that the d i f f e r e n c e s o b s e r v e d in pupation w e r e s ign if ica n t at 1% le v e l . T h e F value f o r in te ra c t io n betw een rad ia tion and d iapause is 6 .1 7 * * . It was o b s e r v e d that the n u m b e r pupating in the ir ra d ia te d g ro u p s tended to d e c r e a s e with in c r e a s e s in d iapause t im e . T h is su g ge s te d in c r e a s e d rad ia t ion d am age in those su b je c te d to the lo n g e r p o s t - i r r a d ia t io n d iapau se . In o u r e a r l i e r stud ies ( l o c . c i t . ) it w as o b s e r v e d that when the i r ra d ia te d la rv a e w e r e m ainta ined cont inu ou sly at 38 °C, pupation o c c u r r e d up to a d o s e o f 12 krad, while in the p r e se n t stud ies it o c c u r r e d on ly up to a d o s e o f 10 krad when a p o s t - i r r a d ia t i o n d iapause in terven ed . T h e se r e s u l t s su g ges ted a m o r e rap id p r o g r e s s io n o f rad iation d am age in the la rv a e s u b je c te d to d iapause at 20°C than in th ose m ainta ined without a d ia pau se . T he fa c to r that con tr ib u ted to this a c c e le r a t io n o f d am àge c o u ld be due to the h igh er m e ta b o l i c ra te o f the la rv a e p re co n d it io n e d to a te m p e ra tu re o f 20°C. The rates o f oxygen co n su m p tion o f the la rv a e r e a r e d at 38°C and those o f the la rv a e in which d iapause was induced at 20°C f o r 10 days w e re d e te rm in e d in a W a rb u rg ap paratus at 38°C. T he oxygen co n su m p tion o f the f o r m e r w as 160 n l/ m g p e r hour.

= very significant

S M - 102/16 153

I o o

DO SECkrad)FIG. 4. E ffect o f 10 (o ), 20 (•) and 30 (A) days' post-irradiation diapause on pupation

w hile that o f the la t te r w as 203 ju l /m g p e r h ou r . T h is d i f f e r e n c e was s ta t is t ica l ly s ign if ican t (P = 0 .01) . The in c r e a s e d rate o f oxygen con su m p tion in d ica ted in c r e a s e d m e t a b o l i c rate which w ould then re su lt in a m o r e rap id p r o g r e s s i o n o f the da m ag e in the i r r a d ia te d la r v a e .T h is cou ld accoun t f o r the ab se n ce o f pupation above 1 0 -k ra d l e v e l when a p o s t - i r r a d ia t io n d iapause in terven ed . The sa m e m e ta b o l i c e f f e c t cou ld a lso explain the d am age d i f fe re n t ia l o b s e r v e d when the p o s t - i r r a d ia t i o n d iapause t im e w as in c r e a s e d . W i l l ia m s [6] studied the e f fe c t s o f g raded e x p o s u r e s o f the d iapausing pupae o f C e c r o p ia s i lk w o r m to 6°C on the in itiat ion o f adult d e v e lo p m e n t a fte r t r a n s fe r r in g them to 25°C. He o b s e r v e d that f o r 5, 10 and 20 w eek s o f d iapause the rate o f de v e lo p m e n t in c r e a s e d m a rk e d ly with the length o f d iapause t im e . T h is su g ges ted an a c c e le r a te d m e ta b o l ic ra te a c c o r d in g to the length o f p r i o r ch i l l in g .Though the authors did not d e te rm in e the ox yg en con su m p tion o f the K hapra bee t le la rv a e su b je c te d to d iapause f o r 20 and 30 days , th e ir r e su lt s f o r the 1 0 -day d iapause p e r io d and th ose o f W il l ia m s ( l o c . cit .) show ed that the m e ta b o l ic ra te w as c o n s id e r a b ly in f lu en ced by the d u ra tion o f d iapause in the c o ld . L a r v a e which had a lo n g e r p e r io d o f d iapause sh ow ed a h igh er m e ta b o l ic rate when the d iapause w as bro k e n . A s the t im e o f s u r v iv a l o f an ir ra d ia te d o r g a n is m is in v e r s e ly p r o p o r t io n a l to the m e t a b o l i c rate , the radiation dam age p r o g r e s s e d m o r e rap id ly in the la rv a e su b je c te d to lo n g e r p o s t - i r r a d ia t i o n d iapause than in th ose e x p o se d f o r a sh o r te r p e r io d . T h is ex p la ined the p r o g r e s s iv e d e c r e a s e in pupation a c c o r d in g to the in c r e a s e in the length o f the p o s t - i r r a d ia t i o n d iapause .

Since high d o s e s o f rad ia t ion tended to l im it pupation, the in flu ence o f p o s t - i r r a d ia t io n d iapause w as bes t d is c e r n ib le at l o w e r d o s e s . At10 krad th is e f fe c t a p p eared m o r e o r l e s s m a sk e d by the e f f e c t o f the rad iation d o s e i t s e l f . T h is , apparently , was the r e a s o n why th ere w as no in flu ence o f the p o s t - i r r a d ia t io n d iapause on the L D 50 f o r la rv a l m o r ta l ity , as no pupation o c c u r r e d at this d o s e le v e l (16 krad).


R E F E R E N C E S

[1 ] NAIR, K .K . , RAHALKAR, G .W . , in Radiation and Radioisotopes applied to insects o f Agricultural im p ortan ce , IAEA, V ienna(19G 3) 4 6 5 -7 7 .

[2 ] BURGES, H .D ., I . Insect physiol. 5 (1960) 317 .[3 ] BACO, Z . M . , ALEXANDER, P . , Fundamentals o f R a d iob io logy , Pergamon Press, New York

(1961) 3.[4 ] DOULL, J . , DUBOIS, K .P . , P roc. S o c . ex p . B iol. M ed . 84 ( 1953) .367.[5] SM ITH , F . , GRENAN, M . M . , S c ien ce 113 (1951 ) 6 S6 . _[ 6] WILLIAMS. C .M . , B iol. Bull, l l j ) (1956 ) 201 .

D I S C U S S I O N

H. E R D M A N : Have you tr ie d m o d ify in g the ga se o u s en v iron m en t, f o r in stan ce by u s in g n itrogen in stead o f a ir, during rad ia t ion e x p o s u re and a f te rw a rd s in o r d e r to d e te rm in e the e f f e c t o f d if fe ren t a tm o s p h e r e s on d iapause and r a d io s e n s it iv i tv ?

G .W . R A H A L K A R : No, we have not.I. A . KANSU: Did you o b s e r v e any d iapause at the e a r ly la r v a l stage

in the K hapra bee t le ?G. W. R A H A L K A R : Diapause in this in s e c t i s in f lu enced by

e n v iro n m e n ta l f a c t o r s and is not p e c u l ia r to any p a rt icu la r dev e lo p m e n ta l stage .

E F F E T S DES RADIATIONS GAM M A SUR L A F E R T IL IT E ET L A LO N G EVITE DES COLONIES DE Dolichoderus quadripunctatus(H Y M E N O P T E R E : FORM ICOID EA DOLICHODERIDAE)

C. TOROSSIAN FACULTE DES SCIENCES.UNIVERSITE DE TOULOUSE ET

• R. CAUSSEINRA, STA TIO N DE ZOOLOGIE,CENTRE DE RECHERCHES AGRONOMIQUES DU SU D -E ST ,M ONTFAVET, FRANCE


THE EFFECT OF G AM M A RADIATION ON THE FERTILITY AND LONGEVITY OF D olichoderus quadripunctatus (HYMENOPTERA: FORMICOIDEA DOLICHODERIDAE). The authors siudied on a batch o f 27 co lon ies o f the ant D olichoderus quadripunctatus the e f fe c t on lon gev ity and fe rt ility o f gam m a irradiation at doses betw een 12 500 and 150 000 rad. The results are presented in the paper. For c o lon ies consisting so le ly o f workers, the m orta lity curve is linear w ith dosesbetw een 150 000 and 50 000 rad. The LD50 is reached in 4 d a y s fo r the 150 000-rad dose and 16 days for the 50 000-rad dose. The lowest dose used in the experim ents (12 500 rad) produced an LD50 after 18 days. For the con trol co lo n ie s , the LD50 occurred within about thirty days.

Fertility , which is norm ally regular and constant in c o lon ies o f workers o f D olichoderus quadripunctatus is expressed by the R .F . ratio [R .F . - reproduction fa ctor = (num ber o f èg g s^ n u m b e r o f workers at the beginning o f the e x p er im en t)]. The R. F. o f the con tro l (unirradiated) c o lon ies was about 1 .6 0 , w hile that o f the irradiated co lon ies ranged from 0 to 0 ,0 6 . These extrem ely low values showed that to a ll intents and purposes the treated co lon ies w ere m ade sterile. This sterility was fin a l, no laying being observed in the irradiated co lo n ie s , by the tim e they died out.

In co lon ies with a queen, on ly the queens were irradiated, with gam m a doses o f 50 000 , 100 000. and 150 000 rad. The results obtained for lon gev ity showed in every case a considerably higher resistance o f the fem ales than the workers under the sam e cond itions. W here fertility is con cern ed , the irradiated fem a les showed a considerable and alm ost im m ed ia te reduction in egg laying. This was fo llow ed in the c o lon ies by a resumption o f laying by the workers, s ince the queen cou ld no longer inhibit this. The fertility o f the irradiated fem ales subsequently dim inished, and b eca m e nil from the eighteenth day.The queens did not theieafter resume laying.

EFFETS DES RADIATIONS G AM M A SUR LA FERTILITE ET LA LONGEVITE DES COLONIES DE D olichoderus quadripunctatus (HYMENOPTERE: FORMICOIDEA DOLICHODERIDÆ). Les auteurs on étudié sur un lot de 27 co lon ies (a v e c ou sans reine) d e la fourm i D olichoderus quadripunctatus les effets , sur la lon gévité et la fertilité , d ’ irradiations gam m a à des valeurs com prises entre 12 500 et 150 000 rad; les résultats obtenus sont donnés dans le m ém oire.

Dans le cas des co lon ies exclu sivem en t com posées d 'ou vrières la courbe d e m orta lité est linéa ire pour des valeurs com prises entre 150 000 et 50 000 rad. La DL 50 est atteinte en 4 jours pour la dose de 150 000 rad, et 16 jours pour la dose de 50 000 rad. Aux valeurs les plus fa ib le s (1 2 500 rad) la DL 50 s ’ obtient après 18 jours. Pour les c o lon ies tém oins la D L50 s 'ob tien t en 30 jours environ. La fé con d ité , qui est habituellem ent régulière et constante dans les c o lon ies d 'o u v riè res , est exprim ée par le rapport QR = quotient re p ro d u c te u r (nom bre d 'œ u fs/n om bre d ’ouvrières au début de l 'e x p é r ie n c e ). Le QR des co lon ies tém oins (n ' ayant subi aucune irradiation) a pour valeur 1 ,6 0 environ . Le QR des co lon ies traitées varie entre 0 et 0 ,0 6 . Ces valeurs extrêm em en t fa ib les montrent que les c o lon ies traitées sont devenues

155

156 TOROSSIAN et CAUSSE

pratiquement stériles. C ette stérilité est d é fin itive , car ju sq u 'à l 'e x t in c tio n de la co lo n ie , aucune ponte n ' a pu être observée dans les c o lon ies irradiées.

Dans les co lon ies avec reine, seules les reines subissent une irradiation gam m a, aux valeurs de 50 000, 100 000 et 150 000 rad. Les résultats obtenus concernant la lon gév ité montrent dans tous les cas une résistance considérablem ent plus é le v é e des fem e lle s que pour les ouvrières p lacées dans les m êm es conditions. Pour c e qui est de la fé con d ité , on observe ch e z les reines irradiées un ralentissement considérable et quasi im m édiat de leur ponte. C e ralentissem ent est suivi dans les co lon ies par une reprise de la ponte des ouvrières: la reine ne peut plus exercer l 'in h ib itio n de leur ponte. Par la suite, lafécon d ité des reines irradiées d im inue et devient nulle 3 partir du 18e jour. Il n 'y a plus de reprise de la ponte des reines par la suite.

INTRODUCTION

D o l ic h o d e r u s quadripunctatus est une f o u r m i a r b o r i c o l e que l ' o n r e n c o n t r e c o u r a m m e n t dans l e s f o r ê ts de chêne de la r é g io n T o u lo u sa in e . E l le constitue des c o lo n ie s p o ly c a l iq u e s . On r e n c o n t re e s s e n t ie l l e m e n t deux typ es de c o lo n i e s : l e s c o lo n ie s e x c lu s iv e m e n t c o m p o s é e s par des o u v r i è r e s , et le s c o lo n ie s c o m p o s é e s par des o u v r iè r e s a c c o m p a g n é e s d 1 une re in e [ 1 , 2 ] .

L o r s q u ' e l l e s évoluent sans re la t io n s e n tre s e l le s , l e s c o lo n ie s du p r e m ie r type ( c o lo n ie s d ' o u v r i è r e s ) pondent abondam m ent, et l e s œ u fs donnent n a is sa n c e a des m â le s . L e s c o lo n i e s du se co n d type ( c o lo n ie s d ' o u v r i è r e s avec une r e in e ) se c a r a c t é r i s e n t au c o n t r a ir e par une ponte e x c lu s iv e de la r e in e . L es o u v r iè r e s en p r é s e n c e de la re in e ne pondent ja m a is [1, 3, 4]. L e s oeufs de re in e donnent n a is sa n ce , so i t à des o u v r i è r e s ( ca s g é n éra l) , so i t à d 'a u t r e s r e in e s (dans des c ond it ion s p a r t i c u l i è r e s que nous avons dé f in ies et e x p o s é e s dans de p r é cé d e n ts m é m o i r e s [5]) .

L e p r é se n t t ra v a i l e st c o n s a c r é a l ' é t u d e de 1' action d e s rad ia t ion s g am m a su r c e s deux types de c o lo n ie s .

C ondit ions de l ' e x p é r i e n c e

V in g t -s e p t co lo n ie s c o n s t itu é e s par des populations d ' o u v r i è r e s c o m p r i s e s en tre 107 et 302 o u v r iè r e s , avec et sans r e in e , sont r é c o l t é e s au s o r t i r de l 'h ib e r n a t io n n atu re lle . Un p r e m ie r lot T de neuf c o lo n i e s (s ix av e c re in e , t r o i s sans r e in e s ) con st itu e le lot tém oin .

Un d e u x iè m e lo t de 18 c o lo n ie s , dont 12 e x c lu s iv e m e n t c o m p o s é e s d ' o u v r i è r e s , constitue le lo t tra ité .

L e s d o s e s de ray on n em en t g am m a r e ç u e s par le s c o lo n i e s d ' o u v r i è r e s sont de 12 500, 25 000, 50 000, 75 000, 100 000 et 150 000 rad.

L e s r e in e s du lo t t ra i té (des co lo n ie s avec fe m e l le ) , r e ç o iv e n t r e s p e c t iv e m e n t 50 000, 75 000 et 100 000 rad.

L e s ir ra d ia t io n s ont été e f fe c tu é e s a v e c un ir ra d ia te u r au 60 Со de 2 000 Ci, au S e r v ice de r a d io a g r o n o m ie du CEN de C a da ra ch e . L e déb it de d o s e a été de 1 040 r a d /m in , c o n t r ô lé par une d o s im é t r ie au su lfa te f e r r e u x .

1. COLONIES E X C L U S IV E M E N T CO M P O SEES D 'O U V R IE R E S

1 .1 . Etude de la m o r ta l it é

L e tableau I ré ca p itu le l e s ré su lta ts que nous avons obtenus. Nous avons c o n s id é r é la d o s e lé ta le 100% (D L 1 0 0 , tem p s à p a rt ir duquel toutes

S M -1 0 2 A 7 157

l e s o u v r i è r e s de la c o lo n ie sont m o r t e s ) . E nsuite , à p a rt ir de c e tableau , nous avons t r a c é le s c o u r b e s des f ig u r e s 1 et 2, qui r e p ré s e n te n t grap h iqu em en t le s r é su lta ts p r é cé d e n ts . La f ig u r e 1 e x p r im e la m o r ta l i t é (D L 50) en fo n ct io n de la d o s e de ra y on n em en t g am m a r e ç u e en une seu le e x p o s it io n . L a f ig u re 2 r e p r é s e n t e la m o r ta l i t é des c o lo n i e s d ' o u v r i è r e s en co n s id é ra n t la D L 50 et la D L 100.

D i s c u s s i o n

L a f ig u re 1 m o n tr e une c o u r b e prat iqu em en t l in é a ire en tre l e s points B et C. La m o r ta l i té e s t donc e x a c te m e n t p r o p o r t io n n e l le à la d o s e de ray on n em en t r e ç u e en tre le s v a le u r s de 50 000 rad et 150 000 rad . A u - d e s s o u s de c e s v a le u rs , c ' e s t - à - d i r e de 12 500 rad à 50 000 rad (partie A B de la c o u r b e ) , la m o r ta l i t é p a ra ît p r o p o r t io n n e l le m e n t plus im portante lo r s q u e le s d o s e s d é c r o i s s e n t . (Une s é r i e d ' e x p é r i e n c e s pour le s v a le u rs in fé r ie u r e s à 12 500 rad e s t en c o u r s d 'é tu d e . )

L a f ig u re 2 e s t t r a c é e pour é tu d ier le s v a r ia t io n s p o s s ib le s de la m o r ta l i t é entre le début et la f in de l ' e x p é r i e n c e . Nous avons c o n s id é r é le r a p p o r t (D L 100 - D L 5 0 ) / D L 5 0 . T ou s le s points du graphique étant s itu és a u -d e s s u s de la l igne p o in t i l lée m arquant le r a p p o r t 1, on peut en c o n c lu r e que la lon gév ité e s t p r o p o r t io n e l le m e n t plus longue lo r s q u e la p r e m iè r e m o i t ié de la population a d isp aru .

1 .2 . Etude de la fé co n d i té

Il e s t b ien connu, à la su ite des travaux de d iv e r s auteurs, notam m ent Cole et al. [6], Narayanan et al. [7], D ru m m on d [8] et H ennebery [9], que, o u tre s e s e f fe ts su r la lo n gé v ité , l ' i r ra d ia t io n g am m a, à des d o s e s v a r ia b le s se lo n le s e s p è c e s , m o d i f ie p ro fo n d é m e n t la fé con d ité des indiv idus i r r a d ié s , et la su p p r im e to ta lem ent a u -d e là de c e r t a in e s v a le u rs .

Nous avons é le v é l e s c o lo n ie s p r é cé d e n te s et avons, dans le m ê m e te m p s , étudié le u r fé co n d i té en dén om b ra nt Íes œ u fs pondus par le s o u v r iè r e s i r r a d ié e s et en suivant le u r d even ir .

P o u r e x p r i m e r plus c o m m o d é m e n t la fé co n d ité d 'u n e c o lo n ie nous con v en on s d ' é t u d ie r le r a p p o r t QR, c ' e s t - à - d i r e le quotient r e p r o d u c teu r (QR = n o m b r e d 'œ u f s p o n d u s /n o m b r e d ' o u v r i è r e s p r é s e n te s au début de l ' e x p é r i e n c e ) . L e s r é su lta ts que nous avons obtenus f ig u ren t dans le tableau II.

D i s c u s s i o n

Etude du Q R . L ' e x am en du tableau II fa it ap para ftre p ou r le s tém oin s un QR n o r m a l c ' e s t - à - d i r e un QR dont la v a le u r e s t s e n s ib le m e n t v o is in e de 1, 6. P a r c o n t r e , l e s t ra i te m e n ts im p o s é s d é te rm in e n t une s t é r i l i t é quas i tota le aux d o s e s de 12 500, 25 000, 50 000 et 75 000 rad , et totale p ou r l e s d o s e s p lus é l e v é e s de 100 000 et 150 000 rad . (L e s t r è s fa ib le s v a le u rs du QR p ou r l e s d o s e s c o m p r i s e s en tre 12 500 et 75 000 rad ne p e rm etten t m ê m e pas une r e p ré s e n ta t io n graphique, s i on l e s c o m p a r e au QR des té m o in s . ) Cette fa ib le fé c o n d i té « r é s i d u e l l e » peut s ' e x p l i q u e r s i l ' o n c o n s id è r e que c e s d o s e s de ray on n em en t g a m m a p e rm etten t une v ie r e la t iv e m e n t b ea u cou p plus lon gu e que le s i r ra d ia t io n s de 100 000 et 150 000 rad.

T A B L E A U I. M O R T A L I T E D E S C O L O N IE S D 1 O U V R I E R E S

158 TO

ROSSIA

N

et C

AU

SSE

SM-102/17 159

FIG. 1. Courbe d e la m orta lité en fon ction d e la dose d e rayonnem ent gam m a reçu e en une seule exposition . Le poin t T représente la DL50 des tém oins.

FIG. 2. M orta lité des ouvrières irradiées.

T e m p s pendant le q u e l l e s œ u fs ex is tent dans la c o l o n i e . L 1 étude de c e c r i t è r e n ' e s t pas s ig n i f i c a t iv e . L e s qu e lques r a r e s œ u fs pondus par le s indiv idus i r r a d ié s re s te n t dans la c o lo n ie un te m p s c o m p a r a b le à c e lu i d es té m o in s .

D even ir du c o u v a in . L e s œ u fs i s s u s d ' in d iv id u s i r r a d i é s ne sont pas s u s c e p t ib le s (g é n é ra le m e n t ) d 'u n d é v e lo p p e m e n t n o rm a l . H abituellem ent, de t e ls œ u fs p résen ten t un a s p e c t f r ip é et p l i s s é qui c o n t ra s te av e c 1' a s p e c t


T A B L E A U II. P R I N C I P A U X R E S U L T A T S O B T E N U S D A N S L ' E T U D E D E L A F E C O N D I T E D E S O U V R I E R E S

D ose d e C o lo n ie N om bre OR Longueur D even ir ORrayonnem ent d 'œ u fs d e la du m oy en

ga m m a ponte co u v a in(rad) ( j)

1 3 0 ,0 2 8 0 24 néant12 500

2 2 0 ,0 1 7 2 9 néant0 , 0 2 2

3 5 0 ,0 2 5 0 16 néant25 000

4 6 0 ,0 3 4 8 18 néant0 ,0 3 0

5 6 0 ,0 3 5 2 9 néant50 000

6 00 ,0 1 8

7 0

75 0008 7 0 ,0 6 1 4 1 2 2 jeunes

larves

0 ,0 3 0

9 0

1 0 0 00 01 0 0

1 1 0

150 0001 2 0

T ém oin s T1 2 1 0 1 ,2 4 2 6 23 nom breuxm aies

T2 190 1 ,5 2 0 0 34 nom breuxm â les

1 ,5 9 2

T3 260 2 ,0 1 5 5 31 nom breuxm âles

d 'u n œ u f n o rm a le m e n t e m b ry o n n é . E x ce p t io n n e l le m e n t , dans une seu le c o lo n i e , l e s deux œ u fs pondus ont donné n a is sa n c e à deux la r v u le s du p r e m ie r stade , qui n 'o n t cependant ja m a is év o lu é a u -d e là de c e stade et ont f in i par d is p a r a î t r e de la c o lo n ie a p rè s qu e lques jo u r s . Dans le m ê m e te m p s , l e s œ u fs des c o lo n i e s té m o in s ont donné n a is sa n c e , suivant le p r o c e s s u s habituel d é jà d é c r i t [3] à de n o m b re u x m â le s (de 50 a 80 par c o lo n ie ) . Il s e m b le donc que l ' i r r a d i a t i o n g a m m a à p a r t ir de 12 500 rad s t é r i l i s e to ta lem en t le s o u v r iè r e s . Si p a r h a sa rd , s e m b l e - t - i l , qu e lques r a r e s œ u fs ou trfes r a r e s la r v e s r é u s s i s s e n t à a p p ara ftre , i l s ne sont pas v ia b le s . En ou tre , ce tte s t é r i l i t é e s t dé f in it ive , c a r ju s q u 'à l ' e x t in c t i o n to ta le d e s c o lo n i e s i r r a d i é e s , aucune r e p r i s e d e s pontes n 'a ja m a is pu ê t r e o b s e r v é e .

SM-102/17 161

Un lo t de 12 c o lo n i e s r é c o l t é e s en un seu l p r é lè v e m e n t dans un m ê m e b io to p e e s t u t i l i sé pou r ce tte e x p é r i e n c e . Six c o lo n i e s constituen t le lo t t é m o in . L e s s ix c o lo n ie s t r a i t é e s r e ç o iv e n t l e s d o s e s su iv an tes : deux r e in e s (p r e m iè r e e x p é r i e n c e ) r e ç o iv e n t 50 000 rad , deux r e in e s (d eu x ièm e e x p é r ie n c e ) r e ç o iv e n t 75 000 rad , deux r e in e s ( t r o i s i è m e e x p é r ie n c e ) r e ç o iv e n t 100 000 rad .

R e m a r q u e . Cette e x p é r ie n c e con st itu e une e x p é r ie n c e p r é l im in a ir e , dont l e s r é su lta ts sont donnés i c i à t i t re p u re m e n t in d ica t i f (pour ê tre c o m p a r é s a ce u x des e x p é r i e n c e s p r é c é d e n te s in té r e s s a n t l e s c o lo n ie s d ' o u v r i è r e s ) . Une e x p é r im e n ta t io n plus p o u s s é e e s t en c o u r s a c tu e l le ment, dont le s r é su lta ts fe r o n t éve n tu e l le m e n t l ' o b j e t de c o m m u n ica t io n s u l t é r ie u r e s .

2. 1. Etude de la m o r ta l i t é

L e s r e in e s ayant r e ç u 50 000 rad sont m o r t e s a p rè s 112 jo u r s d 'é l e v a g e . L e s r e in e s ayant r e ç u 75 000 rad sont m o r t e s a p rè s 35 et 32 jo u r s d 'é l e v a g e (so i t en m oy e n n e 33, 5 j o u r s ) . L e s r e in e s ayant r e ç u 100 000 rad sont m o r t e s a p rè s 35 et 26 j o u r s d ' é l e v a g e ( s o i t en m oy en n e 30, 5 jo u r s ) .L e s r e in e s des c o lo n ie s té m o in s (n 'a y a n t r e ç u aucune ir r a d ia t io n gam m a) sont to u jo u r s v iva n tes a p rè s 172 jo u r s d 'é l e v a g e . R a p p e lon s , a t i tre in d icat if , que le s o u v r i è r e s ayant r e ç u le s m ê m e s d o s e s d ' i r r a d ia t i o n (50 000, 75 000, et 100 000 rad ) avaient une D L 100 de 41 , 5, 27 et 22 jo u r s , et de 78, 7 jo u r s pou r le s t é m o in s .

D i s c u s s i o n

C e s ré su lta ts p a ra is s e n t in d iquer , en v a le u r a b so lu e , une r é s i s t a n c e b ien plus grande d e s r e in e s c o m p a r é e à la r é s i s t a n c e des o u v r i è r e s . On sa it n éan m oin s que le s r e in e s fé c o n d é e s sont de lo in le s ind iv idus le s plus ré s is ta n ts de la c o lo n ie , et ceux qui ont la p lus longue v ie im ag ín a le .Il e s t donc p o s s ib le que la r é s i s t a n c e p lus é le v é e des r e in e s aux rad ia t ion s g a m m a so i t une d e s m a n i fe s ta t io n s de cette r é s i s t a n c e g é n é r a le p lus é le v é e .

2 .2 . Etude de la fé co n d i té

L ' étude de la fé c o n d i té d e s r e in e s i r r a d ié e s nous a p e r m is de r é a l i s e r e x p é r im e n ta le m e n t la l e v é e de l ' in h ib i t i o n de la ponte des o u v r i è r e s en p r é s e n c e de le u r re in e [10].

D 'u n e fa ço n g é n é r a le , l e s r e in e s i r r a d i é e s su b isse n t d e s p e r tu r b a tions p ro fo n d e s dans le u r ry th m e de p on te. Nous avons v é r i f i é au m o y e n de m éth od es b io m é tr iq u e s [10] que, pour d e s tra i te m e n ts de 50 000 et 75 000 rad , la ponte des r e in e s d im inue c o n s id é r a b le m e n t , puis r e p re n d l é g è r e m e n t , p ou r f in a lem en t s ' a r r ê t e r dé f in it ivem en t a p a rt ir du 18e jo u r . Il n 'y a p lus de r e p r i s e de la ponte des r e in e s par la suite.

P a r a l lè le m e n t , 3 à 4 jo u r s a p rè s l ' i r r a d i a t i o n de la r e in e , l e s o u v r iè r e s c o m m e n c e n t à pon d re . La ponte s ' e f f e c t u e dans le v o is in a g e im m é d ia t de la re in e . Il conv ien t de r e m a r q u e r cependant que, e x ce p t io n fa ite de son ry th m e de ponte m o d i f ié , la r e in e i r r a d ié e a un c o m p o r te m e n t abso lum enl identique a c e lu i d e là r e in e n 'a y a n t sub i aucune ir ra d ia t io n .

2. COLONIES AVEC RE IN E

11


En p a r t i c u l ie r , e l le se t ient en p e r m a n e n ce sur le tas d 'œ u f s et de la r v e s , et s e s re la t io n s trop h a lla e t iq u e s avec le s o u v r iè r e s sont n o r m a le s .

La ponte des o u v r iè r e s augm ente par la suite , au fu r et à m e s u r e que c e l l e de la r e in e d im inue.

D i s c u s s i o n

D e ve n ir des œ u fs dans une co lo n ie dont la re in e a subi une ir ra d ia t io n g am m a. A p r è s ir ra d ia t io n , l e s œ u fs de r e in e p r é se n ts dans la c o lo n ie ,id en t i f iab les avec cert itu d e par la m éthod e b io m é tr iq u e , présen ten t qu e lques jo u r s a p rè s la ponte un asp e c t inhabituel. L e s œ u fs p a ra is s e n t « f r i p é s » . L e c h o r io n e s t p l i s s é , a f fa is s é par e n d ro i ts . A p r è s lü à 15 jo u r s le s œ u fs sont fo r te m e n t rata tin és . Aucun de c e s œ u fs n ' e s t v ia b le . L e s œ u fs pondus par l e s o u v r iè r e s ( individus non i r r a d ié s ) p résen ten tl 1 a s p e c t habituel d 1 œ u fs e m b ry o n n é s . Ils donnent n a is sa n c e a l 1 hab itu e lle g én éra t ion de m â le .

CONCLUSIONS

C e s e x p é r ie n c e s r é v è le n t donc une e x ce l le n te r é s i s t a n c e d e s f o u r m is de 1' e s p è c e D o l ic h o d e r u s quadripunctatus au ray on n em en t g am m a, a ins i que le prouve la D L 1 0 0 p ou r des taux d ' i r r a d ia t i o n in fé r ie u r s ou égaux à 50 000 rad. A c e s d o s e s de rad ia t ion s dé jà t r è s é le v é e s , la r é s i s t a n c e d e s indiv idus adultes e s t de 1' o r d r e de 42 à 43 jo u r s pour l e s o u v r i è r e s et 112 jo u r s pour le s r e in e s . La s u r v ie des indiv idus ains i i r r a d i é s es t donc se n s ib le m e n t é g a le à la m o i t ié de c e l l e des té m o in s .

La fécond ité apparaît par co n tre a f fe c té e de fa ço n bea ucou p plus p r o fo n d e : une d o se de ray on n em en t g a m m a de l ' o r d r e de 12 500 rad s t é r i l i s e c o m p lè te m e n t le s o u v r iè r e s pon d eu ses .

R E F E R E N C E S

[1 ] TOROSSIAN', C . , Recherches sur la b io lo g ie et l ’ é th o log ie de D olichoderus quadripunctatus (L )-H ym .Form ico'idea D olich od erid æ , D octorat d'Etat de scien ces naturelles, Toulouse (19 6 6 -1 9 6 7 ) I -280 .

[2 ] TOROSSIAN', C . , Insectes soc . (sous presse).[3 ] TOROSSIAN, C . , Insectes soc. VII (1960 ) 383.14J TOROSSIAN, C . , Insectes soc, (3 paraître, 1967).[5 ] TOROSSIAN, C . , C , r. Séanc. S oc. Biol. (sous presse).[6 ] COLE, M .M ., LA BRECQIJE, G .C . , BURDEN, G . S . , J. e con . Ent. 52 (1959) 448.[7 J NARAYANAN', E. S . , CHAWI.A, S .S . , G MAI,' S, , Proc. natn. Inst. Sci. India В 29 (1963) 613.18] DRUMMOND, R .O ., Int. J. Radial. B iol. 7 (1963) 491.[91 HENNEBERY, T . . ] . , I. e co n . Ent. 3 (1 9 6 3 ) 279.

[10 ] TOROSSIAN, C . , CAUSSE, R ., C . r. hebd. Séanc. A cad . sci. 264 (1967) 2789.

D I S C U S S I O N

W. J. K L O F T : I want to extend, m y congra tu lat ion s to you on y o u r r e s u l t s , which w e r e e s p e c ia l ly in te re s t in g f r o m the point o f v iew o f s o c ia l o rg a n iz a t io n in ants. I w as m o s t s tru ck by the way in which you a ch ieved o v a r ia l inhibit ion — up to tota l s t e r i l iz a t io n — o f the queen without any

S M -1 0 2 A 7 163

dis tu rb a n ce o f the pattern o f beh a v io u r o f the r e s t o f the co lo n y . T he danger o f d is ru p t iv e s i d e - e f f e c t s is a lw ays p r e se n t when c h e m o s te r i la n ts a re u se d with s o c ia l in s e c t s b e c a u s e o f r e g u rg ita t io n o f the s u b s ta n c e s . You m en tion ed that the w o r k e r s s tart to la y e g gs , but that the queen n e v e r th e le s s cont inu es to p lay the dom inant r o l e in d ica t ive o f n o r m a l b eh av iou r .К this can be taken as a de fin it ive r e s u lt it would ind icate the e x is t e n c e o f c e r ta in 1 q u e e n -s u b s t a n c e s 1 (p h e ro m o n e s ) , one o f them linked with f e r t i l i ty o f the w o r k e r s and the o th er with the s o c ia l r o l e o f the queen, perhaps in the f o r m o f s o m e at trac t iv e su bs ta n ce . It would b e in te re s t in g to c a r r y out s im i la r e x p e r im e n ts with ants o f o th er s u b - fa m i l i e s .

■ C. TOROSSIAN: Thank you f o r y o u r in te re s t ; I sh a l l t r y to expand a lit t le on m y su b ject . W o rk on the e f f e c t . o f g a m m a ra y s on ants o f the s p e c i e s D o l ic h o d e r u s quadripunctatus w as in it ia lly undertaken with the o b je c t o f in ves t iga t in g the q u e e n -w o r k e r re la t io n sh ip in r e s p e c t o f e g g - lay in g within the co lo n y . I f e e l I m a y con fid ent ly e la b o ra te on the c o n c lu s io n s r e a c h e d in m y p a p er , p a r t icu la r ly as r e g a r d s the ten den cy to o v ip o s i t io n in the w o r k e r ants.

By su itab le ir ra d ia t io n o f the queen w e s u c c e e d e d in inducing rap id s t e r i l iz a t io n without l o s s o f v ita lity ; a few days a fter i r ra d ia t io n the queen c e a s e s la y in g but h e r beh a v io u r apparently u n d e rg o e s no o th er m o d if ica t io n . In p a r t icu la r , she takes up a perm anen t p o s it io n on the p ile o f eggs and youn g la rv a e p r o d u c e d la r g e ly by the f e m a le w o r k e r s . She i s fed as n o r m a l by the w o r k e r s by s t o m o d e a l and p r o c t o d e a l tr o p h a l la c t ic e x ch an ges ty p ic a l o f this s p e c ie s (s e e [1 - 3]). '

At the sa m e t im e it is found that the w o r k e r s lay e g gs im m e d ia te ly on c o m in g into contact with the queen (I have even p h otographed this b eh av iou r) . T h is r e s u l t w as n e v e r a ch ieved by any p r e v io u s s t e r i l i z in g m ethod . In fa c t , this technique o f i r ra d ia t in g the queen o f a c o lo n y o f in s e c t s is ra th e r in teres t in g , s in c e it i s apparently the on ly one known to date a l low ing s e l e c t i v e s t e r i l iz a t io n o f the queen o r o f a s e l e c t e d group o f ind iv iduals . P r o v id e d that the g a m m a - r a y d o s a g e is c h o s e n c a r e fu l ly the individual i r ra d ia te d can be s t e r i l i z e d without a p p re c ia b le e f fe c t on its l i fe and beh av iou r pattern .

It would ap pear f r o m past and p re se n t w o rk on IX quadripunctatus that inhibit ion o f o v ip o s i t io n in the w o r k e r s in the p r e s e n c e o f th e ir queen w as the co m b in e d r e s u l t o f s e v e r a l se n se s t im u li ( o l fa c to ry , v isu a l , ta c t i le ; s e e [1]) and in p a r t icu la r th o se p r o v id e d by c h e m ic a l su b s ta n ce s s e c r e t e d by the queen and the p r e s e n c e o f e g gs la id as usual by the queen in the nest.

L as t ly , I w ould l ik e to m en tion that w o rk is st i l l go ing on and w il l be continued with the a im o f studying the r e s p o n s e o f ID. quadripunctatus to s m a l l d o s e s o f rad ia tion and the r e a c t io n s o f the queens o f d i f fe ren t s p e c i e s o f ants and oth er s o c ia l in s e c ts to g am m a ir ra d ia t io n .

J. H. G. T IC H E L E R : Do you c o n s id e r that the w o r k e r s a re induced to lay eggs a fter i r ra d ia t io n o f the queen by v irtue o f the fa c t that she c e a s e s lay in g h e r s e l f o r b e c a u s e she s top s s e c r e t in g ce r ta in c h e m ic a l su b s ta n ce s , such as p h e r o m o n e s ?

C. TOROSSIAN: I t r ie d f o r s e v e r a l y e a r s to ca u se tota l inhibition o f o v ip o s i t io n in the w o r k e r s by u s in g r o y a l 1 e x t r a c t s ' f r o m D o l ich o d e ru s queens (this w o rk f o r m s part o f m y D o c t o r a l T h e s is [1]) but was n e ve r s u c c e s s fu l .


In this c a s e , a fter su itab le ir ra d ia t io n the queen w as co m p le te ly s t e r i l i z e d , whereupon (i. e. 3 to 4 days la te r ) the w o r k e r s o f the co lon y , w hile s t i l l behaving quite n o rm a l ly tow ard s h e r , began to lay e g g s . It vould thus s e e m c o n c lu s iv e that the m is s in g f a c to r , a b se n ce o f which in du ces

e g g - la y in g in the 'queen , is the in terru pt ion in n o rm a l a ccu m u la t ion o f ’ e x t r a c t s ’ p ro d u ce d by h er.

T h is naturally lea d s one to think that the p r e s e n c e o f r o y a l 1 e x t r a c t ’ within the group o f w o r k e r s is an im p ortan t fa c to r , p o s s ib ly a d e te rm in in g one, in cau s in g inhibit ion o f e g g - la y in g by fe m a le w o r k e r s .

G. LE M ASN E: What are the r e la t iv e lengths o f the ir ra d ia t io n p e r io d and the p e r io d o f sep a ra t io n o f the queen f r o m h e r c o lo n y ? It w ould se e m im p ortan t that th ese should be as c l o s e as p o s s ib le , s in c e undue ex ten sion o f the sep arat ion p e r io d would p ro b a b ly have an e f f e c t on the q u e e n -w o r k e r re la t ion sh ip .

C. TOROSSIAN: I rra d iat ion t im e v a r ie d f r o m a few m inutes to about th re e h ou rs f o r the la r g e s t d o s e . As soon as the ir ra d ia t io n trea tm en t w as o v e r the queen was put ba ck into the c o lo n y , and it ap peared that the q u e e n -w o r k e r re la t io n sh ip was in no w ay a f fe c ted by this sep a ra t io n , which is in ev itab ly entailed by the ir ra d ia t io n p r o c e s s . In the m o s t in te re s t in g c a s e (50 000 rad ) , the p r o c e s s la s ted about 48 m inu tes ; but o v e r a se p a ra t io n o f 24 h ou rs did not change the q u e e n -w o r k e r b eh av iou r pattern .W e have c o n f i r m e d this m any t im e s .

G. L E M ASNE: Have you in ves t ig a ted the o v a r ie s o f i r ra d ia te d qu e e n s? D oes ir ra d ia t io n d am age show i t s e l f in the p r e v io u s ly f o r m e d eg gs o r on ly in the u pper part o f the o v a r io le s , and what f o r m d o e s it take?

C. TOROSSIAN: I have not yet studied the o v a r ie s o f i r ra d ia te d fe m a le s , hav ing p r e f e r r e d to f o l lo w the d e v e lo p m e n t o f th ese queens throughout the w hole o f th e ir l i f e - s p a n . T h e ir be h a v io u r and b io lo g y was ou r f i r s t o b je c t iv e , though we do eventua lly intend to m ake a h i s t o - c y t o l o g i c a l study o f i r ra d ia te d queens.

REARING TECHNIQUE,BIOLOGY AND STERILIZATIO N OF THE CO FFE E L E A F MINER,Leucoptera coffeella Guer.(LE P ID O P TE R A : LYONETIIDAE)

K .P . KATIYAR AND F. FERRERIN TER-AMERICAN IN STITU TE OF AGRICULTURAL SCIENCES OF THE OAS TRAINING AND RESEARCH CENTER,TURRLALBA, COSTA RICA

Abstract

REARING TECHNIQUE, BIOLOGY AND STERILIZATION OF THE COFFEE LEAF MINER, Leucoptera C o ffee lla G uer. (LEPIDOPTERA: LYONETIIDAE). For tw o years the authors studied the feasib ility o f co n trolling the c o ffe e le a f m iner Leucoptera c o f fe e l la G uer. by the radiation steriliza tion techn ique. During this period a technique for raising large numbers o f tills insect on potted c o ffe e plants in the laboratory was devised. The optim al range for the developm ent o f egg, larval and pupal stages o f the c o ffe e le a f m iner was betw een 20 and 30° C . The pupal stage o f fem a le insects was slightly shorter than that o f the m ale .T h e laying o f fertile eggs began during the first night fo llow in g em erg en ce . During an ov iposition period o f 16 days the average fecu ndity was 68 eggs. T h e m axim um ov iposition by a single fem a le was 131 eggs over the l ife span w hile as many as 34 eggs w ere la id during a single day o f ov ip osition . T o investigate the best stage to induce radiation steriliza tion , pupal and adult insects w ere irradiated with 60C o gam m a rays. S even -da y pupae (c lose to em erg en ce ) showed S8% leth ality in m ales when given 60 krad; the survivors retained som e fertility . Adult fem ales rece iv in g TO krad w ere 100% sterile w hile m ales g iven 90 krad showed0 .027» fertility . Doses as high as 90 krad given to new ly em erged adults did not reduce lon gev ity . Studies are continuing to determ ine i f steriliz ing doses im pair sexual vigour and m ating com petitiveness o f the treated m ales.

1 . IN T R O D U C T IO N

The coffee leaf miner, Leucoptera coffeella Guer., is one of the great destructive pests of coffee plantations in most coffee-growing countries of the Western hemisphere. Recently it has become a very serious pest of coffee in Central American countries, especially in Guatemala [1-3] and Costa Rica [4]. The damage is done by the larvae which feed on the mesophyll layer of the leaves. The heavily mined leaves are easily dislodged, and under severe infestation the plants may be completely defoliated.

The successful eradication of the screw-worm fly, Cochliomyia hominivorax (Coquerel), from Curacao [5] and southeastern portions of the United States [6] by mass release of gamma sterile males has demonstrated the great potential of this new method of insect control. Currently research is underway on several insect pests in various laboratories of the world to study the feasibility of the sterile-male technique for control and eradication of these pests. The exploratory studies to evaluate the gamma sterile-male technique for controlling

the coffee leaf miner started in our laboratory 2 years ago. The preliminary results of these studies are presented in this paper.

165

166 KATIYAR and FERIAR

2. REARING TECHNIQUE

A continuous la b o ra to ry supply o f adequate numbers o f l e a f miners in a l l stages i s e s s e n t ia l f o r ca r ry in g out s t e r i l i z a t i o n s t u d ie s . Techniques to rear the c o f f e e l e a f miner con t in u ou sly in large numbers in a la b o ra to ry have not been worked out p r e v io u s ly .Our f i r s t attempt in th is s tudy , t h e r e f o r e , was to f in d a s a t i s f a c t o r y way to rear th is in s e c t in the la b o r a to r y . We have developed a method which a l low s continuous mass r e a r in g o f th is in s e c t on l i v i n g c o f f e e p la n ts .

A number o f pupae were c o l l e c t e d from the in fe s t e d c o f f e e f i e l d . Upon adult emergence, the moths were tra n s fe r r e d to an o v i p o s i t i o n cage 24" x 22" x 18" c o n ta in in g l i v in g c o f f e e p lants (F ig . 1 ) .The four s id e s and top o f the cage were covered with 52-mesh p l a s t i c s c re e n . The bottom was made o f plywood d iv id e d in to fou r re ctan gu lar s e c t i o n s . Each s e c t i o n had a 9" x 9" window which cou ld be opened or c lo s e d by a s l i d in g plywood panel. These panels were opened to in trodu ce the c o f f e e p lants in to the cage.. On the inner edge o f each panel th ere was a 3 /4 " se m ic ir c u la r h o le that c o in c id e d with a s im i la r h o le in the bottom frame o f the cage when the bottom panel was c l o s e d .

24"

FIG. 1. O viposition ca g e used for rearing the c o ffe e le a f m iner, Leucoptera co ffe e lla Guer.Л = 52-m esh pLastic screen E - Paper ce llu lose w ickВ = Slid ing plyw ood panel . F = Sugar solutionС = Plant stem h ole G = U :shaped stainless steel chann el ■D = O pening fot introduction o f adults H = Vessel for c o lle c t in g excess sugar solution

S M - 102/18 167

The stem o f the c o f f e e p lant used f o r o v i p o s i t i o n passed through th i s h o le . The cage stood on fo u r le g s which cou ld be a d ju sted to a maximum o f 30" depending upon the h e ig h t o f the c o f f e e p la n t . The d es ign o f the cage perm itted the m anipulation o f the c o f f e e p lants so that on ly the upper f o l i a r p o r t io n o f the c o f f e e p la n t was a v a i l ab le f o r o v i p o s i t i o n . The s o i l pot and the lower part o f the c o f f e e p lant were o u ts id e the cage . This arrangement allowed more space f o r moths and c o f f e e le a v e s . A system was d ev ised to prov id e the moths with p le n ty o f f resh sugar s o l u t i o n . Around the in s id e o f the cag e , fou r paper c e l l u l o s e w icks were supported on four U-shaped s t a in l e s s s t e e l channels p laced near the to p . The wicks had been soaked in a sugar s o l u t i o n with one end in s e r te d in t o a beaker o f sugar water p laced on top o f the cag e . The other end o f the wick was p laced over an empty v e s s e l t o c o l l e c t any excess s o l u t i o n .Fresh sugar s o l u t i o n was added d a i l y , and the w icks were changed weekly to insure the moths ample f o o d . The adu lt moths emerging from the pupal h o ld in g cages were t r a n s fe r r e d in to the o v i p o s i t i o n cage through a c i r c u l a r h o le on e i t h e r s id e o f the cage .

The pupal h o ld in g cages were made o f wooden frames covered with heavy b la c k c l o t h . Emerging moths a t t r a c te d t o l i g h t were c o l l e c t e d d a i l y in g la s s beakers in s e r te d at one end o f the cage .The cages were s to re d in a b a t te ry o f s t e e l frame ra ck s .

Four young c o f f e e p la nts (10 -12 months o ld ) were p laced in each o v ip o s i t i o n cage co n ta in in g 2000-3000 moths. O v ip o s i t i o n was allowed f o r 2 days b e fo r e the p la nts were r e p la c e d . I t i s recommended that the p la nts be changed every day to ob ta in pupae o f uniform age and t o in cre a se in s e c t p ro d u c t io n . By leav in g the p lants f o r 2 days in o v ip o s i t i o n ca g e s , sometimes the p la nts were k i l l e d by heavy la rv a l in f e s t a t i o n r e s u l t in g from e x ce s s iv e o v i p o s i t i o n . In such cases the larvae d ied b e fo r e pupation .

The c o f f e e p la n ts in fe s t e d with Leucoptera eggs were p laced in the la b ora tory at atmospheric c o n d i t io n s o f 20 -27° С and 80-90% r e l a t i v e humidity f o r 17 days. During th is p e r io d the larvae matured in the leaves and began pupation . W ithin 4 or 5 days most o f the fu l l -g r o w n la rvae had l e f t the mines, dropped t o the lower leaves by a s i lk e n th read , and pupated in a s i lk e n coco o n . C o f fe e leaves with pupae were removed from the p la n ts and incubated in pupal h o ld in g cag es . Moths began t o emerge on the 2nd day with the m a jo r i ty o f them emerging during the f o l lo w in g 4 t o 5 days . The ad u lts were tra n s fe r r e d to o v i p o s i t i o n cages d a i l y .

Approximately 2 ,000 moths were produced d a i ly w ith th is rear in g technique using three o v i p o s i t i o n cages and 12 c o f f e e p la nts every2 days o f o v i p o s i t i o n . I t would be p o s s ib le to in cre a se prod uct ion t o any d e s ire d l e v e l by augmenting the r e a r in g f a c i l i t i e s .

3 . BIOLOGICAL STUDIES

In form ation on the b i o lo g y and e c o lo g y o f an in s e c t is e s s e n t ia l f o r e v a lu a t io n o f i t s c o n t r o l by the s t e r i l e - m a le tech nique . S tud ies have been c a r r ie d out in our la b o ra to ry on some b i o l o g i c a l phases o f t h i s i n s e c t . A l l the in s e c t s used in the experiments were

168 KATIYAR and FERRER

reared on po t ted c o f f e e p lants in the la b o r a to r y . Temperature- c o n t r o l l e d experiments were c a r r ie d out in s id e the constant-tem perature c a b in e t s . Humidity was not c o n t r o l l e d except in the case o f pupal s t u d i e s . '

The e f f e c t s o f v a r io u s temperatures on the ra te o f d e v e lo p ment and h a t c h a b i l i t y o f the c o f f e e l e a f miner eggs are presented in Table I . The r e s u l t s in d ica te d that the optimum temperature fo r in cu b a tio n o f the eggs was 20 -30° C. Eggs incubated at 30° С gave h ig h e s t percentage o f egg hatch (95.0%) and the sh o r te s t (average o f 4 . 0 days) in cu ba tion p e r io d . At 35° С la r v a l hatch from the eggs was normal but on ly 8.5% o f the larvae were ab le to mine the lea ves .The r e s t o f the newly emerged larvae d ied on the outer su r fa ce o f the l e a f . The reason f o r th is high la r v a l m o r ta l i t y i s not known. E xcess ive heat (35° C) probably k i l l s the newly emerged larvae b e fo r e they are ab le to pen etrate the epiderm is o f the l e a f .

Table I

Temperature e f f e c t on the ra te o f development and h a t c h a b i l i t y o f the c o f f e e l e a f miner, Leucoptera c o f f e e l l a Guer. , eggs

Temperature°C

Number o f eggs

observed

Percenthatch

DevelopmentMax.

per iodsMin.

(days)Avg.

15* 339 0 0 0 0 .0

20 855 80.4 ' 13 10 10.7

25 588 81.1 7 5 5.5

30 806 95 .0 5 4 4 .0

35 282. 8.5 5 4 4 .1

Incubated fo r 60 days.

Data in Table I I show that the optimum temperature range fo r the development o f the c o f f e e l e a f miner larvae l i e s between 25-35° C. Larvae reared at 30° С y i e l d the h igh est percentage o f pupae (92.7%,). At 35° С the la r v a l development was f a s t e s t . The average la r v a l p er iod at 35° С was 7 .2 days. Larvae reared at 15° С f a i l to pupate although 2.7% o f them matured and l e f t the mines. The larvae o f a l l the treatments were reared on i s o la t e d c o f f e e leaves excep t in treatments o f 15 and 35° С whose larvae were reared on potted c o f f e e p la n ts .

Table I I I shows the e f f e c t o f v a r io u s temperatures on the r a te o f development o f the c o f f e e l e a f miner pupae. Temperature between 20° and 30° С was optimum f o r in cu ba tion o f the pupae. The a d u lt emergence from the pupae incubated at th is range o f temperature

SM -102/18 169

Table II

E f f e c t o f temperature on the development o f the c o f f e e l e a f m iner , Leucoptera c o f f e e l l a Guer. , larvae

Temperature Number Percent Larval per iod ( i n days)oc larvae

rearedpupation Max. Min. Avg.

15 564 2.7* 87 68 73.6

20 111 82.2 29 20 23.1

25 92 91.9 16 12 13.1

30 254 92.7 11 8 9 .0

35 295 88.5 8 7 7 .2

Larvae l e f t the mines but f a i l e d to pupate.

Table I I I

E f f e c t o f temperature m iner , Leucopt

on the development o f the c o f f e e era c o f f e e l l a Guer. , pupae*

l e a f

Temperature°C

Number 7o moth Pupal per iod in dayspupae

observedemer- Max. Min. gence

Avg.

15 150 34.7 38 30 33.3

20 100 95 .0 16 13 13.7

25 100 97 .0 7 5 5.3

30 251 88 .8 7 5 5.3

35 100 33 .0 6 5 5.5

*Incubated at 1007o RH.

v a r ie d from 88 .8 t o 97.07». In cu ba tion o f the pupae at 25° С gave h igh est percentage moth emergence (97.07°) and s h o r te s t pupal per iod (5 .3 d a y s ) .

Results summarized in Table IV in d ic a t e that in the c o f f e e l e a f miner the female pupal p er iod was a l i t t l e sh orter than the male pupal p e r io d . The average pupal per iod a t 25° С temperature and 50 to 60% RH f o r the female and male was 6 .7 and 7 .1 days, r e s p e c t i v e ly . Female moths emerged 1 day e a r l i e r than male moths.


Table IV

Duration of male and female pupal periods of the coffee leaf miner, Leucoptera coffeclla Guer.

Pupal period (days)

Percent adult emergence

Male Female

6 0 34

7 92 66

8 8 0

Average pupal period: Male = 7..1 days and Female = 6.7 days.Total number of pupae observed = 860.

•к _At 25 С and 50-607» relative humidity.

FIG. 2 . O viposit ion rate o f the c o f fe e le a f m iner, Leucoptera c o ffe e lla Guer.

Oviposition rate of female coffee leaf miner was studied in small cages by single-paired matings. Daily egg collection was made from each female for a period of 16 days. At the end of this period most of the moths had either died or stopped egg production. The female moths started laying fertile eggs on the first night of emergence. The rate of oviposition increased rapidly reaching a maximum on the 4th day. After the 4th day the oviposition declined sharply until the 7th day and then gradually diminished to almost

S M - 102/18 171

zero by the 16th day (Fig. 2). Thus the female coffee leaf miner moth lays a majority of her eggs during the 1st week of adult life.A female lays an average of 68 eggs during her entire life. The maximum oviposition per female was 131 eggs with 34 eggs the maximum number laid by a female on any 1 day.

4. STERILIZATION STUDIES

Pupae of uniform age for irradiation studies were obtained by spreading small coffee twigs with leaves below the infested coffee plants in the morning between 7:00 and 8:00 A.M; Full-grown larvae left the mines and pupated on these leaves. The twigs were removed 8 hours later in the evening. Thus the age of the pupae used in the irradiation tests varied from 0-8 hours.

The sterilization studies were started by irradiating late-stage pupae (7 days old). The presence of mature sperm was noticed in such pupae. A series of initial irradiation tests conducted on late-stage pupae indicated that the pupal stage of

the coffee leaf miner was not suitable for inducing sterility in the adult moths. Radiation dose of 60 KR applied to 7-day-old pupae (close to adult emergence) was lethal (Tabic V). The adverse effect of irradiation was more pronounced on male pupae than female pupae. Pupae irradiated with 60 KR produced 29.3%, male moths compared with 45.57° male snergence from untreated pupae. The female emergence was normal (41.2%) from pupae irradiated with 60 KR. The majority of the male moths that emerged from irradiated, pupae were sluggish and unable to fly normally. Within 48 hours after the adult emergence 88.1% males were dead. Fertility experiments carried out with the surviving moths indicated that they were not 100%, sterile. In subsequent tests, therefore, radiation was applied during the adult stage. The reason for the severe lethal effect of radiation on male pupae and no or little effect on female pupae is probably that at

the time of treatment female pupae are further developed than male pupae.

Newly-emerged moths were irradiated with several doses of gamma radiation at a dose rate of approximately 2414 R/min. The treated insects were confined in cages together with virgin moths of the opposite sex. Each cage had 20-25 pairs of moths; Since sufficient numbers of insects were not available at one time, the experiment was completed in several steps. A control was used for each experiment. Daily egg collection was made for a period of 7 days. The results of these experiments are summarized in Table VI.The data indicated that the sterilization dose of this insect was ■90 KR. Male moths were more radioresistant than female moths (when sterility is taken as the index of the radiosensitivity). A radia

tion dose of 70. KR induced 100% sterility in the treated females whereas the males receiving 90 KR still retain 0.02%, fertility. The high radioresistance in Lepidoptera is not uncommon. The sterilizing dose for the codling moth, Carpocapsa pomonella L . , is reported to be 40,000 rads [7]; the tobacco budworm, Heliothis virescens г 35,000-45,000 rads fg!; and for the Indian meal moth, Plodia interpunctella (Hübner), and the Angoumois grain.moth, Sitotroga- cereallela (Oliver), as high as 100,000 rads [9].


Table V

Effect of gamma radiation (60 KR) applied to 7-day-old pupae on the adult emergence and mortality of the coffee leaf

miner, Leucoptera coffeella Guer.

TreatmentNumber of pupae studied

Percent moth emergence

Percent motlj mortality"

Male Female Male Female

Normal 1,030 45. 5 39.1 0.0 0.0

Irradiated 1,030 29. 3 41.2 88.1 23.6

*Within 48 hours after adult emergence had started.

Table VI

Effect of gamma radiation" on the fert ility of thecoffee leaf miner, Leucoptera coffeella Guer.

Dosage Matings"" No. Number of eggs Percent(KR) Female X Male pairs Examined Hatched fertility

Check N X N 254 13,932 11,169 80.17

60 N X R 70 4,022 268 6.66R X N 70 1,861 2 0.11R X R 70 2,096 0 0.00

70 N X R 70 3,434 109 3.17R X N 70 944 0 0.00R X R 60 1,248 0 0.00

80 N X R 70 4,216 57 1.35R X N 70 667 0 0.00R X R 65 1,495 0 0.00

90 N X R 75 4,510 1 0.02R X N 75 560 0 0.00R X R 75 1,279 0 0.00

-fçIrradiated at adult stage. Age of the motns at the time of treat

ment varied a few hours to 24 hours in all the crosses except those having both sexes irradiated. Females of the crosses (R female X R male) were approximately 48 hours old and were laying normal viable eggs at the time of treatment.

**The letters R and N designate irradiated and normal moths, respectively.

S M - 1 0 2 / 1 8 173

Since the induction of sterility in this insect requires the application of radiation at the adult stage, it is quite likely that some mating occurred before the radiation was applied. Therefore, a mixed population of normal moths that had laid fertile eggs was irradiated and the subsequent fertility studied. The crosses (R female X R male) in Table VI constitute such treatments. The results indicated that a dose of 60 KR induced 100% sterility in females that had laid normal fertile eggs before irradiation.

It seems that the dose of 90 KR (applied to newly-emerged moths) does not adversely affect the longevity of the treated moths.We are planning to study the sexual vigor and the mating competitiveness of the sterile malas in the near future.

R E F E R E N C E S

[1 ] FISCHER, K. U ., HERRERA, J. C ., M inador del ca fé , estudio té cn ico en G uatem ala , V illanueva,Escuela N acional de Agricultura (1963 ) 5.

[2 ] HERNANDEZ, М. P. , Revta C afet. (G uatem ala) 31 (1964 ) 7 -1 7 .[3 ] HAMILTON, D .W , J. e co n . Ent. 60 5 (1967) 140 9 -1 3 .[4 ] HAMILTON, D. W., unpublished report, USDA, ARS, E ntom ology Research D ivision , Vincennes,

Indiana (1964).[5 ] BAUMHOVER, A .H . , GRAHAM, A .J . , BITTER, B .A ., HOPKINS, D .E ., NEW, W .C . , DUDLEY, F .H .,

BUSHLAND, R .C ., J. e co n . Ent. 48 4 (1 9 5 5 )4 6 2 -6 8 .[ 6] LINDQUIST, A .W ., "Use o f sexually sterile m ales for erad ication o f screw -w orm s, " in Radioisotopes

and Radiation in the Life S cien ces , 2nd In ter-A m erican Sym posium on the P eacefu l A p p lica tion o fN uclear Energy, Buenos Aires 1959, T ID -7 5 5 4 ., W ashington, D .C . , P an-A m erican Union (1960) 2 2 9 -3 5 .

[7 ] PROVERBS, M .D . , NEWTON, J .R ., Can. Ent. 94 11 (1 962 ) 1 1 6 2 -7 0 .[ 8] FLINT, H .M . , KRESSIN, E. L . , J. e co n . Ent., in the press.[9 ] COGBURN, R .R ., TILTOÑ, E .W ., BURKHOLDER, W .E ., J. e co n . Ent. 59 3 (1966 ) 68 2 -8 5 .

D I S C U S S I O N

J . E . SIMON: I con gra tu late you and y o u r c o l le a g u e on the technique you have d e v ise d . One point I am not c l e a r about i s : a c c o r d in g to T a b le s I, II and III the m atura tion p e r io d o f the m oth c o f fe e l la at 30° G is 4. 0 + 9. 0 + 5. 3 = 18. 3 days , so how, with on ly th ree c a g e s , can you m aintain cont inuous p rod u ct ion o f adult m oth s?

K. P . K A T IY A R : At f i r s t , o f c o u r s e , prod uct ion is not continuous, but as soon as the m oths hatched f r o m e g g s la id on the f i r s t day o f o v i p o s it io n r e a c h m aturity the c y c l i c p r o c e s s is su sta ined . S ince th ere is o v ip o s i t io n da ily on the c o f fe e plants th ere is m oth e m e r g e n c e da ily .

E . M . SHUMAKOV: How m u ch d o e s it c o s t to r e a r , say, one m il l ion m oth s in the la b o r a to r y ?

K. P . K A T IY A R : I don 't b e l ie v e we e v e r t r ie d to w o rk out the exact c o s t o f r e a r in g one m i l l io n in s e c ts , but I im ag in e that expend itu re on c o f f e e plants would c o m e to s o m e w h e r e around $ 4 0 - $ 60 p e r m il l ion m oth s . In this w o rk we r e a l ly w e r e not try ing to find a ch e a p r e a r in g method but ra th er to b re e d the in sec t s u c c e s s f u l ly in the la b o ra to ry f o r ou r own e x p e r im e n ta l p u rp o s e s .


M. COHEN: . A r e th ere any data on the le v e ls o f population o f L . c o f f e e l la in the f ie ld , and i f these a re v e r y high cou ld y our r e a r in g p r o c e s s g ive a throughput w hich would enable you to sw a m p the n u m b e rs o f w ild m oths?

K. P. K A T IY A R : The f ie ld population o f L. c o f fe e l la is not known.O ur p re se n t r e a r in g m ethod is c e r ta in ly not su f f ic ien t fo r making la r g e - s c a le s t e r i l e in s e c t r e le a s e s ; o u r a im in this w o rk was ra th er to d e v e lo p s o m e technique which would p rov id e us with enough in se c ts to do s t e r i l i za tion stud ies in the la b o r a to r y ,

M. COHEN: D o e s L. c o f fe e l la mate m o r e than o n ce ?K. P. K A T IY A R : I have no defin ite in fo rm a t io n on this but I b e l ie v e

that it d o e s .C . F . CU RTIS: I think the quest ion o f m ult ip le m atings o f the f e m a le s

m a y actu a l ly be ra th er im portan t b e c a u se o f the v e r y high d o s e - 90 krad - that you r e q u ire d to induce c o m p le te s t e r i l i ty . I b e l ie v e I am right in sa y ing that in H ab ro b ra co n , w h e re 'd o m in a n t lethal mutations can be r e a d i ly d is t inguished f r o m s p e r m d am age im p a ir in g fe r t i l i z in g cap a c ity ,90 krad in d u ces a c o n s id e r a b le am ount o f su ch dam age . If the sa m e ap p lies to y o u r moth, c o f fe e l la , it m ight happen that a fe m a le which had m ated with s e v e r a l s t e r i le m a le s and one fe r t i le m a le would show fu ll fe r t i l i ty .

K. P . KATIYAR-. AU I ca n add i s that we have not yet studied the d a m ag in g e f fe c t o f 9 0 -k ra d d o s e s on s p e r m . A l l we have done so fa r is to d e te rm in e that d o se which c o m p le t e ly s t e r i l i z e s the m a le s and this i s found to be 90 krad . I r e a l iz e that it is quite a high d o s e and th ere is e v e r y p o s s ib i l i t y that it would ca u se so m e s p e r m d a m ag e . We a r e d e fin ite ly planning to in vestigate the e f fe c t o f 90 krad on the sex u a l v ig o u r and m ating ab il i ty and hence on the co m p e t i t iv e n e s s o f s t e r i le m a le s with n o r m a l m a le s , and if this w o r k in d ica tes that da m ag e , su ch as s p e r m in act ivat ion , i s cau sed , then c e r ta in ly the m ult ip le mating habits o f the f e m a le w il l be a v e r y im p ortan t fa c to r , as you have pointed out. In such a c a s e one n o r m a l mating would c a n c e l out a l l the p r e v io u s s t e r i le m atings s in c e the s t e r i le s p e rm w i l l not contr ibute to fe r t i l i za t io n o f the egg .On the o th er hand, i f future stud ies indicated that the s t e r i l i z in g d o s e o f 90 krad did not induce s p e rm d am age o r , in o th er w o rd s , that the s t e r i le s p e r m i s fu lly c o m p e t i t iv e with n o r m a l sp e r m , then the m ultip le mating habits o f the fe m a le would not h am p er a ttem pts to c o n t r o l this in s e c t by the s t e r i l e -m a le technique - p rov id ed , o f c o u r s e , that the o th er fa c to r s w e r e fa v o u ra b le .

D . T . NO RTH : I should l ike to c o m m e n t b r ie f ly on two po in ts . F ir s t ly , I do not b e l ie v e that in p r a c t i c e as m uch as <J0 krad is r e q u ire d to a c c o m p l ish s t e r i l iz a t io n o f the c o f fe e lea f m in e r , s in ce fe r t i l i ty is w e l l under 10% at 60 krad. F o r the p u r p o s e s o f a s t e r i l e - m a le r e le a s e p r o g r a m m e , th is i s a su f f ic ie n t ly high d e g re e o f s t e r i l i ty ; fu r t h e r m o r e , the s iza b le d r o p in r e q u ir e d d osag e w i l l m o s t proba b ly re su lt in r e d u ce d p h y s io lo g ic a l and so m a t ic dam age to the in s e c ts .

S econ d ly , c o n c e rn in g D r . C u r t i s 's c o m m e n t on the e f fe c t o f m ult ip le m ating , f r o m o u r own w o rk with T r ic h o p lu s ia ni. w e 'h av e found that the i r ra d ia te d m a le tends to t r a n s fe r l e s s s p e r m o r at high d o s e s none at a l l .In c a s e s like this , m ult ip le m ating b e c o m e s an im p ortan t f a c to r - s in c e n o r m a l m a le s in the population a r e cont inu in g to con tr ib u te n o r m a l quant i t ie s o f s p e r m . In p r a c t i c e this m eans that n o r m a l m a tin gs fo l low in g

S M- 10 2/1 8 175

s t e r i l e - m a l e m atings would c a n c e l out the e f fe c t o f r e le a s in g the ir ra d ia te d m a le s .

K. P . K A T IY A R : 1 am not c o n v in ce d that 90% s te r i l i ty i s an a c c e p ta b le p r a c t i c a l l e v e l f o r the c o f f e e le a f m in e r in a s t e r i l e - m a l e r e le a s e p r o g r a m m e . If it is , then a d o s e o f 60 krad would c e r ta in ly be m u ch m o r e a c c e p ta b le in v iew o f the re d u c t io n in s o m a t ic dam age c a u se d to the in se c t .

A s to y o u r se co n d point, I a g r e e with what you say but cannot e la b o ra te on this s in c e we have not in fa c t c a r r i e d out any stud ies on the c o f fe e lea f m in e r to tes t the e f f e c t o f s t e r i l i z in g d o s e on the sex u a l v ig o u r o r mating c o m p e t i t iv e n e s s o f the trea ted m a le s .

V . L A B E Y R IE : Is the beh av iou r o f i r ra d ia te d m a le s d i f fe re n t f r o m that o f u n irrad ia ted o n e s ? What s e le c t io n p r o c e s s i s fo l lo w e d by n o rm a l f e m a le s when i r ra d ia te d and n o r m a l m a le s a re in co m p e t i t io n with each o th e r?

K. P. K A T IY A R : I cannot yet say what kind o f s e l e c t io n a n o rm a l fe m a le would m ake in the p r e s e n c e o f both n o r m a l and ir ra d ia te d m a le s but, look in g at c a s e s o f o th er L e p id o p te ra in the l i t e ra tu re , I should im ag in e that the s t e r i le m a le s would be l e s s c o m p e t i t iv e .

V . L A B E Y R IE : In the c a s e o f m ating with an i r ra d ia te d m a le , a r e s p e r m a to z o a t r a n s fe r r e d to the sp e rm a th e c a e o r , i f not, a r e s o m e s e c r e t i o n s f r o m the p a ra gon ia su p p r e s s e d so that the m a le s a r e re n d e re d s t e r i l e although they can s t i l l p ro d u ce s p e r m a to z o a ?

K. P . K A T IY A R : W e have not c h e c k e d the s p e rm a th e c a e fo r the p r e s e n c e o f s p e rm in a fe m a le m ated with ir ra d ia te d m a le s . It would c e r ta in ly be in te re s t in g to study the e f fe c t o f s t e r i l iz a t io n on the s e c r e t o r y a c t iv ity o f the m a le r e p ro d u c t iv e s y s te m and it is ju s t p o s s ib le that the t re a te d m a le d o e s p o s s e s s m ot i le s p e r m but that this is not t r a n s fe r r e d to the fe m a le during m ating b e ca u se the trea ted m a le la ck s se m in a l fluid, w h ich a c ts as a c a r r i e r in the t r a n s fe r o f s p e r m a to z o a f r o m m a le to f e m a le . It i s quite l ik e ly that p ro d u c t io n o f this s e m in a l fluid is a f fe c ted by the s t e r i l i z in g rad ia t ion d o s e .

B. NA G Y : Did you m ake any o b s e r v a t io n s on the e f f e c t o f p r e d a to rs o r p a ra s it e s during y o u r m a s s r e a r in g p r o c e s s ? I should have thought that s in ce you u sed l iv in g c o f fe e t r e e s you m ight w e l l have found so m e p r e d a t o r s o r p a r a s i t e s in the r e a r in g c a g e s .

K. P . K A T IY A R : We did not o b s e r v e any p a ra s it e s o r p re d a to rs a f fe c t in g ou r m oth prod uct ion o th er than ants, w hich p re se n t a threat to the in se c t in the eg g and p r e -p u p a l s ta g e s . We s m e a r e d the le g s o f the r e a r in g c a g e s with tangle foot in o r d e r to r e p e l the ants.

ISOTOPE APPLICATIONS: PHYSIOLOGY AND BIOCHEMISTRY

(Session V, Part 1)

C h a i r m a n : L . K . C U T K O M P

U N I T E D S T A T E S O F A M E R I C A

UPTAKE OF ISOTOPIC ALL Y LABELLED INSECTICIDES IN RELATION TO TEMPERATURE AND OTHER PHYSICAL FACTORS *

. L .K . CUTKOMPDEPARTMENT OF ENTOMOLOGY, FISHERIES AND WILDLIFE, UNIVERSITY OF MINNESOTA,S t. PAUL, MINN. , UNITED STA TES OF AMERICA

Abstract

UPTAKE OF ISOTOPICALLY LABELLED INSECTICIDES IN RELATION TO TEMPERATURE AND OTHER PHYSICAL FACTORS. L abelling in sectic id es with isotopes provides a sensitive m ethod for investigating their uptake in relation to unusual to x ic responses in insects. The uptake o f 14C -D D T was traced at three different exposure tem peratures with m osquito larvae, Aedes ¿eg y p ti. In add ition , explanations o f different to x ic ity responses related to the insect densities w ere studied with U C -D D T on Tribolium con fusum , and with tr itiu m -la b e lled n icotin e on m osquito larvae. In the latter study, the labe lled n ico tin e produced greater tox icity w hich was apparently due to a m ore stable ch e m ica l bonding resulting in fa ilure o f the insects to m eta b o lize the iso top ica lly labe lled ch e m ica l as readily as natural n icotin e .

1. INTROD UCTION

The uptake o r ' p i c k - u p 1 o f an in s e c t i c id e i s one o f the c r i t i c a l f a c t o r s lea d in g to the u ltim ate t o x ic e f f e c t on an in s e c t . The m e t a b o l i s m o f a c om p ou n d , e i th e r d eg ra d at ion o r a c t iva t ion , is the s e c o n d m a jo r c om p on en t in fluenc ing the to x ic r e s p o n s e . The p r e se n t r e v ie w is l im ite d to the u p take o f i s o to p ic a l ly la b e l l e d in s e c t i c id e s , p a r t i c u la r ly as re la te d to te m p e ra tu re d i f f e r e n c e s and population den s ity during the e x p o s u r e p e r io d .

The e x t r e m e ly se n s i t iv e d e te c ta b i l i ty l e v e l a ids in exp la in in g unusual o r u nexp ected r e s p o n s e s to a tox in . E x a m p le s a re p re se n te d as re la te d to te m p e ra tu re and to the p h y s ic a l fa c t o r o f population d en s ity . T h e se fa c to r s b e c o m e h ighly im p ortan t when, with c e r ta in in s e c t i c id e s , the in se c t r e s p o n s e d o e s not fo l lo w the p r e d ic te d pattern .

2. E X P O SU R E T E M P E R A T U R E

D D T exh ib its a n egative te m p e ra tu re c o e f f i c i e n t [1 -4 ] . One p laus ib le explanation is that the uptake o r so r p t io n o f the tox in is g r e a t e r in the c u t ic le o f an in s e c t at a c o o l e r te m p e ra tu re [5] . S ince the stab i l i ty o f D D T w as w e l l known in addition to that o f the p o s s ib le m e ta b o l i t e s , D DE and p o s s ib ly T D E , a 14С la b e l l in g o f the t r ic h lo ro e th a n e p o r t io n o f the m o l e cu le w as u sed to d e te rm in e uptake at th ree d i s c r e t e t e m p e r a tu r e s , 10,20 and 30°C [6] . T he r e s u l t s sh ow ed that the uptake o f 14 C - D D T by

* Paper N o. 6357 S c ie n tif ic Journal Series, M innesota A gricultural Experim ent Station , St. Paul, M inn. 55101.

179

180 CUTKOMP

A e d e s aegypti la rv a e w as p o s i t iv e ly c o r r e la t e d with te m p e ra tu re , and thus did not c o r r e s p o n d to the n ega t iv e ly c o r r e la t e d to x ic i ty r e s p o n s e s . T he uptake o f 14 C - D D T w as 41 . 5 , 1 1 8 .3 , and 157. 4 p g / m g d r y w eight at e x p o s u re te m p e ra tu re s o f 1 0 ,2 0 and 30°C, r e s p e c t iv e ly . O f addit iona l in te r e s t w e r e the c o m p a r a t iv e uptakes by the heads , t h o r a c e s , and a b d o m e n s o f la rv a e as tabulated in T ab le I. The heads had the g re a te s t uptake o f 14C -D D T at the c o o l e r te m p e ra tu re , w hile the th o r a c e s had the g r e a te s t uptake at the w a r m e r t e m p e r a tu r e s . Thus the p h enom enon o f the negative te m p e ra tu re e f fe c t , p a r t icu la r ly ev ident with D D T and r e la te d in s e c t i c id e s , but n ot ’ with m any oth er in s e c t i c id e s , d o e s not ap pear to be attr ibutable to the uptake o f D D T .

T A B L E I. R A D I O A S S A Y O F C O M P O N E N T S O F 14 C - D D T - T R E A T E D A e d e s a e g y p t i L A R V A E

T h e q u a n t i ty ( / j g X 1 0 '4 p e r m g d r y w t . ) o f 14C - D D T d e t e r m i n e d f r o m h e a d s , t h o r a c e s , and a b d o m e n s . L a r v a e e x p o s e d to 0 . 0 0 3 p p m D D T f o r 48 h o u r s . V a lu e s a r e m e a n s o f t h r e e r e p l i c a t i o n s in v o lv in g 50 l a r v a e e a c h

Larval co m p o n e n tT rea tm en t tem peratu res (d e g C )

1 0 ” 2 0 ” 3 0 “

H ead 2 3 .5 6 4 0 .9 3 5 6 .4 0

T h ora x 1 2 .7 0 5 7 .0 6 7 7 .3 3

A b d o m e n 5 .2 3 2 0 .2 6 2 3 .5 3

T o ta l la rvae 4 1 .4 9 1 1 8 .2 5 1 5 7 .3 6

3. P O P U L A T IO N D ENSITY

Is o to p ic a l ly la b e l le d in s e c t i c id e s w e r e used f o r e lu c id at ion o f d i f fe re n t ia l r e s p o n s e s to a tox in as re la te d to population den s ity . D ev ia t ion s ap parently due to n u m b e rs w e r e apparent with a s s a y s o f m o s q u ito la r v a e , A e d e s aegypti and A n op h e les qu ad r im a cu la tu s , and with f lou r b e e t le s , T r ib o l iu m con fu su m .E a rly r e s e a r c h on DDT to x ic i ty to m o sq u ito la rv a e [7 ,8 ] r e v e a le d that changing the n um ber o f la rv a e p er unit v o lu m e by v a ry in g e ith er the v o lu m e o r the n um ber m igh t a f fe c t the per cent m o r ta l i t y . R e su lts ind icated that the g r e a t e r the n u m ber o f la rv a e the l o w e r the p er cent m o r ta l i t y . T h is w as la te r substantiated by Schm idt and W eid haas [9] who u t i l ized i s o to p ic a l ly la b e l le d in s e c t i c id e s to so lv e a p r o b le m . T h e y found a g r e a te r a b so rp t io n ( o r uptake) o f 14C -D D T and a h igher m o r ta l i t y with l o w e r d e n s it ie s o f A n o p h e le s qu ad r im acu la tu s la rv a e than with h igh er d e n s it ie s . C o m p a r is o n s with two 32P - la b e l l e d o rga n o p h o sp h a te s , c o u m ap h os (B a y e r 2 1 /1 9 9 ) and d im eth oate (A . C. 12880) did not show this phenom enon to be r e la te d to the den sity o f m o sq u ito la r v a e . In b r ie f , both p e r cent m o r ta l i t y and uptake

S M - 102/19 181

o r a b so rp t io n o f the d o s e w e r e a p p ro x im a te ly the sa m e even when the n u m ber o f la rv a e p e r b e a k e r w as changed f r o m 25 to 400 (T a b le II). F u r t h e r m o r e , by ca lcu la t in g am oun ts o f la b e l l e d in s e c t i c id e s r e m o v e d f r o m the t rea ted w a te r it w as p o s s ib le to c o n c lu d e that the v a r ia t io n in m o r ta l i t y depended upon the p e r cent r e m o v a l o f the in s e c t i c id e , w hich w as c r i t i c a l in the c a s e o f D D T , but not c r i t i c a l with the two o r g a n o p h o s p h a te s , s in ce the c o n ce n tra t io n o f the a c t ive tox in s w as not d e c r e a s e d enough to a f fe c t the m o r ta l i t y r e s p o n s e .

One cannot c o n c lu d e , h o w e v e r , that population d en s ity m a y not be a fa c t o r in the to x ic i ty r e s p o n s e s o f o rg a n o p h o sp h a te s . In a study witTi parath ion , e . g . the L D 50 value with 10 la rv a e p er b e a k e r w as about half ( 6 . 4 ppb)l that when 300 la rv a e w e r e tes ted ( L D 50 = 11 .3 ppb) [1 0 ] (T a b le III).

The uptake o f 14C -D D T w as a lso u sed to help c la r i f y r e s u l t s o f a b io a s s a y o f d i f fe re n t d e n s it ie s o f the co n fu se d f lo u r b e e t le , T r ib o l iu m con fu su m [10] . T h e se r e s u l t s c o n t ra s te d with those o f the m osqu ito la rv a e b io a s s a y in that a p o s i t iv e c o r r e la t i o n w as found betw een density and p e r cent m o r ta l i t y . The r e la t io n s h ip w as on ly ev ident in c o v e r e d p e tr i d is h e s . The e x p e r im e n t a lso d i f fe r e d in that the am ount o f DDT u sed f o r s u r fa c e e x p o s u re o f the b e e t le s w as a lw ays in e x c e s s , w hile in m o s q u ito la r v a l te s ts D D T w as dep leted by a high d en s ity o f la r v a e . The u se o f 14C -D D T did not r e v e a l s ign if ican t d i f f e r e n c e s in uptake o f the la b e l le d in s e c t i c id e r e la te d to d en s ity , and the c o n c lu s io n s draw n w e re that a c r i t i c a l sh o r ta g e o f ox yg en o c c u r r e d with high d e n s it ie s o f b e e t le s te s te d in c o v e r e d d is h e s , thus r e su lt in g in g r e a te r m o r ta l i t y when they w e r e e x p o se d to D D T .

T r i t iu m - la b e l l e d n ico t in e p ro d u ce d ra th e r u n ex p ected r e s u l t s when tes ted on A e d e s aegypti la rv a e [11] , and these should s e r v e to w arn

T A B L E II. T W E N T Y - F O U R H O U R M O R T A L I T Y , A B S O R B E D D O S E , A N D P E R C E N T O F T O T A L A V A I L A B L E I N S E C T IC ID E R E M O V E D B Y F O U R T H - I N S T A R A n o p h e le s q u a d r im a c u l a t u s L A R V A E E X P O S E D IN 2 50 m l O F W A T E R T R E A T E D W IT H R A D I O A C T I V E D D T ( 0 . 0 0 7 p p m ) A N D C O U M A P H O S ( 0 . 0 3 p p m )

N um ber Per c e n t m o r ta lityA b sorb ed dose

(iJg /L a rv a )Per cen t rem o v e d

la rva e D D T cou m a p h osD DT cou m a p h os

D D T cou m a p h os

25 55 53 0 .0 0 6 9 0 .0 0 2 4 9 .9 0 .9 7

50 33 4 8 0 .0 0 6 4 0 .0 0 2 3 18 1 .5

10 0 14 54 . 0 .0 0 4 6 0 .0 0 2 0 26 2 .7

2 0 0 3 53 0 .0 0 3 3 0 .0 0 1 8 38 4 . 8

400 3 53 0 .0 0 2 6 0 .0 0 2 0 59 1 0

1 ppb = parts per b illion ( I 0 9 ).

182 C U T K O M P

T A B L E III. T H E L D 50 V A L U E S D E T E R M I N E D F O R P A R A T H I O N A N D 4th I N S T A R L A R V A E O F A e d e s a e g y p t i W I T H D I F F E R E N T N U M B E R S O F L A R V A E F O R E A C H T E S T

N um bers o f la rv a e in 1 0 0 m l per b eak er

LD 50

in ppb

•1 0 6 . 4 ± 0 . 3

25 7 . 4 ± 0 .2

1 0 0 7 . 6 ± 0 .2

2 0 0 8 . 6 ± 0 . 1

300 1 1 .3 ± 0 . 2

r e s e a r c h e r s against b l ind ly assu m in g that the beh av iou r o f an i s o to p ic a l ly la b e l le d com pou nd w ould be the sa m e as that o f natural c h e m ic a ls . A s with D D T , p r e l im in a r y s tud ies show ed d i f f e r e n c e s in la rv a e m o r ta l i t y r e la te d to d en sity . The re la t io n sh ip w as a n egative one , as w a s p r e v io u s ly r e p o r t e d f o r D DT and parath ion , although the magnitude o f the d i f fe r e n c e w as l e s s . The l ik e ly explanation was d ep let ion o f n icot ine due to g r e a te r n u m b e rs o f la r v a e . T r i t iu m - la b e l le d n icot in e w as used to test this h ypoth e s is . The r a d io a ct iv e com pound w as p r e p a r e d f r o m n ico t in ic ac id 2 - 3H w hich w as in trodu ced into the nutrient so lu t ion o f the r o o t s o f N icotian a tabacum plants to obtain ra d io a c t iv e n ico t in e (n icot ine 2 - 3H) b io sy n th e t ica l ly . The u se o f the t r i t iu m - la b e l le d n icot in e r e s u lte d in no d i f f e r e n c e s in r e s p o n s e re la te d to d i f fe re n t d e n s it ie s o f the m o s q u ito la r v a e . In addition to this f inding, ra d io a c t iv e n ico t in e was s ign if ican t ly m o r e to x ic to the la rv a e than natural n ico t in e (F ig . 1). The L D 50 va lu es fo r tr it ia ted n icot in e w e r e 178 and 175 ppm with la r v a l popu lations o f 50 and 400, r e s p e c t iv e ly . The L D 50 va lu es f o r natural n icot in e w e r e 202 and 275 ppm with la rv a l populations o f 25 and 400, r e s p e c t iv e ly . S e v e ra l p o s s ib le explanations o f the in c r e a s e d to x ic i ty w e re ru led out, including p o s s ib le contam ination o f a b y -p r o d u c t and the l ib e ra t io n o f r a d io a c t iv e agent. The la tter p o s s ib i l i t y w as tested by usin g tr it ia ted w ater at a m uch h igh er l e v e l o f r a d io a ct iv ity (1 m C i /1 0 0 m l w a ter ) with no m o r ta l i t y fo l lo w in g a 2 4 -h o u r e x p o su re p e r io d . The b e s t explanation o f in c r e a s e d to x ic i t y a p p ears to be the v e r y slow m e ta b o l ic d e c o m p o s i t i o n o f tr it ia ted n ico t in e , the c le a v a g e o f the c a r b o n - t r i t iu m bond be ing m uch m o r e d i f f i cu lt than the n o n - is o to p i c c a r b o n -h y d ro g e n bond.

T he d i f fe r e n c e m u st be re la ted to the s ite o f la b e l l in g (the 2 -p o s i t io n on the p yrid in e r in g o f the n icot in e m o le c u le ) , thus invo lv ing a p o r t io n o f the n icot in e m o le c u le (pyrid ine ring) w hich had not been c o n s id e r e d in p r e v io u s stud ies o f n icot in e m e t a b o l i s m , e ith e r with in se c ts o r v e r t e b r a te s .

The e x p e r im e n ta l study with n icot ine e m p h a s iz e s the value o f i s o t o p i c a l ly la b e l le d co m p o u n d s , but sounds a w o rd o f caution r e g a rd in g a s s u m p tions that the ac t iv ity o f n aturally o c c u r r in g com p ou n ds is the sa m e as that o f th e ir i s o to p ic a l ly la b e l le d cou n te rp a r ts .

SM-lO' j/19 183

150 200 300P P M

FIG. 1. Regression lines showing 48-hour responses o f larvae o f Aedes aegypti to natural n icotin e and tritiated n ico tin e , (adapted from M akky, D octora l thesis, Univ. o f M in n ., 19S5)

4. SU M M AR Y

Is o to p ic a l ly la b e l le d in s e c t i c id e s can be d e tected at v e r y low l e v e l s , and a r e thus v e r y u se fu l f o r de te rm in in g uptake o f the in s e c t i c id e by the in s e c t f r o m the e n v iron m en t . One la b o r a to r y e x am p le is the uptake o f 14C -D D T by m o s q u ito la rv a e at th ree e n v iron m en ta l t e m p e r a tu r e s , 10°,20° and 30°C. A p o s i t iv e c o r r e la t i o n o c c u r r e d betw een uptake and in c r e a s e in te m p e ra tu re . H o w e v e r , the t o x ic i ty r e s p o n s e w as n ega t iv e ly c o r r e la t e d with te m p e ra tu re . 14C -D D T w as a lso u sed to analyse d i f fe re n t ia l t o x ic i ty r e s p o n s e s when the population d en s ity w as v a r ie d . A l o w e r p e r cent m o r ta l i t y o c c u r r e d with h igh er d e n s it ie s o f m osqu ito la r v a e . The e f fe c t w as due to a g r e a t e r d ep letion o f the tox in by g r e a t e r total n u m bers o f la r v a e , r e su lt in g in l e s s tox in p er la r v a . A co n tra st in g study with f lo u r b e e t le s r e v e a le d a h igh er p e r cent m o r ta l i t y with h igh er d en s it ie s in c o v e r e d p e t r i d is h e s . Since the uptake o f 14C -D D T did not d i f fe r at d i f fe ren t d e n s it ie s , a d is t in c t cau se w as in d icated . The e f f e c t a p p eared to be due to ox y g e n dep letion a s s o c ia te d with D DT to x ic ity .

One additional study r e v e a le d that natural n icot in e w as dep leted f r o m so lut ion at a h igh er rate by g r e a te r d en s it ie s o f m o sq u ito la r v a e , an e f fe c t c o r r e s p o n d in g to that d e s c r ib e d f o r D D T . H o w e v e r , when tr it ia ted n icot ine w as e m p lo y e d , an in c r e a s e d to x ic ity o c c u r r e d u nre la ted to population density . E v id e n ce in d ica tes the inab ility o f la rv a e to m e ta b o l iz e the i s o to p ica l ly la b e l le d co m p ou n d s ign if ican t ly during the e x p o su re p e r io d .

R E F E R E N C E S

[ 1 ] FAN, Н .У . , CHENG, Т . Н . , RICHARDS, A . G . , The tem perature c o e ff ic ie n t o f D DT action in insects, Physiol. Z o o l . 21 (1948) 48 .

[ 2 ] VINSON, E .B . , KEARNS, C . W . , Tem perature and the action o f DDT on the A m erican roach ,J. e co n . Ent. 45 (1952) 484 .

[ 3 ] LOFGREN, C , S . , CU TK O M P, L .K . , T o x ic ity ó f DDT to the A m erican cock roa ch when lip id content and tem perature are varied , 1. e co n . Ent. 49 (1956) 167.

[ 4 ] D AS, M . , NEEDHAM, P .H . , E ffect o f tim e and tem perature on tox ic ity o f in sectic id es to insects.I ; Tests o f DDT on larvae o f A edes a eg y p ti, Ann. appl. B iol. 49 (1961) 32.

184 CUTKOMP

[5 ] RICHARDS, A .G . , CUTKOMP, L. K . , C orrelation betw een the possession o f a chitinous cu tic le and sensitivity to D D T , B iol. Bull. 90 (1946) 97.

[6 ] ZUBAIRI, M . Y .C U T K O M P . L. K . , The p ick -u p o f C w - DDT at different tem peratures. Entom ología exp . appl. 7 (1964) 239 .

[7 ] KRUSE, C . W . , LUDVIK, G .F . , HAWKINS, W .B . , Factors a ffectin g evaluation o f insecticides against Anopheles larvae, I. e co n . Ent. 45 (1952) 598.

[ 8] W A T TA L, B. L . , The in fluence o f different numbers o f insects on m orta lity produced by in sectic id es ,as shown with Tribolium spp. and Aedes aegypti L . , Diss. .Abstr. Publ. N o. 3965, Univ. o f M inn.(1952).

[9 ] SCHM ID T, C . H . , WEIDHAAS, D .E ., Absorption and tox icity o f three rad ioactive insecticidesin larvae o f two species o f m osquitoes, J. e con . Ent. 5] (1958) i)40.

[1 0 ] KOLKAILA, A . M . , CU TK O M P, L. K . , unpublished data, Department o f E ntom ology, Fisheries, and W ild life , Univ. o f M inn. (1963).

[1 1 ] W A T TA L, B .L ., CUTKOM P, L . K . , The relationship betw een density and m orta lity o f flour beetles exposed to in sectic id e -trea ted surfaces, J. e co n . Ent. 52 (1959) 1134.

[1 2 ] M A K K Y , A . M . , Radioactive n ico tin e : biosynthesis, t o x ic ity , and m etabo lism , Diss. Abstr. N 0 .6 6 -9 0 3 0 (1965).

D I S C U S S I O N

F . T . P H IL L IP S : What do the quantities shown in T ab le I r e p r e s e n t in t e r m s o f t o x ic i t y ? F o r in stan ce , is th ere any c o r r e la t i o n betw een (a) the uptake at each o f the th ree te m p e ra tu re s , e ith er c o n s id e r in g the v a r io u s bod y co m pon en ts se p a r a te ly o r the la rv a as a w h o le , and (b) a c h o s e n to x ic ity le v e l ( L D 50, L D 90, e t c . ) at the p a rt icu la r te m p e ra tu re which is be ing c o n s id e r e d ?

L . K . C U T K O M P : The va lu es in T a b le 1 w e r e d e te rm in e d a f te r t r e a t m ent at a lethal c o n ce n tra t io n som ew h at l e s s than L D 50 o r L C 5o. T h is c o n c e n tra t io n w as c h o s e n d e l ib e r a te ly , s in c e be low the L C 50 l e v e l a s t r o n g e r e x p r e s s io n o f negative r e s p o n s e is exhib ited and at h igh er le v e l s such as LCgo the amount o f D DT in vo lved would p ro b a b ly be e x c e s s i v e . ■

F . T . P H IL L IP S : Did you m e a s u re ra tes o f uptake over- the 4 8 -h o u r p e r io d and, i f s o , w e r e these the sa m e throughout the p e r io d ?

L . K. C U T K O M P : We did obtain s o m e m e a s u re m e n ts a fte r 24 h ours and, as I r e c a l l , the re la t io n s h ip s at the v a r io u s te m p e ra tu re s w e r e about the s a m e .

F . T . P H IL L IP S : With r e f e r e n c e to T a b le II, does the l o w e r so lu b i l i ty o f D DT in w ater c o m p a r e d with that o f c o u m ap h os in w ater have any b e a r in g on the o b s e r v e d g r e a te r p e r ce n ta g e r e m o v a l o f D D T , c o n s id e r in g that D DT in aqueous so lut ion a c cu m u la te s at in te r fa c e s , such as a i r /w a t e r , con ta in e r w a l l s /w a t e r , and in s e c t c u t i c l e /w a t e r ?

L . K . C U T K O M P : Both the low so lu b i l i ty o f D DT in w ater and its g re a t a ff in ity f o r l ip id s o r l ip o p ro te in s in the in se c t c u t ic l e s e e m to be in vo lved . C ou m aph os is fa r l e s s l ip o p h i l i c and it would thus ap p ear that the m o s q u ito la rv a e can r e m o v e DDT f r o m the aqueous su sp e n s io n m uch m o r e r e a d i ly than an o rg a n o p h o sp h a te .

1. A . KANSU: 1 am c u r io u s about the am ount o f D DT taken up as g iven in T ab le I. Do these data r e f e r to the amount found in the body o f the m osqu ito la rv a e o r that found both in and on the b o d y ? Secon d ly , am I r ight in thinking that T a b le II in fac t show s ' o b s e r v e d m o r ta l i t y ' ra th er than ' c o r r e c t e d m o r ta l i t y ' ba sed on a p r o c e s s such as the Abbot f o r m u la ?

S M - 102/19 185

L . K . C U T K O M P : The am ount r e p o r t e d in T ab le I is that quantity found within the in s e c t , s in c e the ou ts ide o f the la rv a e w as r in s e d with ace ton e b e fo r e a n a lys is w as m a d e . A s to y o u r s e c o n d qu e st io n , I w ould su g g e s t you r e f e r to the paper by Schm idt and W eidhaas (R e f . [9] o f m y p aper) .

P . A . L A N G L E Y : It w as stated that c le a v a g e o f the c a r b o n - t r i t iu m bond in tr it ia ted n ico t in e is m o r e d i f f i cu lt than c le a v a g e o f the n o n - i s o to p i c c a r b o n -h y d r o g e n bond . Have you thought o f using 14C - la b e l l e d tr it ia ted and n o n -t r i t ia te d n ico t in e to substantiate this sta tem en t qu an ti ta t ive ly?

L . K . C U T K O M P : Y e s , w e c o n s id e r e d usin g 14C, but it would not be p o s s ib le , I think, to p re p a re the n ico t in e 14C b io sy n th e t ica l ly as w as done f o r the tr it iated n ico t in e . W e have not yet gone into this p r o b le m in any deta il , but I am o f the op in ion that substitution o f d eu ter iu m instead o f tr it iu m w ould m ake f o r a be t te r c o m p a r is o n .

P . P E L E G R IN : It would be in te re s t in g to d e te rm in e w hether theto x ic i ty o f deu terated n icot in e la y betw een that o f u n la be l led a n d t r i t ia te d n ico t in e , the m a s s o f 2H be ing in te rm e d ia te betw een th ose o f 'H and 3H.

L . K . C U T K O M P : C erta in ly , substitution o f d eu ter iu m fo r ] H o r 3H would be m o s t ap p ro p r ia te fo r p u rp o s e s o f c o m p a r is o n in c a s e s w here c h e m ic a l bonding at the sa m e s ite cou ld be d e te rm in e d and the b io lo g i ca l a c t iv ity c o m p a re d .

II. M A R C H A R T : A s r e g a r d s an explanation f o r the negative t e m p e r a ture c o e f f i c i e n t you found f o r r e s p o n s e to DDT, have you en v isaged the p o s s ib i l i t y o f a change in the rate o f de h yd ro ch lo r in a t io n by the in s e c t ?

L . K . C U T K O M P : Y e s , this had been c o n s id e r e d , but all c h e m ica l tes ts show that d e h y d ro ch lo r in a t io n p r o c e e d s m o r e ra p id ly with in cre a s in g te m p e ra tu re . It s e e m s m o r e l ik e ly , th e r e fo r e , that a c h e m ic a l exp lan ation w ould invo lve c e r ta in m e ta b o l ic com pou n d s and the ir vary in g p r o p o r t ions within the in se c t .

M ETABOLISM OF M ALATHION IN THE DESERT LOCUST Schistocerca gregaria FORSKAL: Distribution and degradation of 32P -labelled malathion in vivo

K. N. MEHROTRA, G .R . SETHIAND Y . PRASADDIVISION OF ENTOM OLOGY,INDIAN AGRICULTURAL RESEARCH INSTITUTE,NEW DELHI, INDIA

Abstract

METABOLISM OF MALATHION IN THE DESERT LOCUST Schistocera gregaria FORSKAL: DISTRIBUTION AND DEGRADATION OF 32P-LABELLED MALATHION IN VIV O . Distribution and degradation o f m alathion in the desert locust (Sch istocera gregaria) was studied in v ivo with S2P -la b e lled m alathion .Results showed that 65 .6 % o f the applied dose was absorbed by the locust treated with 100 /ig m alathion / insect, w ithin 270 m inutes. During the sam e period, the locust excreted 2 4 .5 % o f the total applied dose, or nearly 37% o f the absorbed m alathion , in faeces. The m ajor am ount o f rad ioactiv ity in the faeces was associated with m alathion m eta b o lic products due to phosphate a ction . The m eta b o lic rate and products associated with various tissues w ere also studied and their im portan ce discussed.

1. INTRODUCTION

W ork in o u r la b o r a t o r y p r e v io u s ly esta b l ish e d the p r e s e n c e o f e n zym e s y s te m s in the d e s e r t lo c u s t f o r act iva tion o f m alath ion into a potent a n t ich o l in e s te ra se [1] and f o r the d eg ra dat ion o f m a lath ion into n o n - t o x ic p ro d u c ts [2] . The p u rp ose o f the p re se n t in vest ig a t ion was to e lu cidate the pattern o f o v e r a l l m e ta b o l is m o f m a lath ion by adults o f the d e s e r t lo c u s t S. g r e g a r ia F o r s k a l in v ivo . T h is is im portant, s in c e it is known that two a lternate ty p es o f pathway a re av a i lab le f o r the m e ta b o l is m o f m alath ion (phosphatase type and c a r b o x y e s t e r a s e type) in v iv o . M o r e o v e r , stud ies witli d i f fe ren t in se c t s p e c i e s su g ges ted that the p e r cent o f m alath ion m e ta b o l iz e d through th e se pathways d i f fe r e d sign if ican t ly , both qualita tive ly and quantitatively f r o m one s p e c i e s o f in se c t to another [3, 4] .

2. M A T E R IA L S AND METHODS

Adults o f the d e s e r t lo c u s t S. g r e g a r ia F o r s k a l , obta ined f r o m a la b o r a to r y c o lo n y and m ainta ined a c c o r d in g to M e h r o tr a and Rao [5 ] , w e re t rea ted t o p ic a l ly with 10 /jl so lu t ion o f 1% 32P - la b e l l e d m a lath ion in be n ze n e . T h is d o s e o f m alath ion gave 99% m o r ta l i t y at 8 h ours [5]. T he to p ica l app l ica t ion o f m a lath ion was m a d e on the m e so n o tu m with the aid o f an e l e c t r i c a l l y o p e ra te d m i c r o - a p p l i c a t o r (ISCO M o d e l M) m a nu factu red by the In stru m en ta tion S p e c ia l i t ie s C o . I n c . , L in co ln ,

187

188 MliHROTRA et al.

N ebraska . 32P - la be l led m alath ion was supplied by the A t o m ic E n e rg y E stab lish m en t, T r o m b a y , and was id e n t ica l c h r o m a to g ra p h ica l ly to the known m alath ion . The to x ic i ty o f r a d io a c t iv e m alath ion w as c o m p a r a b le to that o f the standard pure m a te r ia l . M alath ion used as standard was kindly su pp lied by D r. Lallan Rai o f the Cyanam id India L t d . , B o m b a y .

The to p ic a l ly t rea ted in s e c ts w e re r in s e d with a ce ton e to r e m o v e the u n a bsorb ed m alathion rem ain in g on th e ir s u r fa c e . Im m e d ia te ly a fte r r in s in g , the r a d io a ct iv ity in v a r io u s p arts was d e te rm in e d . V a r io u s o r g a n s w e re h o m o g e n iz e d in 10% t r i c h l o r a c e t i c ac id , and the d egradation p r o d u c ts o f m alath ion - c a r b o x y e s t e r a s e and phosphatase - w e re e x trac ted a c c o r d in g to the m ethod o f M atsum ura and H ogendijk [is]. F a e c e s c o l le c t e d at v ar iou s t im e s was e x tracted in the sa m e m ann er as o th er t i s s u e s .

A l l rad ioact iv ity m e a s u re m e n ts w ere m ade with the help o f a G e ig e r - M ü l l e r thin end window tube at a f ixed g e o m e tr y and c o r r e c t i o n s w e re m ade fo r the backgrou n d .

3. RESULTS

3 .1 . A b so rp t io n and d is tr ibu tion o f 3"P - la b e l le d m alathion

Data on the d is tr ibution o f r a d io a ct iv ity in d e s e r t lo c u s ts treated with 100 ц g o f la be lled m alathion are p resen ted in F ig s 1 and 2.T h e se data r e p re s e n t the total rad ioa ct iv ity o f m alathion and its d egradat ion p rodu cts p re se n t at v a r io u s t im e s in v ar iou s t i s s u e s . It i s apparent (F ig . 1, cu rve (a)) that 19 .8% o f the applied rad ioa ct iv ity penetrated within the f i r s t s ix m inutes and that the in terna l r a d io a ct iv ity s te a d i ly in c r e a s e d to 45. 2, 47. (j, and 65. (>% o f the total applied d o s e at 90, 150 and 270 m inutes , r e s p e c t iv e ly , a fte r treatm en t . Rapid and e a r ly penetrat ion o f the in s e c t i c id e c o n f ir m e d the findings o f B u e r g e r and O 'B r ie n [7] . During the o b s e rv a t io n p e r io d o f 270 m inu tes , the in s e c t s e x c r e te d 24 .5% o f the total applied d o se in fa e c e s (F ig . 1, cu r v e (b)). L it t le e x c r e t io n o f ra d io a ct iv ity o c c u r r e d during the f ir s t 150 m inutes , but th e r e a f te r it in c r e a s e d . The th orax (F ig . 1, cu rve ( с )) had the h ighest amount o f ra d io a ct iv ity at 9U m inu tes , then equ il ibr iu m was m a inta ined . The rapid rate o f penetrat ion ended at about 90 m inutes ; th en ce the rate o f e x c r e t io n p re d o m in a te d . In Fig . 2 we se e that d i s t r i bution o f ra d io a ct iv ity in the gut ( including m a lphig ian tubules , m ain orga n r e s p o n s ib le fo r e x c r e t io n ) was s im i la r to that o b s e r v e d f o r fa e c e s .In the gut, as in the fa e c e s , th ere was litt le act iv ity in itia lly , but a fter 150 m inutes the activ ity in c r e a s e d . The amount o f rad ioa ct iv ity in f a e c e s and gut a fter 270 m inutes accounted f o r 3 8 .7 % o f the tota l d o se applied , which is about 5 8 .6 % o f the totai m alathion a b s o r b e d . In the ab dom en (without gut) the amount o f ra d io a ct iv ity in c r e a s e d until 150 m inu tes , at which t im e the abdom en had 7 .8 % o f the total applied dose , and then d e c r e a s e d , being on ly 5. 8% o f the tota l applied do se at 270 m in u tes . Thus, as the r a d io a ct iv ity in c r e a s e d in fa e c e s and gut, th ere was a c o r r e s p o n d in g d e c r e a s e in r a d io a ct iv ity in the abdom en .

L e v e ls o f malathion o r its m e ta b o l i te s in the head (the s ite o f bra in and n erv ou s activity) var ied f r o m 3 .3 to 5. 3% o f the total applied do se at v a r io u s t im e s (F ig . 3, cu r v e (a )) . C a lcu la t ion s o f the p e r cent o f the

S M -102/21 189

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T IM E , M IN .

FIG. 1. Pec cent recovery o f rad ioactiv ity at d ifferent tim es from various parts o f desert locusts top ica lly treated with 100 /jg 32P -la b e lled m alathion . (a) External wash, (b) Faeces, (c ) Thorax.

U<ОÛ<cu

Zыиссыа

TIME, MIN.

FIG. 2. Per cen t recovery o f ra d ioactiv ity at d ifferent tim es from various parts o f desert locusts top ica lly treated w ith 100 yg o f 32P -la b e lled m alathion . (a) Cut. (b) A bdom en.

tota l in ternal m alath ion gave va lu es o f 1 8 .6 , 1 4 .2 , 1 2 .3 and 7 .8 % at LO, 90, 150 and 270 m inutes , r e s p e c t iv e ly ( F i g .3 , c u r v e (b)) , suggesting that a la rg e p e rce n ta g e o f the initia l in terna l d o s e o f m alath ion found its way into the head ( o r brain) and th is con ce n tra t io n o f ac t iv ity rem ained constant f r o m 10 m inutes on w ard . T he constant l e v e l o f .r a d io a ct iv ity in the head ind icated that when the head had attained e q u il ib r iu m with the

190 ME1IROTRA et al .

in terna l le v e ls o f m a lath ion the e x c e s s m a lath ion was d iv e r te d to o th e r o r g a n s , e s p e c ia l ly the g u t .

TIME, MIN.

FIG. 3. Per cen t recovery o f rad ioactiv ity at d ifferent tim es from the head o f desert locusts treated w ith 100 (jg o f 32P -la b e lled m alathion . (a) Per cen t o f total applied dose, (b) Per cen t o f internal a ctiv ity present in the body.

3 . 2 . In v ivo m e ta b o l is m o f m alathion in the d e s e r t lo c u s t

3 . 2 . 1 . M e ta b o l ism in whole in se c ts

S ince m e ta b o l is m o f m a lath ion can take p la ce by two d if ferent pathways, n am ely (a) by c a r b o x y e s t e r a s e action , i . e . h y d r o ly s is o f COO C2 H5 bond o f m alathion, and (b) by phosphatase action , i . e . by- h y d r o ly s is o f PS С bond o f m alathion , the m e ta b o l ic p rod u cts appearing in l o c u s t s and th e ir v a r io u s t i s s u e s w e re analysed to d e te rm in e the p red om in a n t pathways pre se n t in the lo c u s t . The data (T ab le I) in d icated that o f the ra d io a ct iv ity in it ia lly p re se n t (0 -6 m inutes ) inside the lo c u s t , c a r b o x y e s t e r a s e p ro d u c ts p red om in ated o v e r phosphatase p r o d u c ts . H ow ever , with the in c r e a s e in t im e , the p e r cent o f p hosphatase p rodu cts p re se n t in the bod y in c r e a s e d s te a d i ly . T h e se r e su lt s cou ld be in terpre ted in two w ays: (1) that the c a r b o x y e s t e r a s e s w e re a c t ive in degrad in g m alath ion in in itia l s tag es when the o v e r a l l l o w co n ce n tra t io n s o f c h lo r o fo r m -s o lu b le (m a ia th io n /m a la o x o n ) m a te r ia ls w e r e p re se n t in the bod y . L a te r , h o w ev er , with the in c r e a s e in c h lo r o fo r m -s o lu b le m a te r ia l s inside the body, c a r b o x y e s t e r a s e s w ere inhibited and the m ain d egradation route was shifted to phosphatase act ion . T h is v iew was su pported by M atsum ura and H ogendijk [6], who show ed that c a r b o x y e s t e r a s e s w e re inhibited by the high c o n ce n tra t io n s o f o x yg en ana logues o f m a lath ion . (2) S ince the m alath ion ac id fo r m e d as a r e su lt o f c a r b o x y e s t e r a s e action cou ld be fu rth er m e ta b o l iz e d by- p hosphatase action , it was l ik e ly that c a r b o x y e s t e r a s e s w e re act ive throughout but th e ir degradation p rod u cts fa i led to a ccu m u la te b e c a u se o f fu r th e r ph osph atase act ion . The p r e s e n c e o f s ign if ican t am ounts o f c a r b o x y e s t e r a s e p rod u cts at 270 m inutes su ggested that the se co n d explanation w as m o s t p ro b a b ly c o r r e c t . N e v e r th e le ss , the p o s s ib i l i t y that phosphatase action p lays an im portan t r o le in de to x ica t io n o f m a la th ion by the lo c u s t should a lso be kept in v iew .

S M -102/21 191

T he data in T a b le I ind icated that the quantity o f c h lo r o f o r m so lu b le p ro d u c ts continued to in c r e a s e up to 150 m inu tes , su ggest in g that the rate o f p en etrat ion o f the in s e c t i c id e was fa s t e r than the rate o f d e g r a dation and a f te r 150 m in u tes , p erhaps due to l e s s penetrat ion o f the m alath ion , d e g ra d at ive ac t ion was m o r e prom in en t .

3 . 2 . 2 . E x c r e t i o n o f m alath ion and its m e ta b o l ite s

The data p re se n te d in T a b le II show that the m a jo r por t ion o f the ra d io a ct iv i ty in fa e c e s was a s s o c ia te d with the phosphatase p ro d u c ts and o n ly the r e m a in d e r with c a r b o x y e s t e r a s e p ro d u c ts . T h e se f indings a re in a g re e m e n t with the r e su lts obtained with w hole in se c ts , which re ve a le d that phosphatase p ro d u c ts p redom in ated o v e r the c a r b o x y e s t e r a s e p ro d u c ts . H ow ever , the p r e s e n c e o f s ign if ican t am ounts o f c h l o r o f o r m - s o l u b l e m e ta b o l i te s in the fa e c e s was ra th e r su r p r is in g , s in ce it i s g e n e ra l ly thought that in in s e c t s e x cr e t io n is o f l itt le im p o rta n ce in d is p o s in g o f l ip id - s o lu b le com pou n d s [3] .

O ther r e s e a r c h e r s w ork in g with d if fe ren t in s e c ts show ed that l ip id - so lu b le m a te r ia l s , e s p e c ia l ly in s e c t i c id e s , w e re e x c r e te d by the in s e c t s . Bull et al. [8] show ed that e x c r e ta o f the la r v a e o f the b o l lw o rm H elio th is ze a conta ined la r g e quantities o f unchanged d im eth oate as w ell as its o x yg en analogues a fte r be ing trea ted with d im eth oate . S im ila r ly , it has been shown that I le l io th is z e a la rv a e treated with 32P - la b e l l e d d isys ton e x cr e te d a la rg e perce n ta ge o f ra d io a ct iv ity in fa e c e s , and that GO to 70% o f the e x cr e te d r a d io a ct iv ity was due to c h l o r o f o r m - so lu b le com p ou nds [9] . G e ro lt [10] o b s e r v e d that house f l i e s to p ica l ly trea ted with d ie ldrin e x cr e te d n ear ly 11% o f the tota l d o s e as hexane- so lub le com p ou n d s . In co n tra st , the adults o f the b o l lw e e v i l , A nthonom as g ra n d is , w e re not able to e x c r e te the l ip id - s o lu b le and to x ic prod ucts when trea ted with d isys ton [9] . P erh a p s s o m e s p e c ie s o f in se c ts are p e c u l ia r and are able to e x c r e te l ip id - s o lu b le com p ou nds ; i f so , the e x cr e t io n o f in s e c t i c id e s as such m a y be an im portan t p r o te c t iv e m e c h a n ism in so m e in se c t s p e c i e s .

3 . 2 . 3 . M e ta b o l ism o f m alath ion in v a r io u s t i s su e s

Since v a r io u s t i s s u e s o f the body have th e ir own c h a r a c t e r is t i c m e ta b o l ic rate , the m e ta b o l ic p rodu cts a s s o c ia te d with each t i s su e w ere analysed to d e te rm in e the predom in ant pathway(s) in v a r io u s t i s s u e s f o r m alath ion m e t a b o l i s m .

(a) Head

Since the data in F ig . 2 indicated that a la rg e p e rce n ta ge o f the in terna l m alath ion 32P was in the head, we d ec ided to d e te rm in e the f o r m in which it was p r e se n t . T h is was im portan t b e ca u se the head conta ins the bra in (m a in o rg a n o f n erv e activ ity ) and b e c a u se m o s t o f the in s e c t i c id e s a re known to be n e u r o to x ic . Data p resen ted in F ig . 4 show ed that m o r e than half o f the r a d io a ct iv ity in the head was due to c h l o r o f o r m - s o l u b l e m e t a b o l i t e s . The c h l o r o f o r m - s o l u b l e m e ta b o l ite s in the head at v a r io u s t im e s w e re found to be 2 .2 1 , 2 .7 6 , 2 .4 0 , and1. 98 ;ug equ ivalents o f m alath ion at 0 -6 , 90, 150, and 270 m inutes .

T A B L E I . IN -V IV O M E T A B O L I S M O F M A L A T I I I O N B Y D E S E R T L O C U S T ST R E A T E D W I T H 100 цg O F 32P - L A B E L L E D MALATHION^"1)

P ercen ta geP ercen ta ge in tern a l r a d io a c t iv it y present as:

T im e(m in )

o f the totala p p lie d dose

re co v e re dC h lo ro fo rm -

so lu b le products

Phosphatase C a rb ox y estera se products products

0 -6 1 9 .8 . 6 9 .9 9 .7 2 1 .9( 1 4 .0 ) ( 2 .0 ) ( 4 .4 )

90 3 3 .0 6 4 .4 2 1 .5 1 4 .5

( 2 3 .8 ) ( 7 .9 ) ( 4 .3 )

150 3 9 .3 6 5 .2 2 3 .7 1 1 .0

( 2 5 .4 ) ( 9 .2 ) (4 . 3)

270 4 1 .1 3 6 .4 3 5 .1 2 8 .4( 1 4 .9 ) ( 1 4 .3 ) ( 1 1 .6 )

N o te : T h e fig u res in b ra ck e ts represen t ¡ig e q u iv a le n t o f m a la th io n present in s id e the b o d y o f the locu st

A v e ra g e o f tw o sep a ra te d e te rm in a tion s ; e a ch datum represents 8 adu lt m a les o f S. g re g a r ia .

T A B L E I I . E X C R E T I O N O F MA L A TH IO N B Y D E S E R T L O C U S T S T R E A T E DW I T H 100 Mg O F 32P - L A B E L L E D MALATIIIOJSi<a)

T im e(m in )

P ercen tageP ercen ta ge r a d io a c t iv it y r e c o v e r e d as:

r e co v e ry o f the a p p lied

d ose in fa e ce s

C h lo ro fo rm - so lu b le

■ products

Phosphataseproducts

C a rb oxyestera seproducts

0 -6 0 0 0 0

90 8 .2 4 . 2 7 3 .1 2 2 .6( 0 .3 4 ) ( 3 .9 9 ) ( 1 .8 5 )

150 S .3 2 4 .7 5 0 .3 1 6 .0( 2 .6 5 ) (4 .6 7 ) ( 1 .3 3 )

270 2 4 .5 3 6 .2 5 5 .9 7 .8( 8 .8 7 ) ( 1 3 .6 9 ) ( 1 .9 1 )

N o te : Figures in brack ets represent the fjg eq u iva len t o f 32P -la b e l le d m a la th io n .

( a) A v e ra g e o f tw o separate determ in a tion s ; e a c h datu m was ob ta in ed from the fa e ce s c o l l e c t e d from 8 adu lt m a les of_S . g regaria

SM

-102/21 193

194 MKHROTUA et al.

О Ю О 2 0 0 3 0 0

TIME, MIN.

FIG. 4. Amounts o f m alathion and its m etabolites present at various tim es in the head o f desert locuststreated w ith 100 fig o f 33P -la b e lled m alathion . (a) C h loroform -solu b le products, (b) Products due to

■ phosphatase action , (c ) Products due to carboxyesterase action .

r e s p e c t iv e ly . T h is su ggested that at any t im e not m o r e than 2 .7 6 /Jg o f m alathion was p resen t in the head. S ince the a v e ra g e weight o f the head was found to be 181 m g this meant that at any t im e th ere would not be m o r e than 5. OX 1 0 -5 c on cen tra t ion o f malathion p re se n t in the head prov id ed it was equally d is tr ibuted throughout. The p I 50 fo r m a lath ion against in se c t ch o l in e s te r a s e was 4 . 6 [1 1 ] , su ggestin g that no m o r e than 20 to 30% o f the head c h o l in e s te r a s e would be inhibited at that t im e . C h o l in e s te ra se inhibition in the head o f a lo cu s t fo l low in g trea tm en t with 120 /jg o f m alathion was o b s e r v e d to be 28% at one hour and 48% at s ix h ours (M e h ro tra , unpublished). Although no attempt was m a de to est im a te the p e r cent o f rad ioa ct iv ity due to m a laox on in c h l o r o f o r m - s o l u b le m e ta b o l i te s , it was a ssu m ed n eg l ig ib le , s in ce c h o l in e s te r a s e was inhibited m o r e than that o b s e r v e d by M eh ro tra (unpublished). T h e se r e su lts c o n f ir m e d the findings o f M e h ro tra and Sone Lai [1 ] , who found that the head o f S c h is t o c e r c a g r e g a r ia was unable to activate m alath ion to any gre a t extent. In the head, the r a d i o a c t iv ity in the w a te r -s o lu b le m e ta b o l ite s o f m alathion was m a in ly due to phosphatase action and to a l e s s e r extent to c a r b o x y e s t e r a s e act ion .High am ounts o f m e ta b o l i te s due to phosphatase action w e re seen at 90 and 150 m inu tes . Although the degradation p rod u cts f r o m c a r b o x y e s t e r a s e action w e re always p resen t , the con cen tra t ion o f m e ta b o l ite s due to phosphatase action was m o r e than that due to c a r b o x y e s t e r a s e action .

(b) Gut, abdom en and th orax

The d is tr ibu tion o f r a d io a ct iv ity in v a r io u s m e ta b o l i te s in gut, ab d om en and thorax is g iven in T ab le 111. In the gut the c h lo r o fo r m -

13*

S M -102/21 195

so lu b le m e ta b o l i te s continued to in c r e a s e s te a d i ly up to 270 m inu tes at which t im e the gut conta ined 3.65 ng equ ivalent o f m a lath ion . On the o th e r hand, in the ab dom en (without gut) the high co n ce n tra t io n s o f c h l o r o f o r m - so lu b le m a te r ia l which w e re p resen t in it ia l ly d e c r e a s e d with t im e ; the lo w e s t amount (1 .6 1 ng equ ivalent m alathion) was pre se n t at 270 m in u tes . T he tota l c h l o r o f o r m - s o l u b l e m e ta b o l i te s p re se n t in the gut and the abdom en , h o w e v e r , in c r e a s e d stead i ly f r o m 0 to 270 m in u tes , su ggestin g that the gut to o k an ac t ive part in e x cr e t in g the c h lo r o f o r m - s o lu b le m e t a b o l i t e s . S im ila r ly , th e re was a continuous in c r e a s e in m e ta b o l ic p ro d u c ts due to phosphatase action and c a r b o x y e s t e r a s e a c t ion in both gut and a b d om en . A lthough the con ce n tra t io n o f c a r b o x y e s t e r a s e p ro d u c ts w as high in it ia lly in all the th re e t i s s u e s , the ratio was n e a r ly 2 : 1 f o r ra d io a ct iv i ty in c a r b o x y e s t e r a s e p ro d u c ts and phosphatase p rod u cts , r e s p e c t iv e ly . T h is trend r e v e r s e d a fte r 90 m in u te s . The m a in m e ta b o l ic p ro d u c ts w e r e due to p hosphatase action at 270 m inutes and the ra tio o f r a d io a ct iv ity in c a r b o x y e s t e r a s e p ro d u c ts v e r s u s phosphatase p ro d u c ts ap p roa ch ed 1 : 5 . The c h l o r o f o r m - s o l u b l e m e ta b o l i te in the th o ra x v a r ie d f r o m 6 -12% at v a r io u s t im e s .

4. DISCUSSION

R esu lts r e p o r te d in the p re se n t in vest ig a t ion lead to a few tentative c o n c lu s io n s . F ir s t ly , th e re ap peared to be two ph ases o f the in s e c t i c id e pen etrat ion through the cu t ic le a fte r to p ica l treatm ent, an in itia l rapid phase and a la te r s l o w e r ph ase . T h is b ip h a s ic p e r m e a b i l i ty o f the cu t ic le , not on ly in in se c ts , but a lso in m a m m a ls , r e p t i l e s and am phibians, was r e ce n t ly r e c o g n iz e d by B u e r g e r and O 'B r ie n [7] . T he p h y s io lo g ic a l r e a s o n s f o r such beh a v io u r o f the cu t ic le a re s t i l l not fu lly u n d ers tood .

S ince the p h y s io lo g ic a l act ion o f the o r g a n o p h o sp h o r o u s in s e c t i c id e s was supposed to be due to the inhibit ion o f the e n zym e c h o l in e s te r a s e in n e rv o u s t i s s u e [1 2 ] , the d i s c o v e r y that a s m a l l p e r cent (m a x im u m 5 . 0 p e r cent) o f the applied d o s e r e a ch e d the head, the m ain seat o f n e rv o u s activ ity , was s ign if ican t . T h e se r e su lts w e re s im i la r to th ose re p o rte d by F ern a n do et a l . [1 3 ] , B en ja m in i et a l . [1 4 ] , Iyatom i et al [15] and A rth u r and C asid a [1 6 ] , who o b s e r v e d that a sm a l l p e r cent o f the total in s e c t i c id e applied to the A m e r i c a n c o c k r o a c h rea ch e d the head, i . e . the n e rv e t i s s u e . H ow ever , the amount o f actual tox icant reach in g the n e rv o u s t i s s u e ( o r head) was d i f fe re n t f o r d if fe ren t in s e c t i c id e s .Thus F ern a n do et al. [13] o b s e r v e d that 0 .5 5 % o f 5 . 0 ng pa ra o xo n re a ch e d the c e n tra l n e rv o u s s y s te m o f the c o c k r o a c h , w h e re a s 0 .8 2 % o f T E P P and 0. 24% o f parath ion w e re o b s e r v e d in the ce n tra l n e rv o u s s y s te m 60 m inu tes a fte r tre a tm e n t with 5 ng o f th e se in s e c t i c id e s . Our r e su lt s obtained with the d e s e r t lo c u s t S c h is t o c e r c a g r e g a r ia w e re d i f fe re n t in that about 5% o f the applied d o s e w as found in the h ead . The d i f f e r e n c e in o u r r e su lt s and th ose r e p o r te d by F ern a n d o et al. m ight be due to the high d o s e (100 ju g /in sect ) in o u r e x p e r im e n ts and the c o m p a r a t iv e ly low d o s e (5 jug/g) used by F ern a n d o et a l . [13] . M o r e o v e r , th e ir data r e f e r r e d to the in s e c t i c id e in the n e rv e t i s s u e on ly , w h e r e a s , in o u r e x p e r im e n ts , the w hole head, including o th e r t i s s u e s , was taken. The d i f f e r e n c e s o b s e r v e d m a y thus not be rea l o n e s .

196 MKHROTRA et al.

T A B L E 111. I N -V I V O M R T A H O L IS M O F 3“P - L A B E L L E D M A L A T H I O N IK T H O R A X , A B D O M E N A XL) GU'l’ O F D E S E R T L O C U S T S T R E A T E D W I T H 100 ug O F M A L A T H I O N (a>

P ercen ta g e Per c e n t r a d io a c t iv i ty r e c o v e r e d as:

o f tota lT im e

Crnin)a p p lie d

doser e co v e re d

C h lo r o fo r m -so lu b le

products

Phosphataseproducts

C a r b o x y e s te r a se

produ cts

Gut

0 - 6 1 . 4 GO.4 9 . 5 2 5 .0

( 0 .9 ) ( 0 .1 ) ( 0 .4 )

У0 9 . 8 2 3 .1 6 5 . ti 1 1 .5( 2 .3 ) ( 6 .5 ) ( 1 .1 )

150 ' 9 . 6 2 2 . У 6 5 .9 1 1 .2( 2 .3 ) ( 6 .5 ) ( 1 .1 )

■:vj 14 . 2 2 5 .7 6 2 .5 1 5 .4( 3 .5 ) ( » . 4) ( 2 .1 )

A b d o m e n (w ith o u t gut)

0 -n 3 . 6 6 5 .5 1 3 .4 2 1 .0( 2 .6 ) ( 0 .5 ) ( 0 .8 )

90 4 . U •11.3 *13.1 1 5 .5

( 1 .6 ) ( 1 .7 ) ( 0 .6 )

150 7 .4 25.(5 6 -1 .3 1 0 .0

( 2 .1 ) ( 5 .1 ) ( 0 .8 )

270 5 . 3 2 7 .7 G3. 1 9 .1

( 1 .4 ) ( 3 .2 ) ( 0 . 5 )

T h ora x

0 -G 10 . 6 5 8 .6 9 . 2 3 2 .1

( 6 .0 ) ( 1 .0 ) ( 3 .2 )

90 1 7 .7 7 0 .2 2 2 . 0 7 . 5( 1 2 .6 ) (3 .S ) ( 1 .4 )

150 1(3.7 4 3 .5 5 0 .6 5 .8( 7 .0 ) ( 3 .0 ) ( 1 - 0 )

270 1 7 .5 7 1 .3 2 4 .5 4 . 1( 1 2 .1 ) ( 4 .3 ) ( 0 .8 )

N o te : Figures in b ra ck e ts represen t |jg e q u iv a le n t o f m a la th io n present in var iou s tissues.

• a ) A v e ra g e o f tw o sep a ra te d e te r m in a t io n s ; e a c h d a tu m represen ts tissues o b t a in e d from 8 ad u lt m a le s

o f S . g re g a ria .

S M -102/21 197

In co n c lu s io n , the fo l low in g events took p la ce when d e s e r t lo c u s ts w e re trea ted with a lethal d o se oi' m alathion . T h e re was a rapid p e n e tra t ion o f t o p ic a l ly applied ma.lathion and a la rg e p ercen ta ge o f it was e x c r e te d in f a e c e s . Л sm a l l portion o f the applied d o s e r e a d ie d the bead and fluctuated v ery litt le o v e r a Long p e r io d . P erha ps sucli a sm all quantity o f m alathion would be su ff ic ien t to d isrupt the functioning o f the n e rv o u s sy s te m .

А С К N О W L E D G E jV I E N T S

Thanks are due to D r. Lallan Rai o f Cyanam id India L td . , Bom bay, f o r a gift o f pure m alathion and to D r . S. Pradhan, Head o f the D ivision o f E n tom ology , f o r taking a keen in te re s t in the w ork.

R E F E R E N C E S

[ 1 ] MEHROTRA, K. N ; , SON'E LAL, Indian J. Ent. 29 (1967) 44 .[2 ] MEHP.OTRA, K. N ., SETHI, G . R . , BIIAMBURKAR, M . W ., ICAR Seminar on the Use o f Radioisotopes

and Radiations in Agriculture and Anim al Husbandry (1966).|3] O'BRIEN, R. D . , T o x ic Phosphorus Esters, A ca d em ic Press (1961).[4 J O'BRIEN, R. D , , WOLFE, L . , Radiation, B adioactivity and Insects. A ca d em ic Press (1964).'.51 MEIIUOTRA, K .N ., SETHI, CJ.R., Indian I. Ent. 28 (1966) 1 1 4 ..|5a i MEHROTRA, K. N. . RAO, P .J ., Indian I. exptl B iol. 4 (3 9 6 6 ) 56 .[6 ] .VtATSL'Ml/RA, F ., HOGE.VDUK, С . 1 . , Ent. exp. appl. 7 (3964) 179.17 ’ BlIERoLR, A . A . , O'BRIEN, R .D ., I. c e ll . com p . Physiol. 66 (3965) 227 . .IS I BULL, D .U , LINDQUIST, D .A ., HAESKAYI.O, I . . I . e con . F.nt. 56 (3963) 329.:9 1 BULL, D .L . , I. e con . Ent. 58 (1965) 249.

[3 0 ! GEROI.T, P ., I. e con . Ent. 58 (1965) 849.131! O ’ BRIEN, R. D . , J. econ . Ent. 49 (1956) 484.[121 O'BRIEN, R .D ., A . Rev. Ent. 31 (3966) 369.113] FERNANDO, II. E ., ROAN, C . C . , KEARNS, C .W ., Ann. ent. S oc . A m . 44 (3953) 533.[341 BENJAMINI, E. , METCALF, R. I..., FUKUTO, Т .К ., I. e con . Ent. 52 (1959) 94.[1 5 ] IYATOMI, K ., SA I TO , T . , KANEI1ISA, K ., NISII1ZAWA, K. , NARUSE, H. , Botyu Kagaku 22

(1957) 392.[16J ARTHUR, B .W ., CASIDA, I .E ., J. e con . Ent. 5J. (1958) 49.

D I S C U S S I O N

J . E . T R E H E R N E : F i r s t , I am in terested in the b ip h a s ic penetrationo f m alathion which you d e s c r ib e and which ap p ears to p a ra l le l the s i tu ation o b s e r v e d in m a m m a lia n skin. Som e y e a r s ago I d em o n stra te d a r e s e r v o i r e f fe c t in the penetrat ion o f a s e r i e s o f n o n -e le c t r o ly t e s through m a m m a lia n skin and e r e c te d a m a th em a tica l m o d e l to d e s c r ib e this phenom enon (J. P h y s io l . 131 (1956)) . T h is m o d e l in vo lved the pos tu lation o f a p e r ip h e ra l d if fu sion b a r r i e r which had the fo r m o f a i ip id /w a t e r / l ip id sandw ich . It would be in terest in g to s e e whether the k in etics o f m alath ion penetrat ion through the cu t ic le cou ld be accounted f o r by the equations d er iv ed f o r m a m m a lia n skin.

K. N. M E H R O T R A : We o u r s e lv e s w e re s u r p r i s e d at f i r s t at th esefast rates o f pen etrat ion . H ow ever , fu rth er in vest iga t ion s w e re c a r r ie d out by one o f o u r students, M iss S a ro j T u le , who has studied th is p ro b le m

198 MEHROTRA et al .

ra th e r e x te n s iv e ly with a n um ber o f com p ou n d s ; h e r re su lts do not, I am a fra id , fo l low the pattern o f r e su lts you obtained with S c h is t o c e r c a , published in the Journa l o f In sec t P h y s io lo g y (1957). I am not su re w hether the m o d e l you postu late f o r m a m m a lia n skin would w ork fo r in se c t cu t ic le o r not.

J . E . T R E H E R N E : Second ly , I am a litt le trou b led by yo u r assu m p tion that the rate o f ap pearan ce o f ra d io a ct iv ity in the head is an a ccu ra te m e a s u r e o f the a r r iv a l o f la be l led com p ou n d s at the cen tra l n erv ou s s y s t e m . T h is d o e s not, f o r exam p le , take account o f the a r r iv a l o f la b e l le d com pou nd s at the ventra l nerve c o r d o r at the p e r ip h e ra l n e r v e s . F u r th e r m o r e , I think that it is highly d e s ir a b le to e x p r e s s the c o n c e n tra t ion o f t o x ic m a te r ia ls in t e r m s o f unit weight o f n e rv o u s t i s s u e s r a th e r than as total quantity in the head. T h is is e s p e c ia l ly im portant in v iew o f the fact that the c e r e b r a l t i s s u e s contr ib u te on ly a sm a l l f r a c t io n o f the total weight o f the head and a lso that in terpre ta t ion o f the m e c h a n ism o f t o x ic ac t ion in n e u r o p h y s io lo g ica l t e r m s r e q u ir e s a know ledge o f the co n cen tra t ion o f com pou nds within s p e c i f i c n e rv o u s t i s s u e s .

K. N. M E H R O T R A : We did not lo o k at the n e rv o u s t i s s u e s in thes e t o f e x p e r im e n ts re p o rte d h e re b e c a u se o f the d i f f i cu lt ie s in vo lved in d is s e c t in g in se c ts containing la rg e quantities o f r a d io a c t iv i ty . H ow ever ,I can a s s u re you that we have s in ce p r o g r e s s e d m uch fu rth er and in la te r w ork (by M ehrotra , Ph okela and Saasena , unpublished) the n e rv o u s t i s s u e a lone is c o n s id e r e d .

W. J. LE Q U E SN E : M alathion la be l led with su lp h u r-3 5 , c a r b o n -1 4and tr it ium is a lso ava i lab le c o m m e r c i a l l y . A p p lication o f th ese is o to p e s would enable the in s e c t i c id e s to be lo ca te d m o r e a c c u r a te ly in the v a r io u s o r g a n s by m eans o f se c t io n in g and au torad iograp h y . Use o f o th e r is o to p e s , o r even o f double labell ing , would perhaps m ake it p o s s ib le to r e c o g n iz e the two halves o f the m o le c u le p rod u ced by d e tox ica t ion as the resu lt o f phosphatase act iv ity .

K .N . M E H R O T R A : C erta in ly , one cou ld u se m alath ion la be l ledwith 35S, 14C, o r aI-l. Of th èse , 35S would p roba b ly be u n s a t is fa c to ry s in c e it would g ive in su ff ic ien t in form at ion , and tr it iu m labe ll ing , e s p e c ia l ly f o r malathion, m a y not be v e ry usefu l b e c a u s e o f the p r o b le m p osed by exchange react ion , w h ereas 14C has the advantage o f having a lon g h a l f - l i f e and could conven ient ly be u sed to la be l d i f fe ren t p arts o f the m o le c u le . A u torad iograp h y could , o f c o u r s e , be used , but I am afra id that the re su lts would be qualitative ra th er than quantitative. I p e r so n a l ly think that double labe ll ing with 37P and 14C m a y be o f r e a l benefit , but in the last an a lys is it is a m a tte r o f e x p e r im e n ta l o b je c t iv e and o f t im e , equipm ent and m a te r ia l av a i lab le .

D

ISOTOPE APPLICATIONS: CHEMOSTERILANTS

(Session V, Part 2)

C h a i r m a n : L . K . C U T K O M P

U N IT E D S T A T E S O F A M E R I C A

CHEM OSTERILANTS IN EN TO M O LO G Y AND THE S T E R IL E -M A L E TECHNIQUE

A. B. BÓRKOVEC ENTOMOLOGY RESEARCH DIVISION, AGRICULTURAL RESEARCH SERVICE, UNITED STA TES DEPARTMENT OF AGRICULTURE, BELTS VILLE, M d .. UNITED STA TES OF AMERICA

Abstract

CHEMOSTERILAXTS IN ENTOMOLOGY AND THE STERILE-MALE TECHNIQUE. Chemosterilants have been often referred to as agents producing dom inant lethal mutations in the germ ce lls o f treated organisms. Recent studies o f the m ode o f action o f various chem osterilants in insects ind ica te that there are many other pathways by w hich ch em ica l com pounds can induce sterility in insects. Although the gen etic m echanism still appears to be the m ost p lausible explanation o f the m od e o f action o f m any ch e m o sterilants in m ale insects, other alternatives must be considered for explain ing the ch e m ica l sterilization o f fem a le insects. The elucidation o f the b io ch e m ica l processes leading to sterility is important not on ly because ch em ica l mutagenesis was first d iscovered in an insect (Drosophila m elanogaster) ; ch em ica ls offer a f le x ib le , and som etim es the only ava ilab le , m ethod o f inducing sterility in e co n o m ica lly important insects w hich can be utilized in the ste r ile -m a le technique o f insect control. As distinct from radiation, chem osterilants can be u tilized for sterilization o f the naturally occurring pest populations. In sterile - insect release program m es, chem osterilants b e co m e o f im portance whenever secondary radiation e ffects interfere with longevity and sexual com petitiveness o f rad iation -sterilized insects. T h e understanding o f the m echanism and m ode o f action o f chem osterilants w ill aid in the search for new types o f e ffe c t iv e com pounds and in designing new m aterials sp ec ific to insects or even to individual insect species. The sterility principle in pest control has thus far been successfully applied on ly to insects, but vertebrate pests w ill certainly be considered in the future. Chem osterilants m ay be the most e ffe c t iv e tool for steriliza tion o f vertebrate organisms.

By d e f i n i t i o n , c h e m o ste r i la n ts are chem ica l compounds which reduce or d e s t r o y the r e p r o d u c t iv e c a p a c i t y o f an organism to which they are ad m in istered . In s e x u a l l y reprodu cing organisms two main pathways o f induced s t e r i l i t y may be c o n s id e r e d : i n t e r f e r e n c e w ith the fo rm at ion o f a zy go te and i n t e r f e r e n c e with the development o f a z y g o te . A zy g o te cannot be formed i f the male or the female d o e s n ' t produce a f u n c t io n a l gamete or i f the two gametes are p re vented from combining. The death o f a zy go te can r e s u l t from f a u l t y g e n e t i c in fo rm a t io n imparted by e i t h e r o r both o f the gametes or from environm ental c o n d i t i o n s which do not a l lo w normal development.

Because in s e c t ch e m o ste r i la n ts were o f t e n r e f e r r e d to as being mutagenic I would l i k e to d is c u s s f i r s t which o f the in t e r f e r e n c e s which c h e m o ste r i la n ts produce or may produce in in s e c t s a re mutat i o n s in the a c ce p te d sense o f the word. U n fortun ate ly th e re d o e s n ' t seem to be a u n iv e r s a l agreement on what m utation i s . That mutation i s a h e re d i ta r y change in c e r t a in c e l l u l a r components appears to be g e n e r a l ly a c ce p te d but th ere i s some c o n t ro v e rs y whether the c e l l u la r component i s the n u c le u s , a gene, o r simply a m o le cu le o f n u c le i c a c id . With r e f e r e n c e to in d iv id u a l i n s e c t s the key term h e re d i ta r y may r e f e r e i t h e r to the tran sm iss ion o f such changes from the parent (male or fem ale) to the o f f s p r i n g o r , w ith in the i n s e c t ' s organism,

2 0 1

2 0 2 BORKOVEC

to the transmission from a parent cell to daughter cells.- Dominant lethal mutations which are of particular importance in sterilization imply that the offspring die before reaching maturity or that the

daughter cell ceases to be functional. With this concept in mind we can analyze some of the most important effects of chemosterilants in sexually reproducing insects.

Effect on males.

Aspermia and the lack of spermatozoan motility are seldom en

countered in chemosterilized male insects. The most common effect of male chemosterilants is a condition of the sperm which for lack of better expression may be designated as sterility of the mature

spermatozoa. The gametes of treated males appear to be in all re

spects normal [8] but the zygotes which they form die before reaching maturity. This effect corresponds to the classical definition of induction of dominant lethal mutations in the sperm and all of the known male insect chemosterilants which were examined in detail can

be designated as mutagenic agents in the appropriate species of insects.

E f f e c t s on fem ales

The effects of chemosterilants in female insects are variable, and they will be subdivided into several categories.

Treated females do not produce eggs

This type of sterility is usually a result of cytological dam

age to the reproductive organs, and it cannot be called mutagenic in the same sense as the male sterility mentioned earlier. Whether dominant or other lethal mutations were introduced into any cells of

the female organism cannot be decided on the basis of inhibition of oogenesis or oviposition. Nutritional and hormonal factors are known to regulate the functions of the female reproductive system, and if the chemosterilant affects in some nongenetic way these factors its effects cannot be properly called mutagenic. Experimental evidence of such indirect activity of chemosterilants in female mosquitoes was obtained by Akov [1, 2]. Antimetabolites and alkylating agents were found to inhibit the digestion and elimination of blood which is a

nutritional requirement for egg development in mosquitoes. Although Akov didn't prove or claim that the mechanism of sterilizing activity

was nongenetic the evidence she presented suggests strongly that the

primary reason for the sterility in female mosquitoes treated with the chemosterilants was a nutritional deficiency.

Treated females produce eggs which do not hatch

This type of activity is often only a function of the concentration or dose of the chemosterilant administered to the insect [6].

Again, the decision whether or not the mechanism of the sterilization was mutagenesis cannot be made without a detailed analysis of the primary and secondary effects of the chemical in the organism. If

in eggs laid by treated females signs of incipient development are evident but the eggs fail to hatch the induction of dominant lethal

S M - 1 0 2 / 2 4 203

m utations must be c e r t a i n l y su sp ected . In absence o f such ev id e n ce the compound cannot be termed mutagenic w ith r e s p e c t to the whole organism but i t s t i l l may be mutagenic w ith r e s p e c t to some d iv id in g c e l l s o f the organism.

T reated fem ales produce eggs which hatch but the la rv a e d i e b e f o r e pupating

T his typ e o f a c t i v i t y has been observed o n ly in s p e c i a l c l a s s e s o f c h e m o s te r i la n t s , p a r t i c u l a r l y in the d e r iv a t i v e s o f ¿ - t r i a z i n e[ 7 ] . With r e s p e c t to th e t r e a te d in s e c t the death o f immature p ro geny i s a c l e a r s ig n o f dominant l e t h a l mutations a lth ough the p e r io d between the a d m in is t ra t io n o f the chem ica l and the appearance o f i t s e f f e c t s may be q u i t e lo n g .

In t h i s b r i e f survey o f e f f e c t s o f c h e m o s te r i la n ts on in s e c t s I have t r i e d to emphasize the p o in t that chem ica l s t e r i l i z a t i o n cannot be simply equated to m utagenesis w ith ou t a d e t a i l e d study o f the mechanism b y which the c h e m o ste r i la n t a c t s .

A second p o in t which may be even more im portant in p r a c t i c e i s the d i s t i n c t i o n between th e primary and secondary e f f e c t s o f the c h e m o s te r i la n t . In t h i s c o n n e c t io n primary e f f e c t s a re th ose which the ch e m o ste r i la n t i t s e l f (compound A) e x e r t s on the c e l l u l a r cons t i t u e n t s o f the organism ; secondary e f f e c t s a re th o se which are e x erted by a d i f f e r e n t m a te r ia l (compound Б) which i s a d e r iv a t i v e o r m e t a b o l i t e o f th e c h e m o ste r i la n t A. I f , f o r example, the in s e c t organism c o n v e r t s th e compound A to the compound В and i f o n ly the compound В f u n c t io n s as a ' s t e r i l i z i n g a g e n t , the a c t i v i t y o f compound A in o th e r organisms w i l l depend on the a b i l i t y o f th ose o r ganisms to c o n v e r t e f f e c t i v e l y A to B. I t i s im probable that c h e m o ste r i la n ts w ith a broad spectrum o f a c t i v i t y would f u n c t io n v ia such secondary e f f e c t s . I n d i c a t i o n s a r e , however, th a t the h ig h ly s p e c i f i c c h e m o s te r i la n t s , p a r t i c u l a r l y the n o n a lk y la t in g phosphora- mides and t r i a z i n e s , do indeed depend on the a b i l i t y o f the t r e a te d organism to c o n v e rt them to e f f e c t i v e , perhaps a l k y la t in g chem osteri l a n t s . Our c u rre n t work on the mechanism o f a c t i o n o f e f f e c t i v e s t e r i l a n t s hempa and hemel [9] in d i c a t e s that in house f l i e s th ese dimethylamino compounds are s e l e c t i v e l y dem ethylated w ith the e v o lu t i o n o f form aldehyde. Other analogous dimethylamino or methylamino compounds which a re not dem ethylated in the same organisms are i n a c t i v e as c h e m o s te r i la n t s . C o n c lu s iv e e v id en ce su p port in g the h yp o th e s is o f secondary a c t i o n i s not y e t a v a i l a b l e but the p o s s i b i l i t y o f d i s c o v e r in g c h e m o s te r i la n ts which would be s p e c i f i c to o n ly some organisms and r e l a t i v e l y harmless t o o th e r s i s most a t t r a c t i v e and d e s e rv e s fu r t h e r i n v e s t i g a t i o n .

The th ir d p o in t which I wish t o d is c u s s i s the a p p l i c a t i o n o f c h e m o s te r i la n t s . The r o l e which t h i s type o f compound p layed in the h i s t o r y o f g e n e t i c s i s w e l l known although the e a r ly r e p o r t s made r e fe r e n c e s to chem ica l mutagens and not to c h e m o s te r i la n t s . Nevert h e l e s s , the f i r s t r e p o r t on ch em ica l mutagenesis p u b lish e d by Auerbach and Robson [3] mentioned l e t h a l m utations induced in D rosoph i la m clanogaster w ith a l l y l i s o t h io c y a n a t e and with mustard gas [ 4 ] . In the S o v ie t Union, Rapoport d e s c r ib e d the mutagenic and

204 BORKOVEC

s t e r i l i z i n g e f f e c t s o f a ldehydes [12] and carbamates [13] in 1946 and 1947. However, in g e n e t i c l i t e r a t u r e s t e r i l i z i n g e f f e c t s were co n s id e r e d o n ly in pass ing because the main i n t e r e s t was c e n te re d on the g e n e t i c makeup o f th e progeny o f t r e a t e d organism s. Organized e f f o r t d i r e c t e d toward the d is c o v e r y o f e f f e c t i v e male and female in s e c t c h e m o ste r i la n ts s t a r te d o n ly in 1960 in the U.S. Department o f A g r i c u l t u r e [ 5 ] . The main purpose o f t h i s search was t o f in d and to d e v e lo p an a l t e r n a t e method o f s t e r i l i z a t i o n o f i n s e c t s w ith r a d ia t i o n .

As compared to r a d ia t i o n , chem ica ls o f f e r both advantages and d isadvan ta ges . Many s p e c i e s o f i n s e c t s which are k i l l e d by r a d ia t i o n b e f o r e they are s t e r i l i z e d may s t i l l be s t e r i l i z e d ch e m ica l ly w ith ou t undue m o r t a l i t y . N atu ra l ly o c c u r r in g in s e c t s cannot be p r a c t i c a l l y s t e r i l i z e d w ith r a d ia t i o n even i f they are s u s c e p t ib l e to i t . Thus fa r c h e m o ste r i la n ts appear to be the o n ly method f o r s t e r i l i z i n g in s e c t s in n a tu ra l p o p u la t io n s .

On the n e g a t iv e s i d e , c h e m o ste r i la n ts share one o f t h e i r most v e x in g problems w ith o th e r chem ica l p e s t i c i d e s : whenever chem ica l compounds are used in nature the r e s id u e problem a r i s e s . High se l e c t i v i t y o r s p e c ie s s p e c i f i c i t y may ease and minimize the d i f f i c u l t i e s but s p e c i f i c i t y i s a two-edged sword. The more s p e c i f i c a c h e m o ste r i la n t i s the more d i f f i c u l t , t im e-consum ing, and c o s t l y i t i s t o f in d i t .

In c o n c lu s io n I would l i k e to mention one asp e c t o f c h e m o ste r i l a n t s which has remained f o r a l l p r a c t i c a l purposes u nexp lored . The e f f e c t i v e n e s s o f the s t e r i l i t y p r i n c i p l e in p es t c o n t r o l depends on our a b i l i t y t o c on v ert a p o p u la t io n o f l a r g e l y f e r t i l e in d iv id u a ls to a p o p u la t io n c o n s i s t in g l a r g e l y o f s t e r i l e in d iv id u a l s . The f a s t e r we can ach ie ve t h i s c o n v e rs io n the more e f f e c t i v e w i l l be the method. The main proponent o f t h i s method, E. F. K n ip lin g [ 1 1 ] , p o in te d out r e p e a te d ly that v e r t e b r a t e p o p u la t io n s are p o t e n t i a l l y as s u s c e p t ib l e to be ing c o n t r o l l e d by s t e r i l i t y as are the in v e r t e b r a te p o p u la t io n s . The q u e s t io n remains how to s t e r i l i z e v e r t e b r a t e organisms e f f e c t i v e l y and permanently. R a d ia t ion s t e r i l i z a t i o n o f f e r s o n ly very l im i t e d promise p r im a r i ly because the v e r t e b r a te organism c o n s i s t s o f too many systems and organs which are a t l e a s t as s e n s i t i v e t o r a d ia t i o n as are the gonads. A r e le a s e o f a r t i f i c i a l l y r e a r e d , s t e r i l i z e d animals cannot be co n s id e r e d in most in s ta n ce s and thus the s t e r i l i z a t i o n o f the n a tu ra l p o p u la t io n remains as the o n ly p r a c t i c a l p o s s i b i l i t y . C h em oster i lants can be h ig h ly s e l e c t i v e and s p e c i f i c even in h igh er organisms [10] but o f cou rse the p rob lems o f s u i t a b l e treatment and o f r e s id u e s would r e q u ir e much r e se a r c h .

Whatever the u lt im a te a p p l i c a t i o n o f c h e m o ste r i la n ts w i l l be , th e b a s i c r e se a r c h on the chem ica l in t e r f e r e n c e w ith re p ro d u c t io n can stand on i t s own m e r it . Entomology has a lready p r o f i t e d from the i n t e n s i f i e d i n v e s t i g a t i o n s o f in s e c t r e p ro d u c t io n and mating during the past 10 y e a r s . The hormonal c o n t r o l and r e g u la t io n o f r e p ro d u c to ry p r o c e s s e s in in s e c t s a re now o n ly su sp ected or vaguely u nderstood . There i s l i t t l e doubt th a t the r e se a rch on ch e m o ste r i la n t s w i l l c o n t r ib u t e to the knowledge o f in s e c t r e p ro d u c t io n in the y ears to come.

S M - 1 0 2 / 2 4 2 05

REFERENCES

[1] AKOV, S., Inhibition of blood digestion and oocyte growth in Aedes aegypti by 5-fluorouracil, Biol. Bull. 129 3 (1965) 439.

[2] AKOV, S., Retention of the blood-meal in Aedes aegypti following sterilization of chemicals and irradiation, Ann. Trop. Med. Parasitol. ^0 4 (1966) 482.

[3] AUERBACH, C., ROBSON, J.M., The production of mutations by allyl isothiocyanate, Nature 154 3898 (1944) 81.

[4] AUERBACH, C., ROBSON, J. М . , Chemical production of mutations, Nature 157 3984 (1946) 302.

[5] BORKOVEC, A. B., Sexual sterilization of insects by chemicals, Science 137 3535 (1962) 1034.

[6] BORKOVEC, A. B., Insect Chemosterilants, Interscience, New York (1966).

[7] BORKOVEC, A. B., DEMILO, A. B., Insect chemosterilants. V. Derivatives of melamine, J. Med. Chem. (1967) 457.

[8] CHANG, S. C., BORKOVEC, A. B., WOODS, C. W . , Fate of tepa uniformly labeled with C-14 in male house flies, J. Econ. Entomol. 59 4 (1966) 943.

[9] CHANG, S. C., TERRY, P. H . , BORKOVEC, A. B., Insect chemosterilants with low toxicity for mammals, Science 144 3614 (1964)57.

[10] JACKSON; H., Antifertility Compounds in the Male and Female, Thomas, Springfield, 111. (1966).

[11] KNIPLING, E. F., Sterile-male method of population control, Science 130 3380 (1959) 902.

[12] RAPOPORT, I. A., Carbonyl compounds and the mechanism of chemical mutagenesis, Doklady Akad. Nauk USSR 54 (1946) 65.

[13] RAPOPORT, I. A., Derivatives of carbamic acid and mutations, Byull. Eksp. Biol. Med. 2b_ (1947) 198.

D I S C U S S I O N

C. A. P E L E R E N T S : The r e su lt s obta ined in o u r la b o r a to r y withv a r io u s p ro d u c ts (Apholate , M etepa and T ep a ) on H y lem ya b r a s s i c a e show that the d i f fe r in g d e g r e e s o f s t e r i l i ty ( fa i lure to o v ip o s i t ; o v ip o s i t io n w ith out hatching o f the e g g s ; hatching with subsequent death o f the la rv a e ) a re a function o f the applied d o s e . Is this a ch a n ce e f fe c t o r is it c o m m o n ly found?

A. B. B O ftK O V E C : T h is is a ty p ica l d o s e - e f f e c t r e la t ion sh ip .E. H O R B E K : I do not quite a g r e e with y o u r c o n c lu s io n s on the

s te r i l iz a t io n o f in se c ts in natural popu la tion s . We a r e not ab le to r e a r c o c k c h a f e r s (M elo lon th a sp. ) in la r g e quantities within a re a so n a b ly s h ort t im e so o u r tech nique is to c o l l e c t ind iv iduals f r o m a natural pop u lation in an a r e a w h ere they a re abundant and r e le a s e them a fte r i r r a d i a tion in a r e g io n w h ere the population has to be r e d u c e d o r e ra d ica te d .

A. B. B O ftK O V E C : I should perh a ps c la r i fy m y statem ent. When Ir e f e r r e d to in s e c ts in natural popu lations I m eant on ly th ose in s e c ts which, at the t im e o f treatm en t, a r e part o f a natural popu lation . In c o n t r a d is t in ction to th is , y o u r technique u t i l i z e s in se c ts o f natural o r ig in which, at the t im e o f treatm en t , a re not part o f a natural population . I do not, h o w e v e r , en t ire ly ex c lu d e the p o s s ib i l i t y o f a ch iev in g n o n - c h e m ic a l s t e r i l i z a t io n o f naturally o c c u r r in g in s e c t s in the future.

206 BORKOVEC

E. H O R B E R : In the sh ort t im e we have been w ork in g with R o m b u s sp.we noted two c a s e s o f s t e r i l i z in g e f fe c t s through p a r a s i t i s m in s e v e r a l o f th ese s o c ia l in s e c ts . One was ca u se d by a n em atode (Sp h a eru la r ia b o m b i ) in fect in g the o v e rw in te r in g queen b u m b le b e e s ind iv idually . A n oth er c a s e w as the resu lt o f s e v e r a l c o c k o o b u m b le b e e s p e c i e s (T s i th y r u s sp . ) o c cu p y in g bu m b leb ee c o lo n i e s togeth er a f t e r the w o r k e r b r o o d had b e c o m e av a i lab le . I a s s u m e that naturally o c c u r r in g s t e r i l iz a t io n is quite frequent am ong in s e c ts and would t h e r e fo r e su g ges t that the p o s s ib i l i t y be c o n s id e r e d o f analysing su ch c a s e s f o r potent ia l c h e m o s te r i la n t s w hich m ight be m o r e s p e c i f i c f o r in s e c ts and l e s s h arm fu l to v e r t e b r a te s than the o n e s known h itherto .

A. B. B O R K O V E C : T h is is an in te re s t in g ph enom enon and it wouldindeed be w orthw hile knowing whether s t e r i l i t y was ca u se d by p h y s ic a l d am age to the gonads o r by s o m e oth er , p o s s ib ly c h e m ic a l , p r o c e s s .

R a ch e l G ALU N : With r e f e r e n c e to D r. A k o v 's w ork , (R e fs [1] and[2] o f the p a p e r ) , I think one should dis t inguish betw een the e f fe c t o f F lu o r o m a c i l and A pholate . While A pholate s t e r i l i z e s both m a le and fe m a le m o s q u ito e s , F lu o r o m a c i l a f fe c ts on ly the f e m a le s , by in te r fe r in g with sy n th es is o f the p r o te a s e s n e c e s s a r y fo r b lo o d d ig est ion . In f e m a le s t rea ted with Apholate , retention o f the b lo o d m e a l is a s e c o n d a r y e f fe c t , due to inhibition o f o v a r ia n dev e lop m en t .

T . R. O DH IAM BO: At what le v e l , D r. B o r k o v e c , did you c a r r y outy o u r m o r p h o lo g i c a l s tud ies o f the s p e r m f r o m tre a te d m a le s ? Did you , for- e x a m p le , exam ine them with the e le c t r o n m i c r o s c o p e ? Y ou stated that s p e r m f r o m c h e m o s t e r i l i z e d m a le in s e c t s a p p e a r s to be n o r m a l in a l l r e s p e c t s . Could you g ive further in fo rm a t io n about y o u r w o rk on this a s p e c t o f the p r o b le m ?

A. B. B O ftK O V E C : T w o b a s ic p r o p e r t i e s o f s p e r m w e re c o n s id e r e d :m i c r o s c o p i c ap pe a ra n ce and beh a v io u r , that is , g r o s s m o t i l i t y and a b i l ity to f o r m a zygote . How'ever, the w ork we did was v e r y s u p e r f i c ia l and h ence ra th er inadequate. It inc luded exam ination o f e l e c t r o n m i c r o g ra p h s o f t r e a te d and untreated h o u s e - f ly s p e r m , but this r e v e a le d no o b v io u s d i f fe r e n c e s .

T . R. ODIIIAM BO: Have you found any d i f f e r e n c e s in the a c c e s s o r yr e p ro d u c t iv e g lands in the trea ted m a le in s e c t s ?

A . B. B O flK O V E C : We have not gone into this a s p e c t o f the su b jectand, in fact, I know o f no published w o rk on the m o r p h o lo g y o f gonads o f c h e m o s t e r i l i z e d m a le in se c ts .

T . R. O DH IAM BO: Could you su ggest the m e c h a n is m which b r in g sabout death o f the zy go te s a fte r in jec t ion o f s p e r m f r o m trea ted m a le s ?

A. B. BO ÍIK O VEC: I cou ld put fo r w a rd on ly those that a r e c o m m o n lyo f f e r e d to exp lain zy got ic m o r ta l i ty ca u se d by dom inant letha ls . U n fortu nately , none o f them fit o u r e x p e r im e n ta l data.

C . F . CU RTIS: As r e g a r d s possibLe d am age to s p e r m c a u se d byc h e m o s te r i la n t s , it s e e m s to m e that the on ly question o f p r a c t i c a l i m p o r ta n ce is whether ' in a fe m a le m ated to both a c h e m o s t e r i l i z e d and a n o r m a l m a le , w here the two types o f s p e r m a r c thus in d ir e c t c o m p e tition, equal n u m b e rs o f v iab le and inviab le z y gotes a r e p ro d u ce d . Has this point been in vestigated?

A. B. BO RK O VEC : T h is question can be a n sw e re d on ly with r e f e r e n c e to those in se c ts w here the fe m a le m a tes m o r e than o n c e . S ca t t e r e d data in the l i t e ra tu re and unpublished r e p o r t s ind icate that in f e

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m a le s o f s o m e s p e c ie s the s p e r m f r o m trea ted m a le s can c o m p e te s u c c e s s f u l l y with the s p e r m o f untreated m a le s . In o th er in s e c t s , the s e qu en ce o f m ating is the d e te rm in in g fa c to r and, in s t i l l o th e r in s ta n ce s , the s p e r m f r o m trea ted m a le s d o e s not c o m p e te fa v ou ra b ly with the s p e r m f r o m untreated m a le s . Such a la ck o f g e n e ra l i ty is ty p ica l in a n a ly se s o f the e f fe c t s o f c h c m o s te r i la n ts .

F. BERAN : Have you e v e r t r ië d out the e f fe c t o f tr ia z in e c o m p o u n d s ,as used in w eed c o n t r o l?

A. B. B O R K O V E C : Y e s , we have tes ted the e f fe c t on h o r s e f l i e s o fm any o f the im portan t h e r b ic id e s b a se d on s - t r i a z in e ( se e A . B. B o r k o v e c ,G .C . La B requ e and A . B. De M ilo , J. e c o n . Ent. 6£ (1967)). M ost o f th ese co m p o u n d s sh ow ed a weak s t e r i l i z in g a c t iv ity but none was highly e f fe c t iv e .

D. T . NORTH: A s f o r the 'n o r m a l ' a p p e a ra n ce an d-behav iou r o fc h e m o s te r i la n t - t r e a t e d s p e r m , I shou ld like to m ention s o m e r e s e a r c h done by D r. La Chance at ou r M e ta b o l is m and Radiation R e s e a r c h L a b o ra to r y at F a r g o , North Dakota, USA. Using s p e r m f r o m H a b r o b r a c o n , he has shown that treatm en t w'ith the c h e m o s te r i la n t , T e p a , in d u ces both dom inant lethal m utations and s p e r m inactivat ion . S p e rm inactivation o c c u r s at r e la t iv e ly high d o s e s , a s c o m p a r e d with induced dom inant le tha ls , and a p p ears to be som ew h at a g e n t - s p e c i f i c . D r. La C hance found that, w hilst the phosphine o x id e c o m p o u n d s , su ch as T ep a , c a u se s p e r m in activa tion , tr ia z in e c o m p o u n d s a p p ear to b r in g about on ly dom inant le tha ls . It should be b o rn e in m ind that use o f s p e r m inactivation as a type o f m a le s t e r i l i t y cou ld be o f r e a l p r a c t i c a l va lue .

A. B. B O fiK O V E C : S p e rm inactivat ion as o b s e r v e d in H a b ro b ra co nm a y be a cond it ion o f the s p e r m w hich d o e s not a l low fo r m a t io n o f a zygote . On the o th er hand, it m a y not be re la te d to any in teract ion betw een s ter i lan t and s p e r m , but ra th er to an in te ra c t io n betw een ster i lan t and se m in a l fluid. The e x p r e s s io n 's p e r m - in a c t iv a t io n 1 is ra th e r vague and is defined on ly in t e r m s o f the p r o p o r t io n o f u n fe r t i l iz e d e g g s . It is c o n c e iv a b le that s p e r m i t s e l f is not even in vo lved in su ch a p r o c e s s . I know o f no c h e m ic a l which would cau se s p e r m inactivat ion only.

R. C. VON B O R S T E L : W ould the e f f e c t s you a s c r ib e to c h e m ic a l lyinduced inactivation o f s p e r m apply to rad ia t io n - in d u ce d inactivation as w ell?

A. 13. B O RK O VEC : Y e s , I think that rad iation as w e l l as c h e m ic a lscou ld p ro d u ce changes in the se m in a l fluid which would then in te r fe re with fe r t i l iza t ion .

V. DELUCCHI: It was m en tioned that one o f the main l im ita t ions onthe app licat ion o f c h e m o s te r i la n ts in the f ie ld was the r e s id u e p r o b le m .I would ap p re c ia te having yo u r p e r s o n a l op in ion on the fe a s ib i l i ty o f using c h e m o ste r i la n ts in the near future.

A. B . B O R K O V E C : With certa in r e s e r v a t io n s I should e x p e ct c h e m o s ter i lan ts to be put to m o s t im m ed ia te u se in r e a r in g - s t e r i l i z a t i o r i - r e le a s e p r o g r a m m e s , p erh a p s within the next 5 y e a r s . O ther u s e s m a y fo l low if the sa fe ty p r o b le m s can be so lv e d su f f ic ie n t ly rap id ly .

R a ch e l G ALUN: Do c h e m o s te r i la n ts d e c o m p o s e fast enough f o r it tobe fe a s ib le to treat m a s s - r e a r e d in se c ts and r e le a s e them only a fter d e c o m p o s i t io n o f the c h e m o ste r i la n t , so that any p o s s ib le h aza rd is obv ia ted?

A. B. B O R K O V E C : Som e c h e m o s te r i la n ts do d e c o m p o s e at a s u f f i c ie n t ly rapid ra te , but the re s id u e p r o b le m is a constantly r e c u r r in g one,

208 BORKOVEC

which has to be so lv e d fo r each individual com pou nd ap plied to each given s p e c ie s .

A . L E M M A : It has r e ce n t ly been d i s c o v e r e d in Ethiopia that at su b -letha l d o s e s so m e m o l lu s c i c id e s induce s t e r i l i ty in cer ta in sn a i ls which t ra n sm it b i lh a rz ia . Do you know o f any r e s e a r c h done in this d ir e c t io n by o th er w o r k e r s ?

A. B. B O R K O V E C : Not in conn ect ion with m o l lu s c s , but the e f fe c t so f c h e m o ste r i la n ts a re not r e s t r i c t e d to in se c ts and c h e m o s te r i l i z a t io n o f m any oth er an im als has ce r ta in ly been d e s c r ib e d .

J . E . SIMON: I should like to c a l l on Dr. B o r k o v e c 's knowledge o fSouth A m e r i c a to a sk him w hether he thinks that c h e m ic a l s te r i la n ts should be used there o r w hether we should ad v ise ou r young and often s t i l l in e x p e r ie n c e d e n to m o lo g is t s to wait until m o r e is known o f the e f fe c t s o f su ch com p ou n d s .

A. B. BO RK O VEC : C h e m o s te r i la n ts should n e ve r be used u n lesssu itab le p reca u t ion s a r e taken to en su re the sa fety o f the o p e ra t io n . If c h e m o s te r i la n ts a r e used outs ide the la b o r a to r y , that is , against the natural in sec t populations in the fie ld , e x t r e m e p re ca u t io n s a r e n e c e s s a r y and su ch e x p e r im e n ts p erhaps should not be cond u cted when on ly in e x p e r ie n c e d p e r so n n e l a r e av a i lab le . On the o th er hand, r e le a s e o f la b o r a t o r y - s t e r i l i z e d in se c ts does not invo lve handling dangerou s c h e m ic a ls in the f ie ld and y our attention might thus m o r e p ro f ita b ly be c e n t re d on such a p p ro a ch e s to the s t e r i l i ty c o n t ro l m ethod until m o r e is known about the t o x ic o lo g y o f c h e m o s te r i la n ts . In the la b o r a to r y , o f c o u r s e , the use o f c h e m o s te r i la n ts d o e s not p re se n t any p r o b le m s d i f ferent f r o m those n o r m a l ly en cou ntered in handling toxic c h e m ic a ls .

TI. E R D M A N : Until the m e c h a n ism o f the s t e r i l i z in g action o f c h e m o s ter i lan ts is known, these c h e m ic a ls should in fact be t rea ted with the s a m e caution and r e s p e c t as ra d io i s o to p e s . The c a r c in o g e n ic e f fe c t o f s o m e c h e m o s te r i la n ts must prohib it their- w id e sp re a d use in natural en v iro n m e n ts .

R . C . VON B O R S T E L : I should like to e m p h a s iz e the im p o rta n ce o fthis c o m m e n t with r e s p e c t to the de tectab i l ity o f such com p o u n d s . T e c h n ique? a r e w e l l w ork ed out fo r detecting r a d io iso to p e s that have been r e le a s e d into the en v iron m en t but the s a m e is not true fo r alkylating agents though, in g e n e r a l , these induce s im i la r c e l lu la r le s io n s . Ii is h ard ly d e s ira b le to take the o c c u r r e n c e o f a skin le s ion o r a malignant tu m ou r as c r i t e r io n o f the p r e s e n c e o f an alkylating agent. I am su re D r. B o r k o v e c would a g r e e with this.

A. B. BO ftK O VEC : I c erta in ly a g r e e . A l l tox ic c h e m ic a ls should behandled with the utm ost c a r e . The o p e r a t o r should n ever judge the sa fety o f his p r o c e d u r e f r o m the fact that he is s t i l l a l ive .

M. COHEN: In ra is in g the question o f the potential im p o r ta n ce o fr e s id u e s o f c h e m o ste r i la n ts one is drawing on the p r e ce d e n t o f r e s id u e p r o b le m s resu lt in g f r o m the use o f p e s t i c id e s . Many o f the la tter p r o b le m s can be a s c r ib e d to m isu se by fa r m e r s although s o m e , o f c o u r s e , a re due to use by c o n t r a c t o r s and G overnm ent a g e n c ie s .

I suggest that c h e m o ste r i la n ts would p roba b ly be a d m in is te re d so le ly by a c e n tra l agen cy a im in g at erad ica t ion in a p a rt icu la r a r e a o r r eg ion , ra th e r than by individual fa r m s . I should thus im agine that G o vern m en ts would p r o c e e d with g rea t caution in v iew o f past e x p e r ie n c e with the p e s t i c id e res id u e p r o b le m .

ACTION DU TEPA SUR LE DEVELOPPEM EN T NYMPHAL DE Dacus oleæ Gmel. (DIPTERA: TEPIiRITIDAE)

E. FYTIZA S ET A. BACOYANNIS

IN STITU T PHYTOPATHOLOGIQUE BENAKI,KIFISSIA, GRECE

( P r é s e n t é p a r Л/, b7. T za n a k a k is )


ACTION OF TEPA ON T ill: PUPAL DEVELOPMENT OF Dacus oleac G m el. (DIPTERA: TEPHRITIDAE). Third-stage (fully grown) larvae obtained from an artific ia l culture o f Dacus o le æ were dipped, a few hours prior to pupation, into aqueous solutions o f Tepa for 30 m inutes. The fo llow in g results were observed by the authors: even at the highest concentration tried (2%), Te pa had no e ffe c t on the m echanism o f pupation, perhaps because o f the late treatm ent; pupal developm ent was inhibited after the larvae had been dipped in solutions o f above 0 .125% , and death occurred at different stages o f pupal l ife , the tim e o f occurren ce depending on the concentration o f Te pa in the dipping solution ; there was p ractica lly no d ifferen ce betw een the m inim um lethal dose and the m axim um steriliz ing dose, the safety factor being alm ost equal to 1 . The authors discuss these results in com parison with those o f a previous experim ent during w hich Tepa was applied to adults shortly after they had em erged . The safety factor was then o f the order o f 50 . In addition, the results o f the two experim ents m entioned above are com pared with those o f other experim ents by different authors on the sam e insect subjected to gam m a radiation . This com parison shows that, irrespective o f the means o f treatment (gam m a rays or T ep a), the safety factor is large when the insects, at the tim e o f treatm ent, are past the stage o f histolysis-h istogenesis.

ACTION DU ТПРА SUR LE DEVELOPPEMENT NYMPHAL Dl- Dacus oleae G m el. (DIPTERA; TEPHRITIDAE). Des larves du troisièm e stade (stade larvaire co m p lé té ) , issues d'un é levage a r t if ic ie l, furent im m ergées quelques heures avant la pupaison dans des solutions aqueuses de tepa, pendant 30 m in . Les auteurs ont fa it les constatations suivantes: le tepa, m êm e à la plus forte des concentrations utilisées (2%), n 'a pas eu d’ e ffe tsur le m écanism e de la pupaison, peut-être à cause de l'in tervention tardive; le développem ent nym phal fut inhibé après l 'im m ersion des larves dans les solutions utilisées à des concentrations supérieures à 0,125% , la m ort est survenue à différents stades nyrnphaux, le temps de son apparition étant fonction des co n ce n trations de tepa dans les solutions d 'im m ersion ; il n 'y a pratiquem ent pas de m arge entre la dose léta le m in im a le 'e t la dose stérilisante m axim ale , le facteur de sûreté (safety factor) étant presque éga l à l ’ unité.Les auteurs com m en ten t ces résultats et les com parent à ceu x obtenus lors d 'une exp érien ce précédente au cours de laquelle le tepa était adm inistré aux adultes peu après leur ém erg e n ce . Le facteur de sûreté était alors de l'ordre de 50 . En outre, les résultats de deux expériences sont com parés à ceu x obtenus par d'autres chercheurs sur le m êm e insecte après irradiation aux rayons gam m a. De ce tte com paraison il résulte que, indépendam m ent du m oyen de traitem ent (rayons gam m a ou tepa), le facteur de sûreté est é levé lorsque les insectes, au m om ent du traitem ent, ont largem ent dépassé le stade d’ h isto lyse-h istogén èse .

INTRODUCTION

Au c o u r s d 'une e x p é r ie n c e p r é cé d e n te [1 ] , nous avons pu co n s ta te r que le tepa, a d m in is t ré aux adultes de Dacus o l e æ , avait une double action s té r i l is a n te su r le u r sy s te m e r e p r o d u c te u r : d 'une part , i l p rovoqua it des dégâts au m a té r ie l génétique d e s œ u fs et des s p e r m a t o z o a i r e s , et, d 'au tre part, i l inhibait le d év e lo p p e m e n t o v a r ie n ; c e s deux m o d e s d 'a c t io n c o r resp on daient a deux d i f fé r e n te s z o n e s de co n c e n tra t io n du c h im io s t é r i l i s a n t . Enfin le tepa, a une t r o i s i è m e zon e de con ce n tra t io n , provoq u a it des

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2 1 0 FYTIZAS et BACOYANNIS

dégâts a s s e z g r a v e s p ou r en tra în e r la m o r t d es adultes t r a i té s . Cette t r o i s i è m e zon e était a s s e z é lo ig n é e des deux a u tre s : le fa c te u r de sû re té s 'é le v a i t a 50 p ou r la p r e m ie r e zone (d o s e s provoquant d e s dégâts au m a té r ie l génét ique) et a 2 p ou r la d e u x ie m e (d o s e s p rovoquant l ' in h ib it ion du d év e lo p p e m e n t o v a r ie n ) .

L e s su b s ta n ce s c h im io s t é r i l i s a n te s ayant dans le u r m o lé c u le des g ro u p e s d 'a z i r id in y l ont une action s u r le s a c id e s n u c lé iq u e s , et en p a r t i c u l i e r le s ADN [2 - 4 J, qui sont r e s p o n s a b le s du d év e lop p em en t et des d iv e r s e s fon ct ion s de la c e l lu le ; la m i to s e d e s c e l lu le s est inh ibée et, p a r con séqu en t , le d év e lo p p e m e n t d e s t i s su s et des o r g a n e s est b loqu é . L e s m ê m e s auteurs s ignalent que l 'a c t io n d es c h im io s t é r i l i s a n ts es t p lus fo r te l o r s q u ' i l s 'a g i t de t i s su s en p le ine et rap id e évolut ion .

Chez D acus adulte, l e s o r g a n e s en p le ine évo lu t ion sont c e u x du s y s tè m e r e p ro d u c te u r , d 'où la grande se n s ib i l i té de c e s o r g a n e s a l 'a c t i o n cy to tox iqu e d e s c h im io s t é r i l i s a n ts . A l 'e n c o n t r e des im a g o s , l e s pupes de D a c u s , c o m m e c e l l e s de tous le s in s e c te s h o lo m é ta b o le s , su b issen t l 'h i s t o ly s e et l 'h i s t o g é n e s e d e s t i s s u s de la plupart de le u rs o r g a n e s ; le s y s t e m e m u s c u la i r e et l 'e x o s q u e le t te , p a r e x e m p le , su b issen t d e s t r a n s fo r m a t io n s beaucoup plus im p o rta n te s que le s o r g a n e s r e p r o d u c te u r s .

Nous avons c ru opportun d 'e x a m in e r le c a s d 'une in terven tion au tepa au m o m e n t ou le s indiv idus de D acus o l e æ n 'ont pas e n c o r e atteint le stade de l 'h i s t o ly s e - h i s t o g é n e s e et ou l 'a c t io n cy to tox iqu e du tepa su r d iv e r s s y s t è m e s et su r l ' in s e c t e e n t ie r peut ê tre p lus grande qu 'au stade adulte.

M A T E R IE L E T M ETH O D E UTILISES

L e s in s e c t e s u t i l i s é s p roven a ien t de l 'é l e v a g e a r t i f i c i e l de n o tre la b o r a t o i r e et étaient a l im en tés au stade la r v a ir e avec de la n o u rr i tu re p r é p a r é e se lo n la f o r m u le de T zanakakis et E c o n o m o p o u lo s (type M) [5] . L e s la r v e s du t r o i s i è m e stade, peu a p rè s leu r s o r t i e 'a la s u r fa c e su p é r ie u re de leu r m i l ie u nutrit if , étaient t r a n s p o r t é e s dans une f io le d 'E r le n m e y e r contenant une so lut ion aqueuse de te p a 1. A p r e s l ' im m e r s i o n , e l le s étaient égouttées au m o y e n d 'un p a p ie r f i l t r e et p la c é e s dans des b o îte s de P é tr i . L e s la r v e s dont la pupaison ne s 'e f fe c tu a it pas dans le s 5 h eu re s qui suivaient l ' i m m e r s i o n étaient r e je t é e s .

L a s o r t i e des adultes survint entre le 10e et le 14e jo u r a p rè s la p u paison . L e 20® jo u r , le s pupes n 'ayant pas donné lieu a des é m e r g e n c e s d 'ad u ltes furent d is s é q u é e s et e x a m in é e s au b in o c u la ir e ; l e s pupes m o r t e s furent e n suite c l a s s é e s dans l 'une des t r o i s c a té g o r ie s su ivantes : a) pupes m o r t e s avant ou pendant le stade de l 'h i s t o ly s e - h i s t o g é n e s e ; b) pupes m o r t e s a p rè s la fo rm a t io n d 'ad u ltes m a is avant la p igm en ta tion de le u r cu t icu le ; s e u ls l e s y eu x avaient une c o lo r a t io n r o s e - v i o l e t t e pâ le ; le s é b a u ch es des a i le s étaient ap parentes ; c ) pupes m o r te s a p rè s la p igm entation de la c u t icu le des adultes; le s adultes p a ra issa ie n t c o m p lè te m e n t f o r m é s ; q u e lq u e s -u n s de c e s adultes étaient e n c o r e v ivants au m om en t de la d i s se c t io n , m a is non v ia b le s .

1 Après plusieurs essais prélim inaires sur les concentrations du ch im iostérilisant e l la durée d 'im m ersion convenab les pour l 'ex p ér ien ce , nous avons utilisé en d é fin itive des concentrations de 2, 1,. 0 ,5 , 0,25 et0 ,125% e t une durée d 'im m ersion de 30 m in . Des larves im m ergées dans de l'eau pendant 30 m in onr été prises co m m e tém oins.

S M - 10 2/44 211

L o r s q u ' i l y eut é m e r g e n c e d 'adu ltes , c e u x - c i furent t r a n s p o r t é s dans des c a g e s de 30 X 30 X 30 cm (20 c o u p le s pa r ca g e et deux ré p é t it io n s p a r c o n c e n t ra t io n de la so lut ion d ' im m e r s i o n ) . L a n o u rr i tu re était con st itu ée p a r de la le v u r e h y d r o ly s é e , du s u c r e et de l 'eau a ra is o n de 1 : 4 : 5. A p r e s le 10e jo u r , d e s fru its a r t i f i c i e l s de pa ra ffine étaient p la c é s dans le s c a g e s pendant 24 h e u r e s ; le s œ u fs pondus étaient ensu ite t r a n s p o r té s dans des b o î te s de P é tr i p ou r le c o n t r ô le du p ou rce n ta g e d 'é c l o s i o n des œ u fs . T r e n te deux jo u r s a p rè s l ' é m e r g e n c e , le s f e m e l le s étaient d i s s é qu é e s et le u r s o v a i r e s e x a m in é s au b in o c u la ir e p ou r le c o n t r ô le de l 'a c t i o n cy to tox iqu e du tepa; en m ê m e tem p s , l ' e x a m e n d e s sp e rm a th e q u e s s 'e f fe c tu a it au m i c r o s c o p e pou r le c o n t r ô le de la v ig u e u r se x u e l le et de l 'é ta t d e s s p e r m a t o z o a i r e s (m o b i l i t é , f o r m e ) .

L a te m p é ra tu re du la b o r a to i r e o s c i l l a i t en tre 23 et 26°C et l 'hu m id ité re la t iv e en tre 48 et G3%. L a lu m ie r e su r la s u r fa c e en v e r r e des c a g e s était de 500 a 800 lux p ou r une p h otop ér iod e de 3 2 h e u r e s .

R E SU L T A T S

L e s d on nées e x p é r im e n ta le s f iguren t dans le s tab leaux I, II et III. On peut c o n s ta te r que:

1) L e tepa, m ê m e a la co n c e n tra t io n de 2,0%, n 'a pas eu d 'e f fe t su r le m é c a n is m e de la pupaison ; le p ou rce n ta g e de pupaison d e s la r v e s i m m e r g é e s dans d i f f é r e n te s so lu t ion s de tepa o s c i l l e en tre 94 et 100%(tableau I).

2) L e d é v e lo p p e m e n t nym pha l a été inhibé a p rè s l ' i m m e r s i o n des l a r v e s dans des so lu t ion s de tepa aux co n c e n tra t io n s s u p é r ie u r e s a 0,125% (tableau I).

3) L a m o r t des pupes est su rven ue a d i f fé re n ts stad es nymphaux (tableau II). L a v i t e s s e d 'a p p a r i t io n de la m o r t p ara ît ê t r e fon ct ion de la c o n ce n tra t io n de tepa dans la so lut ion d ' im m e r s i o n : la plus grande part ie (79%) des la r v e s i m m e r g é e s dans la so lut ion a 2,0% n 'a pas pu d é p a s s e r le stade de l 'h i s t o ly s e -h i s t o g é r ie s e ; pa r c o n tre , aux c o n ce n tra t io n s de 0,5%

T A B L E A U I. A C T I O N DU T E P A SUR L E S P U P E S D E D a c u s o l e æ A P R E S U N E IM M E R S I O N DE 30 m in D ES L A R V E S ( 3 e S T A D E ) D A N S U N E S O L U T I O N A Q U E U S E DU C H I M I O S T E R I L I S A N T

N om b re de L3 u tiliséesC o n ce n tra t io n du tepa

(%)

Pupaison des L 3

№ )

E m ergen ce des L

(%)

82 2 ,0 93 . 8 0

81 1 .0 100, 0 1 ,4

103 0 .5 100 , 0 2 .9

97 0 ,2 5 1 0 0 .0 4 b , 5

99 0 ,1 2 5 1 0 0 .0 78. 3

110 0 1 0 0 .0 81. 3

T A B L E A U II. S T A D E S N Y M P H A U X P E N D A N T L E S Q U E L S E S T S U R V E N U E L A M O R T , A P R E S U N E I M M E R S I O N D E 30 m in D E S L A R V E S D E D a c u s o le a e ( 3 e S T A D E ) D A N S U N E S O L U T I O N A Q U E U S E D E T E P A

N om b re d e L3 u tiliséesC o n ce n tra t io n

du tepa

(%)

Pupes m ortes ( % des L 3)

A d u ltes m orts pendant l 'é m e r g e n c e (% des L 3)

A vant ou pendant l 'h is to ly s e -h is to g é n è s e

A p rès lafo rm a tio n d 'a d u ltes e t avan t la p ig m e n ta tio n d e leu r c u t ic u le

A près lap ig m e n ta t io n de la c u t ic u le des adu ltes

82 2 ,0 7 9 ,1 1 8 ,4 2 ,4 0

81 1 .0 3 9 ,3 4 3 ,0 1C, 2 0

103 0 . 5 . 2 3 ,3 32 , 8 2 9 ,4 11, 6

97 0 , 25 1 8 ,2 l l i . 2 1 1 .1 6 ,0

99 .0 ,1 2 5 8, 2 B, 2 4 , 2 3 ,1

110 0 10 , 2 1 ,9 2, 9 3, 8

212 FY

TIZA

S et

BAC

OY

AN

NIS

S M - 10 2/44 213

T A B L E A U III. F E C O N D I T E , F E R T I L I T E E T V I G U E U R S E X U E L L E D E S A D U L T E S D E D a c u s o l e æ ISSUS D E L A R V E S I M M E R G E E S P E N D A N T 30 m i n D A N S U N E S O L U T I O N A Q U E U S E D E T E P A (4 0 c o u p l e s d 'a d u l t e s - p a r c o n c e n t r a t i o n )

C o n ce n tra tio n du tepa

(Я

N om bre d 'œ u fs pondus pendant

22 jours

. E closion d 'oeu fs

1%)

Sperm attièques fé co n d é e s

(%)

0 ,2 5 1249 26, 9 67, 0

0 ,1 2 5 1123 7 9 .1 85 , 0

0 986 82, 3 9 0 ,0

et de 0,25%, la p a rt ie d e s pupes m o r te s avant ou pendant l 'h i s t o l y s e - h istogériese ne r e p ré s e n ta i t pas m ê m e le t i e r s des pupes m o r te s .

4) L e p o u rce n ta g e d 'a d u lte s m o r t s pendant la s o r t i e ne p a ra ît pas d ép en dre de la con ce n tra t io n de tepa dans le l iqu ide d ' im m e r s i o n (ta b le a u l l ) ; le fa ib le p ou rcen tag e o b s e r v é ch e z le s adultes is s u s des la r v e s i m m e r g é e s dans le s so lu t ion s de tepa, qui d é p a ss e qu e lq u e fo is c e lu i d e s in s e c te s té m o in s , ne r e p r é s e n t e en r é a l it é que le s adultes ayant d e s défauts qu 'on r e n co n tre bien souvent ch e z le s té m o in s .

5) L e s o r g a n e s r e p r o d u c te u r s d e s fe m e l le s i s s u e s des la r v e s i m m e r g é e s dans des so lu t ion s a 0,25% et 0,125% n 'ont subi aucune action cy to tox iqu e ; le dév e lo p p e m e n t o v a r ie n a été n o rm a l , c o m m e l 'a d ém on tré l 'e x a m e n m i c r o s c o p iq u e 32 jo u r s a p rè s l ' é m e r g e n c e . D 'a u tre part, la fé con d ité d e s f e m e l le s n 'a pas été a f fe c tée p a r le tepa (tableau III).

6) Le p ou rce n ta g e d 'é c l o s i o n d e s œ u fs pondus pa r le s fe m e l le s i s s u e s des la r v e s i m m e r g é e s dans une solution a 0,25% s ' e s t trou vé réduit a 26,9% (tableau III). On ne p o u rra it a t tr ibu er la r é d u ct ion de la fe r t i l i té a la seu le ré d u ct ion du p ou rcen tag e d e s f e m e l le s f é c o n d é e s (67 ,0% ); une pa rt ie de f e m e l le s fé c o n d é e s aurait pondu des œ u fs non v ia b le s .

DISCUSSION E T CONCLUSIONS

On peut c o n c lu r e des ré su lta ts obtenus dans le s c o n d it ion s e x p é r im e n ta le s de c e t ra v a i l que le tepa a une action m a rq u é e au ss i bien s u r le s y s tèm e r e p ro d u c te u r que su r l ' in s e c t e en t ie r l o r s q u ' i l est ad m in is t ré avant que le s in s e c te s atteignent le stade de l 'h i s t o ly s e -h i s t o g é r ie s e . A la c o n - ' cen tra t ion de 0,25%, le s la r v e s im m e r g é e s pendant 30 m in furent à la fo is s t é r i l i s é e s et tu ées (tableaux I et III); ainsi on ne peut pas d is t in g u er le s deux d i f fé re n ts m o d e s d 'a c t io n du tepa, a l o r s que, dans le c a s de notre p ré cé d e n t t r a v a i l [1 ] , la m o r t d e s in s e c te s ne su rven ait que lo r sq u e nous u t i l is io n s des d o s e s 50 f o is p lus fo r t e s que c e l l e s qui p rovoqua ien t la s t é r i l i s a t io n d es adultes.

T zanakakis et T h om ou [6 ] , en ir ra d ia nt aux r a y o n s g a m m a des pupes de D acus o l e æ â g é e s de 9 j o u r s , p rovoqua ien t une s t é r i l is a t i o n c o m p le te ch e z c e s in s e c te s a la d o se de 6 krad ; en é tab lissan t la c o u r b e de to x ic i té , T zanakakis [7] a trou vé qu' a la d o s e de 40 krad le s r a y o n s g a m m a ne

214 FYTIZAS et BACOYANNIS

p rovoqua ien t qu' une fa ib le m o r ta l i té lo r sq u e le s pupes i r r a d ié e s étaient â g é e s de 9 jo u r s . Dans le s deux e x p é r ie n c e s , le s pupes u t i l i s é e s étaient a 1 ou 2 jo u r s de l ' é m e r g e n c e ; le s in s e c t e s avaient la rg e m e n t d é p a ss é le stade de l 'h i s t o ly s e - h i s t o g é n e s e . En utilisant d e s la r v e s de D acus o l e æ un peu avant la pupaison et d es pupes qu e lques h eu re s a p rè s la pupaison , F y t iz a s et T zanakakis ( t ra v a i l non publié ) ont trou vé des ré su lta ts d i f fé r e n ts de ce u x obtenus lo r sq u e le s pupes étaient â g é e s de 9 j o u r s ; a insi, les la r v e s et le s pupes i r r a d ié e s a 2 krad subirent l 'a c t io n in s e c t i c id e a 100% tandis que la to ta lité de c e l l e s qui avaient été i r r a d ié e s a 1 krad é c h a p p èren t a la m o r t et qu 'une pa rt ie d 'e n t re e l l e s é ch a p p èren t a la s t é r i l i sa tion ; la d o s e léta le m in im a le fut in fé r ie u r e a 2 krad et la do se s t é r i l i sante m a x im a le su p é r ie u re a 1 krad. Dans le c a s des i r ra d ia t io n s on ne peut pas non plus d is t in g u er le s deux ac t ions ( in s e c t i c id e et s t é r i l is a n te ) lo r s q u e le s in s e c te s t r a i té s n 'o n pas atteint ou d é p a ss é le stade de l 'h i s t o ly s e - h i s t o g é n e s e .

L e fa c te u r de sû re té (sa fe ty fa c to r ) , rap p ort de la d o s e m in im ale lé ta le a la d o s e m a x im a le s t é r i l is a n te , ne c a r a c t é r i s e r a i t pas seu le m e n t une su bsta n ce c o m m e c h im io s t é r i l i s a n te ou in s e c t i c id e ; la m ê m e s u b stan ce peut ê tre c o n s id é r é e c o m m e c h im io s t é r i l i s a n te ou in s e c t i c id e se lo n le m od e d 'a p p l ica t io n ou le tem ps d ' in terven tion . Le tepa, pa r e x e m p le , c o m m e le s r a y o n s gam m a, ne peut ê tre c o n s id é r é c o m m e s té r i l is a n t l o r s q u ' i l est ad m in istré a des la r v e s ou de je u n e s pupes de Dacus o l e æ .

En ou tre le tepa, aux d o s e s de 0,25%, 0,5% et 1, 0%, a pu in h ib e r le dév e lo p p e m e n t nym phal d 'une p art ie des in s e c te s ayant d é p a ss é le stade de l 'h i s t o ly s e - h i s t o g é n è s e ; p e u t -ê t r e c e produit a u r a i t - i l au ss i une action su r le m é c a n is m e de l ' é m e r g e n c e .

Enfin, l 'a b s e n c e d 'a c t io n du tepa su r la pupaison p o u rra it ê t r e dû a l ' in te r v e n t io n ta rd ive , 2 a 5 h eu re s avant la pupaison. D es travau x su r une action éventue lle su r la pupaison sont en c o u r s , l ' in te rv e n t io n étant e f fe c tu é e à un stade plus p r é c o c e que dans c e trav a i l .

R E M E R C I E M R N T S

Nous tenons à r e m e r c i e r v ivem en t M. M. E. T zanakakis de s e s su g ge s t io n s et des c o r r e c t i o n s q u 'i l a a p p o rté e s au texte de c e m é m o i r e .

R E F E R E N C E S

[1 ] FY TIZA S, E ., A ction du Tepa sur les adultes de Dacus o le æ G m el. (D íptera: T ephritidæ ) en. fonction des quantités du chim iostérilisant utilisées. Annls Inst. phytopath. Benaki (sous presse).

[2] BROOKS, P ., I.AWLEY, P .D ., The reaction o f m o n o - and d ifu nctional alkylating agents with n u cle ic acids, B iocliem . J. 60 (1961) 4 5 6 -5 0 3 .

[3 ] FAHMY, O .G ., FAHMY, N Í.G ., The chem istry and genetics o f the alkylating chem osterilants, Trans. R. S oc . trop. M ed . H yg. 58 (1964) 3 1 8 -2 6 .

[4] FRAENKEL-CONRAT, H ., C h em ica l m od ifica tion o f viral rib on u cle ic acid , 1. A lkylating agents, B ioch em . biophys. Acta 49 (1961) 1 6 9 -8 0 .

[5 ] TZAN AK AK IS, M .E ., ECONOMOPOULOS, A .P . , T w o e ffic ien t larval diets for the continuous rearingo f the o liv e fruit fly , Dacus o le æ (G m elin ) (D iptera: T ep h ritidæ ),]. e c o n . Ent. 60 3 (1967) 66 0 -6 3 .

[6] INTERNATIONAL ATOM IC ENERGY AGENCY, Advances in Insect Population C ontrol by the S terile -M ale Techn iqu e, T ech n ica l Reports Series N o .44 , IAEA, Vienna (1965) 4 .

S M - 10 2/44 215

[7 ] TZAN AK AK IS, M .E ., C ontrol o f the o liv e fruit fly , Dacus o le æ (G m elin ), with radiation or ch e m ica l steriliza tion procedures, T ech n ica l Report, U .S .D .A . , Grant FG -G R -102, P roject EI1-ENT-1 (G February 1967) 80pp.

D I S C U S S I O N

A . B . B O R K O V E C : What w as the la rv a l in s ta r in which the in s e c ts w e r e tre a ted ?

M .E . T Z A N A K A K IS : It w as the end o f the third and last la rv a l stage . T he fu lly g row n la rv a e have then f in ish ed feed ing , and le a v e the la rv a l food m ed ium in s e a r c h o f a m o r e su itab le p la ce to pupate. U nder the con d it ion s in w hich the authors w o rk e d , f o r m a t io n o f the puparium u su a lly took p la ce 2 - 5 hours la ter .

A . B. B O R K O V E C : Do you know w hether s p e r m a to g e n e s is in the m a le la rv a e w as c o m p le te d o r in p r o g r e s s at the t im e o f tre a tm e n t with the c h e m o s te r i la n t ?

M .E . T Z A N A K A K IS : I am a fra id I do not have this in fo rm a t io n to hand, but I b e l ie v e that s p e r m a to g e n e s is w as s t i l l in p r o g r e s s at this stage .

E

S T E R IL E -M A L E TECHNIQUE

(Sessions VI and VII)

C h a i r m a n : E . SH L 'M A K O V

USSR

ТРУДНОСТИ И УСПЕХИ М АССОВОГО РАЗВЕДЕНИЯ НАСЕКОМЫ Х В ЛАБОРАТОРИИ И ВОЗМОЖНОСТИ САМОИСТРЕБЛЕНИЯ НЕКОТОРЫХ ВРЕДНЫХ ВИДОВ

E . М .Ш У М А К О ВВСЕСОЮЗНЫЙ Н АУ Ч Н О -И ССЛ Е Д О ВА ТЕЛ ЬС КИ Й И Н С Т И Т У Т ЗАЩИТЫ Р А С Т Е Н И Й ,Л Е Н И Н Г Р А Д ,С С С Р

Abstract — Аннотация

DIFFICULTIES AND SUCCESSES IN THE MASS REARING OF INSECTS IN THE LABORATORY, AND THE POSSIBILITY OF A U TO CID A L CONTROL OF SOME HARMFUL SPECIES. The p ractica l developm ent o f the ste r ile -m a le release techn ique, as indeed o f a ll methods o f autocidal control o f harmful insects, is lim ited by the d ifficu lties o f mass rearing in artific ia l con d ition s. H ow ever, analysis o f cases o f the successful solution o f this problem for a number o f types o f D íptera, Lepidoptera and Orthoptera gives an indica tion o f possible ways o f setting up la rg e -sca le rearing o f the insects required.

The most d ifficu lt prob lem , that o f ensuring a suitable food supply for the insects at any tim e o f the year, is being successfu lly ov erco m e as a result o f the progress m ade in producing synthetic and sem i- synthetic nutrient m ed ia , which have been deve lop ed both for sem i-saprophytic and for herbivorous and predacious species. T h e ch o ice o f recipes for such nutrients is determ ined by correct se lection o f the necessary ingredients, above a ll vitam ins, free am ino acids and sterols; the proper quantitative proportion betw een these substances and betw een the basic com ponents o f the food — proteins, fats and hydrocarbons — is o f the greatest im p ortan ce . The amount o f work involved in insect rearing depends on what means are used for sterilizing the containers and for preventing the nutrient m edium from d eca y in g . One way in which these problem s have been solved is by the d evelopm en t o f a dried nutrient m edium in pow der form ; this has been successfully used for rearing the m igratory locust in the USSR. For a number o f harm ful species o f Lepidoptera, it has been shown possible to rear the insects in a c lo sed , sealed con ta in er. In this case a supply o f nutrient m edium is provided adequate to ensure the insects* developm en t from, the tim e the eggs or caterpillars are deposited until the im a go o f the new generation em erges. This approach greatly s im p lifies the w hole process o f mass rearing. Many species o f insects can be reared on grain or plant seeds w hich are less subject to decay during prolonged storage.

Anorher important problem is that o f develop ing ways o f overcom in g the diapause in laboratory populations in order to ensure continuous rearing. This can be done either by reactivating the insects by tem perature changes or by instituting a period o f illum ination which prevents die diapause from starting.A further possible m ethod is that o f se lectin g and crossing diapausing and поп-d iapausing strains o f a g iven species. A number o f species o f Orthoptera having a fairly w ide natural habitat have been used to show the possibility o f autocidal control by adding to a natural popu lation which norm ally lias a diapause specim ens o f a non-diapausing population from other parts o f the h ab itat. This possibility has been demonstrated for the crick et T eleogry llu s corn modus W alk , in Australia and for sub-species of Locusta m igratoria L. in the Old W orld . The developm en t o f this form o f autocidal control o f insects merits c lose attention .

The work reported is devoted m ainly to d evelop in g methods o f autocidal con trol and techniques lor mass laborarory breeding o f such harm ful species as Carpocapsa pom on ella L . , Eurygaster in tegriccps Put, and Locusta m igratoria L.

Т Р У Д Н О С Т И И У С П Е Х И М А С С О В О Г О Р А З В Е Д Е Н И Я Н А С Е К О М Ы Х В Л А Б О Р А Т О Р И И И В О З М О Ж Н О С Т И С А М О И С Т Р Е Б Л Е Н И Я Н Е К О Т О Р Ы Х В Р Е Д Н Ы Х В И Д О В . П р а к т и ч е ск а я р а з р а б о т к а м е т о д а н ы п у ск а с т е р и л и з о в а н н ы х с а м ц о в , как и в о о б щ е м е т о д о в с а м о и с т р е б л е н и я в р е д н ы х н а с е к о м ы х , л и м и т и р у е т ся т р у д н о с т я м и м а с с о в о г о р а з в е д е н и я их н и с к у с с т в е н н ы х у с л о в и я х . О д н а к о , а н а л и з у сп е ш н ы х с л у ч а е в р еш ен и я э т о й п р о б л е м ы для р я да п и дов L i p l e r a , L e p id o p te r a и O r t h o p te r a п о з в о л я е т н а м е т и т ь в о з м о ж н ы е п у ти с о з д а ния тех н и к и м а с с о в о г о р а з в е д е н и я н е о б х о д и м ы х о б ъ о к т о п .

219

2 2 0 Ш У М А К О В

Н а и б о л е е тр уд н ы й в о п р о с о б е с п е ч е н и я н а с е к о м ы х с о о т в е т с т в у ю щ и м к о р м о м в л ю б о е в р е м я г о д а у с п е ш н о р а з р е ш а е т с я б л а г о д а р я п р о г р е с с у , д о с т и г н у т о м у в с о з д а н и и с и н т е т и ч е с к и х и п о л у с и н т е т и ч е с к и х п и щ ев ы х с р е д для н а с е к о м ы х . Т а к и е с р е д ы р а з р а б о т а н ы у ж е как для п о л у с а п р о ф и т и ы х , т а к и для р а с т и т е л ь н о я д н ы х и хищ ны х в и д о в . П о д б о р р е ц е п т о в для т а к и х с р е д о п р е д е л я е т с я п р а в и л ьн ы м в ы б о р о м н е о б х о д и м ы х к о м п о н е н т о в , в п ер в у ю о ч е р е д ь в и т а м и н о в , с в о б о д н ы х а м и н о к и с л о т и с т е р и н о в . В а ж н ей ш ее зн а ч е н и е и м е е т п р а в и л ь н о е к о л и ч е с т в е н н о е с о о т н о ш е н и е в п и щ ев ой с р е д е как у к а з а н н ы х в е щ е с т в , т а к и о с н о в н ы х к о м п о н е н т о в к о р м а - б е л к о в , ж и р ов и у г л е в о д о в . Т р у д о е м к о с т ь п р о ц е с с о в р а з м н о ж е ния н а с е к о м ы х о п р е д е л я е т с я с п о с о б а м и с т е р и л и за ц и и п о су д ы и о с а д к о в , а т а к ж е м е т о д а ми п р е д о х р а н е н и я п и щ ев ы х с р е д о т з а г н и в а н и я . П р и м е р о м п р е о д о л е н и я э т и х т р у д н о с т е й я в л я е т с я с о з д а н и е с у х о й , п о р о ш к о о б р а з н о й с р е д ы для п е р е л е т н о й са р а н ч и , на к о т о р о й в С С С Р э т о т ви д у с п е ш н о в о с п и т ы в а е т с я . Для р я д а в р е д н ы х в и д о в L e p id o p te r a " п о к а з а н а в о з м о ж н о с т ь вы р ащ и ван и я о с о б е й в з а к р ы т о й , г е р м е т и з и р о в а н н о й п о с у д е . При э т о м д а е т с я з а п а с п и щ ев ой с р е д ы , о б е сп е ч и в а ю щ и й р а з в и т и е о с о б е й с м о м е н т а п о д са д к и на с р о д у яиц или г у с е н и ц д о в ы х о д а и м а г о н о в о г о п о к о л е н и я . Т а к о й п р и ем в е с ь м а у п р о щ а е т в е с ь т е х н о л о г и ч е с к и й п р о ц е с с п ол у ч ен и я м а с с о в о й п р од у к ц и и . М н о г и е виды н а с е к о м ы х м о ж н о в о с п и т ы в а т ь на з е р н е и с е м е н а х р а с т е н и й , м е н е е п о д в е р ж е н н ы х за гн и в а н и ю npvi д л и т е л ь н ом х р а п е н и и .

В а ж н ей ш и м в о п р о с о м м а с с о в о г о р а з в е д е н и я я в л я е т с я р а з р а б о т к а п у т е й п р е о д о л е н и я д и а п а у з ы у л а б о р а т о р н о й п оп ул я ц и и , ч т о о б е с п е ч и в а е т н е п р е р ы в н о с т ь р а з в е д е н и я . У с т р а н ен и е д и а п а у з ы д о с т и г а е т с я как р е а к т и в а ц и е й т е м п е р а т у р н ы м и в о з д е й с т в и я м и , т а к и с о з д а н и е м ф о т о п е р и о д а , п р е д о т в р а щ а ю щ е г о в о з н и к н о в е н и е д и а п а у з ы . В о з м о ж е н ещ е оди н п у т ь : п о д б о р и ск р е щ и в а н и е г е н е т и ч е с к и р а зл и ч н ы х р а с д а н н о г о ви д а , и м ею щ и х д и а п а у з у и л и ш ен н ы х е е . Н а п р и м е р е р я да в и д ов O r t h o p t c r a , и м ею щ и х д о с т а т о ч н о б о л ь ш и е а р е а л ы , п о к а з а н а в о з м о ж н о с т ь с а м о и с т р е б л е н и я в р е д н ы х в и д ов п у т е м п од м еш и в а н и я к п р и р од н ой п о п у л я ц и и , н о р м а л ь н о и м ею щ ей д и а п а у з у - о с о б е й б е з д и а п а у з н о й п опул я ц и и и з д р у г и х ч а с т е й а р е а л а т о г о же в и д а . Т а к и е в о з м о ж н о с т и вы я в л ен ы для с в е р ч к а T e l e o g r y l lu s c o m - m oclu s W alk в А в ст р а л и и , и для п о д в и д о в L o c u s t a m ig r a t o r ia L . на т е р р и т о р и и С т а р о г о С в е т а . Р а з р а б о т к а т а к о г о пути с а м о и с т р е б л е н и я н а с е к о м ы х з а с л у ж и в а е т б о л ь ш о г о вн и м ан и я .

В н аш и х р а б о т а х о с н о в н о е вн и м а н и е у д е л я е т с я р а з р а б о т к е м е т о д о в с а м о и с т р е б л е н и я и т е х н и к и м а с с о в о г о л а б о р а т о р н о г о р а з в е д е н и я т а к и х в р е д н ы х в и д о в как C a r p o c a p s a р о - m o n e l la L . , E u r y g a s t e r in t e g r ic .e p s P u t , и L o c u s t a m ig r a t o r ia L .

За последние десять лет во многих стр ан а х интенсивно развивались исследования в двух близких направлениях — р а зр а б о т к а м е т о д о в с т е р и лизации н а се к о м ы х и создан и е техники м а с с о в о г о разведения их в л а б о раторн ы х у сл о в и я х . Х отя э т и два направления возникли и развивались н еза ви си м о и опирались на различные ф изи ологи чески е о с н о в ы , очень ск о р о с т а л а ясна орган и ческ ая связь э т и х направлений.

П рактические усп ехи применения стерилизации н а се к о м ы х д о с т и г а лись лишь та м , где была р а зр абота н а заранее адекватная техника м а с с о в о г о получения н а се к о м ы х для их п осл ед ую щ его облучения или о б р а ботки х е м о сте р и л я н та м и .

Р азви ти е исследований в о бл а сти хим ической стерилизации н а с е к о м ы х началось с о в с е м недавно - лишь после 1960 г о д а — но именно после э т о й даты о с о б е н н о интенсивно стали р а зр а б а т ы в а т ь с я и ск у сст в е н н ы е пищевые среды для н а с е к о м ы х . Как п ок азы в ает тол ьк о что вышедший в с в е т о б з о р Х а у с а [1], например для L^epidoptera более 80% р ец еп тов и ск у сс т в е н н ы х ср е д были опубликованы в ра б о т а х , вышедших в с в е т после 1960 г о д а . В р а б о те Х а у с а соверш енно не учтен ы и ссл едован и я , проведенны е в э т о м направлении в С С С Р , г д е первы е публикации о б и ск у сс т в е н н ы х ср е д а х также появились в 1960 г о д у [2] и в последую щ ие годы число их п остепен н о н ар астал о .

Т аки м о б р а з о м , р а зр а б о т к а м е т о д о в воспитания н а се к о м ы х на и с к усств е н н о й пище является очень молодой отр а сл ью э н т о м о л о ги ч е ск и х исследований , о со б е н н о интенсивно разрабатытзающейся лишь в п о с л е д нее д е ся т и л е т и е . Э т о о б с т о я т е л ь с т в о опред еля ет ряд тр у д н о ст е й , н е

S M - i 0 2 / 2 5 221

избеж н о возникающих перед и ссл е д о в а т е л я м и , работающ ими во всякой новой отр а сл и науки. Вполне е с т е с т в е н н о , что те о р е т и ч е ск и е (ф и з и о л о г и чески е) о с н о в ы разведения н а с е к о м ы х на и ск у сствен н ой пище еще не вполне р а зр а б о т а н ы , и х отя осн ова н н ы х на проведенных э к с п е р и м е н т а х обобщ ений вы ска зан о д о ст а т о ч н о м н о г о , они отли ч аю тся значительной ф р а гм е н т а р н о ст ь ю и нуж даю тся в к р и ти ческом анализе и э к с п е р и м е н тальной проверке .

В с л е д с т в и е м о л о д о ст и данного научн ого направления д ает с е б я знать н е д о ст а т о к обобщ ения накопленных данных и небольш ое к ол и ч еств о с в о бодных и о б зо р н ы х р а б о т по э т о м у в о п р о су . Упомянутая выше р а б о т а [11 по с у щ е ст в у я вл яется первым о б з о р о м ,о б ъ е д и н и в ш и м накопленные материалы по рецеп туре и ск у сс т в е н н ы х с р е д для н а с е к о м ы х . Она о х в а ты ва е т преи м ущ ествен н о и ссл едован и я , проведенные в СШ А, Канаде, Великобритании и Японии, и п о к а зы в а е т достиж ения в создании и с к у с с т венной пищи для н а се к о м ы х преим ущ ествен н о из о тр я д о в C o le ó p te r a , D íptera , L e p id o p te r a и O rth op tera . К ол и чество видов, для которы х р а з работаны м е то д ы воспитания на си н тети чески х ср е д а х в каждом из у к а занных о т р я д о в , кол ебл ется о т 10 до 25, а в с е г о Х а у с р а ссм а т р и в а е т с о с т а в пищи примерно для 115 видов н а се к о м ы х . В р а б о т е Х а у с а б о л ь шой и н терес пред ставл я ет библиография об зор н ы х р абот по физиологии питания н а сек ом ы х , данная в приложении. Э т а библиография начинается с и з в е с т н о г о о б з о р а У варова [31 и заканчивается последними работами с а м о г о ав тор а [4].

Х рон ологи чески р а б оте Х а у са предш ествовали лишь о бзорн ы е работы по и ск у сствен н ы м ср е д а м , опубликованные в С С С Р [5, 6].

В начале 6 0 -х го д о в разведение н а с е к о м ы х -ф и т о ф а г о в на и с к у с с т венных с р е д а х только начиналось . Но к э т о м у времени уже были д о с т и г нуты значительные успехи в создании и ск у сст ве н н ы х ср е д для н а с е к о - м ы х -п о л у са п р о ф а го в (D ro so p h i la m e la n o g a s te r , L u c i l ia s e r i c a t a , C o c h l i o - m y a hom in iv и д р . ) , для видов питающихся зерном или мукой , а также пищевыми о ст а т к а м и (например, T e n e b r io m o l i t o r , T r ib o l iu m con fu eum тараканы Blatta и В late 11a и т . д .

Перпые работы о разцедении н а с е к о м ы х -ф и т о ф а г о в на и с к у с с т в е н ных среда х появились лишь в 5 0 - х го д а х и в начале оп ы та не приносили о с о б ы х у с п е х о в , показав значительную трудн ость п робл ем ы .

Лишь после т о г о , как к си н тети ческой с р е д е были добавлены в е щ е с т ва р а ст и т е л ь н о го происхождения в виде р а зм о л о т ы х сухи х л и сть ев или п атен това н н ого препарата из со к о в трав, и з в е с т н о г о под названием "ц е - р оф и л" , были д о ст и г н у т ы первые успехи в воспитании полноценного п о т о м с т в а таких видов как P y r a u s t r a / O s tr in ia /n u b i la l i s и G rapholita m o le s ta (1949 - 1956 год ы ) .

В последующие го д ы , однако, были найдены с о с т а в ы ср е д , б е з д о б а в ления каких-либо в е щ е ств р а ст и т е л ь н о го происхождения; на таких с р е дах удалось выращивать ряд скры тож ивущ их н а се к о м ы х , о т которы х было получено плодови тое п о т о м с т в о в течение нескольких поколений.К таким видам о т н о с я т с я H y le m y ia antigua, P e c t in o p h o ra g o s s ip ie l l a , P e x ic o p ia m a lv e l la , C h lo r id e a o b s o le ta , A nthonom us g ra n d is .

Воспитание н а се к о м ы х , живущих ск р ы т о , внутри р а ст и т е л ь н о го с у б с т р а т а , удавалось л е г ч е , чем выращивание на си н тети чески х средах ф и то ф а го в , живущих о т к р ы т о на п оверхн ости л и с т ь е в .

222 Ш У М А К О В

В о зм о ж н о ст ь воспитания на си н тети чески х среда х открытож ивущ их н а се к о м ы х была показана нами f21 на примере гусениц L o x o stege B t ic t i c a l i s , , а впоследствии и для н ек оторы х других видов L e p id o p le r a .

За последующие 5 лет (1963 — 1967) коли чество р а б о т , посвященных и ск у сст в е н н ы м средам для различных как скрытож ивущ их, так и л и с т о ядных н а се к о м ы х , п р о гр е сси в н о в озр астал о во многих с т р а н а х . При э т о м о с о б о е внимание уделялось технике получения м а с с о в о й продукции н а с е к ом ы х , что сти м ул и ровалось п отр ебн остя м и разработки новых м е т о д о в бор ьбы с вредителями, таких как "S te r i le m a le te ch n iq u e " и други х с п о с о б о в сам ои стреблен и я вредных н а се к о м ы х .

В связи с э ти м в США была предпринята попытка со зд а т ь к апитальное р у к о в о д с т в о по м а с с о в о м у разведению и колонизации н а се к о м ы х [7], которая очевидно, в ближайшее время выйдет в с в е т и б у д е т доступн а заи н тересован н ы м и ссл е д о в а т е л я м .

Перечисленными выше работам и практически и счерпы ваю тся о б з о р ные труды по м етодике воспитания н а се к о м ы х на и ск у сст ве н н ы х ср е д а х .

Н есм отря на м о л одость п робл ем ы , с л е д у е т о т м е т и т ь появление в сам ы е последние годы н ек отор ы х т е о р е т и ч е ск и х обобщ ений, к о т о р ы е , вер оятн о , явятся полезными руководящими принципами в дальнейших иссл едован и ях по р а зр аботк е рецептуры и ск у сст в е н н ы х питательны х ср е д для н а с е к о м ы х .

Одним из таких важных т е о р е т и ч е ск и х исследований я вл яется другая р а б о т а Х а у с а [41, рассм атри ваю щ ая значение н ек отор ы х новых д о с т и ж е ний физиологии питания н а се к о м ы х для р азработк и пробл ем б и о л о г и ч е с к о го м е т о д а борьбы с вредителям и. Х а у с у ста н ав л и в ает три принципа питания н а се к о м ы х , к оторы е он ф ормулирует следующ им о б р а з о м :

1 . Правило т о ж д е ст в е н н о ст и пищевых требований;2. Принцип рационального соотнош ения пищевых к ом пон ен тов ;3 . Принцип компенсирующих д о б а в о к .Э ти обобщения иссл едован и й , проведенных канадскими эн т о м о л о га м и

в Бельвилле, засл уж и ваю т с е р ь е з н о г о внимания.И сследования, проведенные в С С С Р , также позволили сделать н е

к отор ы е обобщ ения . В наших ра ботах [81 показаны пищевые различия - н а се к о м ы х , живущих внутри р а ст и т е л ь н о го с у б с т р а т а (скры тож ивущ ие) и обитаю щ ие о т к р ы т о , питающиеся листвой ра стен и й . В ы ясн ен о , что для видов, развиваю щ ихся в природе на п л од оэл ем ен тах , оптимальными являю т ся среды с большим к оли чеством а з о т и с т ы х в е щ е ст в и жиров, но о т н о си тел ьн о бедных у гл евод ам и и стер и н ам и . Для ли стоядн ы х н а се к о м ы х напротив, необходи м ы среды б о г а т ы е у гл евод ам и и стер и н ам и , в то в р е мя как отн оси тельн ы й и зб ы т о к жиров и а з о т и с т ы х в ещ еств д е й с т в е т на э т и х н а се к о м ы х у гн е т а ю щ е . Указанные различия пищевых требований н а се к о м ы х э т и х двух типов являются отраж ен и ем различий б и о х и м и ч е с к о го с о с т а в а природной пищи э т и х двух групп ф и т о ф а г о в .

Важным обобщ ением с о в е т с к и х и ссл е д о в а т е л е й является также в ы я с нение роли св о б о д н ы х ам инокислот , как н еобх оди м ы х к ом пон ен тов пищи. И зучение питания т у т о в о г о и д у б о в о г о шелкопрядов (B o m b y x m o r i и A n th e ra e a p e r n y i ) , проведенное Филипповичем [9 - 121, показало , ч т о в ы бор пищи в значительной м ер е о п р ед ел я ется содерж ан и ем в ней о п р е д е лен н ого набора своб од н ы х ам и н оки слот . В наших р а б о т а х также п о казан о , что даже у видов, развиваю щ ихся в природе на одном и т о м же р астении (хлопчатнике), п отр е б н о сти в наборе св о б о д н ы х ам инокислот

S M - 1 0 2 /25 223

различны, что видно на примере ср ав н и тел ь н ого изучения пищевых требований G e le ch ia m a lv e l la n C h lo r id e a o b s o le ta [6].

Приведенные выше обобщ ения стали в о зм ож н ы , главны м о б р а з о м , благодаря применению м е т о д о в воспитания н а се к о м ы х на си н т е т и ч е ск и х пищевых ср е д а х ,л и ш е н н ы х продуктов р а ст и т е л ь н о го происхож дения.Э ти и сследования позволили далеко вперед продвинуть наши п р е д с т а в л е ния о роли отд ельн ы х к ом пон ен тов пищи в н а се к о м ы х и бол е е о б о с н о в а н но подбирать рецептуру и ск у сст ве н н о й пищи для м а с с о в о г о разведения н а се к о м ы х в л абор атор н ы х у сл ови я х .

Но пожалуй с а м ы м важным т е о р е т и ч е ск и м д ости ж ен и ем последних лет является о т к а з о т представления о т о м , что вы бор пищи н а се к о м ы х за ви си т о т наличия каки х-л и бо специф ических в е щ е ст в , получивших название акцептан тов , ф а г о ст и м у л я н т о в и т . д . Еще до 1956 г о д а эти представления были очень распростр а н ен ы и защищались н екоторы м и авторам и [13, 14]. Однако, по м ер е того, как были д о ст и г н у т ы успехи в разведении н а с е к о м ы х на чи сто си н тети ч еск и х ср е д а х с т а л о я сн о , что привлекательность пищи зави си т не о т наличия в ней ф а г о ст и м у л я н т о в , а о т соотнош ения и набора осн о вн ы х питательны х в е щ е с т в , к оторы е являются общими для в се х н а с е к о м ы х . О бязательн ы м и компонентами пищевых ср ед для в с е х н а се к о м ы х являю тся а з о т и ст ы е в е щ е ст в а (белки и ам инокислоты ), липиды (жиры и стери н ы ), углеводы ', а такж е набор н еобходи м ы х витаминов и минеральных сол ей .

У сп ех востипания н а се к о м ы х на си н тети чески х ср е д а х зави си т не только о т х и м и ч е с к о г о с о с т а в а с р е д , но и от их конси стен ци и . Для многих скры тож ивущ их н а се к о м ы х , например P e x ic o p ia m a lv e l la или C a r p o c a p s a p o m o n e l la , с р е д а является не только пищевым с у б с т р а т о м , но и средой обитания, в которой гусеницы прокладывают свои ходы , с т р о я т камеру для окукления и т . д . , п о э т о м у она должна обладать со о т в е т с т в у ю щ е й кон си стен ци ей . Для э т о г о в среды д об ав л я ется агар , целлюлоза, а в н е к о то р ы х случаях и альгиновая кисл ота , для связывания влаги . К онсистенция среды в о с о б е н н о с т и должна у ч и т ы в а т ь ся при р а з ведении откры тож ивущ их н а с е к о м ы х . Т а к , например, в наших опы тах о с о б ы е тр удн ости представил подбор пищевых ср ед для гусен иц P orth eti- ia d isp a r и эти трудн ости удалось преодолеть лишь после т о г о , как в с р е ду была добавлен а альгиновая к и сл ота , придавшая ей значительную п л о т н о с т ь .

В к а ч е ст в е примера си н тети чески х с р е д , прим еняемы х в наших р а бо та х для разведения ряда видов L e p id o p te r a , в т а б л . 1 приводится следующий рецепт:

Указанный рецепт п р ед став л я ет ср е д у , обладающую и звестн ой у н и ве р са л ь н о сть ю , так как применяя н ек отор ы е вариации ее с о с т а в а оказа л ось возм ож н ы м воспиты вать на такой среде следующ ие виды н а се к о м ы х : C h lo r id e a o b s o leta , O s tr in ia n ub ila l is , P e x i c o p ia m a lv e l la , L o x o s te g e s t i c t i c a l i s , Iladena s ó r dida и C a r p o c a p s a p o m o n e l la [8].

Ч и с т о -с и н т е т и ч е с к и е ср е д ы , подобны е приведенной выше, конечно, нельзя ра ссм а тр и ва т ь как перспективны е для м а с с о в о г о разведения н а с е к о м ы х в промышленных м а сш та б а х , в сл едстви е их слож н ости и д о р о го в и зн ы . Однако, такие среды служ ат исходны м м о м е н т о м для в ы я сн е ния пищевых требований отд ельн ы х видов и для создания так н азы ваем ы х п ол уси н тети чески х с р е д , в к о т о р ы х определенные индивидуальные х и м и ческие в е щ е ст в а за м ен я ю тся природными продуктами р а ст и т е л ь н о го или ж и вотн ого происхож дения.

ТАБЛИ Ц А 1. С О С Т А В ИСКУССТВЕН Н ОЙ ПИЩЕВОЙ С РЕ Д Ы ДЛЯ Н АСЕКО М Ы Х

T A B L E 1. COM POSITION O F A R T IF IC IA L DIET FOR INSECTS

2 24 Ш У М А К О В

Г руппы в е щ е с т в

G r o u p s o f s u b s t a n c e s

I . А з о т и с т ы е в е щ е с т в а N it r o g e n o u s s u b s t a n c e s

II. Л ипиды L ip id s

III. У г л е в о д ы C a r b o n s

I V . В и та м и н ы V ita m in s

В е щ е с т в аS u b s ta n c e s

К а зеи нC a s e inТ и р о з и нT y r o s in eВалинV a lin eА р ги н и нA r g in in eЛ ейцинL e u c in eЛ изи нL y c ’ ineА сп а р а г и н о в а я к и сл о т аA s p a r -t ic a c idА дани нA d a n in eМ ети он и нM e th iu n in oС ер и иS e r in eФенилаланинP h e n y la la n in eЦ и сти нC y s t in eГ л и ц и нG ly c in e

Р а с т и т е л ь н о е м а с л о V e g e ta b le o i l Х о л е с т е р и н C h o le s t e r in e

С а х а р о з аS a c c h a r o s e

В и та м и н В j BiВ и та м и н ВоВ2В и та м и н Идв .В и та м и н В jo В 12П а н т о т е н о в а я к и с л о т а В Г) (p a n to th e n ic a c id )

К о л и ч е с т в о в е щ е с т в а (г ) в 100 г ср е д ы

(Quantity in g r a m s p e r 100 g n u tr ie n t m e d iu m

8,0

0,015 0 ,015

0,015

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0,015

0,004

0,01

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0,01

0,15

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1,00,1

6,0

0,0012

0,0018

0,0016

0,00006

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S M - 1 0 2 / 2 5 225

Т а б л и ц а 1 (п р о д о л ж е н и е )

Г р у п п ыв е щ е с т в

G r o u p s o f s u b s t a n c e s

П е щ е ст н аS u b s ta n c e s

К о л и ч е с т в о в е щ е с т в а (г ) в 100 г с р е д ы

Q u a n tity in g r a m s p e r 100 g n u tr ie n t m e d iu m

Ф ол и ев а я к и с л о т а B¿¡ ( f o l i c a c id )

0 ,0 0 1

Н и к о т и н о в а я к и с л о т а N ic o t in ic a c id

0 ,0 2 4

А с к о р б и н о в а я к и с л о т а A s c o r b i c a c id

0 ,0 3

V . П р оч и е O th e r s

В и та м и н E (т о к о ф е р о л ) V ita m in E ( t o c o p h e r o l )

0 ,0 3 6

А г а рA g a r

4 ,0

Ц е л л ю л о з а 5 ,0C e l lu l o s eП и вн ы е др ож ж и (а в т о л и з а т ) B r e w e r 's y e a s t (a u t o ly s a te )

8 ,0

С м е с ь м и н е р а л ь н ы х со л е й M ix tu re o f m in e r a l s a l t s

2 ,0

В о д а д о б а в л я е т с я д о 100 r

W a te r a d d e d upto 100 g

Примером у сп е ш н о го создания п р о с т ы х п ол уси н тети чески х ср ед является проведенная в С С С Р р а зр а б о т к а сух ой порош кообразной среды для L o cu s ta m ig r a t o r ia [151. Обычно в лаборатор н ы х у сл ови я х э т о т вид во сп и ты в а е тся на п р о р о ст к а х пшеницы или других злаках, причем з е л е ный корм м о ж е т бы ть частично заменен отр у б я м и или другими ор ган и ческими п родуктам и . В оспитание саранчи L o c u s ta m i g r a t o r ia на с и н т е ти чески х ср е д а х проводилось как в Великобритании [16, 171, так к в С С С Р , и э т о позволило выяснить пищевые требования э т о г о вида.

В р е зу л ь т а т е ряда п о сл ед ова тел ь н ы х упрощений в конечном и тоге была созд а н а очень простая сухая ср е д а , содерж ащ ая в с е г о 7 к ом пон ен т о в : сухой тр остн и к , с у х о е к ор овье м о л о к о , отр у би , сухи е пивные д р о ж жи, с а х а р о зу , аскорбиновую кислоту и холестер и н [8]. На э т о й ср е д е было воспитано н ескол ь ко поколений саранчи, б е з добавления к а к о го - либо зе л е н о го кор м а , и получены нормальные о с о б и с вы сокой пл одови т о с т ь ю .

Сухие п орош кообразн ы е среды о б л а да ю т большими преи м ущ ествам и так как не т р е б у ю т за б о т ы о бо р ь б е с загниванием к ор м а . Нет н е о б х о д и м ости стери ли зовать ср еду или добавлять в нее антисептики для п р е дохранения о т загнивания, что п р ед став л я ет со б о ю одну из наиболее трудны х задач при р а б о т е с и ск у сствен н ы м и ср ед а м и . Однако, сухие среды м о г у т и сп о л ь зо в а т ь ся лишь в ограниченном числе случаев - только для н а сек ом ы х , с п о со б н ы х активно пить поду, подобно саран че .

При р а б о т е с полуси н тети чески м и средам и большие перспективы сулит и спользование природных продуктов о с о б е н н о б о г а т ы х витаминами и ам ин окислотам и.

15


Ряд а в тор ов и ссл едовал пищевую ценность пыльцы для н ек оторы х н а се к о м ы х [ 1 8 - 2 1 ] .

В ы сокая пищевая ценность пыльцы была уста н о вл е н а в о п ы тах с A nthonom us gra nd is [22 ] . Э ти исследования показали, что пищевая ценность и ск у сст ве н н ы х ср ед м ож ет значительно повышена путем д о б а в л е ния с м е с и цветочной пыльцы или зародыш а семян пшеницы. Последний продукт также широко и сп о л ь зу е тся в и сследованиях по и ск у сствен н ы м питательным с р е д а м .

О собы й и нтерес п редставля ет м аточн ое пчелиное мол очко (r o y a l j e l l y ) , к отор ое чрезвычайно б о г а т о некоторы м и жизненно важными для н а се к о м ы х витаминами и ам и н оки слотам и . Недавними исследованиями Р е м б о л д а и е г о сотрудн и ков [23 - 2 5 1 у ста н овл ен о исключительное б о г а т с т в о м а т о ч н о г о м олочка пантотеновой кислотой и фолиевой ки слотой , т . е . витаминами, б е з к оторы х развитие н а се к о м ы х на и ск у сствен н ой пище не м ож ет успеш но п р о те к а ть . В то же время в природе крайне трудно найти другие продукты , которы е были бы столь же б о г а т ы эти м и витаминами. К сож алению , до сих пор м аточн ое мол очко не и с п о л ь з о в а лось при изготовлении и ск у сствен н ы х ср е д .

При использовании в пол уси н тети чески х средах высушенных к о р м о вых растений сл е д уе т иметь в виду, что при сушке растений т е р я е т ся значительная часть витаминов и стор и н ов , жизненно н еоб х оди м ы х для развития н а сек ом ы х , и п о э т о м у они должны в оспол н яться .

В р а б оте Б ор и совой [15] было показан о , что при высушивании т р остн и ка (PhragmiteB communis) для сухи х ср е д , на к оторы х в о сп и т ы валась L o c u ^ t ^ jn lg r a t o r ia п рои сходя т следующ ие изменения в с о д е р ж а нии указанны х в ещ еств :

П о э т о м у для создания полноценной пол уси н тети ческой среды для LocuBta m igratoria в нее ок аза лось н еобходи м ы м добавлять аскорбиновую ки сл оту и хол естери н .

Х отя холестери н является продуктом ж ивотного происхождения и в р а сти тел ьн ы х тканях не в ст р е ч а е т с я , он является ж изненнонеобходимы м к ом пон ен том корма, так как успеш но восполняет п о тр ебн ости н а се к о м ы х в с тер и н ах . П о эт о м у больш инство рецеп тов си н тети чески х и полусинт е т и ч е ск и х ср ед включают холестери н как обязател ьн ую со ст а вн у ю ч а с т ь .

Б олее детальные данные о влиянии отдельны х пищевых ком пон ен тов на развитие н а с е к о м ы х -ф и т о ф а г о в приведены в указанных выше о б з о р а х Э д ельм ан а [5, 6].

Важнейшим в о п р осом при ра боте с и ску сствен н ы м и средам и является предохранение их о т загнивания в течение длительного времени, н е о б х о д и м о г о для развития данного вида н а с е к о м о г о . Э т о д о с т и г а е т с я путем стерилизации посуды до помещения в нее пищевой среды и яиц или личинок н а с е к о м о г о (обы чно с помощ ью автоклавирования), а также д о б а в лением в среду специальных анти септи ков , безв р едн ы х для н а се к о м ы х . Больш ое коли чество различных анти септиков и сп ол ьзов ал ось для п р е дохранения ср ед о т загнивания. Указания о химической природе таких ан ти септи ков , применявшихся в различных стр ан а х , м ож но найти в о б з о ре Х а у са [ 11. В наших р а б о т а х лучшим анти септи ком ок азалась с м е сь м е т и л о в о г о эфира параоксибензойной кислоты с ф о р м ал ьдеги д ом .

Т р у д о е м к о с т ь п р о ц е ссо в воспитания н асек ом ы х в м а с с о в ы х м а сш т а бах оп р е д е л я е тся , в первую оч е р е д ь , затратам и времени на стерилизацию посуды и сад ков и, в о с о б е н н о ст и , на см е н у корма при выращивании н а-

S M - 1 0 2 /25 227

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T A B L E 2. C O N T E N T O F VARIOUS SUBSTANCES (m g) p e r 100 g O F CANE AS P E R C E N T A G E O F D RY WEIGHT

В е щ е с т в а З ол ен ы й т р о с т н и кВ ы су ш ен н ы й

т р о с т н и к

А с к о р б и н о в а я к и с л о т а 1 1 7 ,9 7 5 3 5 ,8 9 3

В и та м и н Е 1 6 ,9 5 14 ,41

С т с р и н ы 0 ,2 3 2 0 ,1 1 4

S u b s ta n c e G r e e n ca n e D r ie d ca n e

A s c o r b i c a c id 1 1 7 .9 7 5 3 5 .8 9 3

V ita m in E 16 .9 5 14 .41

S t e r o l s ( s o l i d a l c o h o ls ) 0 .2 3 2 0.1 14

с е к о м ы х . Упрощение и автом атизация э т и х п р о ц е ссо в является наиболее неотложной задачей в р а зр а б о тк е м е т о д о в м а с с о в о г о разведения н а с е к о м ы х.

В наших и сследованиях д ел ается попытка р а зработа ть такой м е т о д воспитания н а се к о м ы х , который бы устранял н е о б х о д и м о ст ь см ен ы к ор ма в течение в с е г о периода развития полной генерации данного вида.Э т о д о с т и г а е т с я помещ ением пищевой среды в стерилизованную сте к л я н ную или п л а ст м а ссо в у ю посуду , которая после внесения в нее яиц или личинок н а с е к о м о г о , г е р м е т и ч е ск и зк кры в ается на весь период личиночн о го и куколочн ого развития и к он тр оли зуется лишь после вы хода им аго н о в о го поколения. При э т о м у ч и т ы в а е т ся , что коли чество помещенной в п осуду пищевой среды должно не только о б есп ечи ть пищей личинку в т е чение в с е г о периода ее развития , но и сохранить с у б с т р а т , необходимы й для постройки колыбельки для куколки. Опыт показал, что для н орм альн ого развития одной о с о б и т а к о г о р а зм ер а , как C a r p o c a p s a p om on e lla или C h lo r id e a a r m ig e r a д о ст а т о ч н о ге р м е т и ч е ск и за кр ы тое п р о ст р а н с т в о о б ъ е м о м 0 ,030 - 0 , 0 4 0 литра.

Такая м етод и ка м а с с о в о г о воспитания н а се к о м ы х п р е сл е д у е т еще и две другие цели: предотвратить гибель личинок в сл е д ст в и е каннибализма и развития эп и зо о ти й . К онечно, м а с с о в о е воспитание м н оги х о с о б е й в общ ем помещении (садке или стакан е) значительно у п р о ст и л о бы п р о ц е с с воспитания и уменьшило бы затр аты труда при э т о м . Но в э т о м случае предотвратить каннибализм и эпи зооти и в сл е д ст в и е п остоя н н ого контакта о с о б е й почти н е в о зм о ж н о . П о э т о м у в конечном и то ге и зол и рованное воспитание о с о б е й или выращивание их небольшими группами ( 2 —5 о со б е й ) мож ет иметь определенные п реи м ущ еств а . М ожно н аде-


я ть ся , что дальнейшие исследования в э т о м направлении приведут к р а з р аботке таких м е т о д о в индивидуального воспитания личинок, к оторы е бу ду т являться аналогией пчелиных с о т , т . е . той си ст е м ы воспитания, которая созд а н а природой в р е зу л ь т а т е м н о г о в е к о в о г о е с т е с т в е н н о г о о т б о р а .

Еще одним путем упрощения п р оц есса м а с с о в о г о разведения н а с е ком ы х является воспитание их, в т е х случаях, когда э т о в о зм ож н о , на се м е н а х растений, в ч а стн ости на зерне злаков , т . е . на е с т е с т в е н н ы х продуктах , мен ее подверженных загниванию при длительном хранении.В э т о м отношении можно с о сл а т ь с я на опы т Э н т о м о л о г и ч е с к о г о и н сти т у т а Ч ехословац кой Академии Наук, где успеш но р а зво д я т ся в м а с с е на с е м е н а х липы (T i l ia ) клопы P .y r rh o c o r is a p te ru s и на с е м е н а х хлопчатника (G o ss ip iu m ) - D is d c r c u s in te rm e d iu s , а также целый ряд других вредных н а се к о м ы х .

Чехословацкими эн т о м о л о га м и п од готовл ен о к печати р у к о в о д с т в о по м е т о д а м л а бо р а то р н о го воспитания н а сек ом ы х , к отор ое содер ж и т рецепты по культуре 70 .видов н а се к о м ы х [261- Список видов, л а б о р а торная культура к отор ы х о с у щ е ст в л я е т ся в Ч ехословакии , приведен в о т ч е т е Э н т о м о л о г и ч е с к о г о и н сти тута за 1964 - 1965 г г [271.

Аналогичным о б р а зо м в р е зу л ь т а т е мн оголетн и х исследований с о в е т с к и х , иранских и французских э н т о м о л о г о в р азр аботан м е т о д воспитания черепашки (E u ry g a s te r in te g r ice p s ) на зерне пшеницы, который широко прим еняется для размножения паразитов яиц э т о г о вида [281 •

Д ругим важным в о п р о со м м а с с о в о г о разведения н а се к о м ы х я в л я е т ся р а зр а б о т к а путей преодоления диапаузы у л а боратор н ы х популяций, что о б е с п е ч и в а е т н епрер ы вность разведения.

Ч то к а са е т ся видов с факультативной диапаузой , то как показано исследованиями м н оги х а в т о р о в , в о с о б е н н о ст и школой Д ан и л евск ого [29, 301, возникновение диапаузы м о ж е т бы ть предотвращ ено с о д е р ж а нием н а се к о м ы х в у сл ови я х оп ред елен н ого ф отоп ер и ода , т . е . при о п р е деленной длине с в е т о в о г о дня с у ч е т о м влияния тем п ер ату р н ы х у сл ови й .

У спеш ное разрешение э т о г о воп р оса можно проследить на примере плодожорки (C a r p o c a p s a p o m o n e l la ) , для которой пути регулирования диапаузы детально изучены в р е зу л ь т а т е многочи слен н ы х исследований ф отоп ер и оди ч еск ой реакции у э т о г о вида американскими [31 — 3 3 ] , с о в е т скими [29, 34 , 35] и австрийскими [36, 37] и ссл е д о в а т е л я м и .

М ен ее разработан в опр ос устранения диапаузы для видов, имеющих в с в о е м цикле развития облигатную диапаузу , т . е . для н а се к о м ы х с моноволь тинным циклом. При наличии облигатной диапаузы , обы чн о для у ск ор ен и я прохождения э т о г о состоя н и я в реактивации диапаузирующей стади и , д о ст а т о ч н о б ы в а е т выдержать ее в течение о п р ед ел ен н ого в р е мени в у сл ови я х пониженной т е м п е р а т у р ы . Т а к , например, для случая об ли гатн ой эмбриональной диапаузы L o c u s ta m ig r a t o r ia m ig r a t o r д о ст а т о ч н о бы вает выдерж ать яйца э т о й саранчи в течение м е ся ц а при те м п е р а т у р е ниже п о р о га ее развития (16, 7°С ), лучше при 4 - 10°С [38, 3 9 ] . Однако, и зв естн ы нем н оги е виды, как например B o m b ix m o r i , для реактивации диапаузирующих фаз у к оторы х необходи м ы не низкие, а н а о б о р о т вы сокие т е м п е р а т у р ы .

В озм ож ен еще один путь устранения облигатной диапаузы не ж изненного цикла н а се к о м ы х . Э т о т путь практически намечен пока лишь единичными исследованиям и, но те о р е ти ч е ск а я в о зм о ж н о ст ь е г о п о к а

S M - 1 0 2 / 2 5 22 9

зана уже давн о . Он осн ован на изменении г е н е т и ч е ск о й природы вида, путем скрещивания безди апаузн ы х и диапаузирующих рас о д н о го и т о г о же вида. Таки е в о зм о ж н о ст и очевидны лишь для огр ан и ч ен н ого числа видов, имеющ их д о ст а т о ч н о обширные ареалы.

Т е о р е т и ч е ск и е в о зм о ж н о ст и м е т о д а сам ои стр еб л ен и я н а се к о м ы х , о сн о в а н н о го на изменении их ге н е т и ч е с к о й природы, были показаны в р а б о т а х ряда и ссл е д о в а т е л е й [40 - 4 2 ] . В последней р а б о т е [421 были ука зан ы , в ч а ст н о с т и , в о зм о ж н о ст и с а м о и ст р е б л е н и я , осн ован н ы е на введении г е н о в безд и а п а узн о сти в популяции видов, обы чн о развиваю щ ихся в природе с диапаузой ; в р е з у л ь т а т е зимующие ф а зы , теряющие с п о с о б н о ст ь д и ап а узи р ова ть , вы мираю т под в о зд е й ст в и е м низких т е м п е р а тур всл е д стви е их меньшей х о л о д о с т о й к о с т и .

Конкретные в о зм о ж н о ст и т а к о г о пути сам ои стр еб л ен и я н а се к о м ы х п о к а -ч т о выяснены лишь для двух видов O rth o p tcra : с в е р ч к а T e l e o - g ry l lu s c o m m o d u s и саранчи L o c u s ta m i g r a t o r i a .

T e le o g r y l lu s c o m m o d u s широко р аспр остр ан ен по в сей А встр ал и и . Специальными иссл едован и ям и у ст а н о в л е н о , что се в е р н а я , квинслендская р а са э т о г о вида, обитаю щ ая в тр опи ческ и х у сл ови я х , р а зв и в а е т ся б е з д и апаузы . Н апротив, южная р а са , обитаю щ ая в ум е р е н н ы х районах А встр али и , и м е е т эмбриональную ди апаузу . Опыты скрещивания э т и х рас показали, что бе зд и а п а узн о сть является доминантным признаком [43, 4 4 ] . На основании э т о г о Х ога н [45] предложил новый ген ети чески й м е т о д б орьбы с э т и м с в е р ч к о м в ум ерен н ой зоне А встр али и , основанный на подмешивании к природной популяции о с о б е й безд и апа узн ой , т р о п и ч е с кой р а сы . Другими словами он предложил с п о с о б введения новых ген ов в тузем н ую популяцию, как м е т о д снижения численности вредителя, в принципе сходны с м е т о д о м вы пуска стери льн ы х са м ц о в ( "S te r i le m a le te c h n iq u e " ) } тол ь к о не требующий их облучения или о бра ботки х е м о с т е - рилянтами.

Д ругой пример п од об н ого рода дает саранча L o c u s ta m i g r a t o r ia , имеющая очень широкий ареал на в се х континентах С т а р о г о С в е т а . Э т а саранча и м еет целый ряд подвидов и р а с , различающихся б и о л оги ческ и . Обитающая в тропической Африке L o c u s ta m ig r a t o r ia m i g r a l o r i o i d e s , р а зви в ается б е з эмбриональной диапаузы и дает н ескол ько генераций в г о д у . Р аспростран ен н ая в С С С Р и в сопредельн ы х стран ах L o c u s ta m ig r a t o r ia m i g r a t o r i a , напротив, и м еет облигатную диапаузу , б л а г о даря чем у и м е е т лишь одну генерацию в г о д у . На территории Франции о б и та ю т три разн ови дн ости , различающихся ха р а к те р о м эмбриональной паузы . В се э т о о т к р ы в а е т большие в о зм ож н ости для скрещивания рас и для разработки путей сам ои стребл ен и я саранчи, аналогичных т е м , к оторы е предложил Х ога н [451.

П роведенные нами опы ты , а также и сследования Ля Б ер р е [46] во Франции показали, что в се подвиды и расы L. m ig r a t o r ia л е г к о скрещ иваю тся меж ду с о б о й и эти р е зу л ь та ты дают основание н ад ея ться на у с пешную р а зр а б о т к у э т о г о н о в о го м е т о д а борьбы с саранчой в ближайшем будущ ем .

В наших р а б о т а х о сн о в н о е внимание у д е л я е т ся р а зр а б о т к е м е т о д о в сам ои стр ебл ен и я и техники м а с с о в о г о л а бо р а то р н о го разведения таких вредных н а се к о м ы х , как C a r p o c a p s a p o m o n e l la , E u r y g a s te r in te g r i ce p s и L ocu s ta m ig r a t o r ia .

2 30 Ш У М А К О В

Л И Т Е Р А Т У Р АR E F E R K N C E S

[1 ] H O U S E , H. L . , A r t i f i c i a l d ie t s f o r i n s e c t s : a c o m p i la t io n o f r e f e r e n c e s w ith a b s t r a c t s . I n f o r m . B u ll . l i e s . In s t . C a n . D p t. A g r i e . (1 9 6 7 ) 5 .

[2 ] Ш У М А К О В , E . M Э Д Е Л Ь М А Н , H . М . , Б О Р И С О В А , А . Е . , О п ы т в о сп и т а н и я н е к о т о р ы х н а с е к о м ы х -ф и т о ф а г о в на и с к у с с т в е н н ы х с р е д а х . Д А Н С С С Р 130 I (1 9 6 0 ) 2 3 7 . S IIU M A K O V , Е . М . , E D E L M A N , N . M . , B O R IS O V A , А . Е . , E x p e r ie n c e in fe e d in g v a r io u s p la n t -e a t in g i n s e c t s o n a r t i f i c i a l m e d ia , D o k l. A k a d . N au k SSSR 1.30 1 ( I 9 6 0 ) 2 3 7 .

[3 ] U V A R O V , B . P . , I n s e c t n u t r i t io n and m e t a b o l i s m . T r a n s , e n t . S o c . L o n d . 76 (1 9 2 9 ) 2 5 5 .[4 ] H O U S E , II. L . , T h e r o l e o f n u t r it io n a l p r i n c ip l e s in b i o lo g i c a l c o n t r o l . C a n . E n t. 98

(1 9 6 6 ) 1 1 2 1 .[5 ] Э Д Е Л Ь М А Н , H . M . , П и та н и е н а с е к о м ы х на и с к у с с т в е н н ы х с р е д а х . У с п . с о в р .

б и о л о г и и 5J_ 2 (1 9 6 1 ) 204 .E D E L M A N , N . M . , T h e f e e d in g o f in s e c t s o n a r t i f i c i a l m e d ia , U s p . s o v r e m . b i o l . 512 (1 9 6 1 ) 2 0 4 .

[61 Э Д Е Л Ь М А Н , H . M . , О ц ен к а влияния о т д е л ь н ы х к о м п о н е н т о в к о р м а на р а з в и т и ен а с е к о м ы х -ф и т о ф а г о в при в о сп и т а н и и их на и с к у с с т в е н н ы х с р е д а х . З о о л . Ж . 41 7 (1 9 6 2 ) 1 0 1 3 .E D E L M A N , N . M . , A s s e s s i n g th e e f f e c t o f in d iv id u a l f e e d c o m p o n e n t s o n th e d e v e l o p m e n t o f p la n t -e a t in g i n s e c t s f e d on a r t i f i c i a l m e d ia , Z o o l . Z h . £1 7 (1 9 6 2 ) 1 0 1 3 .

[7 ] S M IT H , C . N . , e d i t . . I n s e c t c o lo n iz a t i o n and m a s s p r o d u c t io n , U S A , 1 9 6 6 .[8 ] Ш У М А К О В , E . M . , Э Д Е Л Ь М А Н , H . M . , Б О Р И С О В А , A . E . , Р а з в е д е н и е ф и т о ф а г о в

н а и с к у с с т в е н н ы х с р е д а х . З а щ . Р а с т . 6 (1 9 6 7 ) 2 1 .S H U M A K O V , Е . М . , E D E L M A N , N . M . , B O R IS O V A , А . Е . , R e a r in g p la n t - e a t in g i n s e c t s on a r t i f i c i a l m e d ia , Z a s h c h . R a s t . 6 (1 9 6 7 ) 2 1 .

[91 Ф И Л И П П О В И Ч , Ю . Б . , К в о п р о с у о с в я з и б е л к о в к о р м а с б е л к а м и т е л а и ш елк ад у б о в о г о ш е л к о п р я д а . У ч . з а п . М о е . Г о с . П е д . И н - т а и м . В .И .Л е н и н а 98 (1 9 5 7 ) 7 3 . F IL IP P O V IC H , Y u . В . , T h e c o n n e c t io n b e tw e e n fe e d p r o t e in s and b o d y and s i lk p r o t e in s in th e la p p e t m o th , U ch . z a p . M o s k . O o s . P e d a g o g . I n s t -a irn . V . I . L e n in a 98 (1 9 5 7 ) 7 3 .

[1 0 ] Ф И Л И П П О В И Ч , Ю .Б . , У с в о е н и е , р а сп а д и н о в о о б р а з о в а н и е а м и н о к и с л о т в о р г а н и з м е ш е л к о п р я д а . Б и о х и м и я 24 5 (1 9 5 9 ) 9 0 4 .F I L IP P O V I C H , Y u . В . , U p ta k e , d e c o m p o s i t i o n and s y n t h e s i s o f a m in o a c id s in the s i lk w o r m o r g a n i s m , B io k h im . 24 5 (1 9 5 9 ) 9 0 4 .

[1 1 ] Ф И Л И П П О В И Ч , Ю ..Б . , О ц е н к а п и т а т е л ь н о г о д о с т о и н с т в а к о р м о п ы х р а с т е н и й д у б о в о г о ш ел к оп р я д а при п ом ощ и а м и н о г р а м м , Д А Н С С С Р 131 4 ( 19 6 0 ) 972 .F IL IP P O V IC H , Yu . t í . . U s e o f a m in o g r a m s to a s s e s s the n u tr ie n t v a lu e o f p la n ts fe don b y th e la p p e t m o th , D o k l. A k a d . N auk SSSR 13_1 4 (1 9 6 0 ) 9 7 2 .

[1 2 ] Ф И Л И П П О В И Ч , Ю . Б . , С р а в н и т е л ь н а я о ц е н к а к о р м о в о г о д о с т о и н с т в а для д у б о в о г о ш е л к о п р я д а л и с т ь е в д у б а , и в ы , г р а б а , о р е ш н и к а , б е р е з ы и акации по а м и н о к и с л о т н о м у с о с т а в у и х . В с б . "Б и о х и м и я ш е л к о п р я д а " , 1 9 6 0 , с т р . 1 5 0 .F I L IP P O V I C H , Y u . В . , C o m p a r a t iv e a s s e s s m e n t o f th e fe e d v a lu e d e r i v e d by the la p p e t m o th f r o m l e a v e s o f o a k , w il lo w , h o r n b e a m , n u t - t r e e , b i r c h and a c a c i a , o n th e b a s i s o f t h e ir a m in o a c id c o n te n t , in " B i o c h e m is t r y o f th e S i lk -v v o r m "(1 9 6 0 ) 150 .

[131 D E T H IE R , V . G . , H ost p la n t p e r c e p t io n in p h y to p h a g o u s in s e c t s . 4 ’ r a n s . I X t h Int.C o n g r . E n t. 2 (1 9 5 3 ) 81 .

[141 L I P K E , H . , F H A E N K E L , G . , I n s e c t n u t r i t io n . A n n . R e v . E n t. I (1 9 5 6 ) 17 .[151 Б О Р И С О В А , A . E . , В о с п и т а н и е в о с т о ч н о й и а з и а т с к о й п е р е л е т н о й са р а н ч и на п о л у -

с и н т е т и ч е с к и х с р е д а х . З о о л . Ж . J_4 6 (1 9 6 6 ) 8 5 8 .B O R IS O V A , А . Е , , F e e d in g th e E a s te r n and A s i a t i c m ig r a t o r y l o c u s t on s e m i s y n t h e t ic m e d ia , Z o o l . Z h . J_4 6 (1 9 6 6 ) 8 5 8 .

[1 6 ] D A D D , R . H . , F e e d in g b e h a v io u r an d l o c u s t s . A d v . I n s e c t . P h y s io l . J (1 9 6 3 ) 4 7 .[1 7 ] C A V A N A G H , G . G . , T h e u s e o f the D add s y n t h e t ic d ie t a s a f o o d f o r a d u lts

S c h i s t o c e r c a g r e g a r i a ( F o r s k . ) a n d th e e f f e c t o f s o m e a d d it io n s and m o d i f i c a t io n s t o i t . J . I n s e c t P h y s io l . 9 (1 9 6 3 ) 759 .

[1 8 ] H A Y D A K , M . H . , T A N Q U A R Y , M . C . , P o l l e n and p o l le n s u b s t i tu te s in th e n u tr it io no f th e h o n e y b e e . St. P a u l, 1 9 4 3 .

[191 U A Y D A K , M . H . , V a lu e o f p o l l e n s u b s t i tu te s Гог b r o o d r e a r in g o f h o n e v b e e . J . e c o n .E n t . 3£ 4 (1 9 4 5 ) 4 8 4 .

[2 0 ] И О Й Р И Ш , Н . П . , "П ч е л ы - к р ы л а т ы е ф а р м а ц е в т ы " , 1 9 6 4 .IO IR IS H , N . P . , " H o n e y -b e e s - w in g e d p h a r m a c i s t s " (1 9 6 4 ) .

[2 1 ] L E IU S , K . , E f f e c t s o f p o l le n s on fe c u n d ity and l o n g e v i t y o f a d u lt S c a m b u s b u o l ia n a e ( H t g . ) (H y m e n o p te r a : I c h n e u m o n id a e ) . C a n . E n t. 95 2 (1 9 6 3 ) 2 0 2 .

S M - 1 0 2 / 2 5 231

[221 V A N D E R Z A N T , E . S . , R IC H A R D S O N , C . D . , D A V IC H , T . B . , F e e d in g an d o v ip o s i t i o n b y th e b o l l w e e v i l on a r t i f i c i a l d i e t s . J . e c o n . E n t. 52 (1 9 5 9 ) 1 1 3 8 .

[231 R E M B O L D , H . , Ü b e r d e n W e i s e l z e l l e n f u t t e r s a f t d e r H o n ig b ie n e . V e r h . X I In t. K o n g r . E n to m . 3 (1 9 6 0 ) 77 .

[2 4 ] R E M B O L D , H . , D ie E n ts te h u n g d e r B ie n e n k o n ig in . U m s c h a u 61_ 16 , 17 (1 9 6 1 ) 4 8 8 , 5 2 4 .[2 5 ] R E M B O L D , H . , B i o l o g i c a l l y a c t iv e s u b s t a n c e s in r o y a l j e l l y . V i ta m in s H o r m o n e s 23

(1 9 6 5 ) 359 .[2 6 ] S K U H R A V Y , V . , e d i t . , M e to d y c h o v u h ra y z u , P r a h a ( in p r e s s ) .[2 7 ] R e p o r t on th e a c t iv i t i e s o f th e e n t o m o lo g i c a l in s t itu te o f th e C z e c h o s l o v a k A c a d e m y

o f S c i e n c e s f o r 1 9 6 4 -1 9 6 5 (1 9 6 6 ) .[2 8 ] R E M A U D IE R E , G . , P la n f o r th e in s t a l la t io n an d o p e r a t io n o f a u n it f o r th e p r o d u c t io n

o f e g g s o f E u r y g a s t e r . P a r i s (1 9 6 0 ) Sunn p e s t c i r c . 5 .[2 9 ] Д А Н И Л Е В С К И М , А . С . , Ф о т о п е р и о д и з м и с е з о н н о е р а з в и т и е н а с е к о м ы х . Л , И з .

Л Г У (1 9 6 1 ) .D A N IL E V S K Y , A . S . , P h o t o p e r i o d i s m and s e a s o n a l d e v e lo p m e n t o f i n s e c t s (1 9 6 1 ) .

[3 0 ] D A N IL E V S K II , A . S . , P h o t o p e r i o d i s m a n d s e a s o n a l d e v e lo p m e n t o f i n s e c t s (1 9 6 5 ) .[3 1 ] D IC K S O N , R . S . e t a l . C o n tin u o u s r e a r in g o f th e C o d lin g M o th . J . e c o n . E n t. 45

(1 9 5 2 ) G6.[3 2 ] P R O V E R B S , M . D . , N E W T O N , J . R . , E f f e c t o f h e a t o n th e f e r t i l i t y o f th e C o d lin g

m o th , C a r p o c a p s a p o m o n e l la ( L ) (L e p id o p t e r a : O le t h r e u t id a e ) . C a n . E n t. 94 3 (1 9 6 2 )2 2 5 .

[3 3 ] P U T M A N , W . L . , T h e c o d l in g m o th , C a r p o c a p s a p o m o n e l la ( L . ) ( L e p i d o p t e r a : T o r t r i c i d a e ) : a r e v i e w w ith s p e c i a l r e f e r e n c e t o O n t a r io . P r o c . e n t . S o c . O n ta r io 93 (1 9 6 2 ) 2 2 .

[3 4 ] Ш Е Л Ь Д Е Ш О В А , Г . Г . , З н а ч ен и е длины дня в р е г у л я ц и и ч и сл а п о к о л е н и й и д и а п а у з ы я б л он н ой п л о д о ж о р к и L a s p e y r e s i a p o m o n e l la L . Д А Н С С С Р 147 (1 9 6 2 ) 4 8 0 . S H E L D E S H O V A , G . G . , T h e p a r t p la y e d b y le n g th o f d a y l ig h t in r e g u la t in g th e n u m b e r o f g e n e r a t io n s and th e d ia p a u s e o f th e l e s s e r a p p le w o r m L a s p e y r e s i a p o m o n e l la L . , D o k l. A k a d . N a u k SSSR 147 (1 9 6 2 ) 4 8 0 .

[3 5 ] Ш Е Л Ь Д Е Ш О В А , Г . Г . , Г е о г р а ф и ч е с к а я и з м е н ч и в о с т ь ф о т о п е р и о д и ч е с к о й р еа к ц и и и с е з о н н о г о р а з в и т и я я б л о н н о й п л о д о ж о р к и 'L a s p e y r e s i a p o m o n e l la L . (L e p id o p t e r a , T o r t r i c i d a e ) T p . З оо 'л . и н - т а , Л 3 6 (1 9 6 5 ) 5 .S H E L D E S H O V A , G .G . , G e o g r a p h ic v a r i a b i l i t y o f th e p h o t o p e r i o d i c r e a c t io n and s e a s o n a l d e v e lo p m e n t o f th e l e s s e r a p p le w o r m L a s p e y r e s i a p o m o n e l la L . (L e p id o p t e r a , T o r t r i c i d a e ) , T r . z o o l . I n s t -a 36 (1 9 6 5 ) 5 .

[3 6 ] R U S S , K . , D e r E in f lu s s d e r P h o t o p c r i o d i z i t a t a u f d ie B io l o g ie d e s A p fe lw i c k l e r s ( C a r p o c a p s a p o m o n e l la L . ) . P f l . s c h u t z - B e r . , S o n d e r h e ft (1 9 6 6 ) 2 7 .

[3 7 ] R U S S , K . , N cu e E r k e n n t n is s e ü b e r d ie B i o l o g ie u n d d ie B e k à m p fu n g d e s A p fe lw i c k l e r s . ( C a r p o c a p s a p o m o n e l la L . ) , D e r E r w e r b s o b s t b a u 9 (1 9 6 7 ) 2 1 .

[3 8 ] Я Х И М О В И Ч , Л . А . , С м е н а т р е б о в а н и й к у с л о в и я м с р е д ы в п р о ц е с с е э м б р и о н а л ь н о г о р а з в и т и я а з и а т с к о й с а р а н ч и . Д А Н С С С Р 73 5 (1 9 5 0 ) 1 1 0 5 .Y A K H IM O V IC H , L . A . , A d a p ta t io n t o e n v ir o n m e n t a l c o n d i t io n s in th e e m b r y o n i c d e v e lo p m e n t o f th e A s i a t i c l o c u s t , D o k l. A k a d . N au k SSSR 73 5 (1 9 5 0 ) 1 1 0 5 .

[3 9 ] Я Х И М О В И Ч , Л . А . , Р а з в и т и е яиц и с о з р е в а н и е а з и а т с к о й са р а н ч и в с в я з и с у с л о ви я м и с у щ е с т в о в а н и я . Д и с с е р т а ц и я , Л е н и н гр а д , В И З А Р , 1 9 5 2 .Y A K H IM O V IC H , L . A . , E g g d e v e lo p m e n t and m a t u r a t io n o f th e A s ia t i c l o c u s t a sa fu n c t io n o f e n v ir o n m e n t a l c o n d i t io n s , D o c t o r a l t h e s i s , L e n in g r a d , V I Z A R (1 9 5 2 ) .

[4 0 ] С Е Р Е Б Р О В С К И Й , A . C . , О н о в о м в о з м о ж н о м м е т о д е б о р ь б ы с в р е д н ы м и н а с е к о м ы м и . З о о л . Ж . JJ) 4 (1 9 4 0 ) 6 1 8 .S E R E B R O V S K Y , A . S . , A p o s s i b l e n ew m e th o d o f c o m b a t t in g i n s e c t p e s t s , Z o o l .Z h . Hi 4 (1 9 4 0 ) 6 1 8 .

[4 1 ] K N IP L IN G , E . F . , U s e o f i n s e c t s f o r t h e i r ow n d e s t r u c t io n . J . e c o n . E n t. 5!3 3 (1 9 6 0 ) 4 1 5 .

[4 2 ] L A C H A N C E , L . E . , K N IP L IN G , E . F . C o n tr o l o f i n s e c t p o p u la t io n s th r o u g h g e n e t i c m a n ip u la t io n s . A n n . e n t . S o c . 55 5 (1 9 6 2 ) 5 1 5 .

[4 3 ] B IG E L O W , R . S . , F a c t o r s a f fe c t in g d e v e lo p m e n t a l r a t e s and d ia p a u s e in f ie ld c r i c k e t s . E v o lu t io n 1J> (1 9 6 2 ) 3 9 6 .

[441 H O G A N , T . W . , S o m e d ia p a u s e c h a r a c t e r i s t i c s and i n t e r f e r t i l i t y o f t h r e e g e o g r a p h ic p o p u la t io n s o f T e l e o g r y l l u s c o m m o d u s ( W a lk . ) . A u s t r a l . J . Z o o l . 1_3 3 ^ 1 9 6 5 ) 4 5 5 .

[4 5 ] H O G A N , T . W . , P h y s io l o g i c a l d i f f e r e n c e s b e tw e e n r a c e s o f T e l e o g r y l lu s c o m m o d u s (W a lk e r ) (O r t h o p t e r a : G r y l l id a e ) r e la t e d to a p r o p o s e d g e n e t i c a p p r o a c h to c o n t r o l . A u s t r a l . J . Z o o l . 14 2 (1 9 6 6 ) 2 4 5 .

[461 L E B E R R E , J . R . , C a r a c t è r e s b i o lo g iq u e s d e s L o c u s t a d e la F a u n e d e F r a n c e et é tu d e d 'u n e x e m p le d e d ia p a u s e e m b r y o n n a i r e . A n n a l , é p ip h . H) 2 (1 9 5 9 ) 1 0 1 .


D Т S С U S S I О N

R a ch e l G ALU N : You say that p hagost im ulants a r e l e s s im portant than ap p ropr ia te s e le c t io n and p ro p o rt io n s o f the b a s i c in g re d ie n ts in the nutrient m e d iu m . H ou se , h o w e v e r , e m p h a s ize d in the c o u r s e o f an A g e n cy panel in 1 966* the im p o rta n ce o f phagost im ulants fo r m on op h agous in s e c t s . I think the r e a so n f o r this d i s c r e p a n c y is that the in s e c t s you m en tion have nutrit ional com p o n e n ts as st im ulants — f o r ex a m p le , a s c o r b i c a c id f o r lo c u s ts , c h o le s t e r o l f o r the s i lk w o rm , and g lu c o s e and am ino a c id s f o r the c o r n b o r e r .

I should a lso like to point out that fu rth er e c o n o m y o f m a s s r e a r in g can be obta ined by u sing the m ed iu m on which the in s e c ts w e re r e a r e d to fe e d p igs o r g e e s e , as m uch o f its nutrit ive value is reta ined .

ID. M. SIIUMAKOV: Thank you f o r yo u r c o m m e n ts . T he su g ge s t io n o f r e - u s in g the nutrient m e d iu m is an in te re s t in g one . As the paper points out, the ai’t i f i c ia l m e d iu m is used not on ly as food , but a lso as an en v iro n m e n t f o r pupation, e t c . H en ce a c o n s id e r a b le part o f it is , in fact , not r e a l ly used , and its nutritive, qu a l it ies cou ld indeed be put to fu r th e r u se .

Isotopes and Radiation in Parasitology (Panel Proceedings), IA L A , Vienna (196.S) in the press.

TH E O R E TIC A L AND P R A C TIC A L STUDIES ON A POSSIBLE GENETIC METHOD FOR TSETSE F L Y CONTROL

C. F. CURTISTSETSE RESEARCH LABORATORY,SCHOOL OF VETERINARY SCIENCE,UNIVERSITY OF BRISTOL, LANGFORD, BRISTOL ANDW. G. HILLINSTITUTE OF ANIMAL GENETICS,EDINBURGH, UNITED KINGDOM

Abstract

THEORETICAL AND PRACTICAL STUDIES ON A POSSIBLE GENETIC METHOD FOR TSETSE FLY CONTROL. C hrom osom e translocations m ay b e useful in pest control because they are a com m on type o f m utation in a variety o f organisms and, frequently , the h eterozygote is sem i-s te r ile and the h om o- z ygote fo lly fertile . It m ight b e possible to induce such a translocation in a pest species, to breed from a se lected ancestral pair o f translocation h om ozygotes a large number o f the hom ozygotes and to release these into a wild popu lation . This would cause the production o f heterozygotes in the wild population and hence would reduce the fertility o f the popu lation . This reduction would persist for a num ber o f generations. C a lculations, based on sim p lified assumptions, show ed that this m ethod o f fertility reduction m ight be m ore e co n o m ica l than the use o f sterilized m ales.

In the present paper a th eoretica l com parison is m ade o f the translocation and s te r iliz ed -m a le m ethods for the control o f tsetse flies ( Glossina s p .) . A com puter m od el has been set up which sim ulates, as far as possible, the known facts about birth, m ating and death in a wild tsetse popu lation . T h e predicted effects o f releases o f sterilized m ales and o f translocation h om ozygotes are described and the m odification s which would be caused by density-dependent m ortality, m igration and reduced v iab ility o f the translocation genotypes and sterilized m ales are indica ted . It is con clu ded that to erad icate a w ell isolated wild population the numbers o f translocation hom ozygotes required m ight w ell be considerably less than the number o f sterilized m ales required for the sam e task. H ow ever, im m igration into the populat ion would greatly reduce the e ffic ie n cy o f the translocation m ethod.

The progress so far in attem pting to produce a suitable translocation in Glossina austeni is described. M ales have been treated with 5 -7 krad o f gam m a radiation and a number o f sem i-s te r ile individuals have been se lected from am ong their progeny. The sem i-sterility is inherited and, by analogy with the results in other organisms, is presumed to be due to translocation heterozygosity . T h e m ethods being used to attempt to derive translocation h om ozygotes from the sem i-ster ile lines are described.

L. IN TR OD U C TION

C h r o m o s o m e t r a n s lo c a t io n s can r e a d i ly be induced by rad iation in a wide v a r ie ty o f o r g a n is m s . T r a n s io c a t io n h e te r o z y g o te s a r e frequ ently found to be s e m i - s t e r i l e , w h ereas t r a n s lo ca t io n h o m o z y g o t e s , p ro v id e d that they a re v iab le , a r c g e n e r a l ly found to be fu lly f e r t i l e . The c h r o m o s o m a l b a s is o f these p r o p e r t ie s is w e ll known [ l j . If a su itab le t r a n s lo ca t io n w e re induced in a pest s p e c ie s and if a m a le and a fe m a le h o m o zy g o u s f o r the t r a n s lo ca t io n w e re obtained, a la r g e n u m b e r o f the h o m o z y g o te s cou ld , in p r in c ip le , be d e r iv e d f r o m th is pair. R e le a se o f h o m o z y g o t e s o f both s e x e s into a wild population would lead to m atings

233

234 CURTIS and HILL

betw een t r a n s lo ca t io n h o m o z y g o te s ( T / T ) and wild type h o m o z y g o te s (+ /+ ) which would p ro d u ce t r a n s lo ca t io n h e te r o z y g o te s ( T /+ ) and hence a redu ct ion in the fe r t i l i ty o f the wild population in the next g en era t ion .A s lon g as the t r a n s lo ca t io n and n o rm a l c h r o m o s o m e s p e r s is t e d in the population , h e te r o z y g o te s would continue to be p ro d u ce d in subsequent g e n e ra t io n s and the fe r t i l i ty o f the population would be d e p r e s s e d . Natural s e l e c t io n acts in a p e c u l ia r way in a population contain ing t r a n s lo ca t io n and n o r m a l c h r o m o s o m e s [2, 3] . If the Т / + and T / T indiv iduals a re a s su m e d to have n o rm a l v iab i l i ty and m ating c o m p e t i t iv e n e s s , w h ich e v e r o f the two c h r o m o s o m e types is in the m a jo r i t y when the r e l e a s e p r o g r a m m e is f in ished w ill g ra d u a lly in c r e a s e in f r e q u e n c y until it r e a c h e s f ixat ion . T h e r e is a t h e o r e t i c a l point at exact equality o f the fr e q u e n c ie s o f the two c h r o m o s o m e ty p e s when they would both p e r s i s t in de fin ite ly in the population , but th is is an unstable e q u i l ib r iu m . At this f r e q u e n c y the re d u c t io n in. population fe r t i l i ty is m a x im a l .

C a lcu la t ion s w e re p resen ted in a p r e v io u s p a p e r [3] o f the e f fe c t s on population s i z e o f r e le a s in g v a r io u s n u m b e rs o f t r a n s lo ca t io n h o m o zy g o te s into a g iven population with the ab ove s im p le assu m p t io n s about v ia b i l i ty and m ating c o m p e t i t iv e n e ss as w e l l as the fo l lo w in g o n e s : (a) the g e n e ra t io n s in the tre a te d population do not o v e r la p , (b) th e re is a 1 : 1 s e x ratio , ( c ) no density-dependent fa c t o r s o p e r a te , (d) the trea ted p opu la tion is i s o la te d f r o m m ig ra t io n , and (e) a l l adult f e m a le s a r e in sem in ated . The r e su lt s w e re c o m p a re d with the ca lcu la ted e f f e c t s o f r e le a s in g s t e r i le m a le s into the population and it was conc lu ded that on th ese assu m ption s c o n s id e r a b ly fe w e r in se c ts would have to be r e a r e d to obtain a g iven e f f e c t with the tra n s lo ca t io n m ethod than with the s t e r i l i z e d m a le m eth od . T h is advantage a r i s e s p a rt ly f r o m the long continued e f fe c t o f the t r a n s lo c a t io n s and part ly from the fa c t that the r e le a s e o f fe m a le t ra n s lo ca t io n h o m o z y g o t e s w ou ld c o n t r ib u t e u s e f u l l y t o p o p u la t io n r é d u c t i o n , w h e r e a sthe r e le a s e o f s t e r i l iz e d f e m a le s would not.

We w e re in terested in the p o s s ib le ap p licat ion o f the tr a n s lo ca t io n m ethod to ts e ts e f ly c o n tro l , and it s e e m e d im portan t to repeat th ese th e o r e t i c a l c o m p a r is o n s with m o r e r e a l i s t i c assu m pt ion s about t s e t s e popu la tion s . A th e o r e t ic a l m o d e l o f a t s e ts e population was previously- u sed to p r c d i c t the e f fe c t o f the s t e r i l i z e d m a le m ethod on t s e ts e f l i e s [4].In the p re se n t study we a lso co n s tru c te d a population m o d e l and the r e le a s e o f e ith e r s t e r i l i z e d m a le s o r t r a n s lo ca t io n h o m o z y g o te s could be s im u lated with a c o m p u te r . The e f fe c t s o f r e m o v in g s o m e o f the o th e r assu m p tion s l is ted above have a lso been tes ted .

In the last s e c t io n o f the paper, attem pts to p ro d u ce a su itable t r a n s lo ca t io n in the ts e ts e s p e c ie s G lo ss in a austeni are d e s c r ib e d .

2. TH E M O D E L

We assu m e d that the s i z e o f the population to be trea ted was v e r y la r g e so that a d e te rm in is t i c m o d e l cou ld be u se d .

A s fa r as p o s s ib le the m o d e l was based on data about t s e t s e f l i e s g iven in the standard t e x t -b o o k on the genus [5 ] , but, w here the facts w e re not de f in ite ly known, g u e s s e s have had to be m a d e .

A d ia gram o f the population m o d e l b e fo r e any r e l e a s e s a re m ade is show n in F ig . 1. Without treatm en t we assu m e d that the population s i z e

S M - 102/26 235

stayed constant so the d ia gram r e p r e s e n t s both the h i s t o r y o f a c o h o r t born at one t im e o r the c o m p o s i t io n o f the w hole population at any t im e . Jn F i g . l and throughout the r e s t o f the paper , n um bers o f ind iv iduals a r e e x p r e s s e d in units defined re la t iv e to the tota l in itia l f e m a le popu la tion which c o n s is t s o f 100 units. The continuous p r o c e s s o f b irth and death in the population was a p p rox im a ted by a d i s c r e t e m o d e l with a

FIG. 1. M odel o f a w ild tsetse popu lation .

t im e in terva l o f 10 days , which we ca l l the t im e unit . T h is p e r io d was ch o s e n b e c a u s e it is the a v e r a g e t im e betw een s u c c e s s iv e ovu la tion s in an adult fe m a le , and in the untreated population su rv iv in g f e m a le s o f re p ro d u c t iv e age a re a s su m e d to p ro d u ce one pupa p e r t im e unit and the s e x ratio o f th e se is a s su m e d to be 1 : 1 . The pupal s u r v iv a l c o e f f i c ie n t ,i . e . the p r o p o rt io n o f pupae su rv iv in g at age n t im e units which su rv iv e to n -rl , was a s su m e d constant and w as c h o s e n to g ive an o v e r a l l su rv iv a l o f I f r o m the t im e o f d ep os it ion up to the t im e o f e c l o s io n . Up to age 19 t im e units the adult su rv iv a l c o e f f i c i e n t , i . e . the p r o p o r t io n o f adults o f age n t im é units which su rv iv e to age n+1, was a s s u m e d to be a constant f o r each se x . T he few s u r v iv o r s at age 19 t im e units w e r e assu m e d to d ie o f o ld age . The m a le adult su rv iv a l c o e f f i c i e n t was c h o s e n to c o n fo r m with data on the la te r age g ro u p s o f wild m a le G_. m o r s i ta n s [6] . ■

236 CURTIS and HILL

B e c a ù s e the s i z e o f the untreated population was re qu ired to stay constant it w as n e c e s s a r y to c h o o s e the p r e c i s e va lue f o r the fe m a le adult su rv iv a l c o e f f i c i e n t which s a t is f ie s th is cond it ion . The adult se x ratio gen era ted by the m o d e l is 1 m a le : 1.85 fe m a le s , which c o n fo r m s with the g e n e r a l op in ion that th e re is a c o n s id e r a b le e x c e s s o f f e m a le s in a t s e ts e pop u la t ion . F e m a le s w e re a ssu m ed to be in sem in ated , at the age indicated in F ig . 1, by a random sa m p le o f the su rv iv in g m a tu re m a le s and the s p e r m r e c e iv e d was assu m e d to be used f o r the r e s t o f th e ir r e p r o duct ive l i f e .

T h e s t e r i l i z e d m a le s and tr a n s lo ca t io n h o m o z y g o te s w e re a s su m e d to be r e le a s e d when aged 4 t im e units, and the r e le a s e s w e re a ssu m ed to be d is tr ibuted evenly throughout the trea ted population .

The t r a n s lo ca t io n k aryotyp es , s t e r i l i z e d m a le s and wild typ es w e re a s su m e d to m ate with each o th e r at random and the s t e r i l i z e d sp e rm and the v a r io u s types o f gam ete p rodu ced by the tr a n s lo ca t io n k a ryotyp es w e re a s su m e d to have n o rm a l fe r t i l i z in g ca p a c ity . The la tter assu m ption c o n fo r m e d with o b s e rv a t io n s on r a d ia t io n -s t e r i l i z e d ts e ts e s p e r m [7] and tr a n s lo ca t io n s in D ro so p h i la [8] . The assu m e d o u t c o m e s o f the m atin gs betw een the tr a n s lo ca t io n and n o rm a l k aryotyp es are show n in T a b le I.

W e have assu m e d that the death ol' a zy gote in a fem a le , as a re su lt o f the action o f a t r a n s lo ca t io n o r s t e r i l i z e d sp e rm , does not a f fe c t the t im e o f h e r next ovulation o r pro lon g h e r l i f e . The f o r m e r e f fe c t has been detected [7, 9] but was re la t iv e ly sm a l l and the la tter e f fe c t was not detected in e x p e r im e n ts on young f l i e s [7 J .

T he in flu ence that th ree fa c to r s would have on the e f fe c t iv e n e s s o f the c o n t r o l methods was investigated on the fo l low in g s im p le assu m p tion s about th e ir m o d e s o f op e ra t io n :

T A B L E I. T H E A S S U M E D O U T C O M E O F M A T IN G S B E T W E E N T H E T R A N S L O C A T I O N K A R Y O T Y P E S A N D T H E W IL D T Y P E

M atin g I’rogen y

+./+ T /+ T /T In v ia b le

+ /+ X +/+ 1 0 0 0

T /+ X + /+ i I3 0 i

T /T X +Л 0 1 0 0

T /+ X T /+ 1 /16 3 /1 6 1/16 11 /16

T /+ X T /T 0 4 1.

T /T X T /T 0 0 1 0

S M - 102/26 237

(a) V ia b i l i t y . The adult s u r v iv a l c o e f f i c i e n t s f o r s t e r i l i z e d m a le s o r f o r T / + o r T / T indiv iduals cou ld be m u lt ip lied by a c h o s e n fa c t o r which we c a l l the re la t ive v ia b i l i ty c o e f f i c i e n t .

(b) D ensity dependent f a c t o r s . The m a le and fe m a le adult su r v iv a l c o e f f i c i e n t s at t im e t w e re m u lt ip lied by:

l + d ( l - N , / N 0)

w here N, is the population s i z e at t im e t, Nu is the s i z e o f the untreated population , and d is ca lled the 'd e n s ity d epen d en ce c o e f f i c i e n t ' and maybe sp e c i f i e d at w il l .

When d > 0 the population tends to re tu rn to N0 a fter any te m p o r a r y d is tu rb a n ce .

(c ) M igration-.1 The ten d en cy to m ig r a te was a s su m e d to be independent o f adult age . We defined the 'em ig ra n t p r o p o r t io n ' as the p r o p o r t io n o f adults o f age n t im e units which e m ig r a te d f r o m the popula tion b e fo r e age n + 1 . T h is quantity w as se p a r a te ly s p e c i f ia b le fo r each s e x . In the untreated population the im m ig ra t io n rate was made to ba lance the c h o s e n e m ig ra t io n ra te . A s the population was red uced , the absolute n u m ber o f em ig ra n ts fo i l , but the n u m ber o f im m ig ra n ts , s in c e they w e re assu m e d to c o m e f r o m su rrou nd ing untreated populations, re m a in e d constant. M igrat ion t h e r e fo r e acted as a population s tab i l iz in g in f lu en ce . E m ig ra n ts w e re a ssu m ed to be a random s a m p le o f the adult f l i e s in the trea ted population , but im m ig ra n ts w e re a s su m e d to be fe r t i le wild ty p es on ly and, b e c a u s e f e m a le s are known to be insem inated e a r ly in adult l i fe , f e m a le im m ig ra n ts w e r e all a ssu m e d to be i n s e m i nated with f e r t i le wild type s p e r m .

3. RESULTS O F TH E CO M PU TIN G

A s a ch e ck on the c o r r e c t p e r fo r m a n c e o f the c o m p u te r m od e l , r e l e a s e s o f equ ivalent n u m b e rs o f s t e r i l i z e d m a le s to th o se u sed by S im p son [4] w e r e tr ie d and s i m i l a r r e s u lt s w e re obta ined .

The r e le a s e o f s t e r i l i z e d m a le s would ca u se a r e d u ct ion in fe r t i l i ty o f the population until all f e m a le s in sem in ated with s t e r i l i z e d s p e rm w e re dead, but with the tr a n s lo ca t io n m ethod the fe r t i l i ty r e d u ct ion would continue into la te r g e n era t ion s until the population had b e c o m e h o m o zy go u s e ith e r f o r the n o rm a l c h r o m o s o m e s o r the t r a n s lo c a t io n c h r o m o s o m e s . In s o m e c a s e s it was found that th is p r o c e s s would take s e v e r a l y e a r s a fter the la st r e le a s e o f t r a n s lo c a t io n h o m o z y g o t e s .

The s im u la te d r e le a s e o f a g iven tota l o f s t e r i l i z e d m a le s o r t r a n s lo c a t io n h o m o z y g o t e s was found to have a g r e a te r e f fe c t the m o r e frequent the r e le a s e s and the l o n g e r the to ta l t im e o v e r which r e l e a s e s w e re m a d e . It was d e c id e d in all the s u c ce e d in g c o m p u te r t r ia l s to u se 10 equal r e le a s e s m a d e at in te r v a ls o f f o u r t im e units, which r e p re s e n te d a r e le a s e p r o g r a m m e extending o v e r about one y e a r .

R e le a s e s o f a p p ro x im a te ly equal n u m b e rs o f T / T m a le s and f e m a le s w e re found to g ive a lm o s t the s a m e lo n g -t e rm re su lt as r e le a s e o f the sa m e tota l n u m b e r o f m a le s on ly . It has been show n [10] that the output

1 ’ M igration ' is used in the sense o f diffusion and not orderly m ovem ent.

2 38 CURTIS and HILL

o f p ro ge n y f o r r e le a s e f r o m a capt ive c o lo n y o f t s e t s e f l i e s cou ld c o n s is t o f young adult m a le s and f e m a le s in the ra tio 0 .5 4 : 0 .4 6 without depleting the b r e e d in g s tock , and all r e l e a s e s o f t r a n s lo ca t io n h o m o z y g o t e s w e re a s su m e d to be m a d e in th is ra t io .

T he e f fo r t in vo lved in c o l le c t in g enough t s e t s e pupae f r o m the wild f o r use in a s t e r i l i z e d m a le r e le a s e p r o g r a m m e is v e r y g rea t [1 1 ] , and it s e e m s that with m o d e r n r e a r in g m ethod s [10, 12] it would be m o r e e c o n o m ic a l to r e a r the n e c e s s a r y m a le s in a cap t ive co lo n y . When c o m parin g the t r a n s lo ca t io n and s t e r i l i z e d m a le m e th o d s , c o m p a r is o n should be m a d e betw een the n u m b e rs o f m a le s o f each type that a r e n eeded f o r r e le a s e , s in c e th is in d ica tes the r e s p e c t iv e s i z e s o f the cap t ive c o lo n i e s that would have to be m ainta ined and th e r e f o r e g iv e s a rough in d icat ion o f the re la t iv e c o s t o f the two m eth od s .

N U M B E R OF S T E R I L I S E D M A L E S R E L E A S E D

■FIG. 2 . T ota l numbers o f units o f sterilized m ales or translocation hom ozygotes released plotted against the u ltim ate s ize o f the fem ale popu lation .

R e le a s e s o f v a r io u s n u m b e rs o f s t e r i l i z e d m a le s and t r a n s lo ca t io n h o m o z y g o te s w ere s im u lated , n o rm a l v ia b i l i t ie s and no d en s ity depen d en ce o r m ig r a t io n being a ssu m e d ; the total n u m b e rs r e le a s e d a re plotted in F ig . 2 against the fe m a le population s i z e when it s ta b i l iz e d , which we

S M - 102/26 239

c a l l the u lt im ate n u m b e r o f f e m a le s . The u ltim ate n u m b e r o f f e m a le s d e c l in e d s te a d i ly with in c r e a s e in the n u m b e r o f s t e r i l i z e d m a le s r e le a s e d . With the tr a n s lo ca t io n m ethod th ere is a v e r y sh arp op t im u m n u m ber to be r e le a s e d b e c a u s e o f the p e c u l ia r r e s p o n s e o f t r a n s lo c a t io n s to natural s e l e c t io n which has a lre a d y been d e s c r ib e d . With the a s su m p t io n s used , u n le s s the opt im u m is e x c e e d e d the t r a n s lo c a t io n m ethod sh ow s a g re a t advantage in e f f i c i e n c y o v e r the s t e r i l i z a t io n m e th o d . The s u p e r io r i ty o f the t r a n s lo c a t io n m ethod is s e e n to depend c r i t i c a l l y on r e le a s in g the c o r r e c t n u m b e r o f h o m o z y g o t e s . It would be v e r y la b o r io u s to e s t im a te the s i z e o f the popu lation to be tr e a te d with su f f ic ie n t a c c u r a c y so that the n u m b e rs o f f l i e s to be r e le a s e d cou ld be s p e c i f i e d e x a c t ly in ad vance . H o w e v e r , i f su itab le te ch n iq u e s w e re d e v e lo p e d it m igh t b e p o s s ib le to es t im a te c y t o lo g i c a l ly the f r e q u e n c y o f the tr a n s lo ca t io n c h r o m o s o m e in the w ild population as the r e l e a s e p r o g r a m m e p r o c e e d e d and to adjust the n u m b e rs to be r e le a s e d so as to a p p roa ch the op t im u m fr e q u e n c y o f 0 .5 .

RELATIVE VIABILITY OF STERILISED MALES

RELATIVE VIABILITY OF TRANSLOCATIONHETEROZYGOTE AND HOMOZYGOTE

FTG. 3. T h e numbers o f (a) sterilized m ales or (b) translocation h om ozygotes required to be released to ach ieve the target popu lation reduction plotted against values o f the re la tive v ia b ility c o e ff ic ie n t for sterilized m ales or T /+ and T / T individuals.

It is p ro b a b le that the d ir e c t e f fe c t s o f the r e le a s e o f s t e r i l i z e d m a le s o r t r a n s lo c a t io n s a re on ly re q u ire d to red u ce the population to s o m e v e r y low density and that o th e r fa c to r s , su ch as the inab il ity o f the f l ie s to find m a te s o r the e c o lo g i c a l e f fe c t s o f human o c cu p a t io n o f the land w ill then in terven e to e r a d ic a te the su rv iv in g f l i e s [5] . T h e r e fo r e , to study the e f fe c t s o f v iab il ity d i f f e r e n c e s , density -dependent fa c to r s and m ig ra t io n , we a s su m e d that the ta rg e t was to r e d u ce the population o f fe m a le s , as a d i r e c t r e su lt o f the r e le a s e s , to one unit. F o r each set o f p a r a m e te r s the n u m b e r o f f l i e s r e q u ire d to re a ch this ta rg e t was e s t im a te d . F o r s im p l i c i t y the e f fe c t s o f each o f the th ree fa c t o r s , v iab il ity , d en s ity d epen d en ce and m igra t io n , w e re in vestigated in the a b se n ce o f the o th e r tw o.

240 CURTIS and HILL

F ig u r e 3 show s the n u m b e rs o f s t e r i l i z e d m a le s o r t r a n s lo ca t io n h o m o z y g o te s that would be r e q u ire d to be r e le a s e d at v a r io u s va lu es o f the re la t iv e v iab i l i ty c o e f f i c i e n t (as defined above) o f s t e r i l i z e d m a le s o r the t r a n s lo ca t io n k a r y o ty p e s . The e f fe c t o f any d e c r e a s e in v iab il ity can, in p r in c ip le , be o v e r c o m e by r e le a s in g a su f f ic ien t ly la r g e n u m ber o f f l i e s .

T h e r e is , in fact, e v id e n ce [13] that it is p o s s ib le to s t e r i l i z e t s e ts e f l i e s with T e p a without any l o s s o f v iab il ity in captivity , but data on the v ia b i l i ty o f such f l i e s in the wild have not yet been pu b lish ed . In D r o so p h ila , tra n s lo ca t io n h o m o z y g o te s frequ ent ly show low v ia b i l i ty[1 4 ] ; h o w e v e r , d if fe ren t t r a n s lo ca t io n s v ary in this r e s p e c t . S ince on ly a s ing le t r a n s lo ca t io n would be re q u ire d to e s ta b l ish a capt ive co lon y o f h o m o z y g o te s , it s e e m s re a so n a b ly l ik e ly that one with su itab le p r o p e r t i e s ' c o u ld be se le c t e d in G lo ss in a .

zс5

Dс

FIG. 4. T h e numbers o f (a) sterilized m ales or (b) translocation hom ozygotes required to be released to ach ieve the target popu lation reduction plotted against values o f (1) the density depen dence c o e ff ic ie n t and (2) the im m igration rale.

The n u m b e rs o f f l i e s r e q u ire d to r e a ch the ta rg e t population re d u c t io n in the p r e s e n c e o f v a r io u s le v e l s o f the density d ep en d en ce co e f f i c i e n t a re plotted in F ig . 4. Any d e g r e e o f density d epen d en ce could , in p r in c ip le , be o v e r c o m e by the r e le a s e o f su f f ic ien t s t e r i l i z e d m a le s . H o w ever , i f the c o e f f i c i e n t e x c e e d s about 0 .0 7 5 the r e q u ire d population re d u c t io n cannot be ach ieved by the tr a n s lo ca t io n m ethod , b e c a u s e at h igh er le v e ls o f den sity depen den ce the in c r e a s e in v iab il ity which o c c u r s as the population density fa l ls o v e r c o m e s the m a x im u m red u ct ion in fe r t i l i t y that can be obtained by the act ion o f a t r a n s lo ca t io n . U n fortu nately, l it t le quantitative e v id e n ce e x is t s on which to d e c id e how s t ro n g ly density -dependent fa c to r s o p e r a te in wild t s e ts e populations [15] . H ow -

S M - 102/26 241

e v e r , ITocking [16] red u ced an iso la te d t s e ts e population to 1% o f its in itia l d en s ity with a n o n -r e s id u a l in s e c t i c id e . Human o c cu p a t io n o f the a r e a did not o c c u r . The ts e ts e population den s ity re m a in e d at about

o f that o f a c o n t r o l (u nspraved ) a re a o v e r a s i x - y e a r p e r io d . Л density d ep en den ce c o e f f i c i e n t o f 0 .0 7 5 would have in c r e a s e d the popula tion f r o m 1 unit to 99. 74 units o v e r th is p e r io d ; it a p p e a rs that, at l e a s t in s o m e t s e t s e populations, density -dependent f a c t o r s would not be s t ro n g enough to o v e r c o m e the o p e r a t io n o f the t r a n s lo c a t io n m ethod .

F ig u r e 4 a lso show's the r e l e a s e s r e q u ire d to a ch ie ve the ta rg e t population re d u ct ion at v a r io u s le v e ls o f m ig r a t io n . The sa m e n u m ber o f m a le and fe m a le m ig ra n ts p e r t im e unit w e re a s su m e d , im p ly ing a c o n s id e r a b ly h igh er rate o f m a le m ig ra t io n p e r head o f the adult population . T h is ratio was c h o s e n to r e p r e s e n t the ap parently h ig h e r a c t iv ity o f m a le s in wild t s e t s e popu lations [5] . A s a lre a d y exp la ined , m ig ra t io n has a population s ta b i l iz in g ten den cy so that, as with density -dependent fa c t o r s , th ere is a c r i t i c a l l e v e l o f m ig r a t io n above which it is i m p o s s ib le to ach ieve the d e s ire d ta rg e t with the tr a n s lo ca t io n m ethod .T h e r e is no such c r i t i c a l l e v e l with s t e r i l i z e d m a le s , but the n u m bers re q u ire d r i s e v e r y s h a r p ly with the m ig r a t io n rate . J a ck so n [17] m a d e a quantitative study o f m ig r a t io n f o r wild m a le G. m o r s i t a n s and c o n c luded that about 1. 5 units o f m a le s p e r t im e unit (u sin g the t e r m in o lo g y o f th is paper) w'ere p erm anen t e m ig ra n ts f r o m a 16 sq u a re m i le b lo ck m a rk e d out in a m u ch la r g e r a re a o f t s e t s e o c c u p ie d land. It is c l e a r f r o m F ig . 4 that it would be u s e le s s to t r e a t with t r a n s lo c a t io n s such a s m a l l a re a within a la r g e t s e t s e f ly ' b e l t 1, s in c e w here the im m ig r a t io n rate e x c e e d s about 0 .0 1 8 units o f each s e x p e r t im e unit, the t r a n s lo ca t io n m ethod b e c o m e s in e f fe c t iv e . T s e t s e a re , how 'ever, d is tr ibu ted in a d iscon t inu ou s w ay b e c a u s e o f v a r ia t io n s in the su itab il ity o f the e n v iron m en t .

T he ra tes o f m ig r a t io n a c r o s s unsuitab le e n v iro n m e n ts m u st be v e r y m u ch l e s s than th o se found b y J a ck s o n . A r t i f i c ia l c le a r a n c e o f b a r r i e r s can c e r ta in ly r e d u ce m ig r a t io n to v e r y low r a te s , s in c e the population r e f e r r e d to a lre a d y [1 6 ] , which w as re d u ce d to 1% o f its in itia l density , would have in c r e a s e d se v e n fo ld o v e r the s ix y e a r p e r io d if an i m m i g ra t io n rate o f 0 .0 1 8 units o f both s e x e s / t i m e unit had o c c u r r e d . No in c r e a s e w as , h o w e v e r , d e tected . T h is population o n ly extended o v e r a s m a l l a r e a and wo re m a in u n certa in w hether t s e t s e popu lations o f v e te r in a r y o r m e d ic a l im p o r ta n ce e x is t which a re su f f i c ie n t ly is o la te d , o r which cou ld be m a d e so e c o n o m ic a l ly , to a l low c o n t r o l by the t r a n s lo c a t io n m ethod to be s u c c e s s f u l .

4 . A T T E M P T T O P R O D U C E A S U IT A B L E T R A N SL O C A T IO NIN G lo s s in a austen i

T h e r e a re no g e n e t ic m a r k e r s av a i lab le in G lo ss in a , and so the m ethod used to attem pt to p ro d u ce and s e l e c t t r a n s lo c a t io n s was e ss e n t ia l ly the s a m e as that used f o r the s a m e p u rp o se in m i c e [18] .

Adult m a le G_. austeni w e r e ir ra d ia te d with 5 -7 krad o f 6(t o ga m m a rad ia tion and m ated im m e d ia te ly to u n irrad ia ted f e m a le s . Thus paterna l c h r o m o s o m e s o f the Fj w e re ir ra d ia te d w hile in m a tu re s p e r m a to z o a .

242 CURTIS and HILL

Dom inant ietha ls w e re induced in about 80% o f the ir ra d ia te d s p e rm but the r e m a in d e r gave r i s e to F 1 in d iv id u a ls . The fe r t i l i ty o f the Fj m a le s w as te s te d by m ating each to n o rm a l f e m a le s and r e c o r d in g th e ir pupal p ro d u c t io n f o r a standard t im e ; c a s e s o f r e d u c e d f e r t i l i ty w e re then s e l e c t e d . G lo ss in a has the advantage f o r th is w ork that f e m a le s ovu late on e egg at r e g u la r in te rv a ls , and when the f e m a le s a re w e l l fed and have been m ated to a n o r m a l m a le , the p ro b a b i l i ty that the egg w ill g iv e r i s e to a pupa is high [7, 12] . C a se s w here g e n e t ic a b n o rm a lity c a u s e s deaths o f e m b r y o s a re t h e r e f o r e f a i r ly e a s i ly d is t in g u ish a b le . A bout 20 F j m a le s with re d u ce d fe r t i l i ty have been obtained , and although in m o s t c a s e s the fe r t i l i ty is about one h a lf o f n o rm a l , in a few c a s e s it is v e r y m u ch l e s s than th is . About one half o f the so n s o f th ese s e m i - s t e r i l e m a le s inher ited the s e m i - s t e r i l i t y and by analogy with the r e su lt s in o th e r o r g a n is m s it s e e m s l ik e ly that the s e m i - s t e r i l i t y is due to t r a n s lo c a t io n h e te r o z y g o s i ty . C y to lo g ic a l c o n f i r m a t io n o f th is has not yet b e e n attem pted.

T he m a te s o f one o f the s e m i - s t e r i l e m a le s and th ose o f s e v e r a l o f its so n s p rod u ced a la rg e p r o p o rt io n o f aborted la rv a e . It is su g ges ted that the aneuploid c h r o m o s o m e se ts which this t r a n s lo ca t io n p rod u ced was not a lw ays lethal until la te in e m b r y o n ic d ev e lop m en t .

It was unexpected ly found that a la r g e p r o p o rt io n o f the daughters o f the s e m i - s t e r i l e m a le s show ed total s t e r i l i ty . A few f e m a le s with pa rt ia l s t e r i l i t y w e re , h o w ev er , obtained , and p r e l im in a ry resu lts su g ges t that they tr a n sm it this c h a r a c t e r to th e ir o f f s p r in g in the m ann er ex p e cte d o f a t r a n s lo ca t io n h e te r o z y g o te . If it is a s su m e d that this is c o n f i r m e d , the p r o c e d u r e to attempt to p ro d u ce tra n s lo ca t io n h o m o z y g o te s w ill be to in b re e d the o f fsp r in g o f a s e m i - s t e r i l e fe m a le and to tes t the fe r t i l i ty o f each m ating . One son o f each fully fe r t i le mating will be te s t -m a te d to wild type and if this p r o d u c e s all s e m i - s t e r i l e o f fsp r in g , this would in d ica te that a l ine h o m o zy g o u s f o r a t r a n s lo ca t io n had been p rod u ced , and the r e a r in g o f a c o lo n y o f t r a n s io ca t io n h o m o z y g o te s cou ld then begin.

R E F E R E N C E S

[1] BU RNH AM , C . R . , D is e u ss io n s in C y to g e n e t ic s , B u r g e s s , M inneap olis (1962). Ch. 4.

[2] W RIGH T, S . , A m . Nat. 75_ (1941) 513.[3] CU RTIS , C . F . , Bull. Ent. R e s . (1967) in p r e s s .[4] SIMPSON, H . R . , B i o m e t r i c s U (1958) 159.[5] B U X T O N , P . A . , The Natural H is to ry o f T s e t s e F l i e s , L e w is ,

L ondon (1955).[6] JACKSON, C . H . N . , Bull ent. R e s . 39 (1948) 441.[7] CU RTIS , C . F . , paper in preparat ion .И M U L L E R , H .J . and S E T T L E S , F . Z e i t . ind. A b st . V e r h e rb . 43_

(1927) 285.[9] SAUNDERS, D. S. , T ra n s . R . ent. Soc . Lond. 112 9 (I960) 221.

[10] JO RD AN , A . M . and CU RTIS, C . F . , Bull. ent. R e s . (1967)in p r e s s .

Ü1] D E A N , G. J. W. , p e r so n a l c o m m u n ica t io n .

S M - 102/26 243

[12] JORDAN , A . M . , NASH, T . A . M . and B O Y L E , J . A . , Ann. trop . M ed . P a r a s it . 61_ (1967).

[13] D A M E , D . A . , and F O R D , H . R . , Bull . ent. R e s . 56J1966) 649.[14] IVES, P . T . and FINK, G . R . , G e n e t ics 47 (1962) 963.[15] G L A S G O W , J. P. , The D is t r ib u t io n and Abundance o f T s e t s e ,

P e r g a m o n , O x fo rd (1963).[16] HOCKING, K . S . , XI Int. Kong. Ent. W ien I960 Bd. II (1962) 409.[17] JACKSON, C .H . N. , Bull. ent. R e s . 32 (1941) 53.[18] C A R T E R , T . C . , L Y O N , M . F . and PH ILIPS, R . J . S . , J. Genet.

53_ (1955) 154.

APPENDIX

C O M PU TIN G M ETH O D

It is su f f ic ie n t to s p e c i fy the changes taking p la ce in the population when the time a d va n ce s by one tim e unit.

T r a n s lo c a t io n m ethod

L e t : -

Q , = the n u m b e r o f ind iv iduals o f se x s (s = 1 f o r m a le s ,sknts = 2 f o r f e m a le s ) o f k aryotyp e к (k = 1, 2, 3 f o r +/ +, Т / +, Т / T r e s p e c t iv e ly ) o f age n tim e units at time t t im e units. No T / T f l i e s are r e le a s e d until t > 1.

p = the pupal s u r v iv a l c o e f f i c i e n t ; p = 0 .8 7 3 5 8 0 5 = (■§) 3 .

a = the adult s u r v iv a l c o e f f i c i e n t f o r s e x s : a , = 0 .6 1 4 4 . s 1a 2 = 0 .7 9 7 0 7 3 .

X b = the n um ber o f T / T f l ie s o f s e x s r e le a s e d at t im e t. st

f . = the f r e q u e n c y o f p ro g e n y o f k aryotype к f r o m a mating o f in d iv idu a ls o f karyotyp e i and j ( see T A B L E 1).

v^ = the re la t iv e v iab il ity c o e f f i c i e n t f o r k aryotyp e k.

d = the d en s ity d ep en den ce c o e f f i c i e n t .

m = the e m ig r a n t p r o p o r t io n f o r adults o f se x s.

M = the n um ber o f im m ig ra n ts o f se x s.sN = the population s i z e at t im e t and is g iv e n by

2

Nt = Гs = 1

3 19<~c , . . .sknt s

к = 1 n = 1

24 4 CURTIS and HILL

^ ^ s k , n + l , t + l a s Vk

<2> Q sknt = ° ’

When t > 0 the adult m o r ta l i t y can be e x p r e s s e d by:

[l+d ( 1 - N t/ N 0) ] ( l - m s)

n > 19

and the pupal m o r ta l i t y can be e x p r e s s e d by:

(3) Q . , = Р Q , . 1 « n < 3s k , n + l , t + l sknt,

e x c e p t when T / T indiv iduals a re r e le a s e d when:

Q s 3 4 , t+1 = p Q s33t + X s ,t+ 1

Q s k nt , 4 < n < 18

T o com pu te the n u m b e rs o f new b irths we need to define u., =jkntthe e x p e cte d p r o p o r t io n o f the p ro g e n y o f a fe m a le o f karyotyp e j o f age n (4 < n <. 19) at t im e t w hich have karyotyp e k. T h is quantity depen ds on the f r e q u e n c ie s o f the types o f m a le in the population when the fe m a le m a te s and is g iven by:

3 19f.

Ujk4t

f. 7 - Q + f M . i = 1 l jk n = 5 J

19 3

XT XT Q , , _ , + m ,n = 5 i = 1 lint

Ujk , n+1, t+1 jknt,

The new b irths o f pupae a re th e r e fo r e :

3 1 9

° s k l t ~ 2 У ~ 5 1 Q-,. f u i, f kj = 1 n = 6 2jnt Jknt*

3 2 9

Q s l l t = ± ■ ^ / ^ 2 jnt Uj l n t + j = 1 n = 6 J J

The above fo rm u la e w e re used re p e a te d ly to study long te rm changes in the population and an e le c t r o n i c c o m p u te r was needed to handle the ca lc u la t io n s .

S te r i l is e d m ale m ethod

The above fo rm u la e ca n e a s i ly be m o d if ie d to apply to the s t e r i le m a le m eth od :

к is r e -d e f in e d so that к = 1 f o r n o rm a l indiv iduals and к = 2 f o r s t e r i l i s e d m a le s .

Y is defined as the n um ber o f s t e r i l i s e d m a le s r e le a s e d at timet (t > 0 ) .

S M - 102/26 245

E quations (1) (2) and (3) hold without change e x ce p t that к can on ly take the value 1 in (3). R e l e a s e o f s t e r i l i s e d m a le s g iv e s :

Q 124. - Y t

If we define u (4 «£ n < 19) as the p r o p o r t io n o f f e m a le s o f age n w hich have m ated with a fe r t i le m a le , then:

19 £ n = 5 Q l l n t + M 1

4t2 19

Г Zi = 1 n = 5+ M. lint 1

u , , = un+ 1 , t+ 1 nt, 5 sj n < 19

and:

Q s i l t

19z

n = 6Q

2 1 nt nt + M ,

D I S C U S S I O N

R . C . VON B O R S T E L : The s y s te m you su g ges t cou ld be m adefo r m a l ly iden t ica l to the i r ra d ia t io n -o f -m a le m ethod , by r e le a s in g m a le s (but not fe m a le s ) with m u lt ip le t r a n s lo c a t io n s . Next t im e , m a le s with d i f fe ren t se ts o f m u lt ip le t r a n s lo ca t io n s cou ld be re le a s e d ; in each c a s e m o s t o f the d a u g h t e r s and s o n s would be h e te r o zy g o u s , and thus s t e r i le . A f t e r on e tr a n s lo ca t io n w as m ade h o m o z y g o u s , th is s to ck cou ld be used again to obtain m o r e t r a n s lo c a t io n s , and g ra d u a lly the d e s i r e d l in es cou ld be a ch ie v e d . B y using m a le s f r o m d i f fe ren t s tag es o f the dev e lop in g m u lt ip le t r a n s lo ca t io n l in e s f o r the r e l e a s e s , the advantages o f both the lethal m a le and the tra n s lo ca t io n m a le s y s t e m s cou ld be obta in ed .

C . F . CURTIS: The r e le a s e o f f l i e s f r o m s e v e r a l s t o ck s c a r ry in gd if fe re n t m u lt ip le t r a n s lo ca t io n s would c e r ta in ly be m o r e e f f i c ie n t than the s y s te m I have d e s c r ib e d , p ro v id e d that v iab le m u lt ip le h o m o z y g o te s cou ld be obta ined . I a g r e e that the b e s t way to p ro d u ce m u lt ip le t r a n s lo c a t io n h o m o z y g o te s would be to t r y to a c cu m u la te the tr a n s lo ca t io n s in s u c c e s s iv e s te p s , but b e c a u se o f the lon g g en era t ion t im e s in ts e ts e this p r o c e s s would, o f c o u r s e , take a long t im e . I think it p o s s ib le that the s to ck s with fe r t i l i ty w ell be low 50% that I m en tion ed m ay p erhaps be c a s e s in which two o r m o r e t r a n s lo ca t io n s have a r is e n at o n c e . C yto - l o g i c a l data and e v id e n ce as to w hether th ese 'h igh s t e r i l e 1 l in e s s e g r e g a te 50% s t e r i l e o f f s p r in g are not yet av a i lab le .

V . L A B E Y R IE : Have you te s te d the va lid ity o f y o u r w ork in g h ypothes is that m ating is r a n d o m ? A r e the ir ra d ia te d m a le s not at a d isadvantage c o m p a r e d with n o rm a l m a le s , and d o e s not this d isadvantage depend on

246 CURTIS and HILL

the ratio o f v a r io u s types p r e se n t? If so , s u r e ly the m in o r ity m a le s have an advantage, as has been show n fo r LX m e la n o g a s te r by Petit , and c o n f i r m e d in D obzh an sk y 's la b o r a to r y .

C . F . CURTIS: We have not tested , with the co m p u te r , the e f fe c t so f n o n -ra n d o m m ating . I would gu e ss that an o v e r a l l redu ct ion o f the m atin g co m p e t i t iv e n e s s o f the tra n s lo ca t io n ind iv iduals would g ive m uch the sa m e r e su lt as a red u ct ion in v iab il ity o f th e s e ind iv iduals and, as F ig . 3 o f the pa per sh ow s, th is can be o v e r c o m e by the r e le a s e o f m o r e h o m o z y g o t e s . The o c c u r r e n c e o f a s s o r ta t iv e m ating m ight be m u ch m o r e h arm fu l, s in c e i f ind iv iduals tended to s e l e c t f l i e s o f th e ir own type as m a te s , the p r o p o r t io n o f h e te r o z y g o te s , and hence the s t e r i l i z in g e f fe c t , would be r e d u ce d . T h e r e would be s t ro n g natural s e le c t io n f o r the evo lu t ion o f such a s s o r ta t iv e m ating in a population being attacked by m e a n s o f t r a n s lo ca t io n s , but r e co g n i t io n o f the d i f fe ren t c h r o m o s o m e ty p e s by the in s e c ts m ight be d if f icu lt , b e c a u s e they are not ex p e cte d to d i f f e r m o r p h o lo g ic a l ly .

L>: T . NORTH: Have you c o n s id e r e d ex am in in g y o u r induced t r a n s lo c a t io n s to d e te rm in e w hether any o f them show a bias to w a rd s ad jacent se g r e g a t io n as op p o se d to a lternate s e g r e g a t io n ? M ultip le t r a n s lo ca t io n s a r e not on ly d if f i cu lt to c r e a te , p a r t icu la r ly when genet ic m a r k e r s a re not ava i lab le , but a re o ften d if f icu lt to m ainta in in a h o m o z y g o u s f o r m .It would a p p ear to m e that the se a r c h f o r se g r e g a t io n d ir e c te d to w a rd s ad jacent s e g r e g a t io n o f the tr a n s lo ca t io n h e te r o z y g o te at m e i o s i s would not in vo lve m uch additional w ork , and m ight have m o r e p r a c t i c a l value than the m anipulation and induction o f m u lt ip le t r a n s lo ca t io n s t o c k s .

C . F . CUKTIS: I a g re e that s ing le t r a n s lo ca t io n s with l e s s than50% fe r t i l i ty in the h e te ro zy g o te would be e x t r e m e ly usefu l , but we thought, when setting up o u r c o m p u te r m o d e l , that it would be o v e r - o p t im is t i c to a ssu m e the e x is t e n c e o f such a m utation . If such a t r a n s lo c a t io n could be obtained, it would, o f c o u r s e , help to o v e r c o m e the e f fe c t s o f any d en s ity -dep en den t fa c to r s o r im m ig ra t io n that m ight o c c u r in the wild population it is d e s i r e d to e r a d ic a t e .

D . A . T . B A L D R Y : Although H ocking (R e f . [16] o f yo u r paper) hasr e p o r te d that a t s e ts e population can be red u ced to a low le v e l and can re m a in at that reduced le v e l f o r 6 y e a r s , we know on ly too w ell, ' a fte r m any u n s u c ce s s fu l attem pts to c o n t ro l t s e t s e by in s e c t i c id e m eth od s , that r e s id u a l populations rap id ly in c r e a s e in density . F r o m m y e x p e r ie n c eI think it un likely that human se t t lem ent and d e v e lo p m e n t o f a t s e t s e fly be lt would e l im in ate the rem ain in g 1 o r 2% o f a t s e t s e population that had been red u ced by a c t ive c o n t r o l m e th o d s . Such res id u a l t s e t s e popu la tion s a re e x tr e m e ly d if f i cu lt to e l im in ate by any m ethod .

I a lso have a qu est ion . Could you p le a se d e s c r ib e y o u r technique ■ f o r e st im a tin g the n um ber o f t ra n s lo ca te d t s e ts e f l ie s , and the n u m ber o f e x p e r im e n ta l an im als re q u ire d to m ainta in them , that would be needed to e l im in ate a t s e t s e population o f a known density o c cu p y in g a f l y -b e l t o f known s i z e ?

C . F . CURTIS: If den s ity -d ep end en t fa c to r s o p e r a te in one ts e ts epopulation one would e x p ect them to o p e r a te in a l l . If H ocking found on e population w here they did not o p e ra te , I would be in cl in ed to think, in the a b se n ce o f absolute certa in ty that the populations you r e f e r to a re c o m p le t e ly is o la te d , that the r e c o v e r i e s a fte r in s e c t i c id e attack m a y have been due to im m ig ra t io n . It is ra th e r d if f i cu lt to b e l ie v e that t s e te s

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coznpete f o r food , and the e v id e n ce on p red at ion su g g e s ts that they do not fo r m a substantia l p r o p o r t io n o f the food o f th e ir p r e d a t o r s (R e f . [15] o f the p a p e r ) . I a g r e e , h o w e v e r , that on p re se n t data it is not p o s s ib le to ex c lu d e the p o s s ib i l i t y that de n s ity -d e p e n d e n t fa c to r s do o p e r a te in t s e t s e at a low l e v e l .

I w o n d e r w hether the lo w -d e n s i t y r e s id u a l popu lations , p ro v id e d they do not in c r e a s e again, a re o f m e d ic a l o r v e te r in a r y im p o r ta n ce in t r y p a n o s o m e t r a n s m is s io n . It m ight be found u se fu l a fte r a population has been r e d u c e d to a low l e v e l by the tr a n s lo ca t io n m ethod to e r a d ica te the s u r v iv o r s by r e le a s e o f s t e r i le m a le s . Of c o u r s e the n u m b e r s o f s t e r i l e m a le s re q u ire d would then be re la t iv e ly low ; with a low popula tion it would p r o b a b ly be d if f i cu lt to a s s e s s the c o r r e c t n u m b e r o f t r a n s lo c a t io n s to be r e le a s e d , but in the c a s e o f s t e r i l e m a le s it would not do any h arm to r e le a s e an e x c e s s . If we c o n s id e r a f l y - b e l t c o v e r in g 1000 square m i le s with the population d en s ity found by J a ck s o n [6 ] , th e re would be a l tog eth er 3 . 5 X 106 f e m a le s , pupae and adults. On the o p t im is t i c a ssu m pt ion o f F ig . 3, th is would r e q u ire 6. 3 X 106 h o m o z y g o te s to be r e le a s e d f o r e r a d ic a t io n . T o p ro d u ce th is n u m ber in on e y e a r a c o lo n y o f 250 000 adults [10] would have to be m a in ta ined . At the p resen t f ly feed in g ra te s that we use this would r e q u ir e a c o lo n y o f 4000 rabb its , but about half this n u m b e r o r even l e s s o f goats m igh t be adequate.

IRRADIATION E F F E C T S ON RESPIRATION AND BLOOD DIGESTION IN THE TICK Ornithodoros tholozani AND. THEIR IM POR TAN CE FOR THE S T E R IL E -M A L E TECHNIQUE

RACHEL GALUN AND M. WARBURGISRAEL IN STITU TE FOR BIOLOGICAL RESEARCH,NESS-ZIONA, ISRAEL

Abstract

IRRADIATION EFFECTS ON RESPIRATION AND BLOOD DIGESTION IN THE T IC K Ornithodoros tholozan i AND THEIR IMPORTANCE FOR THE STERILE-MALE TECHNIQUE. Soft ticks feed exclu sive ly on blood at very long intervals. The ingestion o f b lood is a ccom p a n ied by a m an ifo ld increase in oxygen consum ption. This increase in m eta b o lic rate is not a ffe c ted by irradiation up to 16000 R. Irradiation doses m uch low er than that required to produce 99% dom inant lethals in m ales reduce the rate o f digestion o f b lood taken by the t ick . Thus at 4000 R the rate o f digestion in m ales is slow ed dow n, w hile 16000 R is required to produce 9Э% dom inant lethals. In fem a les, a dose o f 4000 R is necessary to prevent oogenesis, but the rate o f digestion is slow ed down even at 1000 R. This e ffe c t on digestion causes ticks w hich were fed im m ed ia te ly after irradiation to feed m uch less frequently thereafter, m ales and nymphs exposed to 16 000 R took a first b lood m eal after irradiation and did not feed again be fore death. Adult m ales are very litt le dependent on the b lood m ea l for survival and successful m ating, h en ce the irradiated m ales can be useful in controlling tick populations. Furthermore, s ince they do not take a second b lood m eal, they w ould not introduce any harm ful e ffe c t into the environm ent.

1. INTRODUCTION

When d ev e lop in g a radiation s t e r i l iz a t io n method f o r the c o n t r o l o f O rn ith o d o r o s tholozani (L aboulbéne and M cgnin) t i ck s , the authors foundthat a d ose o f 16 000 R w as n e c e s s a r y to p ro d u ce 99% dom inant ietha ls in m a le s . F e m a le t ick s , h o w e v e r , w e r e m uch m o r e se n s i t iv e to s te r i l iza t io n by ir ra d ia t io n - a fte r e x p o s u re to 4000 It no e g gs w e re la id , w hile t r e a t ment with lOUO R s lo w e d down o o g e n e s i s c o n s id e r a b ly [1].

I r ra d ia ted t ick s s e e m e d to re ta in the b lood m e a l that they had taken a f t e r ir ra d ia t io n m uch lo n g e r than untreated t i c k s . T i c k s r e q u ire a b lood m e a l to d e v e lo p e g g s . T h e r e f o r e , the authors in vestigated the p o s s ib i l i t y that the h igh er s e n s it iv ity o f f e m a le s w as due to the in h ib itory e f f e c t o f i r ra d ia t io n on b lood d ig est ion .

Since the inhibit ion o f b lood d ig e s t io n can a lso be ex p e cte d to a f fe c t the v ia b i l i ty o f the t i ck s , the r o le o f this phenom enon in the c o n t r o l o f t i ck s by the s t e r i l i ty method w as studied .

2. M A T E R IA L S AND METHODS

T ic k s w e re r e a r e d a c c o r d in g to a m ethod d e s c r ib e d in a p re v io u s pub lica tion [1]. V irg in t i ck s w e r e obtained by keeping b l o o d - f e d nym phs in d iv idu a lly in v ia ls and sex in g them a fte r their moult .

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T he rate o f b lood d ig est ion w as studied in the fo l low in g m ann er . T ic k s w e r e w eighed , a l low ed to feed on ra ts and then w eighed again im m e d ia te ly a f t e r feed ing , b e fo r e th ere was any opportunity to e x c r e te any c o x a l fluid. A f t e r w eighing , each t ick w as p la ced in a sep arate v ia l upon which the am ount o'f b lood taken by the t i ck w as r e c o r d e d . At a p p rop r ia te in te rva ls the t ick s w e r e .d i s s e c te d , the gut content w as w ashed into 5 m l d is t i l le d w ater and the d e b r is and d ig e s te d in so lu b le f r a c t io n s w e re r e m o v e d by c e n tr i fu g at ion . The c o n ce n tra t io n o f the h a em og lob in in the supernatant w a s d e te rm in e d in a B eck m ann s p e c t r o p h o to m e te r at 540 m ( im . Since the am oun t o f h ae m o g lo b in in it ia lly in gested by the t ick w as known, the p e r c e n ta g e o f d ig est ion cou ld be ca lcu la te d . F o r each d e term ination , th ree t i ck s w e r e used .

M e a su re m e n t o f oxygen uptake w as c a r r i e d out using a W a rb u rgconstan t vo lu m e r e s p i r o m e t e r , with cups o f about 2 0 - m l ca p a c ity . The r e s p i r o m e t e r contained in the c e n t ra l w e l l 0. 2 ml o f 20% KOH solution a b s o r b e d on f i l t e r paper, and 0. 2 m l w ater in the s ide a r m . T en t ick s w e r e p la ce d in each cup and th ree g ro u p s w e re used fo r each determ in a tion . M e a su re m e n ts w e re c a r r i e d out at 22 ° , 26° and 30° С . H eadings w e re taken at 30 -m in u te in te rv a ls f o r th ree c o n s e cu t iv e h ours . The m e a s u r e m en ts w e re repeated da ily f o r the f i r s t two w eek s , and then at w eek ly in t e r v a l s .

F o r i r r a d ia t io n , a 60Co s o u r c e o f the type G am m acell . 20U w as used, em itt in g 7 000 R /m in , F o r a d ose o f 1000 R, a 3 0 - k C i eoCo plate i r r a d ia to r w a s u ti l ized . The t ick s w e r e held in p la st ic petri d ish es during ex p o su re to the rad ia tion .

F o r h is t o lo g ica l stud ies, t i ck s w e r e f ixed in C a r n o y 's f ixat ive f o r three h ou rs and c lean ed in ce d a r w o o d o i l overn igh t . E m bedding in para ffin fo r 48 h ou rs w as n e c e s s a r y b e ca u se o f the la rg e amount o f b lood in side the gut. T ic k s w e re se c t io n e d at 7 jum and stained in G ie m sa .

3. R E SU L T S AND DISCUSSION

Adult t i ck s can su rv iv e f o r up to s e v e r a l y e a r s under con d it ion s o f starva t ion . D uring this p e r io d they maintain v e ry low m e ta b o l is m . In th e ir natural habitat, the s o i l o f c a v e s , w h ere the a v e ra g e te m p e ra tu re i s about 22° C, the fe m a le t ick r e q u ir e s l e s s than 1 m m 3 O ^ /h , which i s l e s s than 0. 1 m m 3 0 2 / m g l ive weight (F ig . 1). No e g gs a re p ro d u ce d by the fe m a le without a b lood m e a l . When o f f e r e d a rat, the fem a le takes an a v e ra g e o f 7 0 mg b lood within l e s s than 1 h.

T h is la rg e blood m e a l c a u s e s an in itia l instantaneous in c r e a s e in m e t a b o l i s m , fo l lo w e d by a cont inuous in c r e a s e which r e a c h e s a peak within 8 - 1 2 days . At the peak, the r e s p i r a t o r y ra te is about ii- fo ld h igh er than that in the state o f starva tion (F ig . 1).

T h e se o b s e rv a t io n s d i f fe r f r o m th ose r e p o rte d by K ozan ch ikov [2] w ho found that O. tho lozan i d isp la y s a re d u ce d oxygen dem and a fte r a b lo o d m ea l. The r e s p i r a t o r y pattern m e a su re d by the authors is v e ry s i m i l a r to the pattern show ed by nym phs o f D e rm a c e n to r m arg inatus and Ix o d e s r i c i n u s , w here an even g r e a te r in c r e a s e in r e s p i r a t o r y rate due to feed in g was o b s e r v e d [3]. A c o n s id e r a b le in c r e a s e in m e ta b o l ic rate a f t e r a b lood m e a l was a lso o b s e r v e d in m o s q u ito e s [ 4 ] . B e l o z e r o v c la im s that it i s im p o s s ib le to expla in this m a rk e d in c r e a s e in m e ta b o l i s m so le ly

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WEEKS AFTER FEEDING

FIG. 1. O xygen consum ption by ticks

by the "d y n a m ic e f f e c t o f p ro te in ou s f o o d " . He attr ibuted it to in ten sive grow th and de v e lo p m e n t o f the integum ent, in testine, gonads and other o r g a n s which a c c o m p a n ie s the p ro lo n ge d feed ing o f the ixod id nym phs [3].

T h is explanation is quite in a p p lica b le in the p re se n t c a s e w here adult f e m a le s w e re u sed . . In these f e m a le s the main change fo l low in g feed ing is in the o v a r ie s . Indeed, the peak o f r e s p ir a t io n c o r r e s p o n d s n ice ly to the peak o f o o g e n e s is (F ig . 2) and, th e r e fo r e , one tends to c o r r e la t e the two phenom ena .

H ow ever , when r e s p ir a t io n o f ir ra d ia te d fe m a le s w as m e a su re d , the sa m e in c r e a s e in m e t a b o l i s m a fte r feed in g w as o b s e r v e d ( F ig .3 ) . Even a d ose o f 16-000 R, w hich c a u s e s c o m p le t e deg e n e ra t io n o f the o v a r ie s , did not a f fe c t the pattern and the rate o f r e sp ir a t io n .

It thus s e e m s that b lood in ge s t io n d o e s have quite a s tron g ' s p e c i f i c d yna m ic e f f e c t 1. If a n on -n u tr i t iv e so lut ion c o n s is t in g o f 1 0 _3 M g lu ta th ione in 0. 15 M N aCl i s o f f e r e d to the t i ck s , they e n g o r g e it to the sa m e extent as ЫоосГТэ] ; but this m e a l i s not a c c o m p a n ie d by any in c r e a s e in oxygen uptake. W hether the in c r e a s e is due to the b lood p ro te in s o r to s o m e oth er c om p on en t r e m a in s u n c lear .

F u rth e r e v id e n ce that the in c r e a s e in r e s p i r a t o r y rate i s independent o f any d e v e lo p m e n ta l p r o c e s s can be found in c o n s id e r in g the r e s p ir a t io n o f .th e m a le s . O xygen uptake in c r e a s e s 2 - 3 fo ld a f t e r a b lo o d m e a l (F ig . 1), although no c o n s id e r a b le d e v e lo p m e n t o f any o rg a n fo l lo w s the m e a l . The i n c r e a s e is in deed m uch s m a l l e r than that found in fe m a le s , but so is the am ount o f b lood taken: an a v e r a g e b lood m e a l o f 11 m g as c o m p a r e d with 70 m g taken by the f e m a le .

In addition to t r ig g e r in g an in c r e a s e in the m e t a b o l i c ra te , the in gestion o f the b lood m e a l s ta r ts a v e r y s t ro n g d ig e s t iv e p r o c e s s . In f e m a le s , o v e r 7 0% o f the b lood p ro te in s a re d ig e s te d within one month, and a f t e r 2 months

252 G A I,UN and WARBURG

IOr

FIG. 2. Rate o f o o cy te developm ent

- 68 D

8

• control О 4 0 0 0 R

□ 1 * 0 0 0 R

_ L .....4 8 12 16 20

W E E K S A F T E R FEFDING FIG. 3. O xygen consum ption by irradiated fem ales

90% o f the p ro te in s have been u t i l ized . At th is stage , the rate o f d ig e s t io n d e c r e a s e s so much that the rem a in in g 1 0 % o f undigested food can m aintain the t ick f o r m onths o r y e a r s though at a v e r y low m e ta b o l ic r a t e .

T h is pattern o f d ig e s t io n s e e m s to be quite w aste fu l . An adult t i ck w e igh in g 10 m g c o n s u m e s within two w eek s o v e r 7 m g prote in , m o s t o f it be ing u t i l ized as an e n e rg y s o u r c e ra th e r than f o r egg prod uct ion .T h is point i s i l lu s tra te d in v i r g in f e m a le s which show s c a r c e l y any o o g e n e s i s a f t e r a b lood m e a l [G], but n e v e r th e le s s d ig est o v e r 50% o f the b lo o d p r o te in s within one month (F ig . 4).

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FIG. 4. Blood digestion in fem ales

If the c u r v e s o f d ig e s t io n f o r m ated and v irg in t i ck s in F ig . 4 a re c o m p a r e d , it can be se e n that on ly about 20% o f the b lo o d p ro te in s in gested by the t i ck s is u ti l ized f o r egg p rodu ct ion .

T hus the rate o f d ig e s t io n i s regu la ted on ly to a s m a l l extent by the am ount o f p r o te in s n eed ed f o r the de v e lo p in g o o c y t e s . A m a jo r p art o f p ro te in u ti l iza t ion not c o n c e r n e d with egg p rodu ct ion i s t r ig g e r e d by the b lo o d m ea l, a s can be seen f r o m the in c r e a s e in ox y g e n uptake and d ig e s t io n w hich o c c u r s in m ated , v irg in and ir ra d ia te d t i ck s .

F u r th e r e v id e n ce f o r the r o le o f the b lood m e a l in t r ig g e r in g d ig e s t iv e p r o c e s s e s in depen den tly o f p ro te in r e q u ir e m e n t s is obta ined f r o m the pattern o f b lo o d d ig e s t io n by the m a le s . Although th e ir ra te o f d ig e s t io n i s s l o w e r than that o f the fe m a le s , the m a le s a l s o u t i l ize c l o s e to 70% o f the b lood m e a l within two m onths (F ig . 5).

Nym phs d ig e s t about 40% o f th e ir b lood m e a l until they m oult . D ige s t io n cont inu es at the in it ia l rate fo r two m onths. D uring th is p e r io d about 80% o f the b lood p r o te in s a r e u t i l ize d . H ere a l s o the n u trit ion a l r e q u ire m e n ts o f the body d e te rm in e the rate o f d ig e s t io n on ly to a s m a l l extent. N ym phs e x p o s e d to 4000 R b e f o r e the b lo o d m e a l do not m oult [1 ] . At th is d o s e the ra te o f d ig e s t io n during the f i r s t month is a lm o s t equ a l to that o f the n on- i r ra d ia te d c o n t r o l s . O nly in the s e c o n d m onth d o e s d ig e s t io n s lo w down in the ir ra d ia te d n ym phs , in d icat ing p o s s ib le d am age to the gut ep ithe l ium . A f t e r e x p o s u r e to 16 000 R, d ig e s t io n i s s lo w e d down in the nym phs f r o m the f i r s t days a f t e r feed in g (F ig . 6). At this d o s e su r v iv a l is a lso c o n s i d e r a b ly a f fe c te d .

% di

gest

ion

% di

gest

ion

254 G ALUN and WARBURG

months FIG. 5

monlhs FIG. b.

. Blood d igestion in m ales

Blood digestion in nymphs

S M -102/27 255

T he e f f e c t o f i r r a d ia t io n on the rate o f b lo o d d ig e s t io n in f e m a le s w a s studied in m o r e d e ta il . It w as found that, at a low d o s e o f 1000 R, th e r e i s c o n s id e r a b le inhibit ion o f the d ig e s t iv e rate (F ig . 4). At th is d o s e o o g e n e s i s i s a l s o s lo w e d down. T he a v e r a g e t im e r e q u ire d f r o m feed in g to o v ip o s i t io n is 7 5 days as c o m p a r e d with 23 days in the n o n - i r r a d ia te d c o n t r o l t i ck s . It i s d i f f i cu lt to d is t in gu ish w hether o o g e n e s i s i s r e ta r d e d by the s l o w e r d ig e s t io n rate o r w hether d ig e s t io n is s lo w e d down as a re s u lt o f a f e e d - b a c k m e c h a n ism regu la ted by the rate o f p ro te in uptake o f the o o c y t e s .

In ir ra d ia te d m o s q u ito e s a d e la y in b lood d ig e s t io n w as o b s e r v e d .T h e d e la y in th is c a s e w as not due to a la ck o f p r o te o ly t i c e n z y m e s and cou ld be re la ted to e g g d e v e lo p m e n t [7]. At low ir ra d ia t io n d o s e s t ick s a l s o show s o m e c o r r e la t i o n betw een o o g e n e s i s and b lood d ig e s t io n .F e m a le s t rea ted with 4000 R, which p reven ts o o g e n e s i s c o m p le te ly , d ig e s t b lood at the sa m e rate as v irg in s (F ig . 4).

S ince the gut o c c u p ie s m o s t o f the vo lu m e o f the body, it is im p o s s ib le to sh ie ld the gonads and ir ra d ia te on ly the gut, as w as done with b e e s [8], o r with the b o l l w e e v i l [9 ] . Thus, a d i r e c t a n sw e r as to w hich o f the two is the m o r e se n s i t iv e t i s s u e cannot be obtained. H o w ever , even i f the gut ep ithel ium is a f fe c te d , the am ount o f d ig est ion that s t i l l takes p lace a fte r ir ra d ia t io n is quite high. T h e r e f o r e , the p r im a r y r e a s o n f o r la ck o f d e v e lo p m e n t o f the gonads at 4000 R couLd not be la ck o f the pro te in n e c e s s a r y f o r o o g e n e s i s . T he h igh er sen s it iv ity o f f e m a le s to s t e r i l iz a t io n by ir ra d ia t io n cannot be exp la ined on this b a s i s . At a d o s e o f 8000 R (not shown in the f ig u re ) and at 16 000 R the rate o f d ig e s t io n w as lo w e r in the ir ra d ia te d f e m a le s than in untreated v irg in s - ind icat ing h ere too p o s s ib le dam age to the gut ep ithe l ium .

T o in vest igate th is h yp oth es is fu rther , p r e l im in a r y h is t o lo g i c a l o b s e r v a t ion s w e re c a r r i e d out, in w hich the gut e p ithelium fo l lo w in g a b lood m e a l was c o m p a r e d in m ated t i ck s , v irg in s and t ick s i r ra d ia te d with 16 000 R.

When a fe m a le e n g o r g e s b lood the w a lls o f the gut a r e stron g ly s t re t ch e d . The s t re t ch e d ep ith e l ia l c e l l s r e s e m b le a thin m e m b r a n e with sl ight th ickenings w hich conta in the c e l l n u c le i . A f t e r s lo w feed in g the d ig e s t iv e c e l l s s tart to e n la r g e . T h r e e days a fter the b lood m e a l the m ated t i ck s show a few d ig e s t iv e c e l l s reach in g a d ia m e t e r o f 20цтп. At th is stage the v irg in and ir ra d ia te d t i ck s reta in m o s t o f the ep ith e l ia l c e l l s in a r e s t in g state . T h e d ig e s t iv e c e l l s continue to e n la rg e , s o that in m ated f e m a le s sev en days a fte r the b lood m ea l, the c e l l s r e a c h a d ia m e te r o f 100/J m . At th is stage s o m e o f the c e l l s bud o f f into the lum en o f the gut. The f loa ting c e l l s , w hich a s su m e a round shape, conta in haem atin p a r t i c l e s . The haem atin i s the end p rod u ct o f the in t r a c e l lu la r d ig est ion o f the in ges ted h ae m o g lo b in . T h is p r o c e s s is de layed in v ir g in s and in ir ra d ia te d t i ck s , w h ose d ig e s t iv e c e l l s a r e on ly 30-40Aim in d ia m e te r and conta in no haem atin ; they a re a l l s t i l l attached to the gut w all . O nly 14 days a f te r the m e a l d o e s the d ig e s t iv e p r o c e s s o f the v ir g in s and ir ra d ia te d t i ck s r e a c h the stage a r r iv e d at by the m ated o n es on the seventh day.

A l l the d ig e s t iv e c e l l s o f the m ated f e m a le s conta in m any haematin g ra n u le s 14 days a f t e r the b lood m e a l . Many o f the c e l l s a r e a lre a d y detached and f loat in the lum en o f the gut. T h is stage i s attained in the ir ra d ia te d and v irg in t i ck s on ly a f t e r 30 days .

The detached d ig e s t iv e c e l l s a re n o rm a l ly r e p la c e d by c e l l s which d i f fe re n t ia te f r o m r e s e r v e c e l l s . T he r e s e r v e c e l l s a r e s m a l l e r than the

256 GAI.UN and WARBURG

d ig e s t iv e c e l l s and th e ir d is ta l ends a r e u su a lly c o v e r e d by the g row in g d ig e s t iv e c e l l s . M ito s is o f the r e s e r v e c e l l s is r a r e ly seen in adult t i ck s .It i s m u ch m o r e c o m m o n in nym phs [Ю ], the h is to lo gy o f w hich we have not y e t stud ied . Add it ion a l h is t o lo g i c a l stud ies a r e planned to d e te rm in e the ra te o f r e p la c e m e n t o f d ig e s t iv e c e l l s in i r ra d ia te d t i ck s . It w as found in B la b e r a that ir ra d ia t io n d e s t r o y s the r e g e n e ra t iv e c e l l s o f the m id -g u t , but d o e s not dam age the s e c r e t o r y c e l l s w h ich have a lre a d y p a sse d the d i f fe re n t ia t io n stage , and w hich continue to function n o r m a l ly a fte r i r r a d ia t ion [11 ] . It s e e m s that in the f e m a le t i ck the d i f fe re n t ia te d d ig e s t iv e c e l l s a r e a lso undam aged by ir ra d ia t io n . T he s l o w e r rate o f e n la rg e m e n t o f the c e l l s m a y be d icta ted by la ck o f d ev e lop m en t o f the o v a r ie s , s in ce the sa m e phenom enon w as a lso o b s e r v e d in the v irg in s . It is su sp e cte d that the additional de lay in the rate o f d ig e s t io n in ir ra d ia te d f e m a le s , w h ich i s s een s e v e r a l w eek s a fte r feed in g , i s due to d am age c a u se d to the r e s e r v e c e l l s r e p la c in g the d ig e s t iv e c e l l s .

W e have seen that, to u ti l ize s t e r i le t i ck s f o r c o n t ro l , a d o s e o f 16 000 R i s n e c e s s a r y to p ro d u ce 99% dom inant letha ls in m a le s . T h is d o s e a l s o d e s t r o y s a l l the g on ia l c e l l s , so that no r e c o v e r y in fe r t i l i ty ta k es p la ce . H ow ever , it i s o f g rea t im p o r ta n ce that the trea ted m a le s m aintain their v igo u r , lon gev ity and sex u a l c o m p e t i t iv e n e s s . T h e se s e e m to be a f fe c ted to a c e r ta in extent by the ir ra d ia t io n [1], and this p h enom enon m ay p erhaps be attributed to the inhibition in d ig e s t io n d e m o n s tra te d in the m a le s even a fter e x p o s u r e to on ly 4000 R (F ig . 5).In this r e s p e c t the m ale O. tho lozan i beh av es s im i la r ly to the m a le b o l l w e e v i l , w h ere the ir ra d ia t io n dose p roduc in g a v e r y high le v e l o f s te r i l i ty i s about 4 - 5 t im e s that which p r o d u c e s d am age to the gut ep ithel ium [9]. T h e r e f o r e , ir ra d ia te d m a le b o l l w é e v i l s cannot be used fo r c o n t r o l p u r p o s e s . F ortu nate ly , unlike the b o l l w e e v i l , O. tho lozani m a le s depend to a v e r y s m a l l extent on a m e a l f o r su r v iv a l and s u c c e s s f u l mating.T h us , in sp ite o f the inhibit ion in the rate o f d igest ion , m o r e than 50% o f i r ra d ia te d m a le t ick s su rv iv e fo r o v e r 5 - С m onths, during which p e r io d they cou ld be used f o r c o n t r o l p u rp o s e s . Though they a r e not fu lly c o m p e t i t iv e , th e ir e f fe c t on the extin ct ion o f the population is quite h ig h [ l ] .

I r ra d ia t ion did not a f fe c t the pe rce n ta ge o f t i ck s e n gorg in g im m e d ia t e ly a f te r the e x p o s u re . T h is is quite d i f fe re n t f r o m the beh av iou r o f the fe m a le t i ck A m b ly o m m a which d o e s not e n g o r g e a fter e x p o s u re to 5000 R [12].

Since b lood i s d ig ested m o r e s lo w ly by the ir ra d ia te d tick , a m uch lo n g e r in te rv a l i s r e q u ire d b e f o r e a se co n d b lood m e a l is taken. I r ra d ia ted m a le s w hich to o k a b lood m e a l a fte r trea tm en t did not e n g o rg e again b e fo r e death; n o r did nym phs e x p o se d to 16 000 R. H ow ever , about 30% o f the i r r a d ia te d f e m a le s did take a se c o n d b lood m e a l . Although this p e r c e n ta ge is low when c o m p a r e d with the c o r r e s p o n d in g value o f about 90% in the n o n - i r r a d ia te d fe m a le s , it is high enough to w arra n t se p arat ion o f m a le s and f e m a le s b e fo r e r e le a s in g the s t e r i le m a le s . Separation o f the s e x e s is e a s y due to the b ig d i f f e r e n c e in s ize between m a le and fe m a le . B e c a u s e o f th e ir s m a l l e r s i z e , nym phs w i l l contam inate the fr a c t io n which c o n ta in s the m a le s and w ill , t h e r e fo r e , be r e le a s e d tog eth er with them.T h e fac t that m a le s and nym phs do not take a se co n d b lood m e a l a fte r i r ra d ia t io n m a k es th e ir r e le a s e into natural populations p o s s ib le without adding any hazard to the en v iron m en t . T he on ly preca u t ion r e q u ir e d is to p ro v id e a m e a l b e fo r e r e le a s e .

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R E F E R E N C E S

[1 ] GALUN, R ., WARBURG, М ., AV1VI, A . , Ent. exp . app l. 10 (1967) 143.[2] KOZANCHIKOV, I. V . D okl. Akad. Nauk SSSR 32 (1941 ) 515.[3 ] BELOZEROV, V .N . , Ent. O bozr. 43 (1964 ) 288.[4 ] GALUN, R ., KINDLER, S .H . , S c ien ce 147 (1 965 ) 166.[5 ] GALUN, R .. Nature 185 (1960) 391.[6 ] GALUN, R ., WARBURG, М ., Proc. Int. Sym p. Insect Endocrines Brno (1 9 6 6 ) in the press.[7] AKOV, S . , Ann. trop. M ed. Parasit. 60 (1 966 ) 482 .[8 ] LEE, W .R ., J. a grie . Res. 3 (1964 ) l l T[9 ] RIEMANN, J .G ., FLINT, H .M ., Ann. ent. S oc. A m . 6 0 (1 9 6 7 ) 298.

[10] BALASHOV, J .S . Parazit. Z b . 20 (1 9 6 1 ) 185. _[11] MORTREUIL-LANG LOIS, М ., C . r. Séan c. S o c . Biol. 154 1769.[12 ] DRUMMOND, R .O ., MEDLEY, J .G ., GRAHAM, O .N . , Int. J. Radiat. B iol. 10 (1 966 ) 183.

D I S C U S S I O N

V. NIGON: Y o u r r e s u l t s on the e f fe c t o f d iet on r e s p ir a t io n in ticks a r e v e r y s im i la r to th ose obta ined at Lyon U n ivers ity on m e ta b o l ic changes in in s e c t la rv ae d e p r iv e d o f food . I r e f e r to F o u r c h e 's w o rk on D ro so p h i la m e la n o g a s te r , and that by F o u rc h e and C oulon on B om by x rnori. R ecent e x p e r im e n ts c o n f i r m that this regu lat ion o f the r e s p i r a t o r y m e ta b o l is m is dependent on a h o rm o n e m e ch a n ism .

W .J . K L O F T : When d e term in in g the r e sp ir a t io n quotient, D r. Galun, did you m e a su re C O 2 output as w e l l as O 2 c o n su m p tion ? I ask this beca u se o f the continuous u ti l iza t ion o f ox yg en which i s bound in la rg e quantities to the b lood e r y t h r o c y te s in the gut, even when venou s b lood is used . The t im e f o r which the t ick s t o r e s oxygen in its gut thus depends on the rate o f breakdow n o f the e r y t h r o c y te s during b lood d ig est ion . T h is oxygen may be a b s o r b e d by the body t i s s u e s o r r e le a s e d through the gut, but in any c a s e it r e m a in s in the w ork in g v e s s e l s and w il l in flu ence yo u r read in gs .

In Rhodnius p r o l i x i s , it s e e m s p o s s ib le to l ib e ra te a l l the oxygen f r o m the h aem og lob in r e la t iv e ly soon a f t e r feed ing , which leads to e x c e s s iv e p r e s s u r e in the v e s s e l s . Rhodnius h as -en d osym bion ts in the m id -gu t which a re se n s i t iv e to high oxygen p r e s s u r e , and the in s e c t th e r e fo re n e e d s a b lood deoxygena tion m e c h a n is m . We a i e at p resen t studying this p r o b le m at the U n iv ers ity o f Bonn.

R a ch e l G ALUN : W e attr ibuted the strange r e s u l t s o f o u r r e sp ira t io n quotient m e a s u re m e n ts to the fact that the COo is r e le a s e d in large bu rsts ra th e r than cont inu ou sly . We did not c o n s id e r the p o s s ib i l i t y o f 0 2 being bound to the b lood h aem og lob in .

K. K. NAIR: If you e x p r e s s e d y o u r data on oxygen consum ption per unit weight in stead o f per tick , would you find any d i f f e r e n c e between m a le and fe m a le ? If the sa m e w e re done a fte r a b lood m e a l , would there be any d ro p in O 2 c on su m p tion o r would it r e m a in m o r e o r l e s s at a constan t le v e l?

R a ch e l G ALU N : F e m a le s a re on ly 1. 5 t im e s h e a v ie r than m a le s and thus, p e r m g body weight, they take up m o r e o x yg en . A f t e r the b lood m ea l, the true weight o f the adult fe m a le d o e s not change; one should not c o n s id e r the b lood s t o re d in the gut as a part o f the body weight.

H. M A R C H A R T : W ould you p le a se s p e c i fy in what w ay a secon d b lo o d m e a l o f y o u r ir ra d ia te d t ick s , a fte r r e le a s e o f the t i ck s into natural popu lations , would add any hazard to the en v iron m en t?

25 8 G ALUN and WAKBURG

R a ch e l G ALUN: T h rou gh the ir r i ta t in g e f fe c t o f biting by la rg e num b e r s o f t i ck s on the one hand, and the p o s s ib i l i t y o f tran sm itt in g re la p s in g f e v e r on the o th er .

P . A . L A N G L E Y : la th ere no e x t r a - c e l lu la r en zym e activ ity in the gut o f the t ick?

R a ch e l GALUN: No.

17'

NEW SITE FOR THE E R A D I C A T I O N O F A WIIITE GRUB POPULATION (Melolontha vulgaris F abr. AND Melolontha hippocastani F a b r .)B Y RELEASE OF IRRADIATED ADULTS

E. IIORBERSWISS EXPERIMENT STA TIO N FOR AGRICULTURE,ZURICH-OERLIKON, SWITZERLAND

Abstract

NEW SITE FOR THE ERADICATION OF A WHITE GRUB POPULATION (M elolontha vulgaris Fabr.AND M elolontha h ippocastani Fabr.) BY RELEASE OF IRRADIATED ADLJLTS. T h e area considered (about 1300 ha) is situated on the Swiss shore o f Lake Constance and is infested with two cock ch a fer species ( M elolontha vulgaris Fabr. and M_. hippocastani F abr.). The dynam ics o f the cock ch a fer population were fo llow ed from ЗУ55 to 1967, Estimates o f the cock ch a fer population were obtained by: (1 ) soil sam pling for adults; (2 ) so il sam pling for white grubs; (3 ) observation o f flight, a ctiv ity and assessing dam age on preferred food trees; (4) capture-recapture m ethod with the aid o f m arked adults in spring 1967. Kstimates were m ade several lim es with the isotop ic dilution technique. Throughout the flight period 1967 the occu rren ce o f adults was observed along the border o f the Guttingerwald. Preferred food trees were regularly inspected and changes o f p referen ce wilh season w ere noted. Crepuscular flight activ ity o f adults was w atched at exposed com ers o f the w ood. Changes in the flight d irection during the period o f observation w ere related to sex ratios in sam ples taken the fo llow in g m orning and com pared with the developm ental stage o f the ovaries. Beetles were co lle c te d by shaking the host trees at dawn, and weight and volum e o f the sam ples were registered. Four therapeutical X -ra y units potentially u tilizab le for irradiating beetles are a v a ila b le within 35 km . The nearest fa c ility , 5 km aw ay, was tested for its daily capacity .

1 . IN TROD UCTION

In a p r e v io u s ly r e p o r te d f ie ld t r ia l c a r r i e d out in n o r t h -w e s t e r n Sw itzer lan d (1 9 5 8 -1 9 6 2 ) it w as d e m o n s tra te d [1, 2] that the s t e r i l e - m a l e tech n iq u e can s u c c e s s f u l l y e r a d ic a te a white g ru b popu lation . S ince this f ie ld t r ia l a r o u se d in te re s t a m on g f a r m e r s and app lied e n to m o lo g is t s , a new s ite f o r the ap p lica t ion o f th is tech nique was e x p lo r e d in n o r th e a s te r n Sw itzer lan d .

2. D E SC R IP T IO N O F THE REGION

T he r e g io n is s ituated in the Canton o f T hurgau on the S w iss sh o re o f L ake C on stan ce , s o m e 8 km east o f the town o f K on sta n z . The t e r r a in in c lu d e s the n o rth e rn s lo p e o f a range o f h ills with a ltitudes ranging f r o m 398 to 495 m above s e a l e v e l . T h is range is topped by an extended wood ca l le d the G iit t ingerw ald . In tensive g e n e r a l fa r m in g is p r a c t i s e d in the w hole a re a , with fru it t r e e s , g r a s s , c e r e a l s , p o ta toes , su g a r b e e ts , rape and g re e n v e g e ta b le s cu lt ivated on m o s t o f the ara b le s u r f a c e . T h re e p red o m in a n t ly ru r a l v i l la g e s , Altnau, Güttingen and K e s s w il , a re lo ca ted betw een the G üttingerw ald and L ake C on stan ce ( s e e F i g . 1 ) .

259

260 HORBER

FIG. 1. Map o f Sw itzerland showing the area explored for cock ch a fer (M elolontha sp. ) eradication by the release o f irradiated adults.

3. D E L IM IT A T IO N O F TH E IN F E ST E D A R E A

The in fested a r e a within the d e s c r ib e d r e g io n was outlined a c c o r d in g to the m ethod d e s c r ib e d by S ch n e id e r [3] . On 17 May 1967, when m a x im a l d e fo l ia t io n o f the oak t r e e s ap p eared at the e x p o se d b o r d e r s o f the w oods , the b o u n d ar ies o f the a r e a s m o s t at trac t ive to c o c k c h a f e r s w e re d e te rm in e d with the aid o f an in cl in ation m e t e r , and th ese poin ts w e re then f ix ed on a m a p ( s c a l e 1 : 25 000). The l in e s conn ect in g th e s e points f o r m e d the bou n d ary around the in fested a r e a . T h is a r e a m e a s u r e d 1128 ha includ ing tw o v i l la g e s , Altnau and Giittingen.

4. S U R V E Y ON P O P U L A T IO N DYNAMICS

The re g io n c o n s id e r e d was in fested with two c o c k c h a fe r s p e c ie s , M e lo lon th a v u lg ar is F a b r . and M . h ip pocastan i F a b r . The fl ight p e r io d within the tes t a re a o c c u r r e d s im u lta n e o u s ly with that o f the 'U r i c y c l e ' ( f l ight in 1967), w h ereas in the ad jacent n e igh bou rh ood s to the east and w est the flight o c c u r r e d s im u lta n e o u s ly with the 'B e r n e s e c y c l e ' (flight in 1966). Population d y n a m ics w ere stud ied in con n ect ion with a su r v e y

SM -102/32 261

s tarted in the cen tra l part o f the Canton o f Thurgau fo l low in g an in s e c t i c id e ca m p a ig n against th is pest in 1 949 n e a r Sulgen. In 1 952 the su rrou n d in g s o f Altnau w e re included in th is s u r v e y . F r o m 1.955 to 1967 this s u r v e y was continued e v e r y th re e y e a r s in the su rrou n d in g s o f the th r e e ad jacen t v i l la g e s o f Altnau, Güttingen and K e s s w i l ( s e e T a b le s I and II) and c o m pared with s e v e r a l a r e a s o f the ce n tra l part o f the Canton o f Thurgau .

T A B L E I. C O C K C H A F E R P O P U L A T I O N D E T E R M I N E D B Y T H E S A M P L I N G T E C H N IQ U E

L oca lity 1952 1955 1958 1961 1964 1967

Altnau 3 . 2 4 . 6 1 .2 3 .2 0 .9 2 .0

G tlttingen - - 1 .5 1 .8 0 .9 1 .0

T A B L E II. W H IT E G R U B P O P U L A T I O N D E T E R M I N E D B Y T H E S A M P L I N G T E C H N IQ U E

L oca lity 1952 1955 1958 1961 1964 1967

A ltnau 8 .9 2 .4 6 .2 1 .4 5 .2 1 5 .3

GUttingen - 6 .6 5 .8 1 .8 1 .3 5 .5

5. SA M PLIN G TECHNIQUES

5 . 1 . So i l sa m p l in g f o r adults

In autumn o r sp r in g b e fo r e the flight s tar ted , the population density was es t im a te d by digg ing and se a rc h in g through sa m p l in g units o f 0 .2 5 m 2 o f s u r fa c e a r e a down to a depth o f 20 - 40 c m . F r o m 4 to 11 p lots o f g ra ss la n d o f about 0 .4 to 0. 6 ha w e r e c h o s e n in e a ch a r e a . In each plot 16 random 0 . 2 5 - m 2 sa m p lin g units w e re a v e r a g e d . The n u m ber and se x o f b e e t le s w e re r e c o r d e d se p a r a te ly f o r e a ch plot.

5 . 2 . So i l sa m p lin g f o r white g ru bs

T h is p r o c e d u r e was re p eated during late s u m m e r and autumn o f the f l ight y e a r . The sa m p l in g units w e r e dug in the s a m e p lots as in the

262 HORBER

p r e v io u s s u r v e y s . F o r id e n t i f ica t ion p u rp o s e s the p lo ts w e re n u m bered on a sk e t ch -p la n f o r each a r e a and a lso on a 1 : 25 000 s c a le su rv e y m ap l'or the w hole r e g io n . T he counts o f c o c k c h a f e r s and white g ru bs s e r v e d f o r the es t im a t io n o f :

( a) the population d en sity , se x ratio , and d is tr ibu t ion o f c o c k c h a f e r s just b e fo r e the flight p er iod ;

(b) the population d en s ity and d is tr ibu t ion o f white g ru bs a fte r the flight p e r io d ;

(c ) the rate o f r e p ro d u c t io n a fte r the flight p e r io d , obtained by div id ing the n u m b e r o f white g ru bs counted in a g iven plot o r a re a by the n u m b e r o f c o c k c h a f e r s counted in the s a m e plot o r area b e f o r e the f light p er iod ;

(d) the m o r ta l i t y in the th re e y e a r s betw een s u c c e s s iv e white gru b o r c o c k c h a f e r cou n ts .

5 . 3 . O b s e rv a t io n o f flight act iv ity

F o r the flight p e r io d during sp r in g 1967, th re e points o f o b s e r v a t io n w e re c h o s e n at an e x p o se d angle on the w est b o r d e r o f the G üttingerw ald , o v e r lo o k in g the su rrou n d in g s o f Altnau. R e c o r d s w e re taken o f m e t e o r o l o g i c a l con d it ion s , o f the n u m b e r o f f ly ing b e e t le s at 5 -m in u te in te rv a ls and o f .th e d ir e c t io n o f th e ir f light to o r f r o m the wood. T h e se o b s e r vations w e re m ade e v e r y even in g at one point on ly , beginning on 8 May, o n e day a fte r the f i r s t m a s s flight, until 24 May, when m o s t o f the f e m a le s had returned f r o m the f i r s t o v ip o s i t io n flight (s e e T ab le III).At the two o th e r points o b s e r v a t io n s w e re c a r r i e d out i r r e g u la r ly .

5 . 4 . A s s e s s in g d am age on p r e f e r r e d food t r e e s

At w eekly in terva ls , beginning on 20 A p r i l , the b o r d e r s o f the Güttingerw ald as w ell as th ose o f the neigh bou rin g w oods w e re in sp e c te d f o r feed ing by c o c k c h a f e r s . YVe in sp ected Q u e r cu s R obur L . , F agus s i lv á t i ca L . , A c e r cam pest re L . , Juglans r e g ia L . , P runus c e r a s u s L , . , P . d o m e s t i c a L . , and Popu lus tré m u la L . which are p r e f e r r e d by c o c k c h a f e r s . On 21 June, when the last in sp e c t io n to u r was c a r r ie d out, the oak t r e e s w e re pa rt ia lly , and at s e v e r a l points a lm o s t c o m p le te ly , d e fo l ia ted by T o r t r ix v ir id an a L . The en t ire 1 3 .2 - k m length o f the b o r d e r o f the w oods was c la s s i f i e d as shown in T a b le IV.

5 . 5 . C a p tu r e -r e c a p tu r e o f m a rk e d adults

T o est im a te the s i z e o f the c o c k c h a fe r population and to fo l low its d i s p e r s a l , b e e t le s w ere captured e a r ly in the m o r n in g f r o m 9 to 13 May,19 to 20 May arid 23 to 24 M ay. A g rou p o f th ree m en was equipped with a la d d e r , a hooked rod, a 4 X 4 m can vas and a s e r i e s o f w ire c a g e s and sh ook the t r e e s to c o l le c t the b e e t le s on the can va s and confine them in the c a g e s . T he captured b e e t le s w e re t ra n sp o rte d to a c o o l in g fa c i l i ty at the l o c a l f a r m e r s ' c o o p e r a t iv e . S a m p les w e re taken f r o m each lo t to d e te rm in e the weight o f the b e e t le s , the se x ra tio and the o c c u r r e n c e o f the two s p e c i e s o f M e lo lo n th a . A ft e r the r e le a s e and re ca p tu re o f m a rk e d b e e t le s , ind iv iduals w e re in sp e c te d by a Raytech u .v . la m p M od el LS-7

SM - 102/32 263

T A B L E III. F L I G H T A C T I V I T Y A N D S E X R A T I O O B S E R V E D N E A R A L T N A U IN S P R IN G 1967

D ate ( in M ay)

Flight d ir e c te d tow ards:

w ood fi e ldS ex ra tio

m a le / fe m a leO bserv ation s on o v ip o s it io n

8 2620 12 1 .3

И 2846 6 1 .8

10 2431 27 -

11 1719 34 1 .8

12 536 273 2 .1 O v ip o s it io n starts

13 3S7 1880 -

14 266 16«Г>

15 12 3 -

16 99 ' 73 -

17 452 40 -

18 202 30

19 1769 1852 3 .1 F em ales return to the w oods

20 214 109 1 .9

21 У10 553 -

22 1084 350 2 .0

23 - - -

24 504 100 In creasin g fligh t a c t iv ity during d a y t im e

264 HORBER

T A B L E I V . C L A S S I F I C A T I O N O F D A M A G E A L O N G T H E B O R D E R O F T H E W O O D S

D a m a ge c la ss if ie d as

D egree o f d e fo lia t io n Length o f the b order (km)

S ev ere Oaks c o m p le t e ly d e fo lia te d , exp osed b e e ch e s h ave 1 in o f branches d e fo lia te d 4.2

M ed iu m Oaks 1/4 - 1/2 d e fo lia te d , exp osed b e e ch e s have 1/2 - 1 m o f branches d e fo lia te d 2.0

Slight Oaks d e fo lia te d on ly at the ends o f exposed branches 7.0

T o ta l len gth o f in sp ected border 13.2

T A B L E V . C O C K C H A F E R S C A P T U R E D IN A L I G H T T R A P

D ate op era ted ( in M ay)

C o ck ch a fe rs trapped:

m ales fem a lesT o ta l M arked in d iv id u als

18 84 84 1

19 44 322 366 2

22 77 25 102 -

24 10 65 75 1

T o ta l: 131 496 627 4 = 0.64"/o

(R a y te ch Equ ipm en t C o . , S o m e r s , C o n n . , USA). S a m p le s o f 20 to 30 f e m a le s w ere d is s e c te d to fo l low the d ev e lop m en t o f o v a r i e s and o v ip o s i t io n .

5 . 6 . M arking o f b e e t le s with f lu o re s ce n t dyes

T he two d y es u tilized f o r the bulk o f the b e e t le s w e re 'N it r o s te l la ' Red and Y e l low (Stella SA, Chatelaine, G en èv e ) . The red dye was based on BASF 'L u in og en LT fe u e r r o t ' and the y e l lo w dye on BASF 'L u m o g e n

S M - 102/32 265

L T h e l lg e ib 1. F r o m 9 to 11 M ay and on 18 M ay a total o f 1.5 kg (about 1 5 000 ind iv iduals ) o f r e d - m a r k e d b e e t le s w e r e r e le a s e d n e a r the c e n tra l o b s e r v a t io n point. On 11 to 12 M ay and 17 to 18 May a to ta l o f 21 kg (about 21 000 indiv iduals ) o f y e l lo w -m a r k e d b e e t le s w e re r e le a s e d at p t .5 01.

A s m a l l e r lot o f 1937 indiv iduals handpainted with a w h ite -b lu ish le a th e r dye was r e le a s e d on 18 M ay near the th ird o b s e r v a t io n point, w here a light t rap was o p e r a te d .

5 . 7 . Captu res in a light t ra p

A light t ra p was o p e ra te d n ear the o b s e r v a t io n point A . T w o b e d - sh eets w e re fasten ed 4 m in fron t o f two 125-w att m e r c u r y v ap ou r la m p s . In tercepted b e e t le s w e re c o l le c t e d m anually on both s id e s o f the sheets ( s e e T ab le V) f r o m 1 8 - 1 9 May and on 22 and 24 May.

6. PO P U L AT IO N E ST IM AT ES WITH THE ISOTO PIC .DILUTION TECHNIQUE

At two o b s e rv a t io n poin ts , the population w as est im a te d by the i s o to p ic dilution m ethod . At the cen tra l o b s e rv a t io n point A, w here r e d - m a rk e d b e e t le s w e re r e le a s e d , the population den sity w'as h igh er than at the p e r ip h e ra l point B, w here y e l lo w -m a r k e d b e e t le s w e re re le a s e d ( s e e T ab le VI). T h is e s t im a te was c o m p a re d with the da ily flight o b s e r vations and the dam age a s s e s s m e n t on p r e fe r r e d food t r e e s .

The population e s t im a te obta ined by the i s o to p ic d ilution m ethod fo r two s e le c t e d points was e x trap o la ted fo r the whole length o f the 1 3 .2 -k m b o r d e r o f the in fested w o o d s . T h is was done a c c o r d in g to the d e g r e e o f o b s e r v e d de fo l ia t ion and c la s s i f i e d in se c t io n 5 . 4 . The c o c k c h a fe r popu la t ion within the d e l im ited area was est im ated to be betw een 5 1 / 2 to 25 l / 2 m i l l io n b e e t le s (s e e T a b le VII).

fi. 1. O b se rv a t io n o f f light beh av iou r

The fluctuation o f the population e s t im a te s m a y be exp la ined by the flight be h a v io u r . The f i r s t o v ip o s i t io n f l ights began on 12 May. During the even in gs o f 1 7 to 19 May the d ir e c t io n o f the m a in fl ight act iv ity r e v e r s e d , ind icat ing that f e m a le s w e re returning f r o m the f i r s t o v i p o s it io n . A f t e r 24 May flight ac t iv ity g radually sh ifted to d is p e r s a l during dayt im e .

6 . 2 . Sex ratio

F r o m 12 to 19 May the ratio o f m a le / f e m a l e in c r e a s e d to 3. 1 and then drop ped to 1. 9 ( s e e T a b le III). T h is trend was o b s e r v e d f o r all o b s e r vation points and was c o r r e la t e d with the b eh av iou r o f f e m a le s as they returned to the population a fter o v ip o s i t io n .

T A B L E V I . E S T I M A T E D C O C K C H A F E R P O P U L A T I O N O F M A R K E D B E E T L E S A T T W O R E L E A S I N G P O IN T S

R eleasingpoint

P eriod o f o b se rv a tio n ( in M a y )

Individualsreleased

M arkedin d iv id u a lsrecaptu red

Proportionm arked

in d iv id u a ls

№ )

C o rre c t io nfa cto r

P op u la tione s tim a te

A

H angeten w est 10 4032 15 /2688 - 0 . 56 0 .9 5 5 679 000

11 8736 3 8 /2 5 2 0 1 .5 0 . 965 55 0 400

north 12 6020 8 /4 5 1 2 0 .1 8 0 .9 3 3 100 000

13 10820 3 6 /2 6 4 0 1 .3 6 0 ,9 8 6 77 7 000

W est + north 13 19556 6 4 /2 9 5 7 ■2. 16 0 . 965 85 4 000

В

Pt. 501 19 2327 2 8 /4 8 0 0 0 .5 8 0 .9 8 7 3 9 4 000

20 7240 1 1 4 /3 0 2 0 3 .7 8 0 .9 2 8 169 500

2 4 10832 7 9 /4 2 3 1 1 .8 0 .9 2 8 547 000

266 H

OR

BE

R

T A B L E V II . E S T I M A T E D C O C K C H A F E R P O P U L A T I O N A L O N G W O O D S IN T H E I N F E S T E D A R E A

E x a m p le

L ength o f

in fested b order

fm )

P op u la tion es t im a te

( is o t o p ic d ilu tio n m eth od )

m in im u m m a x im u m length(k m )

E xtrapolated e s t im a te on to ta l

len gth o f borders o f w oods

m in im u mp op u la tion

m a x im u mp op u la tion

H an geten 600 5 5 0 0 0 0 3 1 0 0 0 0 0 4 . 2 3 740 000 2 1 1 0 0 000

Pt. 501 300 170 000 500 000 ■ 2 .0 1 200 000 3 5 0 0 000

H orb en 700 50 000 100 000 7 .0 500 000 1 0 0 0 000

■ 1 3 .2 5 400 000 25 600 000У

SM - 1U 2,32

267

2 68 IIORBER

6 . 3 . O v a r ia l dev e lop m en t

On 13 M ay th r e e - q u a r t e r s o f the f e m a le s captured in the m o rn in g and d is s e c te d had been read y to o v ip o s i t . On 23 May, 5% o f the fe m a le s w e re ready f o r the se co n d o v ip o s i t io n .

6 . 4 . D istr ibu t ion o f the two s p e c ie s (М . v u lg a r is F a b r . and M . h ippocastan i F a b r . )

M . h ippocastan i r e p re s e n te d 3 .3 to 13 .6% o f the c o c k c h a fe r s c o l le c t e d , and 1. 5 to 4 .2 % at points A and B, r e s p e c t iv e ly .

7. P O P U L A T IO N E S T IM A T E S WITH THE SA M PLIN G TECHNIQUES IN THE F IE L D

In sp r in g 1967, we found an av e rag e o f 2 c o c k c h a fe r s ready to e m e r g e p e r m 2 at Altnau and 1 p e r m 2 at Güttingen (s e e T a b le I). B a sed on th ese f ig u r e s the e st im a ted population would be 1 7 .5 m i l l i o n b e e t le s within the d e l im ite d in fested a r e a o f 1128 ha.

8. IR R A D IA T IO N OF B E E T L E S

Of fo u r th erapeu tic X - r a y units potent ia lly ava i lab le at d i f fe re n t h o sp ita ls within d is ta n ce s o f between 5 to 35 km f r o m the in fested area , the n e a r e s t on e at the Canton H osp ita l M ün ster l in g en w as te s te d f o r its ca p a c ity . T w o 'S ie m e n s S tab il ivo lt ' w e re o p e ra te d at 163 and 156 kV,6 m A and 2 m m AI f i l t ra t ion . T hey p ro d u ce d 94 and 8G R / m i n with a d is ta n ce o f 30 cm betw een tube and su b je c t . The rad ia tion f ie ld was 15 X 19 c m . T w o c a r d b o a rd b o x e s o f th is s i z e , each with a 6 cm la y e r

T A B L E V III . W E I G H T O F IN D IV ID U A L C O C K C I I A F E R S (a)IN T H E C A N T O N O F T H U R G A U

Flightyear

N um berw e igh ed

M ales

(g )

N um berw eigh ed

Fem ales

(g )

1958 249 1 .0 4 6 0 229 1 .1 9 1 3

1961 90 1 .0 7 1 6 86 1 .2 4 1 8

1964 13 1 .2 1 2 4 17 1 .3 8 9 3

1967 49 1 .1 1 9 2 38 1 .3 3 7 9

M e lo lo n lh a vu lgaris Fabr. w e igh ed b e fo re flight

SM - 1 02/32 2 69

o f c o c k c h a f e r s o r about 4 l i t r e s o f b e e t le s , w e re e x p o se d at a t im e to 5000 R m e a s u r e d on top o f the b o x e s . The c h o ic e o f th is d o s e was ba sed on la b o r a t o r y te s ts (1955 - 1958) and a f ie ld e x p e r im e n t (1959 - 1965) p r e v i o u s ly r e p o r te d [1, 2] . D o se m e a s u r e m e n t s taken on top , betw een and be low the b o x e s w e re 9 4 .3 , 5 6 .0 and 3 9 .0 R / m i n . T o a s s u r e h o m o g e n e o u s i r ra d ia t io n o f the b e e t le s , the p os it ion o f the two b o x e s w as r e v e r s e d at half e x p o s u r e t im e . A tota l o f 60 l i t r e s o r about 21 000 b e e t le s w e r e ir r a d ia te d and r e le a s e d a fte r be ing m a rk e d with f lu o r e s c e n t d y es as d e s c r ib e d in s e c t i o n 5 . 6 .

9. HANDLING AN D R E L E A S E O F B E E T L E S

The handling o f c o c k c h a f e r s destined to be i r ra d ia te d and r e le a s e d in v o lv e d the fo l lo w in g o p e r a t io n s at v a r io u s l o c a l i t i e s and t im e s :

O p era t ion s L o c a l i t ie s T im e

Capture o f B e e t le s F ie ld M orn in g

P r o c e s s in g : C oo l in g

D e te rm in a t io n o f

F a r m e r s ' c o o p e r a t iv e M orn ing

weight and v o lu m e F ie ld la b o r a t o r y M orn in g

Sex ratio F ie ld la b o r a to r y M orn in g

O v a r ia l d e v e lo p m e n t F ie ld la b o r a to r y M orn in g

S ep arat ion o f s p e c ie s F ie ld la b o r a to r y M orn in g

M arking F ie ld la b o r a to r y M orn in g

P ack in g F ie ld la b o r a to r y M orn in g

Ir ra d ia t io n H osp ita l A fte rn o o n

R e le a se F ie ld Even ing

O b s e rv a t io n o f flight act iv ity F ie ld Even ing

C o c k c h a fe r s a re robu st and can e a s i ly be handled without being d a m ag ed . An ind iv idual w eighs a p p ro x im a te ly 1 g, but weight v a r ie s with lo c a l i ty , y e a r and se x ( s e e T a b le VIII). T he 1967 c o c k c h a f e r s w e re o f a v e r a g e s i z e and w eight. An av e rag e o f 338 b e e t le s p e r l i t r e was o b s e r v e d (ran ge : 310 - 380) in c o o le d condition ; the v o lu m e was g r e a te r when the s a m p le s w ere w arm ed and ag itated .

During t ra n sp o rta t io n b e e t le s w e re left at about 4°C . D ry v e r m ic u l i t e o r sawdust was f i l le d into the b o x e s to a b s o r b e x c e s s iv e m o i s tu r e and e x c r e t i o n s . Surplus b e e t le s w e re kept in w ire c a g e s at the f a r m e r s ' c o o p e r a t iv e fru it c e l l a r with + 1°C and 9C% re la t iv e h um idity .

10. CONCLUSIONS

In the r e g io n s u r v e y e d the c o c k c h a fe r popu lation c o n s is t e d p r e d o m i nantly o f M elo lon th a v u lg a r is F a b r . In festation was lo c a l i z e d in an area

2 70 MORDER

o f ] 128 ha bounded by the 1 3 . 2 km b o r d e r o f the w ood . During flight th e se b e e t le s aggregated on a s t re tch o f 6. 2 km o f the b o r d e r l in e . T he m a in con ce n tra t io n o c c u r r e d on a length o f 4. 2 km,' w here oaks w ere c o m p le t e ly de fo l ia ted in the f i r s t 10 days o f flight.

The s i z e o f th is population was es t im a te d by two independent m eth od s to be betw een 5 1 /2 and 25 1 /2 m i l l io n and 17 . 5 m il l ion , r e s p e c t iv e ly .The n e a r e s t i r ra d ia t io n fa c i l i ty was in a h osp ita l 5 km f r o m the in fested a re a with two th erapeu tic X - r a y units and had a daily output o f 20 l i t r e s . T h is insta llation p roved inadequate to trea t th is amount o f b e e t le s within on e flight p e r io d . It would be n e c e s s a r y to p rov id e a s o u r c e with a ca p a c ity o f 2000 - 3000 l i t r e s / d a y n ear the s ite o r to s u b -d iv id e the a re a arid t re a t the d if fe ren t s e c t i o n s in c o n s e cu t iv e flight p e r io d s .


T h is r e s e a r c h was su p ported by a s p e c ia l grant r e c e iv e d f r o m the D iv is io n o f A g r i cu l tu r e o f the F e d e r a l D epartm ent o f P u b lic E c o n o m y ,B e r n . T he author is m uch indebted to D r. W'alter and to M is s jVi. Chiot and M is s S. B o s s a r d f o r th e ir a s s is ta n c e during the i r ra d ia t io n e x p e r im e n ts at the Canton H osp ita l M ü n ste rü n g e n . M o s t o f the sa m p l in g and o th e r p r a c t i c a l f ie ld w ork was c a r r i e d out by M e s s r s A . Fekti , H. I lausw irth and H. R ie d e r . The author takes this op portu n ity to thank them f o r th e ir c a r e fu l w'ork.

R E F E R E N C E S

[1 ] HORBFR, E. , In Radiation and Radioisotopes Applied to Tnsects o f Agricultural Im portance, IAL’A, Vienna (1963) 313 -32 .

[•21 HORBER, E ., W aldhygiene 6 6 (1966) 161-70 .[3 ] Schneider. F . , M itt, sch w eiz . Landw. 2 2 (1954) 17 -34 .

D I S C U S S I O N

B. NAGY: A r e th ere d i f f e r e n c e s in the r a d io s e n s it iv i ty o f the twoc o c k c h a f e r s p e c ie s ( M elo lontha v u lg a r is and M_. h ip p o ca s ta n i) ? T h is m ight p resen t d i f f i cu lt ie s in the use o f the s t e r i l e - m a le tech n ique in the c a s e o f a m ix e d population o f the two s p e c i e s . A ls o , f r o m the data you obta ined f r o m i ig h t - t r a p s , have you found any d i f f e r e n c e s in light se n s i t iv i ty

(a) o f young and o ld c h a fe r s r e s p e c t iv e ly , i . e . during the f i r s t and la s t part o f the flight p e r io d , and

(b) betw een adults that have not yet fed and th ose that h ave? 1E . H O R B E R : We set up an e x p e r im e n t to d e te rm in e the d o s e l e v e l

f o r s t e r i l iz a t io n o f both s p e c ie s , but unfortunately th is was not s u c c e s s ful b e c a u s e o f the ab il i ty o f the b e e t le s to detect X - r a y s . Som e o f the b e e t le s m anaged to lea ve the phantom and to m o v e out o f the X - r a y beam during e x p o su re , and the r e m a in d e r which r e c e iv e d the planned d o s e was to o s m a l l to r e p re s e n t the w hole s p e c i e s . A s a p r e l im in a r y re su lt we obta ined a h igh er d o s e l e v e l f o r M. h ip p o ca s ta n i . T he r e s u l t s w e re hard to in terp re t , b e c a u se a fte r a sh arp d r o p in hatchability at 3000 R,

S M - 102/32 271

a lon g tail o f r e s id u a l hatching at h igh er d o s e s was o b s e r v e d ; even at 12 000 R s o m e la rv a e hatched, but fu rth er d e v e lop m en t fa i led .

The ca p tu re s in the l ig h t - t r a p w e re l e s s than th o se ex p e cte d on the b a s i s o f p r e v io u s t r i a l s . F e m a le s w e re m o r e abundant, which was a lso in c o n tra st to p r e v io u s e x p e r i e n c e . A s th e se r e su lts a re not r e p re s e n ta t iv e , I cannot r e a l ly a n sw e r the quest ion .

W. J. K L O F T : Y o u r p la n t o s t o r e adult c o c k c h a f e r s at low t e m p e r a tu re f r o m one flight s e a s o n to the next s e e m s v e r y p r o m is in g , and cou ld i n c r e a s e the e f f i c i e n c y o f y o u r m ethod , which is at p re se n t unique in that it u se s on ly natural p op u la tion s . Can you s t o r e the in s e c ts both b e fo r e and a fter feed in g has s ta r te d ?

E . ITOJ.ÎBER: No. It is e a s y to s t o r e c o c k c h a f e r s f o r m o r e thana y e a r at +1°C and m o r e than 90% re la t iv e hum idity , p r o v id e d that they a re s t i l l in d iapause when c o l le c t e d , but they cannot be s t o re d f o r m o r e than a few w eek s under th e se cond it ion s i f feed in g has s ta r te d . We are now try ing to a s c e r ta in w hether they can be s to re d i f c o l l e c t e d during the f i r s t f light a fte r e m e r g e n c e , but b e fo r e feed in g .

E X P L O R A T O R Y STUDIES ON THE ERADICATION OF THE KOREAN .PINE C ATER PILLAR Dendrolimus spectabilis BUTLER BY MEANS OF RADIATION*

K .B . LEE, U .K . HWANG AND K .S . LEE BIOLOGY DIVISION,A TO M IC ENERGY RESEARCH INSTITUTE,SEOUL, REPUBLIC OF KOREA

Abstract

EXPLORATORY STUDIES ON THE ERADICATION OF THE KOREAN PINE CATERPILLAR D endrolim us Spectabilis BUTLER BY MEANS OF RADIATION. Experiments to develop suitable a rtific ia l diets to rear the larvae o f the Korean pine caterp illar D endrolim us spectab ilis Butler w ere carried out in con n ection with studies on eradication by the ste r ile -m a le tech n iqu e. Several nurritient m edia w ere tested on larvae o f various instar stages, and one was found to be most successful, rearing 2nd instar larvae without loss o f body w eight. M ortality at 30 days after a rtific ia l feed in g was less than on e-th ird . This m edium consisted o f sucrose, L -ascorb ic acid , 0 -sitosterol, yeast hydrolysate, brew er's yeast, Butoben, ch o lin e ch loride , residue o f pine leaves and sodium glutam ate. The com position o f the pine leaves as w ell as o f the larvae was analysed to obtain inform ation 011 the nutritional requirem ent o f the larvae. The larval m etabolism o f ca rb o hydrate, sterol and protein» was also studied. The radiosensitivity o f the larvae o f the 1st through4th instar stages is dose-dependen t and radioresistance increases with instar a ge . Doses o f 100 krad or less did not produce m ortality in the 4th instar, and could be used as the steriliz ing dose for irradiation o f o lder larvae or pupae. The number o f eggs laid by a norm al fem a le m ated with m ales irradiated with a dose o f betw een 1 .5 and 100 krad during the pupal stage did not ch an ge. T h e hatchab ility , how ever, was a ffected by irradiation o f 15 krad or m ore where co m p le te absence o f hatching was observed. With 5 krad or less hatchab ility was not reduced from norm al (5 5 .8% ) w hile 10 krad resulted in partial reduction . The sexual com petitiveness o f m ales irradiated with 15 and 25 krad was about o n e -h a lf o f the norm al m ales and the hatchab ility o f the eggs was reduced by about 50% to 37 and 31% resp ective ly . It was conclu ded that the use o f the s ter ile -m a le method in the eradication o f the pine caterp illar is feasib le i f the m ethod for mass rearing o f the larvae is successful. Work in this con n ection is in progress.

1. IN TROD UCTION

The pine c a te r p i l la r D en d ro l im u s sp e c ta b i l is B u tler is a se r io u s d e s tr u c t iv e pest in K o re a n fo r e s t s and the dev e lopm en t o f m ethod s to c o n t ro l it is a task o f s o m e u rgency , s in c e the pine is one o f the m ost im portant f o r e s t r y p ro d u c ts . The in se c t was f i r s t d e s c r ib e d by Hutler in 1877 [1], and m any r e p o r t s have been published s in ce then, mainly in Japan ese and K o re a n s c ie n t i f i c jou rn a ls , on its e n to m o lo g ic a l d e s c r ip t i o n [1 - 3 ] , the da m ag e it d o e s to t r e e s , its b io n o m ic a l beh av iou r [4 -6 ] and p o s s ib le m ea n s o f e x term in at ion and preven tion . Though

* Experiments perform ed under IAEA Contracts N o.27 7 /R B , 277/R 1/R B and 277 /R 2/R B during the period 1 9 6 5 - 1967. Som e o f the results were presented at the Panel on Radiation, Radioisotopes and Rearing M ethods in the C ontrol o f Insect Pests, held in T e l-A v iv , Israel, 1 7 - 2 1 O ctober 1966.

273

27 4 LEE et a l .

many attem pts have been m ade to c o n t ro l the in sec t , no r e a l ly e f fe c t iv e l o n g - t e r m method has as yet been e v o lved .

S ince the e rad ica t ion o f the s c r e w - w o r m fly by the s t e r i l e - m a le technique [ 7 - 1 1 ] on the is land o f C u raçao , many w o r k e r s around the w o r ld have been applying this method to o th er in se c ts , e . g . the fruit fly , the bo l l w eev il , and the cod lin g m oth. T h is p r o je c t is the f i r s t a ttem pt to use this p r o c e d u r e f o r c o n tro l o f the pine c a te r p i l la r .

B a s ic know led ge o f the pine moth indicated that it was fe a s ib le to c o n t r o l the in se c t by m ea n s o f gam m a rad iation . T he pest has one host and h as sh ort pupal (about 15 days) and adult s ta g e s . The la tter stage is about 8 days , so that any rad iation e f fe c t on fe r t i l i ty is s oon evident.T he egg p e r io d is 6 days and the la rv a l stage is about 10 m on th s. A ft e r the e c o l o g i c a l c h a r a c t e r i s t i c s had been re v ie w e d , e s p e c ia l ly th ose c o n c e rn in g re p ro d u c t iv e beh av iou r , the fe a s ib i l i t y o f applying the s t e r i l e - m a le technique to e ra d ica t io n o f the in s e c t by gam m a rad ia tion was e x a m in e d . In v iew o f the p r o m is in g r e su lt s o f ou r p r e v io u s e x p e r im e n ts on rad ia tion e f fe c t s on the pine moth [12], we dec id ed to d e v e lo p an a r t i f i c i a l diet f o r m a ss r e a r in g o f the in se c t f r o m the la rv a l stage, which could lead to s u c c e s s fu l app lica t ion o f the s t e r i l e - m a le tech nique . Due to the c o m p le x ity o f the p r o b le m and no in form at ion pertinent to a r t i f i c ia l r e a r in g , w e analysed the c o m p o s i t io n o f pine le a v e s which f o r m the natural nutrit ional s o u r c e o f this in se c t . T he ra d io se n s it iv i ty , the fe r t i l i ty and the sexu a l c o m p e t i t iv e n e ss o f i r ra d ia ted m a le s w e r e a l s o stud ied .

2. E X P E R I M E N T A L PR O C E D U R E S

2 . 1 . L i f e c y c l e o f the pine moth

B e f o r e and during the e x p e r im e n ts , data gathered on the l i fe c y c l e and b e h a v io u ra l a s p e c ts o f the m oth c o n f i r m e d th o se published s e v e r a l d e c a d e s ago [3, 8].

2 . 2 . R a d io se n s it iv ity o f youn g la rv ae

T he la rv a e o f 1st through 5th in s ta rs w e r e c o l l e c t e d (August and D e c e m b e r ) f r o m the b r a n c h e s and bark ( fo r h ibernating 5th in s ta rs ) o f p ine t r e e s , and the la rv a e o f each in star w e r e d iv ided into equal l o t s . In a la r g e r o o m (25 °C , 60% r e la t iv e hum idity), s c r e e n e d against d i r e c t sun ra y s , each e x p e r im e n ta l lot c o n s is t in g o f 30 to 50 la rv a e was p la ce d in a c y l in d r i c a l v iny l m esh ca g e (height 60 c m , d ia m e t e r 20 c m ) . T o s im u late the natural en v iron m en t , f r e s h tw igs o f pine w e r e cont inu ou sly supplied in the c a g e . A ft e r a few days o f r e a r in g by this s e m i - a r t i f i c i a l m ethod , the la r v a e o f each in star w e r e again d iv ided into nine e x p e r im e n ta l g ro u p s f o r i r ra d ia t io n , ranging f r o m O(control) to 600 kR . T he i r ra d ia t io n was g iven by a SOCo p a n o r a m ic i r r a d ia t o r (cap acity 1000 C i, d o s e ra te 2500 R /m i n , ta rg e t d is ta n ce 7 c m ) . The ir ra d ia te d la rv a e w e r e retu rn ed to the ca g e and r e a r e d as b e f o r e . M o rta l ity was r e c o r d e d da ily until 10 days a fter t rea tm en t .

SM-102/29 275

2 . 3 . F e r t i l i t y o f adult m a le s i r ra d ia te d during the pupal stage

T h e la rv a e o f 7th and 8th in s ta r s w e r e c o l l e c t e d f r o m A p r i l to July and r e a r e d as d e s c r ib e d . A f t e r the la r v a e b e c a m e pupae, the m a le s w e r e se p a ra te d f r o m the f e m a le s . At 10 to 13 days o f pupal age , the m a le s w e r e d iv ided into e le v e n e x p e r im e n ta l g ro u p s , each c o n s is t in g o f 40 to 80 pupae, f o r i r ra d ia t io n ran g in g f r o m 0 to 100 kR . T he ir ra d ia te d m a le pupae w e r e caged as b e f o r e until e m e r g e n c e , which took p la c e a w eek to ten days a fte r tre a tm e n t . Upon e m e r g e n c e , w hich o c c u r r e d in late a f t e r noon, an indiv idual m a le adult m oth w as p la ced in a s m a l l e r ca g e c o n taining a v irg in fe m a le adult m oth . C opu la tion w as o b s e r v e d and a few days a fte r the m ating o v ip o s i t io n began . T he e g gs la id by the fe m a le on the tw igs o f the p ine w e r e c o l l e c t e d da i ly in p y re x t e s t - t u b e s ( 1 5 X 2 . 5 cm ) and counted . A f t e r the c o l l e c t i o n w as co m p le te d , the m ouths o f the t e s t - tubes w e r e c o v e r e d by p ie c e s o f nylon net c lo th and s t o re d in the r o o m .T he p e r cent hatchabil ity o f the eggs in each e x p e r im e n ta l g ro u p w as then d e te rm in e d .

2 . 4 . F e e d in g e x p e r im e n t with the a r t i f i c i a l diet

P in e - c a t e r p i l l a r la rv a e in v a r io u s in s ta r s tag es w e r e c o l le c t e d f r o m the lo c a l f o r e s t and d iv id ed ra n d o m ly into e x p e r im e n ta l and c o n t r o l g ro u p s . D uring the e x p e r im e n ta l p e r io d , the la rv a e w e r e housed in g la s s ja r s (15 c m in d ia m e te r , 14 c m high), o r in v iny l net c a g e s (20 X 20 X 20 cm ) , each o f w hich conta ined one to twenty la rv a e , depending on the in star stage . The m ouths o f the ja r s w e r e c o v e r e d with nylon net c lo th . A l l the a r t i f i c ia l d iets w e r e g iven in the f o r m o f p i e c e s o f thin f i lm sp re a d o v e r p a r affin p a p e r sheet to p reven t the u n n e c e s s a r y ev a p o ra t io n o f m o is tu r e f r o m the d ie t . In the c o u r s e o f the fe e d in g tes t , grow th , v ita lity and oth er d e v e lo p m e n ts w e r e o b s e r v e d . A l l the in gred ien ts excep t L - a s c o r b i c ac id and ch o l in e c h lo r id e s w e r e m ixed with pine le a f d is t i l la te , and h o m o - genated in d is t i l le d w ater , then au toc laved at 15 l b / i n 2 f o r 20 m in to k il l the m i c r o - o r g a n i s m s . The pH o f the m ed ium w as ad justed to 3. 1 with 20% a c e t i c a c id i f n e c e s s a r y . A f t e r the m ix tu re had been a l low ed to c o o l to about 50 °C , L - a s c o r b i c ac id and ch o l in e c h lo r id e w e re added and m ixed th orough ly . D urin g the feed in g e x p e r im e n ts , the m ed ia w e r e not a llow ed to d r y . In fu r th e r stud ies on r e a r in g la rv a e o f d i f fe re n t in s ta r stages , v a r io u s m o d i f ie d m e d ia w e r e tes ted , in which p ro te in h y d r o ly s a te s f r o m v a r io u s s o u r c e s and b a c t e r i o - and fu n g i - s ta t i c agents w e r e supp lem ented o r om itted .

2 . 5 . Sexual c o m p e t i t iv e n e s s o f the ir ra d ia te d m a le pine m oth

Irra d ia ted ( s t e r i le ) m a le s , n orm al m a le s and n o rm a l f e m a le s w e re p la ced in a v iny l m e s h ca g e ( 3 X 3 X 3 m) with ra t io s o f 1 :1 : 1 to 50 : 1 : 1.T he hatchability o f the eggs p ro d u ce d by the fe m a le was r e c o r d e d . In this ex p er im en t , on ly two d o s e s , 15 and 25 kR, w e r e u sed .

T o d e te rm in e the am ino ac id re q u ire m e n t , the in d ir e c t m ethod with g l u c o s e - U - 14C was e m p lo y e d [19], and the individual a m in o a c id was identi f ied and d e te rm in e d quantitatively a c c o r d in g to the m ethod o f Stein and M o o r e [ 20].

276 LEE et a l .

3. RE SU LTS AND DISCUSSION

3 . 1 . O b s e rv a t io n s on the l i fe c y c le o f the pine moth

T he d e v e lo p m e n ta l stages and their durations a r e i l lu s tra te d in F ig . 1. T he findings on m o r p h o lo g ic a l d ev e lop m en t and r e p r o d u c t iv e b eh av iou r w e r e in a g re e m e n t with th ose f r o m the c o m p r e h e n s iv e stud ies o f p r e v io u s authors [3, 13]. M ost o f the l i fe c y c le o f the m oth was c o v e r e d by the la r v a l (pine c a te r p i l la r ) p er iod , which, in turn, w as d iv ided into eight in s ta r s ta g e s . . About 200 d ays o f the la rv a l p e r io d , h o w e v e r , w e r e spent in h ibernation du ring w in ter m onths. The sh ort l i fe span o f the adult m oths o f both s e x e s and the e ss e n t ia l ly m o n o g a m o u s nature o f the fe m a le w e r e both advantageous and d isadv an ta geou s . One o f the d isadvan ta ges w as that the a v e r a g e adult l i fe span o f 6 - 8 days m ade the rep e t it io n o f an ex p e r im e n t , e s p e c ia l ly the fe r t i l i ty test, e x t r e m e ly d i f f i cu lt . The w e a th e r cond it ion s du ring the e x p e r im e n t w e r e a l s o an u n p red ic ta b le f a c to r .

M onth 1 2 3 4 5 G 7 3 <) IU U 12

1 s t y r . * -!- *

_ _

i a* * * *

FIG. 1. L ife c y c le o f Korean p ine caterp illar D endrolim us speclab ilis Butler.* Adult ( li fe span av. 7 clays; copu lation soon after em ergence). Egg (hatching in 10 clays)- - Larva (about 320 days)1st instar: av. 10 days; 2nd instar: av. IS days; 3rd instar: av. 15 days; 4th instar: av. 25 days;5th instar: av. 1Ü0 clays (hibernation): 6th instar: av. 10 days; 7th instar: av. 23 days; Sth instar: av.34 days. (End o f the larval period iden tifiab le by change o f body co lou r from blu e-red to b lack -brow n , body length ; m ale av. 7.8 cm , fem a le av. 9.5 cm ). Pupa (20 days), m ale sm aller than fem a le .

3 . 2 . R ad iosen s it iv ity o f y o u n g e r la rv ae

R a d io se n s it iv ity in t e r m s o f m o r ta l i ty at 5 and 10 da ys a f t e r the i r rad ia t ion o f la rv a e is shown in F ig s 2 and 3, r e s p e c t iv e ly .

In each o f the e x p e r im e n ta l in s tars (1st through 5th), m o r ta l i t y was d o s e -d e p e n d e n t and r a d io r e s i s t a n c e in c r e a s e d with la rv a l a g e . T he d i f f e r e n c e in m o r ta l i t y betw een the m o s t r e s is ta n t (1st instar ) and the le a st r e s is ta n t (5th instar) la rv a e w as c o n s id e r a b le . At 5 days a fter i r r a d i ation (F ig . 2), f o r in stan ce , a d o s e o f 50 kR, which r e su lte d in about 20% m o r ta l i t y in the 1st in star , p ro d u ce d no e f fe c t in the 5th in s ta r . A m o i— ta lity o f 100% w as o b s e r v e d at 175 kR in the 1st in star , but this d o s e did not in f lu ence the 5th in s ta r . At 250 kR m o r ta l i t y o f the 4th and 5th in s ta r s began to' a p p ear . An in te rm e d ia te re su lt w as noted with both 2nd and 3rd in s ta r s , although the f o r m e r a lw ays had h igh er m o r ta l i ty . Up to about 125 kR, th ese two ir ra d ia te d in s ta rs show ed sl ight d i f f e r e n c e s in m o rta l ity ,

SM- 102 /29 277

D o se (k R )

FIG. 2. M ortality o f various instar stages o f irradiated pine caterp illar larvae at 5 days post-irradiation о--------o : 1st instar; •-------• : 2nd instar; x-------к : 3rd instar; о-------о :4 t h instar; в-------в : 5th instar.

D o se ( k-R )

FIG. 3. M ortality o f various instar stages o f irradiated pine caterp illar larvae at various instar stages at 10 days post-irradiation ; sym bols are the sam e as in Fig. 2.

but a b o v e th is d o s e m o r t a l i t y b e c a m e g r e a t e r and r a p i d : 90% in th e 2nd in s ta r at 200 kR, w hile the 3rd in s ta r re m a in e d at l e s s than 40%. In the 1st in star , with 250 kR, 100% m o rta l i ty was obtained within 24 h ours a fter the trea tm en t . A l o s s o f v ita lity usually p r e c e d e d the death o f the la rv a .In the la te r in s ta r s tag es , the la rv a e w e r e m o r e r a d io r e s i s ta n t . A d o s e o f 300 kR, which r e su lte d in about 95% m o rta l ity within 5 days in the 3rd in star , p ro d u ce d no e f fe c t in the 5th in star (h ibernating) la r v a e . H ow e v e r , with a d o s e o f 400 kR r e m a r k a b le in c r e a s e s in m o r ta l i t y o f la rv a e w e r e o b s e r v e d .

At 10 days p o s t - i r r a d ia t io n , although the indiv idual v a lu e s o f m o rta l ity n aturally w e r e h igh er than th o se at 5 days a fte r treatm en t , e sse n t ia l ly s im i la r patterns o f m o r ta l i ty w e r e o b s e r v e d ( F ig .3 ) . No resu lt was a v a i l ab le f o r the 1st in s ta r b e c a u s e o f high m o r ta l i ty b e f o r e 10 days p o s t t rea tm en t . The m o r ta l i t y o f the 2nd in star was h igh er than that o f the 3rd in s ta r at e v e r y d o s e applied , but the d i f f e r e n c e betw een them at h igher d o s e s was not s o g rea t , ind icat ing a h igh er death ra te o f the la tter betw een 6 and 10 days a f t e r the trea tm en t .

When a c o m p a r is o n was m a de betw een the data o f 5 days and 10 days p o s t - i r r a d ia t io n , at d o s e s l e s s than 200 kR the m o r ta l i t y in the f o r m e r was a lw ays a p p ro x im a te ly o n e -h a l f that in the la tter . T h is in d ica ted that the

278 LEE et a l .

T A B L E I. F E R T I L I T Y O F A D U L T P I N E - C A T E R P I L L A R M A L E S G A M M A I R R A D I A T E D D U R IN G P U P A L S T A G E

D ose (kR)N um ber o f pairs

(RM X N F )(a)A v e ra g e n u m ber o f

eg g s per fe m a leH a tch a b ility

Л

0 80 312 5 5 .8

1 .5 78 330 5 4 .4

2 . 5 79 313 5 5 .1

3 71 297 7 3 .0

5 60 286 5 4 .8

10 58 309 1 9 .8

15 46 267 0

25 51 328 0

50 75 316 0

75 59 338 0

100 39 287 0

(a) N M : n orm a l m a le , RM : irrad ia ted m a le , N F; n orm a l fe m a le

ra te o f la rv a l death a fte r ir ra d ia t io n w as even and g radua l during the f i r s t 10 days a f t e r treatm en t .

In v iew o f the r e su lt s p re se n te d h e re , it ap p ears that the r a d i o s e n s i t iv ity o f the h ibernating la rv a e o f pine c a te r p i l la r s d e c r e a s e s as d e v e lo p m ent p r o g r e s s e s f r o m the e a r l i e r in star stage to the la te r . F o r v a r io u s s p e c i e s o f in se c ts , many in v e s t ig a to rs r e p o r te d that young la rv a e w e r e m o r e r a d io s e n s it iv e than o ld la rv a e [ 1 4 - 1 5 ] .

F r o m the p r a c t i c a l point o f v iew , the p r o s p e c t o f u sing ir ra d ia te d la rv a e in the fe r t i l i ty test was not an a t trac t iv e one . S ince d o s e s o f about 100 k R o r l e s s w e r e not lethal f o r the 4th and 5th instar , 100 kR would be the m a x im u m s te r i l i z in g d o s e ap p licab le in the ir ra d ia t io n o f pupae to p r o d u ce ir ra d ia te d adult m a le m o th s . T h e ir ra d ia t io n o f eg g s and la rv a e to p r o d u c e se x u a lly s t e r i l e m oths was attem pted, but many authors r e p o rte d v a r io u s u n d e s ira b le s ide e f fe c t s which m a d e the ov a l o r la rv a l ir ra d ia t io n im p r a c t i c a l [14, 16]. D o s e s ranging f r o m 2, 5 to 10 kR w e r e app lied to the la rv a e o f the c o d l in g moth, the m a llow m oth and the cu tw o rm m oth.

3 . 3 . F e r t i l i t y o f the ir ra d ia te d m a le pupae

The a v e r a g e n u m ber o f eggs laid by a n o rm a l fe m a le m ated with a n o r m a l m a le was 312, as p r e v io u s ly published [13]. In each g rou p o f i r rad ia ted m a le s m ated with n o r m a l fe m a le s , r e g a r d l e s s o f d o s e between1 .5 and 100 kR, the a v e r a g e n u m bers o f eggs p rod u ced by the fe m a le w e r e not m a rk e d ly d i f fe ren t f r o m n o rm a l (T a b le I). D uring the c o u r s e o f o b s e r v a t io n s , no n o t iceab le a d v e r s e e f fe c t s w e re o b s e r v e d on the lon gev ity o f pupa o r adult, on the co p u la to r y and o v ip o s i t io n a l b e h a v io u r in e ither

S M - 1 0 2 / 2 9 279

m a le and fe m a le , o r on the v ita l i ty . T he hatchability o f the e g gs in th e se i r ra d ia te d g ro u p s show ed a c l e a r ir ra d ia t io n e f fe c t . T he n o r m a l h a tch a b i l i ty w as 5 5 .8 % and, with d o s e s o f 5 kR o r le s s , no r e d u c t io n w as o b s e r v e d . At d o s e s betw een 15 and 100 kR, h o w e v e r , c o m p le te s t e r i l i t y o f the m a le w as noted in e v e r y ir ra d ia te d g ro u p . The 1 0 -k R ir ra d ia te d g ro u p show ed a s ign if ican t re d u ct ion o f hatchability (1 9 .8 % ) . At 3 kR, the 17% in c r e a s e o f hatchabil ity without change in egg pro d u c t iv ity w as c o n s id e r e d a s t im u la to ry e f fe c t . Such an e f fe c t was found in the ra te o f e m e r g e n c e in the g ra n a ry w e e v i l [17].

T he r e su lts su ggested that the t im ing ( la ter h a lf o f the pupal stage) o f i r ra d ia t io n in this e x p e r im e n t to obtain s t e r i l e m a le m oths w as su itable b e c a u s e no s e r io u s s ide e f fe c t w as n o t ice d . Ir ra d ia t io n o f pupae that w e re about 5 days o ld was not s u c c e s s f u l . The la test pupal s tag e w as r e c o m m ended as an e f fe c t iv e ir ra d ia t io n t im e and applied to the b o l lw o r m [18]. D o s e s betw een 15 and 100 kR on the pupae in this e x p e r im e n t s e e m e d a d e quate as s t e r i l i z in g d o s e s . C o m p a r a b le e f fe c t s w e r e o b s e r v e d in the pink b o l lw o r m , the c o d l in g moth, the cu tw o rm m oth and the C o lo r a d o bee t le with d o s e s o f 55, 40, 9 - 1 1 , and 8 - 1 0 kR, r e s p e c t iv e ly , [16, 18].

3 . 4 . Sexual c o m p e t i t iv e n e s s o f ir ra d ia te d m a le s

In this e x p e r im e n t , the hatchability o f the e g gs p ro d u ce d by n o rm a l m a le s and n o r m a l fe m a le s (1 : 1) was 48. 24% (c f . v a lu es o f 54. 90% in 1966 and o f 5 5 .8 0 % in 1965) which w as not d i f fe re n t f r o m the p r e v io u s e x p e r im e n t . When the r a t io of n o rm a l m a le s to n o r m a l f e m a le s w as in c r e a s e d to 2 : 1 , about 10% re d u ct ion in hatchability w as noted . T h is p r o b ably w as ca u se d by the h a ra ssm e n t o f the fe m a le by the m a le s [9], which w as u ndetected during o b s e rv a t io n .

When m a le s ir ra d ia ted with 15 kR w e r e p la ced with n o rm a l m a le s and f e m a le s with the r a t io 1 : 1 : 1 , hatchability w as 38. 06% (the p r e v io u s y e a r ' s va lue was 37. 5% with the s a m e ratio ) which is l e s s than the n o r m a l value o f 4 8 .3 % . S im ila r ly , m a le s ir ra d ia te d with 25 kR with the s a m e ra t io fu r th e r re d u ce d hatchability to 3 6 . 3 % (the p r e v io u s y e a r ' s va lue was 31 .9% ) (T a b le II).

S ince th e o r e t i c a l hatchabil ity f o r equal c o m p e t i t iv e n e s s o f ir ra d ia ted m a le s to n o r m a l on es w as 23. 8%, the d e g r e e o f r e d u ct ion in hatchability o f i r ra d ia te d m a le s in this e x p e r im e n t indicated that the se x u a l c o m p e t i t iv e n e ss o f ir ra d ia te d m a le s w as about half o f n o rm a l . T h is f inding a g reed with m any published data on v a r io u s f l ie s and on the pink bo l lm oth [18].

Thus, s t e r i l e m a le s con f in ed with n o rm a l m a le s and f e m a le s at v a r io u s r a t io s ( 1 : 1 : 1 to 1 : 50 : 1) ca u se d a re d u ct ion in the n u m b e r o f p ro g e n y . (Th is r e f e r s to the ratio o f n o n - ir r a d ia te d m a le s to i r ra d ia te d m a le s to n o n - ir r a d ia te d v irg in f e m a le s used in co m p e t i t iv e m ating t e s t s . )

I f one a s su m e d an equal se x ra t io in natural popu la tion s , m a le s treated with 15 kR would be fu lly c o m p e t i t iv e when r e le a s e d in ra t i o s o f 1: 30 : 1.In a mating co m p e t i t iv e n e s s study on the h orn fly , it w as r e p o r te d that e f f e c t iv e s t e r i l i ty w as obtained when the r a t io o f s t e r i l i z e d m a le s to wild m a le s w as o f the o r d e r o f 20 : 1 [21].

T A B L E 11. E F F E C T OF P I N E C A T E R P I L L A R

S T E R I L E M A L E R E L E A S E S O N T H E R E P R O D U C T I V E P O T E N T I A L O F T H E

D o se (kR)T h e ra t io o f irrad ia ted

m a le s t o n o n -irra d ia te d m a les to v irg in fe m a le s

N um ber o f irrad iated

m a les tested

T o t a l n u m b er o f e g g s la id

N u m b er o f

eg g sh a tch ed

H a tch a b ility

m

0 RM : N M : NF

(co n tro l) 1 : 1 150(a) 23 736 11 450 48.24

1 : 1 1 100 15 824 6 144 38.06

5 : 1 : 1 250 12 423 2 842 22.89

10 : 1 : 1 aoo 7 193 .1 078 15.00

20 : 1 ' : 1 300 3 156 125 3.9S

30 : 1 : 1 300 2 959 0 0

50 : 1 : 1 300 601 0 0

1 : 1 : 1 100 20 041 7 230 36.32

5 : 1 1 250 9 633 1 803 18.71

25 10 : 1 : 1 300 9 2 5 5 702 7 .5 8

2 0 : 1 : 1 300 2 901 90 3 .1 0

30 : 1 1 300 1 632 0 0

50 : 1 : 1 300 1 130 0 0

( a ) N u m b er o f n o n -ir r a d ia te d m a le s

280 LEE

et al

SM- 1 02 /2 9 281

3 . 5 . R e a r in g la rv a e on a r t i f i c i a l m ed ia

3 . 5 . 1 . Initia l attem pts to feed the la rv a e

T o s im u la te the natural d iet , f r e s h pine le a v e s w e r e c ru sh e d and the r e s id u e w as ly o p h i l iz e d a fte r r e m o v a l o f the fat so lu b le f r a c t io n s . The final diet w as m a de by m ix in g this ly o p h i l iz e d fr a c t io n with le a f m a te r ia ls obtained by steam d is t i l la t io n (M edium 1). T h is m e d iu m w as not s u c c e s s ful; no la rv a e o f the 3rd through 6th in s ta r stages ap p ro a ch e d it. Next, a m o d i f ic a t io n o f an a r t i f i c i a l d iet f o r the s i lk w o rm [22] w as used as a ba sa l d iet . T o m ak e the c o m p le t e m ed ium , v itam in and m in e r a l m ix tu re s and p u lv e r iz e d pine le a v e s w e r e added to this ba sa l d iet . A lthough this was ap p roa ch ed by the la rv a e o f the 5th in star at the beginning , a fte r about5 days they stopped feed ing .

It was found that the a s c o r b i c ac id content o f the f r e s h pine le a ve s was r e la t iv e ly high, 0 .4 m g / g o f f r e sh lea f . In the th ird tr ia l , the vitam in and m in e r a l m ix tu re s o f M edium 2 w e re re p la c e d by 40 m g o f a s c o r b i c a c id . Although this w as not s u c c e s s f u l f o r feed ing 1st and 2nd instar la rv a e , a p art ia l s u c c e s s was brought about with the 7th and 8th in s ta rs . L a r v a e which fed on M edium 3 w eighed l e s s than the co n t ro l ; n e v e r th e le ss , about 40% o f them pupated and fo r m e d c o c o o n s . The on set o f the pupal stage in the e x p e r im e n ta l la rv a e se e m e d sl ightly e a r l i e r and the c o c o o n was th inner and c o a r s e r than the c o n t r o l . T h e se pupae died e a r ly . One r e a s o n f o r the d i f f e r e n c e in r e s p o n s e to M edium 3 betw een the e a r l ie s t and the la test stage la rv a e ap p eared , am ong oth er things, to be the inab ility o f the young la rv a e to d ig e s t the c a s e in . It w as a lso felt that it m ight be u s e ful to use ca se in h y d ro ly sa te instead o f c a s e in i t s e l f as the p ro te in s o u r c e . O ther r cp o i 't s ind icated that the a s c o r b i c ac id was a n e c e s s a r y nutrit ional r e q u ire m e n t in the grow th o f in se c ts [23] . T he biting and sw a llow in g ac t io n s o f s i lk w o rm la rv a e w e re pa rt ly dependent on the ag a r contained in f r e s h m u lb e r r y le a v e s .

3 . 5 . 2 . Studies on the c o m p o s i t io n o f pine le a v e s and la rv a e

In v iew of the r e su lts o f the fo r e g o in g initia l e x p e r im e n ts , the d e v e lo p ment o f a su itab le m ed iu m s e e m e d to r e q u ire ex te n s iv e in fo rm a t io n on the c o m p o s i t io n o f both pine le a v e s and la rv a e to understand the nutrit ional r e q u ire m e n ts o f the la r v a e . The fo l lo w in g e x p e r im e n ts w e r e c a r r ie d out f o r this p u rp o s e : P in e lo a v e s w e re taken f r o m Pinus d e n s i f l o r a S et Z . The an a lyt ica l m ethod s o f c a r b o h y d r a te s , l ip ids , p ro te in and o th e r su bsta nces a r e i is ted in T a b le III, and s p e c ia l p r o c e d u r e s pertinent to the treatm ent o f the pine le a v e s a re a lso s u m m a r iz e d .

3 . 5 . 3 . T he se c o n d feed in g e x p e r im e n t with d i f fe re n t m ed ia

F r o m r e su lts o f the fo r e g o in g e x p er im en t , and a l s o f r o m kn ow led ge o f a r t i f i c ia l d iets f o r a r i c e in se c t [24], the le o p a rd moth [25] and the o l iv e f ly 126], a m o d if ie d m ed iu m w as p ro d u ce d as M ediu m 4. The r e s u l t s with this m ed iu m w e r e not s u c c e s s f u l with the f i r s t in s ta r s . Many c o m binations o f v a r io u s am ounts o f the in gred ien t did not im p r o v e the re su lt . M edium 7, in which d is t i l la te and h om ogen a te o f pine le a f r e s id u e and sod iu m g lutam ate w e r e added (T ab le III), was on ly s u c c e s s f u l in r e a r in g

282 LEE et a l .

T A B L E III. C O M P O S I T I O N O F N U T R I E N T M E D IU M F O R D e n d r o l i m u s s p e c t a b i l i s L A R V A E

Ingredients M ed iu m N o . 7 M ed iu m N o . 8

Sucrose 1 .0 g 1 .0 g

L -a s c o r b ic a c id 4 0 0 .0 m g 4 0 0 .0 m g

Y ea st h yd ro lysa te e n z y m a tic 1 .0 g 1 .0 g

B eta -s ito s te ro l 2 . 0 m g 2 . 0 m g

B rew er 's y ea st, U .S .P . f*3) 2 . 0 g 2 . 0 g

P otassium sórbate 2 6 .0 m g 2 6 .0 m g

C h o lin e c h lo r id e 2 . 0 m g 2 . 0 m g

P ine l e a f d is t illa te 1 5 .0 m l 1 5 .0 m l

P ine l e a f residue 2 . 0 g 2 . 0 m l

S od iu m g lu ta m a te W) 2 0 .0 m g 8 0 .0 m g

N ip a g in ( e ) 30 m g

N ip a so l 30 'm g

N o te : T h e pH o f the m ed iu m w as ad justed to 3 . 1 by the a d d ition o f a c e t ic a c id .

( a ) N a tion a l B io c h e m ica l C o rp o ra tio n , C le v e la n d , O h io ,(b ) N a tion a l B io c h e m ica l C o rp o ra tio n , C le v e la n d , O h io .( c ) H oech st, Leverku sen, F ed era l R ep u b lic o f G erm a n y .(d ) S igm a C h e m ic a l C o . , S t . Lou is, M o .( e ) M eth y l p -h y d r o x y b e n z o a te , M e r c k C o . , D arm stadt, F ed era l R ep u b lic o f G erm a n y . ( 0 Propyl p -h y d ro x y b e n zo a te , M e r c k C o . , D arm stadt. F ed era l R ep u b lic o f G e rm a n y .

the 2nd in star la rv a e by cau s in g a gain in body weight c l o s e r to that o f the pine le a f fed c o n t r o l and m o d ify in g the m o r ta l i t y o f the e x p e r im e n ta l la r v a e . The appetite o f la rv a e on this d iet s e e m e d en cou ra g in g , b e c a u se they not on ly ingested the d iet i t s e l f but a l s o the para ff in sheet on w hich the d iet w as sp re a d .

The p a rt ia l s u c c e s s o f the g lutam ate in the m e d iu m was not im m e d i ately ev ident. G lu tam ic ac id , although not e s s e n t ia l f o r m o s t in se c ts , o c c u p ie s a cen tra l r o le in am in o ac id m e t a b o l i s m . T h is am in o ac id is r e q u ired as a phagost im ulant by the pine c a te r p i l la r .

As r e g a r d s the p ro te in s o u r c e s , the c o m m e r c i a l l y av a i lab le p ro te in h y d r o ly s a te s , i . e . b a c to -p e p to n e , y e a s t en z y m a t ic h y d ro ly sa te , so y e n z y m a t ic h y d ro ly sa te and am in o ac id m ix tu re (16 c r y s t a l l in e am in o a c id s ) w e r e su pp lem en ted in the p re se n t s tu d ies . The re su lts show ed that the c a s e in was not a p r e fe r a b le p ro te in fo r the la rv a e . A m on g the above p r o te in en zy m a t ic h y d r o lv sa te s , y e a st en zym a t ic h yd ro ly sa te was the m os t p r e f e r a b le p ro te in h y d r o lv sa te under a se p t ic cond it ion s [ 8 - 1 0 ] .

O ur r e ce n t e x p e r im e n ts with g l u c o s e - U - 14С to d e te rm in e the am ino ac id r e q u ire m e n t r e v e a le d ten e ss e n t ia l am in o a c id s , i . e . tryptophan,

S M- 1 02 /2 9 283

T A B L E I V . D I S T R I B U T I O N O F 14 С IN F R E E A M IN O A C ID S O F T H E P IN E C A T E R P I L L A R F O L L O W I N G I N J E C T I O N O F G L U C O S E - U - 14C

A m in o a c id s C o u n ts /m in

T h reon in e 103G

A sparagin e 245

T a u rin e 197

G ly c in e 183

G lu ta m ic a c id 138

I s o - le u c in e 1 0 3 .6

H y d rox y p ro lin e 99

S erin e 6 9 .3

A la n in e 1 4 .8

T yrosin e 1 2 .5

P rolin e 1 1 .9

A rg in in e 1 0 .3

L eu cin e 10

C y stin e 7 .2

V a lin e 5 .3

H istid in e 1 .6

M eth ion in e 0 .6

P h en y la la n in e 0

L ysine 0

T ryptoph an 0

ly s in e , phenyla lanine , m eth ion ine , h ist id ine , valine , le u c in e , arg in ine , p ro l in e and ty r o s in e . A lanine and cy s t in e w e re a lso c o n s id e r e d e ssen t ia l (T a b le IV).

A m ong the ca r b o h y d r a te s , s u c r o s e cou ld be the m o s t su itab le one fo r the la rv a e o f the pine c a te r p i l la r . It w as r e ce n t ly d e m o n s tra te d in our l a b o r a to r y that s u c r o s e and m a lto s e m e ta b o l iz in g e n z y m e s w e r e p resen t in the la rv a l gut.

A s d e s c r ib e d in a p r e v io u s p r o g r e s s r e p o r t (supplem ent to the p r o g r e s s r e p o r t , C o n tr a c t No. 2 2 7 /R 1 /R B , 1966), am ong the e x p e r im e n ts conducted last y e a r , w e s u c ce e d e d in r e a r in g la rv a e with a synthetic diet ca l le d M ediu m 7. In the p re se n t feed in g ex p e r im e n t , the la rv a l m ed ium (M ediu m 8) was m o d i f ie d by in c r e a s in g g lutam ate and supplem enting fu n g ic id e s , i . e . , N ipagin and N ip a so l (T ab le III).

The l a r v a - r e a r in g e x p e r im e n ts with M ediu m 8 w e r e s u c c e s s f u l in m od ify in g m o r ta l i t y as w e l l as bod y weight o f the la rv a e (F ig . 4). D if fe ren t

284 LEE et a l .

1.5-

FIG. 4. T im e course changes in the survival number and body w eight o f pine caterp illar larvae under various experim ental conditions. Л - l : fed on fresh leaves; Л -2 ; fed on m edium 8 ; Л -3 : fed on m edium 8 ( - yeast hydrolysate + am ino acid m ixture): Л -4 : fed on m edium 6 ( - yeast hydrolysate + soy hydrolysate): Л -5 : fed on m edium 8 ( + yeast hydrolysate + blended carrot); A - t-j ; fed on water on ly ; B - l : body w eight curve, fed on fresh leaves; B -2 : fed on m edium 8.

m g

3

FIG. 5. T im e course changes in the survival number and body w eight o f 2nd instar p ine ca terp illa r larvae under various experim ental cond itions; sym bols are the sam e as in Fig. 4.

co m b in a t io n s o f f o rm u la e , changing the p r o p e r t i e s o f each in gred ien t , w e r e te s te d . R e a r in g e x p e r im e n ts with M ed iu m 8 on the la rv a e o f 2nd and 3rd in s ta r s tag es gave good r e su lt s . The la rv a e gained body weight and m o d i f ie d the m o r ta l i ty (F ig . 5). The fe e d in g ex p e r im e n t is continuing.

4. SU M M A R Y AND CONCLUSION

T he r a d io s e n s it iv i ty o f the la rvae o f the K o re a n pine c a te r p i l la r 1J. sp e c ta b i l i s B u tler , 1st through oth in s ta r s tag es , was d o se -d e p e n d e n t ,

S M- 10 2/2 9 285

and r a d io r e s i s t a n c e in c r e a s e d with age . A d o s e o f 100 kR o r le s s did not p r o d u c e m o r ta l i t y in the 5th in s ta r and th e se d o s e s cou ld be used as the s t e r i l i z in g d o s e in the e r a d ic a t io n p r o g r a m m e s .

F e r t i l i t y and se x u a l c o m p e t i t iv e n e s s o f the ir ra d ia te d m a le s w e r e d e t e r m in e d . M ale pupae ir ra d ia te d with 15 kR would be fu lly c o m p e t i t iv e when r e le a s e d in a r a t io o f 1 : 3 0 : 1 .

E x p e r im e n ts w e r e c a r r i e d out to d e v e lo p an a r t i f i c ia l m e d iu m to r e a r la rv a e o f the K o re a n pine c a t e r p i l la r in co n n e ct io n with e r a d ic a t io n by the s t e r i l e - m a l e te ch n ique . S e v e r a l m ed ia w e r e tested with la rv a e o f v a r io u s in s ta r s ta g e s . T h e se m ed ia c o n s is t e d o f s u c r o s e , L - a s c o r b i c ac id , y east h y d ro ly sa te , b e t a - s i t o s t e r o l , b r e w e r ' s ye a st , p o ta ss iu m só r b a te , cho line c h lo r id e , le a f d is t i l la te and r e s id u e and sod iu m glutam ate .

N utr it iona lly e s s e n t ia l and n o n -e s s e n t ia l am in o a c id s f o r the la rv ae w e r e d e te rm in e d by the in d ir e c t method with g l u c o s e - U - 14C .

R E F E R E N C E S

[1] BUTLER, A .G . , A nn. M ag. nat. Hist. 20 (1877) 481.[2] UCHIDA, T .J . , Fac. A gr. H okkaido Im p . U niv. 25 (1930) 254 .[3] K AM IYA, K ., Studies on the M orphology, B ionom ics and H ym enopterous Parasites o f the P ine-C aterpillar

(D endrolim us speciab ilis BUTLER), M onograph publ. by G ov . o f Korea (1934 ).[4] EZAKI, T . , Jap. Forest Bull. 16 (1922) 480 .[5] NAGANO, K ., W orld o f Entom ol. 20 (1916) 489 .[6] SAITO, K .I . , Forest. S o c . U (1933) 912.[7] BUSHLAND, R .C . , HOPKINS, D .E ., J. e co n . Ent. 44 (1951) 725.[8] BUSHLAND, R .C . , HOPKINS, D .E ., ] . e co n . Ent. 46 (1953) 648 .[9 ] BAUMHOVER, A .H . , GRAHAM, A . ] . , HOPKINS, D .E ., DUDLEY, F .H ., NEW, W .D . , BUSHLAND. R .C . ,

J. e co n . Em. 48 (1955) 459 .[10] KNIPLING, E .fT, J. e co n . Ent. 48 (1955) 459 .[11] LINDQUIST, A .W ., J. e co n . Ent. 48 (1955) 467 .[12] LEE, K .B ., HWANG, U .K ., PAIK, W .H ., unpublished data, 1963.[13] TAKAGI, G ., -Report o f Forest Exptl Station 2 , publ. by G ov . o f Korea (1925 ).[14] PROVERBS, M .D . , NEWTON, J R ., C an . J. Z o o l . 40 (1962) 400 .[15] HALBERSTAEDTER, L ., GOLDHAIiER, G ., HECHT, O ., Growth 7 (1943 ) 413 .[10] LABRECQVE, G .C . , KHLLER, J .C . , A dvances in Insect Population Control by the S ter ile -M a le T e ch

nique, T ech n ica l Reports Series N o .44, IAEA, Vienna (1965J .1.[17] JEFFERIES, D .J . , in Radioisotopes and Radiation in Entom ology, IAEA, Vienna (1962) 213 .[18] OUYE, M .T . , GARCIA, B .S ., MARTIN, D .F . , J. e co n . Ent. 57 (1964) 387 .[19] KASTING, R ., McGINNIS. A .J . . C an . J. B ioctieni. Physiol. 3S (1960) 1229.[20] MOORE, S . . STEIN, W . Il, J. b io l. C h em . 192 (1951) 663 .[21] HENtfEBERRY. T .J . , J. e co n . Ent. 56. (1963) 279 .[22] FUKUDA, T . , SlITO, M ., KAM EYAM A, T . . KAWASUGI, S . , Nature (L ou d .) 196 (1962) 53.[23] ITO, T . , Nature, (L on d .) 192 (1961) 951.[24] ISHII, S . , Sym posium on m ajor insect pests o f r ice at the International R ice Research Institute, Los

Baños, Philippines Septem ber 14 - 18, 1964.[25] MOORE, I . , NAVEN, A . , personal com m u n ication .[26] HAGEN, K .S . , SANTAS, L ., TSECOURAS, A . , Radiation and Radioisotopes A pplied to Insects o f

Agricultural Im portance, IAEA, Vienna (1963) 333 .

D I S C U S S I O N

G . W . R A H A L K A R : I understand you p e r fo r m e d y o u r e x p e r im e n t with v a r io u s s e x - r a t i o c o m b in a t io n s in in sec t c a g e s . Would th e re be any change in yo u r o b s e r v e d r e s p o n s e i f c a g e s o f la r g e r s i z e w e re used?

286 LEE et a l .

K . B . L E E : We have not e x p er im en ted with c a g e s o f d i f fe ren t s i z e s , and the n um ber o f in se c ts p e r cag e w as a lw ays the sa m e .

M. AN W AR: In F ig . 1 o f y o u r p a p e r you m ention that the l i fe span o f the adult is 7 days and in the sa m e f ig u re you g iv e the o v ip o s i t io n p e r io d as 20 d a ys . Could you p le a se explain?

K . B . L E E : The p e r io d o v e r which adults a r e found in the f ie ld o v i p os it in g is about 20 days (m id -J u ly to e a r ly August). The o v ip o s it io n p e r io d o f each adult is , o f c o u r s e , l e s s than 7 days .

I. A . KANSU: Y our T a b le I show s that hatchability o f e g gs is at its h ighest (73 .0 % ) a fter 3 kR ir ra d ia t io n . Do you have any explanation o f this f ig u r e (in the s a m e ex p er im en t , c o n t r o l eggs show only 5 5 .8% hatchabil ity )?

K . B . L E E : In two se ts o f e x p e r im e n ts out o f th ree , we h ave o b s e r v e d a s t im u la to ry e f fe c t on hatchability at this d o s e . J e f f e r i e s (R ef . [17] o f our p aper) has noted a s im i la r e f fe c t in the ir ra d ia t io n o f g ra in w e e v i l pupae.

J . H . G . T IC H E L E R : I note f r o m y o u r T ab le II that ir ra d ia te d m a le s a r e on ly one q u a r te r as co m p e t i t iv e as n o rm a l m a le s . Did you try to i m p r o v e th e ir p e r fo r m a n c e by lo w e r in g the ir ra d ia t io n d o s e , o r by applying the ir ra d ia t io n at a la te r stage in the in s e c t s ' d e v e lop m en t?

K . B . L E E : W e ir ra d ia te d the pupae at the la test p o s s ib le stage .T e s t s with a l o w e r d o s e a re planned.

CRIA MASAL, D E Dysdercus peruvianus G. Y SU ESTER ILIZACION M EDIANTE RAYOS G AM M A

J . E. SIMON ESTACION EXPERIMENTAL AGRICOLA DE LA MOLINA,

LIMA, PERU

Abstract — Resumen

LARGE-SCALE BREEDING OF Dysdercus peruvianus G. AND ITS STERILIZATION BY G AM M A RAYS.A m ethod o f la rg e -sca le breeding o f Dysdercus peruvianus G. (fa m ily Pyrrhocoridae) w ith natural food and giving a hundred-fold popu lation increase each m onth has been developed in the Entom ology Laboratories o f the Experim ental A gricultural Station at La M olina, Lim a, Peru.

Cages 90 x 60 x 60 cm are used; 2000 adults are p laced in each , care being taken that there are not m ore m ales than fem ales (tw o m ales to each three fem ales are su ffic ien t). T h e ca g e floor consists o f wire mesh w ith nine strands per sqare in ch ; the roof, m ade o f c lo th or mesh with 1 4 -18 strands per square inch, is fitted with a 1 5 -W fluorescent ligh t or a 6 0 -W incandescent light, and the walls are o f wood or som e other washable m ateria l. Inside the cages are p laced two containers w ith levant cotton shoots, and about 500 g o f seed are scattered on the floor . Under the mesh is a sloping floor o f p lex ig las or som e other sm ooth m aterial w hich allow s the eggs to slide down to a c o lle c t in g channel ending in a tube marked in centim etres (1 c m 3 = 4250 eggs).

Sim ilar cages, but with a w ooden or brass floor, are used to contain £ c m 3 o f eggs, w hich are scattered on the floor covered w ith levant cotton seed. The eggs hatch and the nymphs grow in this cage until they reach the adult stage, when they are taken out in batches o f 2000 and transferred to tanks for adults or irradiated.

Doses o f 1000, 2500, 5000 and 10 000 R w ere used, and sterility was obtained from 2500 R, at w hich dose eggs were produced and developm en t occurred to the first nymph stage. At 5000 and 10 000 R the eggs did not d eve lop . At 1000 R d evelopm en t was norm al, and in addition the c y c le was accelera ted and there were m ore eggs. Irradiated m ales and fem a les accep ted , and were a ccep ted by, norm al insects w ithout d iscrim ination . C om p eten ce leve ls o f 2, 8, 32 and 128 m ales were tested.

CRIA MASAL DE Dysdercus peruvianus G . Y SU ESTERILIZACION MEDIANTE RAYOS G AM M A.Un m étodo de crfa m asal de Dysdercus peruvianus G . (Fam . : Pyrrhocoridae) m ediante a lim entación natural, que perm ite centuplicar la p ob la c ión m ensualm ente, ha sido desarrollado en los laboratorios de en tom olog fa de la Estación Experim ental A g rfco la de La M olina (L im a-P erd ).

Se em plean jaulas de 90 x 60 x 60 cm donde se liberan 2000 adultos procurando que el núm ero de .m achos no sea superior al de hem bras (dos m achos por cada tres hembras son su ficien tes). El piso de las jaulas es de m alla m étá lica de nueve h ilos por pulgada cuadrada; e l te ch o de te la o m alla de 14 a 18 hilos por pulgada cuadrada está provisto de una luz fluorescente de 15 W (o incandescente de 60 W ), las paredes son de m adera u otro m aterial la v a b le . Dentro de las jaulas se c o lo ca n dos recip ientes con brotes term inales de algodonero y sobre el p iso se esparcen unos 500 g de sem illa . D ebajo de la m alla hay un piso inclinado de plex ig lás u otro m aterial liso que perm ite a los huevos deslizarse hasta una canaleta de r e co le c c ió n term inada en una probeta graduada en centím etros (1 c m 3 = 4250 huevos).

En jaulas sim ilares pero con piso de m adera o latón se pone i c m 3 de huevos que se esparcen en el piso cubierto de sem illa de algodonero . Los huevos eclosionan y las ninfas se desarrollan en esta jaula hasta a lcanzar e l estado adulto, de donde se las extrae en grupos de dos m il y se las pasa a jaulas de adultos o irradiados.

Se probaron dosis crecien tes de 1000, 2500, 5000 y 10 000 R, obteniéndose esterilidad desde 2500 R en que hubo producción de huevos y desarollo hasta ninfa I. Con 5000 y 10 000 R los huevos no se desarrollaron . Con 1000 R é l desarrollo fue norm al e inclusive e l c ic lo fue ace lera d o y e l núm ero de huevos m ayor. Los m achos y las hembras irradiados aceptaron y fueron aceptados, indistintam ente, por los norm ales.Fueron probados n iveles de com p eten cia de 2, 8, 32 y 128 m achos por pareja norm al.

287

2 8 3 SIMON

IN TROD UCCIO N

E l D y s d e r c u s peruv ianu s G. (F am . P y r r h o c o r id a e ) está c o n s id e r a d o c o m o la plaga m á s p e r ju d ic ia l para e l cu lt iv o d e l a lg o d o n e r o en e l P erú , p ues lo s daños que o c a s io n a son de tal magnitud que p r o v o c a n no s ó lo una r e d u c c ió n de la c o s e c h a sino tam bién una d is m in u c ió n de la ca l id ad d e l a lgod ón . En e fe c to , cuando e l in s e c to ataca a f ru to s pequeños , o bien é s t o s caen y se p ierden , o bien se p a ra l iz a su d e s a r r o l l o o, f in a lm ente , la s s e m i l l a s p icad as de jan e s c a p a r a c e i t e que m ancha la f ib ra c re a n d o , a d e m á s , c o n d i c io n e s fa v o r a b le s para e l d e s a r r o l l o de hongos que é l m is m o in tro d u ce con su t r o m p a y que acaban por d e s t r u ir la f ib ra .

Si a lo a n te r io r a g r e g a m o s que e l in s e c to e s m ig r a t o r io , que no tiene e n e m ig o s n atu ra les de im p o r ta n c ia e c o n ó m ic a y que cada h e m b r a es c a p a z de o v ip o s i ta r 500 huevos , p o d r e m o s c o m p r e n d e r e l p e l ig r o que r e p r e s e n t a e l in se c to .

S u m e m o s a e l lo que e s r e s is te n te a to d o s lo s in s e c t i c id a s c l o r a d o s , que de lo s f o s f o r a d o s s ó lo e l Parath ion lo d e s tru y e , y e l lo m ed ian te c o n s tantes y c o s t o s a s a p l i c a c io n e s de in s e c t i c id a s , y que de lo s c a r b a m a t o s s ó l o Sevin e s e m p le a d o co n éx ito p a ra c o m b a t ir lo , p e r o que, en to d o s lo s c a s o s la s h e m b r a s o v ip le n a s in to x ica d a s vac ian su ú tero antes de m o r i r y lo s huevos , que son f é r t i l e s , dan lu gar a n infas s ó lo d e s p u é s de 5 ó6 d ías , cuando ya e l p o d e r d e l in s e c t ic id a ha d ism in u ido o d e s a p a r e c id o , y nos d a r e m o s cuenta de la n e ce s id a d de b u s c a r n uevos m é to d o s de c o m b a te .

P u es bien, e sta ch in che , c o n o c id a co n lo s n o m b r e s c o m u n e s de " a r r e b i a t a d o " , rabiatado , c u l i - c u l i o ch in ch e m a n ch a d o ra de la f ib ra de l a lg o d o n e r o , ataca a la c a s i totalidad de la s 200 000 h e c tá r e a s cu lt iv ad a s c o n a lg o d o n e r o en e l P e rú , cau san do daños p o r v a r io s m i l l io n e s de s o le s , a lo s que hay que a g r e g a r io s g astos que o c a s io n a la lucha co n t ra e l m i s m o .

ID E N T IF IC A C IO N

E s to s in s e c t o s p e r te n e ce n a l o rd e n H em ip tera , fa m il ia P y r r h o c o r id a e , g é n e r o s D y s d e r c u s y E u ryoph th a lm u s, s ien d o las e s p e c i e s m á s c o m u n e sD. p eruv ianu s G . , D. r u f i c o l l i s L . , D. m im u s S a y . , D. fe rn a ld i B a llou ,D. in c e r tu s D ist . , _D_. r u s t ic u s Stal . , D . im ita to r B lote y E u ryop h th a lm u s h u m il is Stal [1]. De e l la s la s t r e s p r im e r a s están d is tr ib u id a s en toda la re p ú b l ic a , s ien d o la m á s abundante D. peruv ianu s G. En c a m b io , la s o t r a s c in c o e s p e c i e s s ó lo se encu entran en la hoya A m a z ó n ic a , no a s i en la C o s ta de l P a c í f i c o que es donde se cu lt iva e l 95% d e l a lg o d o n e r o .

BIOLOGIA

L o s huevos , de c o l o r b la n co , son d e p o s ita d o s s o b r e e l su e lo deb a jo de la h o ja r a s c a o bien en la s c o m is u r a s de la s b e l lo ta s ( fru tos ) de l a lg o d o n e ro , que c o m ie n z a n su d e h is c e n c ia . A l cu a rto o quinto día d e sp u é s de p u e sto s , l o s huevos se tornan g radua lm en te a m a r i l l o s y an aran jad os y un día d esp u és de a d q u ir i r c o l o r an aran jado , c c lo s io n a n .

SM-102/30 28 9

L a s l a r v a s r e c ié n n ac id as son de c o l o r anaranjado , tono que se va in ten s i f i ca n d o a m e d id a que pasan lo s d ías , hasta que al c u a r to día mudan a su segundo e s ta d io ninfal, cuando son de c o l o r r o jo in tenso . E s te e s tadio dura de 4 a 7 d ías. L u ego mudan y a p a r t ir de l t e r c e r e s ta d io son de c o l o r r o j o c l a r o c o n l ín ea s t r a n s v e r s a l e s b la n ca s . E s te e s ta d io dura de 4 a 6 d. Hasta a q u í la s la r v a s o n infas p e r m a n e c e n g r e g a r ia s , p e r o al m u dar a su cu a rto e s ta d io , que dura de 4 a 7 d, c o m ie n z a n a s e p a r a r s e unas de o tra s y, f in a lm ente , en su quinto e stad io , que t iene una du ra c ión de 6 a 12 d ías , se m o v i l i z a n en f o r m a autónoma. A l f inal de l quinto estad io la s p r o tu b e r a n c ia s a la r e s son b ien v i s ib le s y la s n infas se t r a n s fo rm a n en adultos.

L as c h in ch e s adultas tienen sus a las bien f o r m a d a s y son de c o l o r a m a r i l le n to , anaran jado y r o j i z o . L as h e m b r a s son m á s g ra n d e s y v o lu m in o sa s que l o s m a c h o s , m id ien d o de 13 a 14 m m de la r g o p o r 3 a4,5 m m de ancho. La m a d u ra c ió n sexu al, c o m o e s f requ ente en tre lo s h e m íp te ro s , se a lca n z a en e l ú lt im o e s ta d io n infal, de tal m o d o que el m i s m o día que a lcanzan e l e s ta d o adulto y apenas se han e n d u r e c id o la s a las , se p r o d u c e la cópu la , que puede d u ra r d esd e unas p o c a s h ora s hasta 4 ó 5 d. N o rm a lm e n te una h e m b r a copu la de 5 a 7 v e c e s en su vida y o v ip o s i ta de 3 a 5 g ru p o s de huevos . Una o v ip o s i c ió n n o rm a l se co m p o n e de a l r e d e d o r de 100 huevos , de donde r e su lta que cada h em bra e s capaz de p o n e r en tre 300 y 500 huevos . E l p o r ce n ta je de r e c u p e r a c ió n (r e la c ió n en tre e l n ú m e ro de huevos o v ip o s i ta d o s y e l n ú m e ro de adultos o b ten idos de e s a cam a d a ) e s , en c o n d ic io n e s de la b o r a to r io , de a l r e d e d o r de 30%, lo que in d ic a r ía que e l ín d ice de r e p r o d u c c ió n de e s t o s in s e c to s e stá entre 1:90 y 1:150.

DANOS

L a s ninfas, en su s t r e s p r i m e r o s e s ta d io s , se a l im entan de sav ia de la s plantas y, a p a r t i r d e l cu a rto e s ta d io , son c a p a c e s de a lca n z a r y p e r f o r a r la c á s c a r a de la s s e m i l l a s a l im e n tá n d o se d e l a ce i te conten id o en e l la s . La fe r t i l id a d de la s h e m b r a s está en ra zó n d ir e c ta a su a l im e n ta c ión con s e m i l l a s .

A l p e r f o r a r la s b e l lo ta s p a ra a l im e n ta rs e causan l e s io n e s tanto en l o s c a r p e lo s c o m o en la s s e m i l l a s . De a m b o s e scap an ju g o s que m anchan la s f ib ra s .

Si la in c is ió n se p r o d u c e antes de lo s 20 d ías de edad de lo s f ru tos , é s t o s ca e r á n o p a ra l iz a rá n su d e s a r r o l l o ; s i e l la se p r o d u c e en c a m b io en tre lo s 20 y 30 d, h abrá p r o d u c c ió n de f ib ra p e r o é sta s e r á dura y de in f e r i o r ca lid ad . F in a lm en te , s i la in c is ió n se p r o d u c e en tre lo s 30 y 50 d de edad del fruto , é s te s ó lo a c u s a r á e l daño en e l l ó c u l o atacado , a lcanzando lo s o t r o s d os o t r e s su d e s a r r o l l o n o rm a l ; s i y a e l a lgodón está en fr a n ca d e h is c e n c ia , no habrá daño.

L a ra zó n de la im p o rta n c ia de l o s p e r ju i c i o s que ca u sa e s te in se c to e s t r ib a no tanto en e l daño d ir e c to , que a c a b a m o s de d e s c r ib i r , s ino en e l daño in d ir e c to que o c a s io n a a l t r a n s m it i r hongos p a tó ge n o s , ta le s c o m o A lte r n a r ía sp. , A c r e m o n iu m s p . , N e m a to s p o r a sp. y b a c t e r ia s .

E l a r r e b ia ta d o no s ó l o ataca a l a lg o d o n e r o s ino ta m b ién a plantas s i lv e s t r e s de la s fa m il ia s B o m b a c a c e a e , M a lv a ce a e , S o lan aceae y A m a r y l la d a c e a e . A d e m á s puede v iv i r , sin c a u s a r daño, en plantas

2 9 0 SIMON

cu lt ivadas ta les c o m o e l guayabo (P s id iu m gua java) , e l n ís p e r o (M esti lus g e r m á n ic a ) , la b e re n je n a (Solanum m e lon g en a ) y a lgunos c í t r i c o s .

E s to , unido a su c a p a c id a d m ig r a t o r ia , da al in se c to una gran p o s i b i l id a d de su p e r v iv e n c ia a ún en la s m á s v a r ia d a s o a d v e r s a s c o n d ic io n e s p a ra su vida.

C R IA N Z A A R T IF IC IA L DE D y s d e r c u s peruv ianu s G.

M étod o W il le

E l a r re b ia ta d o IX peruv ianu s G. e s un in se c to de f á c i l c r ia n z a en la b o r a t o r io , lo que e x p l i c a que en e l D eparta m en to de E n to m o lo g ía de la E s ta c ió n E x p e r im e n ta l A g r í c o l a de La M olin a , L im a , P e r ú , se r e a l i c e su c r í a desde 1926, cuando se c r e ó e l D ep ar ta m en to , y se m antenga ac tu a lm en te una c r í a in ic iad a en 1930 p o r e l D r . J . E . W il le , sin que se hayan in trodu cid o , en lo s 37 años que e l la se r e a l iz a , nuevos in s e c to s . D eb id o a e l lo e s t o s indiv iduos son su s c e p t ib le s a tod os a q u e l los in s e c t i c id a s a lo s que lo s indiv iduos de c a m p o han adqu ir ido o d e s a r r o l l a d o r e s i s t e n c i a , p o r lo que tienen un in co n m e n s u ra b le v a lo r , c o m o te s t ig o s , en la s p ru eb as de in s e c t ic id a s [2].

E l m é to d o de c r í a segu id o p o r W il le c o n s is t e en r e c lu i r 100 a 150 ad ultos en un f r a s c o de v id r io v e r d e o azu l de 30 c m de alto p o r 15 ó20 c m de d iá m e tro . D entro d e l f r a s c o , que se tapa con un p e d a zo de te la b lan ca a m a rra d a al c u e l l o d e l m is m o , se pone un p o c o de a ren a en e l fondo, un b r o te te r m in a l de a lg o d o n e r o co n dos o t r e s h o jas y unas20 ó 30 s e m i l l a s (f igura 1).

FIG. 1. Paca la cria de D. peruvianus G. por e l m étodo W ille se em plean placas de Petri para guardar los huevos y los dos primeros estadios ninfales. . Las ninfas III, IV y V, así co m o los adultos, se guardan en frascos d e v idrio d e b oca ancha.

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L o s adultos se a limentan p ican do y chupando lo s ju g o s tanto de las h o ja s c o m o de la s s e m i l l a s y la s h e m b r a s ov ipos itan en la a ren a de l fondo, en tre la s s e m i l l a s .

D ia r ia m e n te se ca m b ia e l b r o te y se sacan l o s huevos , l o s que se ponen en p la ca s de P e t r i , con una h o ja de a lg o d o n e r o f r e s c a . E s ta hoja se c a m b ia cada dos d ías. A l quinto día lo s huevos b la n co s tornan a a m a r i l l o o naran ja y al día s igu iente e c lo s io n a n la s ninfas que p e r m a n e c e n agrupadas s o b r e la c a r a in fe r i o r de la hoja .

Cuando la s n infas a lcanzan su t e r c e r es tad io se la s c o l o c a en un f r a s c o p a ra adultos donde se le s ca m b ia e l a l im en to y se. l im p ia d ia r ia m e n te hasta que a lcanzan e l e stad o adulto [3].

La atención de cad a p la ca de P e t r i tom a de 2 a 3 min y la de cada f r a s c o de ninfas de 4 a 8 min, dependiendo d e l n ú m e r o 'd e indiv iduos y la habilidad de l o p e r a d o r .

En cada p la ca pueden g u a rd a rs e hasta 500 huevos o n infas y en cada f r a s c o 500 ninfas o 200 adultos. De cada f r a s c o de adultos se obtiene un p r o m e d io de 30 000 huevos , de l o s que se pueden r e c u p e r a r entre 9 y 10 000 adultos en la b o r a t o r io s m a nten id os en c o n d ic io n e s n a tu ra les y em p lean d o 12 h d ia r ia s de tra b a jo .

D ispon iendo de s e i s f r a s c o s p a ra adultos se puede o b te n e r una p r o d u c c ió n d ia r ia in in terru m p ida de 2700 adultos o sea 81 000 m e n su a les em p lean d o 40 h o ra s d ia r ia s , 200 p la ca s y 200 f r a s c o s .

C R IA N Z A M A SA L DE D y s d e r c u s peruv ianu s G.

M éthodo s e m ia u to m á t ico

E l M étodo W il le , s i bien e s s e g u r o y s e n c i l lo , no p e r m ite p r o d u c ir , en f o r m a e c o n ó m ic a , g ra n d e s can t id ades de a r r e b ia ta d o s p o r lo que durante e l año 1966 nos p r o p u s im o s d e s a r r o l l a r un s i s t e m a de c r ía m a s a l y e c o n ó m ic a , que c r e e m o s h aber lo g ra d o en la f o r m a que d e s c r i b im o s a cont inuac ión .

En una jau la de 9 0 X 6 0 X 6 0 c m con sus cu a tro p a r e d e s la te r a le s c o n s tru id a s de m a d e r a u o t r o m a te r ia l la vab le , e l techo de m a l la p lá s t i c a de 14 h i lo s p o r pu lgada y e l p is o de m a l la m e tá l i ca de nueve h ilos p o r pulgada, se in trod u ce , p o r cu a lq u ie r a de las dos ventanas c i r c u la r e s de 15 a 20 c m de d iá m e tro , que tiene una p a re d fronta l , d o s f l o r e r i t o s contenien do agua y b r o te s te r m in a le s de a lg o d o n e r o cu y as b o c a s se han tapado con f ib ra de a lgodón a fin de que lo s in s e c to s no se ahoguen y sos ten ga n l o s b r o te s e rg u id o s . A d e m á s se in troducen dos p la tos de15 c m de d iá m e tro con s e m il la de a lg o d o n e r o y 2000 adultos de D. p e r u v ianus G. s e x a d o s y en la p r o p o r c ió n de dos m a ch o s p o r ca d a t r e s h e m b r a s ( f igura 2).

D eba jo de la jaula hay un p lano in cl in ad o de m a d e r a , latón, p le x ig lá s , o cu a lq u ie r o t r o m a te r ia l l i s o que p e r m ite a lo s huevos , p u estos p o r las h e m b r a s , r o d a r hasta una p r o b e ta graduada donde se r e c o le c ta n y cuentan v o lu m é tr ic a m e n te , tom ando c o m o unidad 4250 huevos p o r c e n t ím e tro c ú b ic o ; s o b r e la jaula hay un f o c o de luz in ca n d escen te de 60 W o f lu o r e s c e n te de 15 W ( f igura 3).

292 SIMON

FIG. 2 . Los adultos de Lh peruvianus G. ovipositan en las sem illas co loca d a s en e l fondo d e los frascos. D iariam ente se extraen los huevos pasándolos por tam ices.

E1G. 3. En el m étodo sem iautom ático d e cria m asal d e D. peruvianus G. se em plean dos tipos de jaulas. La superior con tien e los adultos y la in ferior las ninfas.

P o r cada jaula de adultos se deben te n e r 15 jau las p a ra ninfas, la s que son igua les a la s de adultos, con la única d i fe r e n c ia de l p is o que es ta m b ién de m a d e ra o d e l m i s m o m a te r ia l la va b le de la s p a re d e s .

En cada jaula p a ra ninfas se in trodu cen 5 c m 3 de huevos , de l o s que e c lo s io n a r á n la s ninfas que d e s a r r o l l a r á n toda su v ida den tro de la jaula

SM-102/30 293

a l im en tá n d ose de lo s t e r m in a le s de a lg o d o n e r o , que se c a m b ia r á n cad a d o s d ías o a m e d id a que sea n e c e s a r i o , y de las s e m i l la s de lo s p la tos . D ia r ia m e n te , o cada v e z que se ca m b ie n lo s te r m in a le s , se hará una l i g e r a l im p ie z a p a ra r e t i r a r c a d á v e r e s y exhuvias.

C ada jaula de adultos p r o d u c e , durante un p e r io d o de 21 d con tad os a p a r t i r d e l se x to día de edad de l o s ad ultos , a l r e d e d o r de 5 c m 3 de h u evos d ia r io s , con lo que se l len a rá n entre 15 y 20 ja u las p a ra ninfas y, c o m o la r e c u p e r a c i ó n con este m é to d o e s de a l r e d e d o r de 50%, se p r o du cirán entre 150 000 y 200 000 adultos, o l o que e s lo m i s m o 10 000 in d i v idu os d ia r io s durante 20 d ías , a p a r t ir d e l m e s de la p r im e r a o v ip o s i c ió n .

Cada jaula r e q u ie r e ap ro x im a d a m e n te 4 min de a tenc ión d ia r ia , a l r e d e d o r de una h ora p a ra tod o e l s i s t e m a , lo que qu ie re d e c i r que un so lo h o m b re puede m a n ten er o c h o s i s t e m a s o, lo que e s lo m i s m o , p r o d u c ir 80 000 indiv iduos d ia r i o s . E s d e c i r que, con las 40 h o ra s d ia r ia s de a tenc ión que n e c e s i ta e l s i s t e m a W il le p a ra p r o d u c i r 2700 indiv iduos d ia r i o s , n o s o t r o s p o d r ía m o s l o g r a r , t e ó r i c a m e n t e , 400 000 indiv iduos d ia r io s .

E S T E R IL IZ A C IO N DE D y s d e r c u s p eruv ianu s G.M ED IAN T E RA YO S G A M M A

L o s t r a b a jo s se r e a l iz a r o n en e l i r r a d ia d o r de 137C s , insta lado en lo s L a b o r a to r io s de E n to m o lo g ía de la E s ta c ió n E x p e r im e n ta l A g r í c o l a de L a M olina , c e d id o p o r la USAEC p o r in te r m e d io de la Junta de C o n tro l de E n e rg ía A t ó m ic a d e l P e r ú , ten iendo c o m o an teceden te un trab a jo s im i la r e je cu ta d o p o r e l autor y e l In gen iero G o d o fr e d o G a r c ía B aca , en 1961, en un i r r a d ia d o r de 60Co durante la ex h ib ic ió n « A t o m o s p a ra la P a z » [4].

M a te r ia le s y m é th od os

E l i r r a d ia d o r de 137Cs de 20 000 Ci, dob le m e n te e n ca p su la d o en a c e r o in oxidab le s o ld a d o en a t m ó s fe r a de h e l io , t iene la fuente de i r ra d ia c ió n c o lo c a d o en una « j a u l a » que r o d e a la cav id ad de i r r a d ia c ió n , cuando e stá en p o s ic i ó n de « i r r a d i a r » . La i r r a d ia c ió n e x tern a p r o m e d io r e su lta a s i m e n o r de 1 m R /h a 30 c m de la unidad.

La c á m a r a de i r r a d ia c ió n de s e c c i ó n pentagonal de 15 c m de d iá m e t r o p o r 20 c m de la r g o , e stá tam bién c o n s tru id a de a c e r o in ox idab le y e stá equipada con p u e rta de c la u su r a m ontada p a ra d e s l i z a r s e h o r iz o n t a l m en te de la p o s i c i ó n de « c a r g a » a la de « i r r a d i a c i ó n » en m e n o s de 4 s.

Un r e lo j e l é c t r i c o de 20 m in, de p r o g r a m a c ió n a u tom át ica , detiene la i r ra d ia c ió n a u tom át ica m en te al f inal d e l t ie m p o p r e d e te r m in a d o de e x p o s ic ió n . P a r a p e r ío d o s m á s la r g o s de e x p o s ic ió n , s e puede anular e l r e lo j au tom át ico y p r o c e d e r m ed ian te c o n t r o l m anual. F in a lm en te en c a s o que fa l le la c o r r ie n t e e l é c t r i c a se puede m o v i l i z a r la c á m a r a m e diante un v o lan te que se halla en un e x t r e m o d e l equ ipo .

L o s in s e c to s e m p le a d o s fu eron e x t r a íd o s de la s ja u las de c r ia n z a m a s a l de D y s d e r c u s p e ru v ia n u s G . , donde n u m e r o s a s n infas estaban m udando en adultos e s e día, de ta l m o d o que aún no habían cop u la d o .

L o s in s e c to s fu eron c la s i f i c a d o s a s im p le v is ta (las h e m b r a s son m á s g ra n d e s y v o lu m in o s a s que lo s m a c h o s ) y con f in a d o s en la c á m a r a

294 SIMON

de i r r a d ia c ió n , en g ru p o s de 10 que lu e go de i r r a d ia d o s fu eron con f in ad os en f r a s c o s de v id r io , d á n d o s e le s una le t ra pa ra cad a intensidad de i r r a d ia c ión y un n ú m e ro p ara cad a p a re ja , lo que se anotaba en e l f r a s c o r e s p e c t iv o .

L o s f r a s c o s e m p le a d o s fueron de v id r io o p a co v e r d o s o , de 20 cm ' de a lto p o r 15 c m de d iá m e tro , con b o c a ancha. D entro d e l f r a s c o se puso a re n a en e l fondo y s o b r e e l la un tubito con agua conten ien do un te r m in a l de a lg o d o n e r o con t r e s h o jas que se m antenía e r e c t o g r a c ia s a un c o p o de a lgodón que a la v e z tapaba la b o c a d e l tubito , ev itando que l o s ad u ltos se ahogaran en e l agua. A d e m á s , se puso una cáp su la de 4 c m de d iá m e t r o conten ien do s e m i l l a de a lg o d o n e r o . E l f r a s c o se tapó m ediante una c u b ie r ta de to cu yo e n m a r c a d o en a c e r o .

D ia r ia m e n te se o b s e r v a r o n lo s f r a s c o s y se anotó en un cu a d e rn o l o s a c o n te c im ie n to s de l día, ta le s c o m o cóp u la , o v ip o s i c i o n e s , n ú m e ro de h uevos , m u e r to s , n ú m e ro de ninfas de cada e s tad io , etc .

L o s h u evos fueron e x t r a íd o s de l f r a s c o y c o l o c a d o s en una p la c a de P e t r i conten ien d o una hoja t ie rn a de a lg o d o n e r o . La p la ca se m a r c ó con la le t ra m a y ú scu la y n ú m e ro c o r r e s p o n d ie n te a l f r a s c o , seg u id o de la le t r a m in ú scu la « a » p a ra la p r im e r a p o s tu ra , « b » p a ra la segunda, « c » p a ra la t e r c e r a y a s í s u c e s iv a m e n te . La h o ja se c a m b ió d ia r ia m e n te .Si l o s huevos d ieron ninfas é s ta s se m antuv ieron hasta su t e r c e r estad io en las p la ca s y al cu a rto e s ta d io pa saron a un f r a s c o p r e p a r a d o en la m i s m a f o r m a que e l de adultos.

En un p r im e r e n say o se ir ra d ia ro n tanto a lo s m a ch o s c o m o a las h e m b r a s , con las s igu ien tes in tensidades A-.1000 R; B :2500 R; C :50 00 R; D :10 000 R, y T :te s t ig o n o rm a l sin ir ra d ia c ió n .

En un segundo e n say o se ir ra d ia ro n s ó lo io s m a ch o s , e x ce p to en el t e s t ig o , y se lo s con f in ó con una h em b ra n orm al. Las in tensidades e m p le a d a s fueron : A-.IOOR; 1Ï:200 R; C :40 0 R; D:800 R; E : ] f i0 0 I Í ; F :32 00 R; G :6400 R; 11:12 800 R; y T : t e s t ig o n o rm a l, sin ir ra d ia c ió n .

En un t e r c e r e n sa y o se ir ra d ia ro n con 5000 R y 10 000 R m a c h o s y h e m b r a s v írg e n e s , que lu e go se r e c lu y e r o n , cad a uno, con una p a re ja n orm al con e l fin de c o n o c e r si e l m a ch o o la h em bra i r ra d ia d o s eran r e c h a z a d o s p o r la h em bra o e l m ach o n o r m a le s o si é s t o s tenían p r e f e r e n c ia p o r lo s n o r m a le s s o b r e lo s e s t e r i l i z a d o s .

F inalm ente , en un cu a rto en sayo se . irrad iaron m a c h o s con 10 000 R y se r e c lu y e r o n con una p a re ja n o rm a l en las p r o p o r c io n e s de 1:1:2;1 :1 :8 ; 1 :1 :32; y 1 :1 :128, a d e m á s de un te s t ig o con 1: 129.

RE SU L T A D O S Y DISCUSION

En lo s cu a d ro s I .y II p r e s e n ta m o s lo s re su lta d o s obten id os en el p r i m e r e n say o , cuando a m b o s s e x o s fueron s o m e t id o s a i r r a d ia c ió n , y de su estud io puede d e d u c i r s e , en tre o tra s c o s a s , que la cantidad de huevos , aún cuando se p rod u z ca n , e s in fe r io r en las p a r e ja s i r ra d ia d a s que en las n o rm a le s . Se o b s e r v a tam bién que e l d e s a r r o l l o de lo s in s e c t o s fue p rá c t ic a m e n te n orm al con 1000 R (tratam iento A); p e r o con 2500 R (13) s ó lo unos p o c o s huevos l le g a ro n al e s tad io de ninfa I y lu ego m u r ie r o n . Con 5000 R (С) y 10 000 R (D) hubo p r o d u c c ió n de huevos y aunque e l l o s no e c lo s io n a r o n , e l lo nos in d icó que la h e m b ra del a r r e - b iatado no fue e s t e r i l i z a d a ya que, de a c u e rd o a la s n o rm a s u n iv e r s a l -

SM-102/30 295

m en te a cep tad as , una h e m b ra e s t é r i l no p r o d u c e huevos; o b ien , de a c u e r d o a lo s r e su lta d o s o b ten idos p o r e l autor y G a r c ía B aca en 3 961, en que no se obtuvo e c lo s ió n de lo s huevos i r r a d ia d o s con 1000 R,10 000 R y 100 000 R, la h e m b ra no s e r ía v irg e n o e l la s e r ía cap a z de

C U A D R O I. IN K L U K N C IA DE D OSIS C R E C IE N T E S DE R A D IA C IO N E N LA V ID A DE D y s d e r c u s p e ru v ia n u s G . C U A N D O A M B O S SE X O S F U E R O N IR R A D IA D O S (L A M O L IN A , P E R U , 1907)

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p r o d u c i r huevos sin in terven c ión del m a ch o , en cuyo c a s o la i r r a d ia c ió n de h e m b ra s y su l ib e r a c ió n junto con l o s m a ch o s e s t é r i l e s no s e r ia p e r ju d ic ia l .

En cuanto a l o s r e su lta d o s del segundo en sayo que p r e s e n ta m o s en lo s c u a d ro s III y IV y V y en el que s ó l o lo s m a ch o s r e c ib ie r o n ir r a d ia c ió n , p o d e m o s c o m e n ta r que ap arentem en te la ra d ia c ió n , en in ten s idades in f e r i o r e s a 10 000 R, a fec ta a lo s in s e c to s ob l ig á n d o lo s a r e a l i z a r c ó p u la s m á s p ro lo n ga d a s y d ism in u yen d o el n ú m e ro de huevos p u estos en cada lo te , con la única e x c e p c ió n de lo s m a c h o s que r e c ib ie r o n 400 R, lo que p o d r ía d e b e r s e a m a y o r fecundidad de la s h em b ra s . T a m bién se pudo o b s e r v a r que la longevidad de lo s m a ch o s i r r a d ia d o s se r e d u jo en un 88% p a ra la s ba ja s in ten s idades y en (¡G, G2 y 39% para la s in ten s id ad es de 1600 y 3200, 6400 y 12 800 R, r e s p e c t iv a m e n te , y que la in cu ba c ión de lo s huevos e s a c e le r a d a p o r la s ba ja s in ten sidades y re tard a d a p o r las a l tas , s i bien e s c i e r t o que la du ración d e l c i c l o b i o ló g i c o no e s a p a r e n te m en te a fectada p o r la s ba ja s in ten sidades , p e r o si" e s p ro lo n ga d a hasta c a s i en un 50% p o r las altas.

En e l t e r c e r en sayo las h e m b ra s y l o s m a ch o s ir ra d ia d o s con 5000 Ro con 10 000 R copu laron indistintam ente con lo s m a ch o s y h e m b r a s n o r m a le s , lo que in d ica r ía que estas in ten sidades no reducen la c a p a c id a d de c o m p e te n c ia de ninguno de lo s dos s e x o s .

En cuando a lo s n iv e le s de c o m p e te n c ia , s i bien en este p r im e r e n sa y o , en las p r o p o r c io n e s de 32:1 y 128:1, l o s m a c h o s n o r m a le s no e n co n tr a r o n a la s h e m b ra s , c r e e m o s que debe r e p e t i r s e la e x p e r ie n c ia an tes de p o d e r señ a lar un n ive l de c o m p e te n c ia p a ra este in se c to .

CONCLUSIONES

1. E s p o s ib le c r i a r en f o r m a m a sa l y e c o n ó m ic a el a r re b ia ta d o o ch inche m a n ch a d o ra del a lg o d o n e ro , D y s d e r c u s peruv ianu s G. en la b o r a t o r io s a c o n d ic io n a d o s a 27°C, 5°C y 80% H. R. 5%.2. La ch in che m anch ad ora de la f ib ra del algodón , peruv ianu s G. e s fá c i lm e n te e s t e r i l i z a b le con 5 a 10 000 R de rad ia c ión gam m a.3. La lon gev idad de lo s m a ch o s e s t e r i l i z a d o s m ediante r a y o s ga m m a se r e d u c e hasta un 61% en r e la c ió n c o n l a de m a ch o s n o r m a le s .

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4. L as h e m b r a s de IX p eruv ianu s G. , ir ra d ia d a s hasta con 12 800 R,son c a p a c e s de o v ip o s i ta r , aunque s ó l o en un c a s o lo s huevos e c lo s io n a ro n .5. D o s is entre 1600 R y 12 800 R pro lon ga n c o n s id e r a b le m e n te e l c i c l o b i o ló g i c o de E). peruv ianu s G.

R E F E R E N C I A S

[1 ] WILLE, T . , J. E . , Entom ología A g ríco la d e l Perú (2a. E d .), Junta de Sanidad V egeta l, Lima,Perú (1952).

[2 ] SIMON, F . , J. E . , Resistencia del arrebiatado de Dysdercus peruvianus G. a los insecticidas orgánicos,Inf. Men. N °368, E. E. A. La M olina (M arzo 1958).

[3] OHOSHI, J . , SIMON, F . , J. E . , D eterm inación de las dosis leta les d e l Sevin para Dysdercus peruvianus G . en strains resistentes y susceptibles al BHC, La M olina (1963).

[4J SIMON, F. , J. E. , G arcfa B. G. , Efectos d e la radiación gam m a en e l arrebiatado, Bol. de la S oc. Ent. Agr. d e l Perú, 3 (Junio 1983).

D I S C U S S I O N

W. J . K L O F T : What da ily p h otop er iod did you use in yo u r m a ss r e a r in g o f D y s d e r c u s p eru v ia n u s? We found f r o m ou r e x p e r im e n ts with re la ted H e te r o p - te r a that 16 h ou rs o f light to 8 h ours o f dark w as the opt im u m fo r d e v e lo p ment and r e p ro d u c t io n .

J . E . SIMON: We il lum in ated the in s e c ts f o r 14 h ours daily . D espite the fac t that D. peruvianus is quite able to l ive a l l y e a r round under P e ru v ia n cond it ion s , we d i s c o v e r e d that a da ily e x p o su re o f m o r e than 12 h ours o f light s ign if ican t ly im p r o v e d the r e p ro d u c t io n rate .

W. J . K L O F T : Did you en co u n te r the p r o b le m o f mutual can n ib a l ism in y o u r c ro w d e d m a s s r e a r in g s ? E ven s e e d - fe e d in g bugs like O ncope ltus s o m e t im e s show a ten den cy to w a r d s this pattern o f beh av iou r .

J . E . SIMON: W ell , we used the m e s h at the bottom o f the a d u lts ' cage e x p r e s s ly to avoid any dam age to the eg gs , and d esp ite th ere being 2000 adults p e r ca g e we did not o b s e r v e any can n ib a l ism .

3 0 0 SIMON

W. J . K L O F T : A r e you su re that it i s n e c e s s a r y to g ive the in se c ts the additiona l food supply o f co tton le a v e s ? We found that ou r s e e d - f e e d e r O n cop e ltu s fas c ia tu s needed no g re e n plant com p on en t in its diet, but cou ld be r e a r e d on s e e d s and w ater on ly . T h is might p ro v e a m eans o f red uc in g the c o s t o f y o u r m a s s r e a r in g s .

J . E . SIMON: In fact, during the eight w eek s p r e v io u s to m y v is i t to Vienna we w e r e not a l low ed to c r o p the cotton le a v e s by v irtue o f ou r P lant Quarantine R egu lat ions and so we have had to r e a r the bugs on seed and w a te r on ly . I had fe a r e d that on return to L im a I would find that m y c u ltu re o f D y s d e r c u s had pe r ish e d , but yo u r r e m a r k s have g iven m e s o m e e n co u ra g e m e n t to b e l ie v e that this diet w il l be adequate, a fte r a l l .

A P P L IC A T IO N A U X O ryctcs (C O LEO PTER ES: SCARAB AEIDAE) DE LA METHODE DU LACHER DE M ALES STERILES'"

B. HURPININRA, STA TIO N DE RECHERCHES DE LU TTE BIOLOGIQUE

ET DE BIOCCENOTIQUE LA MINIERE, VERSAILLES, FRANCE


RESEARCH INTO TI-IE APPLICATION OF THE STERILE-MALE RELEASE TECHNIQUE TO THE RHINOCEROS BEETLE (COLEOPTERA:SCARABAELDAE). The control o f the Rhinoceros b eetle , w hich causes considerable dam age in palm and cocon u t plantations in the trop ical regions o f the w orld, meets w ith d ifficu lties resulting from the habits o f the insects. C h em ica l and b io lo g ica l control m ethods have not yet produced e co n o m ica lly worthw hile results. It was therefore proposed, under the Joint P roject established by the UN S pecia l Fund and the South P a cific C om m ission for devising a m ethod o f con trollin g the m ain pest in this group, Oryctes rhinoceros, that the possibility be investigated o f using against these co leop tera the technique o f releasing m ales sterilized by radiation. Studies have been m ade involving the two most im portant species O. m onoceros O l. and O. rhinoceros L. T h e first stage was the developm en t o f a continuous breeding m ethod, w hich had not hitherto been possible because o f the la rg e -sca le slaughter arising from fungus attacks by M etarrhizium anisopliae M etsch. For the larvae the technique used involved first in d ividual rearing, fo llow ed by group rearing tests w ith 100 or so insects in tanks. The average tem perature in both cases was c lo se to 30eC and the insects w ere fed on a m ixture o f rotten w ood and le a f m ould m ixed w ith the faeces o f d om estic anim als. The adults, fed on banana slices, are p laced in troughs or tanks contain in g sieved -earth. It was thus possible to rear Oryctes in several successive cy c le s , avoid ing the e p iz o o ic stage o f M etarrhizium , w ith a m u ltip lica tion rate from one generation to the next o f c lo se on 10, Tests on radiation steriliza tion procedures taking as variables the gam m a-ray dose, the stage o f the insect and the ph ysio log ica l state o f the im a go , showed that O. m onoceros adults w ere sterilized by a single a p p lica tion o f 4000 rad, w ith a slight reduction in their length o f l ife . The sam e was true o f O. rhinoceros, w h ich how ever required a dose greater than 6000 rad to ster ilize it. T h e m ating urge and sexual vigour o f the irradiated insects are, how ever, redu ced ; c o m p e tit iv e trials betw een irradiated and norm al m ales are described and com m en ted on. Apart from the im m ed ia te e ffe cts o f gam m a-rays, a possible deferred e ffe c t on the progeny o f fem a les m ated w ith partially sterilized m ales was also envisaged.

APPLICATION AUX O ryctes (COLEOPTERES: SCARABAEIDAE) DE LA METHODE DU LACHER DE MALES STERILES. La lutte con tre les O ryctes, qui com m etten t des dégâts considérables dans les plantations d e palm iers et d e co co tie rs dans les régions trop ica les du g lobe , se heurte à des d ifficu ltés dues aux mœurs d e ces insectes. Les m éthodes ch im iques d e m êm e que les essais de lutte b io log iq u e n ’ont pas abouti jusqu'à présent à des résultats écon om iq u em en t valables. Aussi il a é té proposé, dans le cadre du Projet con jo in t du Fonds spécia l des Nations Unies et d e la C om m ission du P acifiqu e Sud, chargés de m ettre au point la lutte contre le principa l ravageur d e c e groupe, O. rh inoceros, d 'é tu d ie r les possibilités d ’a p p lica tion à ces co léop tères des procédés d e lutte a u tocide par lâchers d e m âles stérilisés par irradiation.Les recherches e ffectu ées par l'auteur et son équipe ont porté sur les deux espèces les plus im portantes;O. m onoceros O l, et O. rhinoceros L. Dans une prem ière étape on a ch erch é à réaliser un é lev a g e perm anent qui n 'ava it jam ais pu être obtenu du fa it des h écatom bes provoquées par la m uscardine verte à M etarrhizium anisopliae M etsch. Pour les larves, la techn ique utilisée a été d 'abord l 'é le v a g e in d iv id u el; ensuite il a é té procéd é à des essais d 'é le v a g e par groupes d ’ une cen ta in e d ’ insectes dans des bacs. La tem pérature m oyenne dans les deux cas était voisine d e 30°C et l'a lim en ta tion assurée par un m éla n ge

* Recherches e ffectu ées sous contrat (Projet con jo in t du Fonds spécia l des Nations Unies et de la C om m ission du P acifiqu e Sud, «R h in o ce ro s Beetle P r o je c t » , A p ia , Sam oa occid en ta les ).

301

302 HURPIN

d e bois d écom p osé ou d e terreau de feu illes a v e c des fè ce s d 'an im au x dom estiques. Les adultes a l i m entés a v ec des rondelles d e banane sont m is en ponte dans des seaux ou des bacs renferm ant du terreau cr ib lé . L 'é lev a g e des O ryctes a pu ainsi être réalisé en plusieurs cy c le s successifs, sans é p iz o o t ie à M etarrhizium , avec un taux d e m u ltip lica tion voisin d e 10 d ’une génération à l'au tre . Les essais sur les m odalités d e stérilisation par irradiation en fon ction d e la dose d e rayons gam m a, du stade de l 'in se c te et d e l 'é ta t physiologique des im agos ont m ontré que les adultes de O. m onoceros étaient stérilisés par une seule app lica tion d e 4000 rad a v ec une fa ib le réduction d e la lon g év ité des individus. Les m êm es phén om ènes s'observent ch ez O. rh inocéros, qui dem ande toutefois une dose supérieure à GOOO rad pour aboutir à la stérilisation. La tendan ce à l 'a cco u p le m e n t, la vigueur gén ita le des insectes irradiés apparaissent toutefois am oindries; les essais d e com p étition entre m âles irradiés et m âles normaux sont exposés et com m en tés. En dehors des effets im m édiats des rayons gam m a, une éven tu e lle a ction d iffé ré e sur la d escen dan ce d e fe m e lle s a ccou p lées avec des m âles partie llem en t stérilisés a é té recherch ée.

1. D IF F IC U L T E S DE I A L U T T E C O N T R E LES O r y c t e s

L e s O r y c t e s constituent un des p r in c ip a u x e x e m p le s de ra v a g e u r s d ' im p o r ta n c e é c o n o m iq u e c o n s id é r a b le c o n t re le s q u e ls , en dépit des p r o g r è s r é a l i s é s depuis 20 ans dans le dom ain e p h ytosa n ita ire , i l n 'e x is t e p as e n c o r e de m éthode de lutte ra t ion n e l le et e f f i c a c e , a ins i que nous avons eu l ' o c c a s i o n de le r a p p e le r p r é c é d e m m e n t [1|. On connaît au m o in s 25 e s p è c e s d 'O r y c t e s n u is ib les aux p a lm ie r s et aux c o c o t i e r s , sans p a r l e r d es au tres g e n r e s de D ynastides te ls que le s Strategus, Scap anes et a u tres X y lo t ru p e s répandus dans toutes le s r é g io n s t r o p ic a l e s et é q u a to r ia le s .L e s deux e s p è c e s le s plus im p ortan tes sont O. m o n o c e r o s d 'A fr iq u e et O. r h in o c e r o s d 'A s ie et d 'O cé a n ie qui re p ré s e n te n t , surtout la d e r n iè r e , uri v é r i ta b le f léau dans c e r ta in e s p lantations . D 'a p r è s C u m b e r [2], en e f fe t , deux in s e c te s pa r a r b r e su ff isent pou r p r o v o q u e r des dégâts t r è s im p o rta n ts , et H in ck ley [3] p en se que, dans le s con d it ion s du P a c i f iq u e , l o r s q u ' i l y a un in se c te pou r 19 c o c o t i e r s ce tte cu ltu re n 'e s t p lus ren ta b le .

L e s d if f i cu lté s de la lutte c o n t re c e s in s e c te s sont l i é e s à le u r m o d e de v ie et leu r c o m p o r te m e n t . L e s la r v e s v ivent dans le s m a t iè r e s v é g é ta les en d é c o m p o s it io n , notam m ent le s p a lm ie r s m o r t s , tandis que le s adultes ne so r te n t que la nuit p ou r a l le r s 'a t ta q u e r à la b a se des jeu n es p a lm e s , dans le s q u e l le s i ls fo ren t une g a le r ie où i ls s 'a b r i te n t pendant p lus ou m o in s lon gtem p s avant de r e to u r n e r p on d re dans le s g î te s la r v a i r e s . C e u x - c i sont d i s p e r s é s à la fo is dans le s plantations et dans la b r o u s s e environnante . La d e s tru c t io n de c e s g i le s est une des m e s u r e s e s s e n t ie l l e s p o u r l im i t e r la p r o l i f é r a t io n des O r y c t e s ; a u ss i e s t - e l l e p r e s c r i t e pa r la lé g is la t io n dans bea ucou p de p a ys . M ais e l le n 'e s t pas tou jou rs fa c i le à r é a l i s e r .

En d e h o rs de p r o c é d é s e m p ir iq u e s te ls que l 'e x t r a c t io n des adultes des g a l e r i e s et la d es tru c t io n des la r v e s dans le s g îtes n atu re ls ou dans des b û c h e s p iè g e s d is p o s é e s à c e t e f fe t , la m éth od e de lutte la p lus c o u r a m m e n t e m p lo y é e est le t ra i tem en t des c o u ro n n e s pa r un m élan ge d ' in s e c t i c id e et de s c iu r e de b o is p la cé à l ' a i s s e l l è des p lus jeu n es f e u i l le s . Alais i l est n é c e s s a i r e de r é p é te r ce tte o p éra t ion t r è s souvent (deux ou t r o i s f o is par m o i s en C o t e - d 'I v o i r e , pa r e x e m p le ) [4], de s o r t e que le p r ix de rev ien t est é le v é , de l ' o r d r e de 4000 ou 5000 F C F A / h a ’ an, p ou r une e f f i c a c i t é d ou teu se .

De m u lt ip les e s s a i s de lutte b io lo g iq u e ont été e n tr e p r i s depuis le début du s i è c l e p ou r o b ten ir un c o n t rô le n a tu re l de c e s r a v a g e u r s , so it p a r l ' in tr o d u c t io n d ' in s e c t e s p a r a s i t e s , t e ls que d iv e r s e s e s p è c e s de

SM-102/31 303

S c o l ia (H y m é n o p tè re s S c o l i id æ ) , ou p r é d a t e u r s , te ls que des C o lé o p tè r e s E la té r id e s ou S c a r i t id e s et des H é m ip tè r e s R édu v id es , so i t pa r t r a i t e m ent de c e r ta in s g îte s la r v a i r e s p a r le cham pign on M e ta r r h iz iu m anisopliae .

Dans le p r e m ie r c a s , l 'a c c l im a ta t io n de c e r ta in s en to m o p h a ge s a été obtenue à p lu s ie u r s r e p r i s e s dans d i f fé re n te s î l e s du P a c i f iq u e (F id j i , P a lau , Sam oa ), m a is ju sq u 'à p r é se n t aucun de c e s in s e c te s n 'a p e r m is de r é d u ire le s dégâts d 'O r y c t e s . Dans le d e u x ièm e c a s , qu e lques r é s u l tats e n co u ra gea n ts ont été e n r e g i s t r é s a v e c le cham pign on , m a is i l n 'a pas été p o s s ib le de le s g é n é r a l i s e r en dépit des t r è s n o m b r e u s e s tentatives d 'u t i l isa t ion de c e pathogène e f fe c tu é e s dans d iv e r s e s r é g io n s de l 'a i r e g é o grap h iq u e de O. r h in o c e r o s et m a lg r é la f r é q u e n c e des é p iz o o t ie s n a tu re l les dues à ce tte m u s c a r d in e .

2. POSSIBILITES O F F E R T E S P A R L A L U T T E AU T O C ID E

Devant c e s é c h e c s des m é th od es ch im iq u e s et b io lo g iq u e s et du fait du c o m p o r te m e n t de c e s in s e c te s , dont le stade im a g in a i est le se u l v r a i m ent a c c e s s i b l e , au m o in s dans c e r t a in e s p é r io d e s , nous avons p r o p o s é d 'e x a m in e r le s p o s s ib i l i t é s d 'a p p l ica t io n aux O r y c t e s et n otam m en t àO . r h in o c e r o s des m é th od es de lutte au toc ide qui ont fait le u r preuve p o u r d 'au tres r a v a g e u r s , depuis le s s u c c è s r e n c o n t r é s aux E ta ts -U n is dans la lutte c o n t re la m ou ch e du bé ta i l , C o c h l io m y ia h o m in iv o ra x .

D 'autre part , l 'e x p é r im e n ta t io n de I l o r b e r [5, 6] su r le hanneton com m u n , e s p è c e de la m ê m e fa m i l le , constitua it un p r é cé d e n t fa v o ra b le p ou r le r e c o u r s aux lâ c h e r s de m â le s s t é r i l e s en vue de ten ter une é r a d ica t io n de O. r h in o c e r o s .

Cet o b je c t i f a été p r i s en c o n s id é r a t io n p a r le C o n s e i l de d ir e c t io n du P r o je t con jo in t du F on d s s p é c ia l des Nations Unies et de la C o m m is s io n du P ac i f iq u e Sud, é ta b l i depuis 1064 en vue de m e t tr e au point la lutte c o n t re O . r h in o c e r o s et le s in s e c te s ap pa re n té s . A u s s i l 'Institut de r e c h e r c h e s p ou r le s h u iles et o lé a g in e u x (IRHO) et la Station de r e c h e r c h e s de lutte b io lo g iq u e et de b iocoen otiqu e de La M in iè re (INRA) b é n é f i c i e n t - i l s d 'un contra t de r e c h e r c h e dans le c a d r e de ce P r o je t , en vue de p r é c i s e r le s p o s s ib i l i t é s d 'a p p l ica t io n aux D yn astides de la lutte au to c id e p a r ir ra d ia t io n .

Dans le s c a s des O r y c t e s , c o m m e p ou r le s a u tres in s e c te s , a in s i que F é ro n [7] l 'a sou lign é p r é c é d e m m e n t , l ' e m p l o i de ce tte m éthod e de lutte su pp ose des r e c h e r c h e s p r é a la b le s dans t r o is p r in c ip a le s d i r e c t io n s : lam is e au point d 'un é le v a g e p e rm a n en t , la c o m p a r a is o n du c o m p o r te m e n t des in s e c te s s t é r i l i s é s pa r ra p p o r t à c e lu i des in s e c te s n o rm au x , l 'é tu de en plantation de l ' é th o lo g ie et de l ' é c o l o g i e p o u r p a r v e n ir à une m éthode d 'e s t im a t io n des popu la tion s .

Nous nous s o m m e s attachés depuis deux ans à la r é a l is a t io n des deux p r e m i è r e s con d it ion s : m i s e au point de l 'é l e v a g e et e x a m e n en la b o r a to i r e des e f fe ts de l ' i r r a d ia t io n , le s r e c h e r c h e s é c o lo g iq u e s étant condu ites p a ra l lè le m e n t , so i t dans le P a c i f iq u e Sud p a r le s so in s des c h e r c h e u r s du P r o je t ou de l 'O f f i c e de la r e c h e r c h e s c ien t i f iqu e et technique d 'o u t r e m e r (ORSTOM ) dans le c a s de O. r h in o c e r o s , so it en C ô t e - d ' I v o i r e pa r l 'IRH O dans le c a s de O. m o n o c e r o s .

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3. R E A L IS A T IO N D 'U N E L E V A G E P E R M A N E N T D 'O r y c t e s

La m éthode d 'é le v a g e de c e s D yn astides tro p ica u x a été adaptée à p a r t i r des résu lta ts de l 'e x p é r im e n ta t io n a n té r ie u re [8] que nous av ion s condu ite su r deux e s p è c e s e u ro p é e n n e s de la m ê m e fa m i l le , O r y c t e s n a s i c o r n is L. et Phyllognathus s i len us F . C e l l e - c i avait m is en é v id e n ce qu 'e n d e h o rs d 'une te m p é ra tu re su ff isan te (25 à 30°C) le fa c te u r e s s e n t ie l p o u r é v i te r le s h é ca to m b e s dues à M. an isop liae était l 'a l im e n ta t io n des la r v e s a v e c un m é la n ge de m a t iè r e v é g é ta le en d é c o m p o s i t i o n ( te r re a u ou b o i s p o u r r i ) et d 'e x c r é m e n t s d 'o r ig in e a n im a le . La ponte des ad ultes is s u s des la r v e s é l e v é e s dans c e s co n d it ion s était obtenue sans d i f f i cu lté dans un seau de 10 l i t r e s en p o lyéth y lène r e m p l i aux deux t i e r s de t e r r e a u c r ib l é et f e r m é p a r une fe u i l le de polyane p ou r c o n s e r v e r l 'h u m id ité du m i l ie u , c a r c e s in s e c te s ne s 'a l im e n te n t pas à l 'é ta t im ag ina i.

P o u r le s O r y c t e s t r o p ic a u x , dont le s ad ultes se n o u r r i s s e n t , i l a été n é c e s s a i r e de p r o c é d e r à un ce r ta in n o m b r e d 'e s s a i s portant n otam m en t s u r l 'a l im e n ta t io n et la nature du ré c ip ie n t d 'é le v a g e p o u r le s la r v e s et p o u r le s im a g o s , a in s i que su r l ' in f lu e n c e du g ro u p e m e n t des in d iv idu s , avant de p a r v e n ir à une m éthod e é p r o u v é e . C e s e s s a i s ont p o r té e s s e n t i e l l e m ent su r O . m o n o c e r o s à p a r t i r d'un m i l l i e r d 'ad u ltes et de la r v e s e x p é d ié s en 13 e n v o is s u c c e s s i f s de la p lantation de l 'IR H O à P o r t Bouet ( C ô t e - d ' I v o i r e ) de m a i 19G4 à s e p te m b r e 1965, et su r O . r h in o c e r o s à p a r t i r de p lus de 4000 œ u fs r e ç u s d 'A p ià (Samoa o c c id e n t a le s ) de m a r s à n o v e m b r e 1966.

Deux p u b lica t ion s [9] ont fait le point des ré su lta ts a c q u is dans l 'o b te n t io n en la b o r a to i r e , en toute sa is o n , de c e s deux e s p è c e s , et p r é s e n té le s p r in c ip a le s c a r a c t é r i s t iq u e s de l 'é v o lu t io n de c e s in s e c te s .La p o u rsu i te de l 'e x p é r im e n ta t io n nous a p e r m is d 'a p p o r te r depuis c e r ta in es a m é l io r a t io n s , en p a r t i c u l i e r dans la fé co n d i té des f e m e l l e s , de s o r t e que l ' e n s e m b le des d on nées a c tu e l le s en c e d om ain e peut se r é s u m e r de la fa çon suivante.

3 .1 . E le v a g e des la r v e s

Il peut ê t r e e f fe c tu é , s o i t en b o i te s in d iv id u e l les de 500 m l de c a p a c i t é , so i t dans des r é c ip ie n ts en po ly é th y lèn e de 35 l i t r e s contenant 20 à 30 la r v e s , so it en b a c s de C00 l i t r e s re n fe r m a n t une centaine d ' in d iv id u s .L a p ré ca u t io n e s s e n t ie l l e e st d 'u t i l i s e r le m é la n g e de m a t iè r e s en d é c o m p o s i t io n et de f è c e s en re sp e c ta n t le s e x ig e n c e s de te m p é ra tu re et d 'hu m id ité (30°C et 65% H R). De m u lt ip le s e s s a i s c o m p a r a t i f s de la v a le u r de d i f fé re n ts su bs tra ts ( te r re a u , b o i s d é c o m p o s é , s c iu r e ) et des e x c r é m e n t s des p r in c ip a u x an im au x d o m e st iq u e s (cheval, v a c h e , p o r c , m ou ton ) ont m o n tré que la c o m b in a is o n b o i s de feu il lu à f ib r e c o u r te (h ê tre , ch â ta ign ie r , o r m e ) + b o u se de v a ch e constitua it le m e i l l e u r m i l ie u p o u r le d év e lop p em en t la r v a i r e , en p a r t i c u l i e r p ou r l im ite r la m o r ta l i t é p a r m y c o s e , a in s i qu 'en té m o ig n e le tableau I récap itu lant 45 e s s a i s portant chacun su r 50 à 200 indiv idus.

L e s é le v a g e s de g ro u p e s de O. r h in o c e r o s donnent des r é su lta ts t r è s h é té r o g è n e s et v a r ia b le s d 'un lot à l 'a u t r e à c a u se de la p lus gra nd e du rée du c y c l e é v o lu t i f de ce tte e s p è c e , qui dem ande 6' à 7 m o is de l ' é c l o s i o n de l 'œ u f à la fo r m a t io n de l ' im a g o , a l o r s que p ou r O. m o n o c e r o s i l suffit de

S M -102/31 305

T A B L E A U I. D E V E L O P P E M E N T D E S O r y c t e s S U I V A N T L E M I L I E U D 'E L E V A G E

E sp èce E lev a g e SubstratLarves L j

en é le v a g e

Im agos

(IP) fo rm és

Ip /1 0 0 L i

M etar-rh iz iu m

№ )

0 . m o n o In d iv id u e l b o is + bou se 87 4 6 0 4 70 i , 6

cerosEn groupe sc iu re + bou se 7 8 4 329 42 19

En groupe b o is + bouse 328 24 3 74 1

O . rh in o In d iv id u e l terreau + bou se 735 271 36 25

cerosb o is + bouse 989 526 53 6

En grou pe terreau + bouse 252 30 12 60

sc iu re + bouse 200 60 30 20

bois + bouse 512 1 64 32 20

Ip = In sectes parfaits.

3| m o i s à 4 m o i s . L e s 3 m o is s u p p lé m e n ta ire s n é c e s s a i r e s p ou r O. r h i - n o c e r o s sont le s p lus d i f f i c i l e s p o u r le m a in tien de la s t ru c tu re et de l 'h u m id ité du m i l ie u , qui tend à se d e s s é c h e r p r o g r e s s iv e m e n t . Dans ce c a s , de m ê m e q u 'a p r è s un t r a n s fe r t des la r v e s dans un m i l ie u d 'hum id ité bru squ em en t trop é le v é e , la m y c o s e se m a n i fe s te et détru it la q u a s i - totalité de l ' é l e v a g e c o n s id é r é , d 'où , p ou r ce tte e s p è c e , des re n d em en ts t r è s d i f fé re n ts d 'un b a c à l 'a u tr e p ou r un m ê m e su b s tra t . C e s d i f f i cu ltés sont en c o u r s de so lu t ion , en p a r t i c u l i e r p a r le r e c o u r s à des en ce in tes de v o lu m e plus r e s t r e in t p ou r la fin de la v ie l a r v a i r e et la n ym p h ose .En utilisant le b o is e i la b o u se de v a ch e on obtient p a r c e p r o c é d é un rendem ent de 70% d 'adu ltes f o r m é s p a r rap p ort au n o m b r e de la r v e s n ou v e l les n ées m i s e s en é le v a g e , de la m ê m e façon que p ou r O .m o n o c e r o s .

Nous nous p r o p o s o n s d 'a n a ly s e r le s fa c te u r s p h ys iqu es ou c h im iq u e s des su bstrats e x p é r im e n té s d éterm inant le m e i l l e u r d é v e lo p p e m e n t des la r v e s et la l im ita tion de la m y c o s e . Il s e m b le que le s deux constituants in terv iennent, c a r des tes ts p r é l im in a i r e s ont m o n tr é que le b o is d é c o m p o s é se u l ou le t e r r e a u se u l ne p e rm e tta it pas l 'é l e v a g e de c e s in s e c t e s . D 'a u tre part , 11 e s s a i s e f fe c tu é s av e c O. m o n o c e r o s a l im en té en é le v a g e ind iv iduel av e c des f è c e s de d i f fé re n ts an im aux d o m e st iq u e s (vache , ch ev a l , p o r c , m outon) m é la n g é e s , so i t au b o is d é c o m p o s é , so it au te r re a u , ont m is en é v id e n ce la s u p é r io r i t é du b o i s , notam m ent à l ' é g a r d de M. an isop liae (tableau II).

20

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T A B L E A U II. I N F L U E N C E D E L A N A T U R E DU S U B S T R A T SU R L E D E V E L O P P E M E N T L A R V A I R E D E O. m o n o c e r o s

Substrat L] en é le v a g e Ip fo rm és R en dem en t

%

M eta r -rh iz iu m

(%)

Bois + fè c e s 270 218 80 3

T errea u + fè ce s 311 136 43 36

3. 2.__E le v a g e des ad u ltes

Dans l 'é ta l actu e l de nos e x p é r i e n c e s su r l ' in f lu e n c e de d iv e r s e s c a g e s et de d i f fé re n ts su b s tra ts , i l apparaît que le t e r r e a u de c o u c h e de deux ans r e p r é s e n t e le m i l ie u le p lus fa v o r a b le au dépôt des œ u fs des deux e s p è c e s , dont l 'a l im e n ta t io n est a s s u r é e p a r des r o n d e l le s de banane d i s p o s é e s en s u r fa c e et r e n o u v e lé e s deux fo is pa r se m a in e . L e s f e m e l le s sont aptes à la r e p ro d u c t io n dans le s j o u r s qui suivent le u r s o r t i e de la d ép ou il le nym phale, de s o r t e que p ou r c e s deux D ynastides la ponte peut c o m m e n c e r au bout de 2 s e m a in e s . C h ez O. m o n o c e r o s , e l le peut se p r o lo n g e r pendant 4 m o i s , c h e z O . r h in o c e r o s pendant C m o i s , dé la is qui c o r r e s p o n d e n t a p p ro x im a t iv e m e n t à la lon gév ité des f e m e l l e s . Dans l e s d is p o s i t i f s e x p é r im e n té s , la fé co n d i té de l ' e s p è c e a f r i c a in e est p lus de deux fo is m o in d r e que c e l l e de l ' i n s e c t e as ia tique (30 œ u fs en m oy en n e p a r fe m e l le c o n t re 74 dans le s m e i l l e u r e s co n d it io n s ) , av e c des pontes pouvant atte indre 70 oeufs p ou r c e r t a in e s f e m e l le s de O. m o n o c e r o s et 150 à 180 œ u fs p o u r O. r h in o c e r o s . L e g ro u p e m e n t des indiv idus dans des r é c ip ie n t s d 'un c e r ta in v o lu m e p ara it f a v o r i s e r la su r v ie et la ponte des f e m e l l e s , surtout c h e z O. m o n o c e r o s , c o m m e l ' in d iq u e le tableau III. P o u r O. r h in o c e r o s , l e s d i f f é r e n c e s en tre le s d iv e r s types d 'e n c e in te s de ponte sont bea ucou p m o in s nettes . Il en est de m ê m e p ou r la f e r t i l i t é des œ u fs e x p r im é e pa r le p o u rce n ta g e de la r v e s é c l o s e s à p a r t i r des p on tes ob te n u e s .

Dans la p rat iqu e , le bac de 50 l i t r e s a v e c 10 c o u p le s est le p r o c é d é le p lus c o m m o d e p o u r ob te n ir la ponte des O r y c t e s , s ' i l n 'e s t p a s n é c e s s a i r e de fa i r e des études à l ' é c h e lo n indiv iduel.

4. E T U D E DE L A R E P R O D U C T IO N DES O r y c t e s

La m i s e au point d 'un é le v a g e de c e s D yn ast ides nous p e r m e t de d i s p o s e r d ' in s e c t e s d 'â g e et d 'é ta t p h y s io lo g iq u e connus et, a in s i , d 'e n t r e p r e n d r e l 'e x a m e n des p r in c ip a u x c a r a c t è r e s de la fé con d a tion et de la r e p ro d u c t io n des in s e c te s n o rm au x , de façon à p o u v o ir in te r p r é te r l e s p h é n o m è n e s p rod u its p a r l ' i r r a d ia t io n .

L e s e x p é r i e n c e s en c o u r s ont p ou r but de d é te r m in e r le rythm e d 'é vo lu t io n des o v o c y te s à 30°C et l ' in f lu e n c e de l 'â g e des indiv idus des

S M -1 0 2 /3 1 307

deux s e x e s su r le s d é la is et le n o m b r e d 'a c c o u p le m e n t s . L à d is s e c t io n de 4 f e m e l le s tous le s 2 ou 5 jo u r s indique l 'é ta t du t issu ad ipeux, le n o m b r e et la ta i l le des o v o c y te s et la p r é s e n c e ou non de s p e r m a to p h o r e s dans la poch e c o p u la t r i c e . En ou tre , le s fe m e l le s de m ê m e o r ig in e sont é l e v é e s ju sq u 'à la ponte p o u r é v a lu e r la fé co n d ité des in s e c te s c o n s id é r é s .Il a été p r o c é d é à l 'é tu d e de c o u p le s co n s t i tu é s , so it d ' in s e c t e s f o r m é s depu is qu e lques jo u r s , so it d 'an im a u x v ie r g e s a l im e n té s pendant 1 m o i s avant la m is e en é le v a g e et m aintenus e n s e m b le pendant des d é la is v a r i a b le s de 2 se m a in e s à 2 m o i s . D 'a u tre part , le s r é su lta ts e n r e g i s t r é s a v e c des c o u p le s i s o lé s ont été c o m p a r é s à ce u x obtenus dans le s b a cs re n fe rm a n t un g ro u p e de 10 c o u p le s .

Il ap para ît que la m a jo r i t é des a c c o u p le m e n ts s 'e f f e c t u e 8 à 12 jo u r s a p rè s la m is e en p r é s e n c e des deux s e x e s , au m o m e n t où le t i s su ad ipeux des f e m e l le s c o m m e n c e à ê tre bea ucou p m o in s abondant et à p re n d re l 'a s p e c t de ba llon n ets p le in s d 'a i r , p a ra l lè le m e n t à une o v o g e n è s e a c t ive se m anifestant p a r l 'e x i s t e n c e de 3 à 5 o v o c y te s dont le p lu s g r o s et le p lus an c ien e s t en début de v i t e l l o g e n è s e . L e n o m b r e de m â le s et de f e m e l le s dans la m ê m e en ce in te ne p a ra ît pas in terven ir .

Un e s s a i portant su r l 'é l e v a g e de O. r h in o c e r o s des deux s e x e s d 'â g e s d i f fé re n ts (m â les de qu e lques jo u r s a v e c des f e m e l le s m aintenues 6 s e m a in e s à jeun à 20°C et v i c e - v e r s a ) a m o n tr é q u ' i l n 'y a pa s de d i f f é r e n c e de fé con d ité (40 œ u fs p a r f e m e l le en m o y en n e ) ni de f e r t i l i t é (85%), ni de d é la is p ou r la p r e m iè r e ponte (2 s e m a in e s à 30°C) en tre le s deux lo ts .P a r c o n t re , l e s m â le s â g é s de 6 s e m a in e s sont su s c e p t ib le s de fé c o n d e r l e s f e m e l le s dès le s p r e m i e r s jo u r s a l o r s que pou r des m â le s jeu n es le s p r e m i e r s a c c o u p le m e n ts ne s 'o b s e r v e n t qu 'à p a r t i r du 10e jo u r , a v e c p o s s ib i l i t é de deux c o ï t s p ou r le m ê m e co u p le dans le s 15 jo u r s qui suivent la m is e en é le v a g e . En o u tre l ' o v o g e n è s e est p lus rap id e et p lus h om o g è n e dans le c a s des f e m e l le s jeu n es .

A la d i f f é r e n c e de M. m e lo lon th a , l e s fe m e l le s d 'O r y c t e s sont donc aptes à la r e p ro d u c t io n dans le s jo u r s qui suivent la s o r t i e de la nym phe, tandis que p o u r le s m â le s une p é r io d e de m atura tion d 'au m o in s 1 se m a in e est n é c e s s a i r e p ou r p e r m e t t r e l 'a c c o u p le m e n t . C e s o b s e r v a t io n s é th o lo - g iqu es devron t n a tu re l lem en t ê t r e c o n f i r m é e s et p r é c i s é e s p a r une étude h is to log iqu e .

L 'a c c o u p le m e n t n 'e s t pas in d isp e n sa b le à l 'é v o lu t io n des o v o c y t e s : 10 f e m e l le s v i e r g e s de O. r h in o c e r o s en é le v a g e g ro u p é à 30°C ont d éposé un tota l de 165 œ u fs en 2 a 6 s e m a in e s . M ais la fé co n d ité (16. œ u f s pa r fe m e l le ) et la lon gév ité ( in fé r ie u r e à 2 m o i s ) de c e s in s e c te s sont in fé r ie u r e s à c e l l e s de f e m e l le s a c c o u p lé e s , c e qui am èn e à p e n s e r que des p h é n o m è n e s an a logu es à c e u x m is en é v id e n ce p a r Landa [10] c h e z le hanneton c o m m u n se p ro d u ise n t é g a lem en t c h e z le s D yn astides : u t i l isa tion des r e s t e s de s p e r m a to p h o r e s c o m m e s o u r c e c o m p lé m e n t a ir e de p r o té in e s p ou r l 'é la b o r a t io n du v ite l lu e .

Cette h ypothèse e s t c o n f i r m é e pa r la d i f f é r e n c e de fé co n d ité e n r e g i s t r é e dans un e s s a i p r é l im in a i r e su r le p o u v o i r fécondant du m â le de O. r h i n o c e r o s . Dans t r o i s lo ts de 12 fe m e l le s é l e v é e s a v e c 1, -3 et 12 m â le s , la f é c o n dité m oyen ne pa r fe m e l le a été , r e s p e c t iv e m e n t , de 35, 30 et 63 œ u fs .

D 'a p r è s l e s n o m b r e u s e s o b s e r v a t io n s que nous avons eu l ' o c c a s i o n de fa i re dans nos é tu des su r la ponte , 1еб s p e r m a to z o ïd e s c o n s e rv e n t leu r p o u v o ir fécondant dans le s v o ie s gén ita les f e m e l le e pendant 2 à 3 m o i s ch e z O. m o n o c e r o s et ju sq u 'à 5 m o i s c h e z O. r h in o c e r o s , à en

T A B L E A U III. I N F L U E N C E D E S C O N D IT IO N S DE P O N T E S U R L A F E C O N D I T E E T L A F E R T I L I T E DES O r y c t e s à 3 0°C

E sp èce D is p o s it if

N om b re d e c o u p les par e n ce in te

N om b re to ta l d e f e m e lle s

N om b red 'oeu fs

F é c o n d ité m o y e n n e

par f e m e l le

(n o m b re d 'œ u fs )

F ertilité

m o y e n n e des œ u fs

• 0o)

O . m o n o ce ro s seau d e 10 litres 1 85 1150 13, 5 26

seau d e 10 litres 3 33 512 15, 5 64

b a c d e 50 litres 10 113 3331 30 78

O . rh in océros b a c d e 5 litres 1 14 101 4 72 72

seau d e 10 litres 1 7 390 56 70

seau d e 10 litres 3 15 410 28 80

b a c d e 35 litres 5 14 830 60 75

b a c d e 50 litres 10 37 2611 74 80

308 H

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SM-102/31 309

ju g e r p a r la fe r t i l i t é des œ u fs d é p o s é s dans le s é le v a g e s de co u p le s dont le m â le est m o r t p ré m a tu ré m e n t .

5. ST E R IL ISA T IO N DES M A L E S P A R IRRADIATION

5. 1. D éfin ition des d o s e s s t é r i l i s a n te s

L ' i r r a d ia t io n a été r é a l i s é e p a r un ir ra d ia te u r c h a rg é de u0C o dont l 'a c t iv i t é to ta le était de 1500 Ci, ayant un débit de d o se au m om ent de nos e s s a i s de 1100 r a d /m in . En fon ct ion des données de H o r b e r [5, 6] p ou r M. m elo lon th a une d ose de 9000 rad a été u t i l i s é e , en p r e m iè r e a p p rox im a tion de la se n s ib i l i té de c e g e n r e d ' in s e c t e s , pou r des m â le s de O. m o n o c e r o s , dont nous avons eu l 'é l e v a g e avant c e lu i de O. r h in o c e r o s . P o u r l ' i r r a d ia t io n , le s in s e c te s étaient p la c é s p a r g ro u p e s de t r o i s dans une bo îte en p o ly s ty r è n e de 8 c m de haut su r 7,5 c m de d ia m è t r e r e m p l ie de tou rbe h um id if iée . I ls .é ta ient ensuite m is en d is p o s i t i f de ponte à 30°C a v ec des f e m e l le s n o r m a le s , so it pa r c o u p le s , so it par- g ro u p e s de 10 c o u p le s . D es adultes de m ê m e âge et de m ê m e o r ig in e , non i r r a d ié s , se r v a ie n t de té m o in s p ou r chaque d o s e . Dans une p r e m iè r e étape nous avons c h e r c h é à d é t e r m in e r la d o s e m in im a le de ra y o n s gam m a s té r i l is a n t le s m â le s et réduisant le m o in s p o s s ib le le u r lo n gév ité , en fon ct ion de l 'â g e des i n s e c t e s : im a g o s f o r m é s depuis qu e lques jo u r s , et im a g o s c o n s e r v é s à jeun à 20°C pendant 3 s e m a in e s . L e tableau IV r é cap itu le le s r é su lta ts c o n ce rn a n t O . m o n o c e r o s et le tableau V le s données r e la t iv e s à O. r h in o c e r o s .

C es tab leaux font r e s s o r t i r la se n s ib i l i t é m o in d r e de O. r h in o c é r o s à l ' i r r a d ia t io n . A l o r s que 4000 rad a ssu re n t la s t é r i l i s a t i o n de O. m o n o - c e r o s et que 3000 rad se r a ie n t su f f isan ts p ou r ce tte e s p è c e , i l faut au m oin s 6000 rad p ou r O. r h in o c e r o s , dans l 'é ta t a c tu e l de nos r e c h e r c h e s . Des e s s a i s sont en c o u r s en utilisant des d o s e s s u p é r ie u r e s , allant ju sq u 'à 10 000 ra d , p ou r f ix e r la d o se o p t im a le . Dans le s deux c a s l 'â g e des adultes ne pa ra ît pas a v o ir d ' in f lu e n ce m a rq u é e su r la se n s ib i l i t é aux ray o n s g a m m a et la lon gév ité des m â le s t r a i té s de O. m o n o c e r o s est peu a f fe c té e p a r le s d o s e s s t é r i l i s a n te s , tandis que 6000 rad rédu isen t le s d é la is de s u r v ie des adultes de O . r h in o c e r o s pa r ra p p o r t aux in s e c te s n orm au x .

Dans t r o i s lo t s de 10 c o u p le s de O. r h i n o c e r o s , le s deux s e x e s ont été i r r a d ié s s im u lta n ém en t à r a is o n de 3000 rad, 4000 rad et 5000 rad. Seu les le s f e m e l le s s o u m is e s à 3000 rad ont d é p o s é de6 œ u fs , en n o m b re trè s r e s t r e in t (23 au total p ou r 10 f e m e l le s ) et aucune é c l o s i o n n 'a été e n r e g i s t r é e . La lo n gé v ité des f e m e l le s t r a i té e s a été analogue à c e l l e des m â le s : 6 s e m a in e s e n v iro n . D es e x p é r i e n c e s c o m p lé m e n t a i r e s p e r m ettron t de p r é c i s e r c e s p r e m i è r e s données su r la s e n s ib i l i t é des deux s e x e s aux ray on s g a m m a .

De p r e m i e r s e s s a i s d ' i r r a d ia t io n aux r a y o n s X ont été e f fe c tu é s p a ra l lè le m e n t p ou r O. r h in o c e r o s . D es s é r i e s de 5 im a g o s ont été p la c é e s dans des b o î te s en p o ly s t y r è n e de 10 c m X 7 c m , au contact du g é n é ra te u r so u s une ten s ion de 200 kV, à 12,5 m A , à un débit de d o s e de 111 r a d /m in , en in terposant un f i l t r e en c u iv r e de 0,5 m m . D eux d o s e s ont été u t i l i s é e s p ou r des lo ts de 10 m â le s : 3000 rad et 5000 rad . L a fé co n d ité des f e m e l le s et la fe r t i l i té des oeufs (67% d 'œ u fs f e r t i l e s p ou r 3000 rad et 48% p ou r 5000 rad) sont, du m ê m e o r d r e de g ra n d e u r que p o u r le s m ê m e s d o s e s

T A B L E A U IV . A C T I O N D E S R A Y O N S G A M M A SU R O . m o n o c e r o s

A g e du m â leD ose(rad)

N om b re d e m â les

L on g é v ité m o y e n n e du m â le

(sem a in es)

F é c o n d ité m o y e n n e par

f e m e l l e (n o m b re d 'oeu fs )

F e r t ilité m o y e n n e d es œ u fs

m

3 sem a in es 0 7 8 16 50

3 sem a in es 9000 17 6 10 3

3 jours 9000 16 7 20 3

3 jours T é m o in 6 8 17 80

3 sem ain es T é m o in 12 8 40 72

3 jours 600 0 30 6 12 0

3 sem ain es 600 0 25 5 12 0

3 jours T é m o in 10 10 2 4 62

3 sem a in es T é m o in 15 7 10 59

3 jours 400 0 11. 8 30 2

3 sem a in es 40 0 0 20 7 2 4 0

3 jours 3000 10 5 35 5

3 sem a in es 300 0 10 8 51 4

3 jours 2 00 0 10 6 36 12

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T A B L E A U V . A C T I O N D E S R A Y O N S G A M M A S U R O . r h i n o c e r o s

A g e d u m â leD ose(rad)

N om b re d e m â les

L o n g é v ité m o y e n n e du

m â le (sem a in es )

F é co n d ité m o y e n n e par

fe m e l le (n om b re d 'œ u fs )

F e r t ilit é m o y e n n e des œ u fs

(%)

3 jours T é m o in 12 10 74 86

3 sem a in es T é m o in 10 9 75 72

3 jours 3000 10 9 95 76

3 sem a in es 3000 10 5 15 27

3 jours 4000 20 9 55 62

3 sem a in es 40 0 0 20 C 56 66

3 jours 5000 20 9 55 17

3 sem a in es 5 00 0 2 5 7 48 5

1 s e m a in e 6000 10 6 27 2

SM

-102/31 311

T A B L E A U V I . C O M P E T I T I V I T E D ES M A L E S IR R A D I E S P A R R A P P O R T A U X M A L E S N O R M A U X ( P o n t e à 3 0°C p a r l o t s d e 9 o u 10 c o u p l e s )

E sp èceD o se(rad)

M âlesirradiesnorm aux

N om b re to ta l d e c o u p le s

F é c o n d ité m o y e n n e

par f e m e l l e (n o m b re d 'œ u fs )

F e r t i lit é m o y e n n e d e s .œ u fs

(%)

0 . m o n o c e r o s 4000 i 218 49 62

1 4 30 47 6 4

T é m o in - 10 22 73

O . rh in océros

5000 ( 2 36 29 60

l 4 10 62 81

60 0 0' I 2 36 (51 76

1 4 10 52 82

T é m o in - 10 52 80

312 H

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SM-102/31 313

de r a y o n s g a m m a . M ais nous p o u rs u iv o n s la c o m p a r a is o n des deux m o d e s d ' i r r a d ia t io n afin de v é r i f i e r que la nature du ray on n em en t n ' in terv ien t p a s , a in s i que A m y [11] l 'a d é m o n tré su r le s œ u fs de H a b ro b ra co n .

5. 2. C o m p é t i t io n en tre m â les n o rm a u x et m â le s i r r ad iés

Un des po in ts im p ortan ts à é ta b l i r p ou r le c a lc u l th é o r iq u e du n o m b re d ' in s e c t e s s t é r i l e s à lâ c h e r dans la popu lation c o n s id é r é e est la v igu eu r gén ita le et la c o m p é t i t iv i té se x u e l le des m â le s i r r a d ié s . Dans c e but, des lo t s de 10 co u p le s de O. m o n o c e r o s ont été c o n s t itu é s en a s s o c ia n t 10 f e m e l le s n o r m a le s a v e c 8 (ou fi) m â le s i r r a d ié s à 4000 rad et 2 (ou 4) m â le s non t r a i té s . Dans le c a s de O. r h in o c é r o s nous a v on s o p é r é , so it av e c 9 c o u p le s (C m â le s i r r a d ié s et 3 m â le s n o rm a u x ) , so it a v e c 10 c o u p le s (8 m â le s i r r a d ié s et 2 m â le s n o rm a u x ) , dont le s in s e c te s tr a i té s avaient été s o u m is pou r p a rt ie à 5000 rad et p ou r pa rt ie à 6000 rad . L e tableau VI m o n tr e q u ' i l e s t n é c e s s a i r e d 'a u g m e n te r la p r o p o r t io n de m â le s s t é r i l e s p ou r a g ir su r la fe r t i l i t é des œ u fs , c e qui est en a c c o r d a v e c le s données de la l i t t é r a t u r e : P r o v e r b s et Newton [12] indiquent q u ' i l faut 20 m â le s s t é r i l e s de c a r p o c a p s e pa r m â le n o r m a l p ou r o b ten ir une ré d u ct ion rapide des popu lations eh v e r g e r , et la m ê m e p r o p o r t io n a été t r o u v é e par H owland et al. [13] p o u r T r ie h o p lu s ia ni.

5. 3. E ffe ts de l ' i r r a d ia t io n su r la d e s c e n dance

En plus de l ' in f lu e n c e d ir e c te des r a y o n s g a m m a su r la fé con d ité et la fe r t i l i té des ad u ltes , nous avon s a b o rd é l 'é tu d e des effets , d i f f é r é s éventue ls sur la gén é ra t io n is su e des œ u fs d é p o s é s p a r des f e m e l le s a c c o u p lé e s av e c des m â le s i r r a d ié s . P lu s i e u r s ce n ta in es de la r v e s provenant d 'ad u ltes de O. r h in o c e r o s s o u m is à 3000, 4000 ou 5000 rad sont é l e v é e s in d iv id u e l lem en t . En ra is o n des d é la is de d é v e lo p p e m e n t et des d i f fé r e n c e s in d iv id u e l les c e s é le v a g e s ne sont pas t e r m in é s . Cependant, un grand n o m b r e d 'ad u ltes sont maintenant f o r m é s et en c o u r s de ponte . Dans l 'e n s e m b le i l ne p a ra ît pas y a v o i r de d i f f é r e n c e m a rq u é e dans la du ree de l 'é v o lu t io n des la r v e s , le p o id s des im a g o s f o r m é s et la fé co n d ité de c e u x - c i p a r r a p p o r t aux in s e c te s is s u s des é le v a g e s n orm au x .

CONCLUSIONS

Un c e r ta in n o m b r e de don nées p o s i t iv e s ont pu ê t r e a c q u is e s c e s deux d e r n iè r e s années au c o u r s de n otre e x p é r im e n ta t io n su r le s m o d a l i té s d 'a p p l ica t io n aux O r y c t e s des m é th o d e s de lutte a u toc id e p a r ir ra d ia t io n . .Un des points fondam entaux, non seu le m e n t p ou r c e s r e c h e r c h e s m a is a u s s i p ou r toute étude p h y s io lo g iq u e ou pa tho log ique p r é c i s e de c e s D yn as t id es , e st la m i s e au point d 'une technique d 'é le v a g e en la b o r a to i r e assu ra n t la d isp o n ib i l i té en p e r m a n e n c e d ' in s e c t e s d 'â g e b ien d é te rm in é . C 'e s t g r â c e à c e s é l e v a g e s , dans le s q u e ls la m y c o s e à M e ta r r h iz iu m est c o n t r ô lé e , que nous avons pu e n tr e p re n d r e l 'e x a m e n de la b io lo g ie de la r e p ro d u c t io n des deux e s p è c e s le s p lus im p o rta n te s au point de vue é c o n o m iq u e , a in s i que la dé fin it ion des n o r m e s de s t é r i l i s a t i o n et des p r o c e s s u s de co m p é t i t io n en tre indiv idus i r r a d ié s et in s e c te s n o rm au x .

3 1 4 HURPIN

L e s r é su lta ts r é s u m é s dans c e m é m o i r e ont e n c o r e un c a r a c t è r e p r é l im in a i r e du fait de la du rée du c y c l e év o lu t i f des e s p è c e s c o n s id é r é e s et de la n é c e s s i t é de p r o c é d e r à des ré p é t it io n s en n o m b r e su ff isan t pou r p e r m e t t r e des c o n c lu s io n s d é f in i t iv e s . La p ou rsu ite de nos r e c h e r c h e s a p o u r ob je t d 'a b o u t ir à de te l le s c o n c lu s io n s notam m ent en c e qui c o n c e r n e : - l e s d i f fé re n te s étapes de la v ie s e x u e l le des m â le s et des f e m e l le s n orm au x

et i r r a d ié s , pa r une étude h is to log iqu e a p p r o p r ié e ,- l a c o m p a r a is o n de l ' e f fe t s t é r i l is a n t des d iv e r s e s s o u r c e s de rad ia t ion s

(X et g a m m a ) av e c d i f fé re n ts déb its ,- l a dé l im ita t ion de la p r o p o rt io n d 'in d iv id u s s t é r i l i s é s n é c e s s a i r e s p ou r

p r o v o q u e r une réduction su ff isan te du taux de p r o l i f é r a t io n de la population .M a is , en d eh ors de c e s études de la b o r a to i r e , l 'a p p l ica t io n de cette

m é th o d e de lutte aux O r y c tes su p p o se l 'a m é l io r a t io n de nos c o n n a is s a n c e s su r le c o m p o r te m e n t des adultes et su r la dynam ique des p op u la tion s en m i l ie u naturel, et surtout la m is e au point d 'une m éthod e de cap tu re par p ié g e a g e des im a g o s .

Cette m éthod e est n é c e s s a i r e à la fo is p ou r su iv re le s n iveaux de p o p u la t ion et pou r r é c o l t e r en grand n o m b r e le s in s e c te s qui s e ro n t so u m is à l ' i r r a d ia t i o n avant d 'ê t r e r e lâ c h é s . Dans le c a s des O r y c t e s , en e f fe t , i l nous p a ra ît t r è s d i f f i c i l e d 'e n v is a g e r la s t é r i l is a t io n d ' in s e c t e s proven an t d 'é l e v a g e s p ou r deux r a is o n s : l e s é le v a g e s de m a s s e dem an d era ien t des m o y e n s c o n s id é r a b le s , en p a r t i c u l ie r à ca u se de la lon gu eu r du c y c l e é v o lu t i f de c e s C o lé o p tè r e s qui n 'a r ien de c o m m u n av e c c e lu i des D ip tè r e s . D 'a u tre part , l e s dégâts sont o c c a s io n n é s par le s adultes dont i l y a lieu , p a r con séqu en t , d 'a c c r o i t r e le m o in s p o s s ib le le n o m b r e , m ê m e de fa çon t e m p o r a i r e .

Ce sont de te l le s é tudes , qui sont a c t ive m e n t cond u ites par le s s p é c ia l i s t e s du p r o je t « R h in o c e r o s B e e t le » à A p ia , n otam m en t pa r la r e c h e r c h e d 'a t t ra c t i f s c h im iq u e s , qui doivent p e r m e t t r e d 'a b o u t ir à l 'e x p é r im e n ta t io n su r le te r r a in de p r o c é d é s de lutte a u to c id e , m éthod e qui nous p a ra ît ê tre une des p o s s ib i l i t é s o f f e r t e s p a r l ’ e n to m o lo g ie m o d e rn e p ou r fa i re fa ce au g r a v e p r o b lè m e des O r y c t e s .

R E M E R C IE M E N T S

L 'a u te u r r e m e r c i e le C o n s e i l de d ir e c t io n du p r o je t c o n jo in t U N /S P Cet la D ire c t io n de l ' IR H O p ou r le s fa c i l i t é s a c c o r d é e s et la c o n f ia n ceté m o ig n é e .

R E F E R E N C E S

[1 ] HURPIN, B . , Résultats et perspectives d e la lutte b io log iq u e contre les O ryctes, O léagineux 21 2 (1966) 77 -82 .

[2J CUMBER, R. A . , Le rhinocéros du c o c o t ie r aux Sam oa O cciden ta les, C om m ission du P acifiqu e Sud, D oc. T echn . n° 107 (1957) 1-76 (M im éo).

[3] HINCKLEY, A. D . , Les dégâts causés par l 'O ryctes rhinoceros (L. ) dans les co co tera ies des îles du P a cifiqu e, Bull. Pacif. Sud. lfi 4 (1966) 4 3 -4 4 .

[4 ] MARIAU, D . , Lutte ch im iqu e contre l 'O ryctes, Résultats prélim inaires, O léagineux 22 3 (1967)155 -15 8 . ~ ■

[5 ] HORBER, E ., "E radication o f w hite grub (M elolontha vulgaris F .) by the ste r ile -m a le techn ique” , Radiation and Radioisotopes Applied to Insects o f Agricultural Im portance" (Proc. Sym p. Athens, 1963), IAEA, Vienna (1963) 313 -32 .

S M -102/ 31 315

[6 ] HORBER, E ., M aikáfer gegen Maikafer.* Ein Versuch zur T ilgung eines Engerlingsherdes des Feld - m aikáfers (M elolontha vulgaris Fabr. ) m it rtfntgenbestrahlten M annchen, W ald hygiene б 6 (1966) 161 -70 .

[7 ] FERON, М ., La lutte contre les insectes par les m éthodes autocides, Revue Z o o l . agrie , appl. 26 (19ü’ 3) 3 7 -4 8 .

L8J HURPÏN, B . , FRESNEAU, M . , Elevage d e deux dynastides, O ryctes nasicornis L. et Phyllognathus silenus F. (C o léop t. S ca ra b æ id æ ), Revue Path. v é g . Ent. agrie. Fr. 43 2 (1964) 7 5 -9 6 .

[9 ] HURPIN, B . , MARIAU, D . , Contribution à la lutte contre les O ryctes nuisibles aux palm iers, M ise au point d 'u n é lev a g e perm anent en laboratoire, C . r. hebd. Séan c. A cad . A grie. Fr. (1966) 178 -18 6 . . HURPIN, B . , FRESNEAU, M . , Contribution a la lutte contre les Oryctes nuisibles aux palm iers,2. E levage en laboratoire de O. rhinoceros L . , O léagineux 22 11 (1967).

[10 ] LANDA, V . , O rigin, deve lop m en t and function o f the sperm atophore in c o ck ch a fer (M elolontha m elolontha L. ) , A cta S oc. Ent. C ech oslov . 57 (1960) 297 -31 6 .

[11] AM Y, R. L . , A com parative study o f the e ffe c t o f 0 -rays, y -rays and X -rays on d evelopm en t in H abrobracon, Radiat.Res. 3 (1955) 166 -81 .

[12] PROVERBS, M. O . , NEWTON, J. R . , Suppression o f the reproductive potentia l o f the cod lin g m oth by gam m a irradiated m ales in ca g ed orchard trees, J. e con . Ent. 55 (1962) 934 -36 .

[13] HOWLAND, A .F . , VAIL, P . , HENNEBERRY, T . J . , Results o f c a g e experim ents w ith sterile m ale releases and a chem osterilant technique for con trol o f ca b b a ge looper populations, J. e con . Ent. 59 1 (1966) 194-9G.

D I S C U S S I O N

G. L E M ASN E: Do you have any in fo rm a t io n on b e h a v io u ra l a s p e c tsin fluenc ing c o m p e t i t io n be tw e e n i r ra d ia te d and n o n - i r r a d ia te d m a le s ?

B. HURPIN: No, I 'm a fra id not.

E F F E C T OF \iEL EASING STERILE Scirpophaga nivella F. ON CROP DAM AGE AND INSECT POPULATION DENSITY IN SUGAR CAN 1C PLANTATIONS

S. HATMOSOEWARNOENTOM OLOGICAL AND PHYTOPATHOLOGICAL LA BORA TO RY,STATE SUGAR A C A D E M Y ,JOGJAKARTA, INDONESIA

Abstract

EFFECT OF RELEASING STERILE Scirpophaga n ive lla F. ON CROP DAMAGE AND INSECT POPULATION DENSITY IN SUGAR CANE PLANTATIONS, Successful use o f the ste r ile -m a le technique to control insect populations was reported in several countries. Certain characteristics o f a candidate insect must be co n sidered before em ploym ent o f this techn ique. Since 1965, experim ents have been perform ed at the State Sugar A cad em y in Jogjakarta, Indonesia, to exam in e these characteristics in the w hite top borer (Scirpophaga n ive lla F . ) t an important sugar can e pest, and to determ ine the suitability o f the sterile- m a le m ethod . Several important characteristics w ere found su itab le, i . e . copu lation ab ility did not decrease after radiation , and rearing on an artific ia l m edium is possible for a ll l ife c y c le stages. Radiation at the pupal stage induced sterility in the adult. Prelim inary experim ents in a sugar can e plantation w ere perform ed to determ ine the e ffe c t o f sterile insect release on the attack intensity and population density. The number o f released sterile insects approxim ately equa lled that o f the natural population.Release was carried out at a tim e when cane was susceptible to attack in an area w hich had previously reported 10% annual crop loss due to this in sect ’ s activ ity .

Released sterile insects caused a decrease in the attack intensity and the popu lation density. An exam ination carried out just before harvesting, after release o f the insects had been term inated , revealed that the insect population density at the control plots was roughly equal to that at the release plots. This ind ica ted that the release o f the sterile insects would be e ffe c t iv e in suppressing the increase o f the population i f it w ere constantly done. Another fact that supports this opin ion is that although the figures shown by the con trol plots and release plots just before harvesting were alm ost equ a l, the dam age in the release plots was on ly to the leaves. Thus, i f we considered that the dam age caused by this pest on sugar can e 10 months old or o lder m ay be ignored , con trol cou ld be a ccom plish ed by releasing sterile insects during the sensitive period o f sugar can e growth. If how ever, this pest is to be com p le te ly erad ica ted , the release o f the sterile insects must be done constantly during the grow ing period o f the sugar can e.

1. IN TROD UCTION

The white top b o r e r is the m o s t d e s tr u c t iv e native su g ar cane p est in Indones ia . A c c o r d in g to r e s e a r c h c a r r i e d out in Indones ia in 1957 at 16 su gar fa c t o r i e s , the a v e r a g e l o s s w as 8. 9%. If the total su g ar p r o duction in Indones ia w as 1 000 000 ton s , then 89 000 tons o f su g ar w e re d e s tr o y e d by the white top b o r e r . The value o f the l o s s , c a lcu la te d a c c o rd in g to the c u r r e n t su g ar p r i c e in In dones ia , w as a lm o s t two b i l l io n rupiahs [8] . The top b o r e r , in co m b in a t io n with the s t e m b o r e r , ca u se d 12% d am age to the su g ar c r o p . O ther c o u n tr ie s a r e c o n ce n tra t in g the ir attention on the s tem b o r e r , w h ich , f o r in s ta n ce , c a u s e s an annual l o s s o f $6 000 000 in the United States o f A m e r i c a [1] .

317

3 1 8 HATMOSOEWARNO

T he in c r e a s in g su g ar l o s s in Indones ia is p a rt ly ca u se d by a planting s y s t e m w hich fa v o u rs the e x is t e n c e o f the white top b o r e r and part ly by ou tm od ed c o n t r o l m eth od s . The p re se n t c o n t r o l m ethod is the so c a l le d 1 r o g e s a n 1 m ethod , i . e . cutting the top o f the in fested su g a r can e .

C o n tro l by in s e c t i c id e w as not s a t is fa c to r y b e ca u se the g r e a t e r part o f the in s e c t 1 s l i f e is spent within the s t e m , beyond the re a ch o f in s e c t i c id e s .

T he s u c c e s s o f the s t e r i l e - m a l e technique in c o n t ro l l in g d i f fe re n t p e s ts in v a r io u s c o u n tr ie s p ro m p te d the fo l low in g e x p e r im e n ts to d e te rm in e w hether this technique cou ld be used to c o n t r o l the white top b o r e r in su g ar can e . T h e se in s e c t s can e a s i ly be r e a r e d by an a r t i f i c i a l m ethod out o f their natural cond it ion in the f ie ld , in 1965, r a d io b io lo g i c a l stud ies in d icated that the copu la t ion ab il i ty w as not r e d u c e d .

Pupae w e r e ir ra d ia te d with a 60Co s o u r c e ow ned by the F a cu lty o f M a th e m a t ic s and P h y s i c s , Gadjah Mada U n iv e rs ity , A v e r y s m a l l d o s e ca u se d the m oths to b e c o m e s t e r i l e [5 ,6 ] .

T h is p a p e r d e s c r ib e s p r e l im in a r y e x p e r im e n ts on the attack intensity and population den s ity a fte r a r e l e a s e o f s t e r i le Sc irp oph ag a n ive l la F . F u r th e r r e s e a r c h su g ge s te d in the X VIth D ies Natalis sp e e ch o f the State Sugar A c a d e m y [8] is a l s o d e s c r ib e d .

2. M A T E R IA L S AND M ETH ODS

T he fo l lo w in g e x p e r im e n ts w e r e p e r fo r m e d to c o m p a r e the 1 r o g e s a n 1 c o n t r o l m ethod with the s t e r i l e in s e c t r e le a s e .

2 . 1 . O rien ta tion o f su g a r can e plantations

Six is o la te d f ie ld s with ca n e s about two m onths o ld which had n e v e r been treated f o r any p est w e r e u sed . T h e se f ie ld s w e r e s im i la r with r e g a r d to the v a r ie ty o f c a n e s , the kinds o f s e e d l in g , the s e v e r i ty o f p e s t in festation , the intensity o f the e ra d ic a t io n a t tem pts , the a r e a in vo lved ,e tc . T h re e p lots w e r e u se d f o r the r e l e a s e of. s t e r i l e in s e c ts and the o th e rsw e r e used as c o n t r o l p lo ts .

2 . 2 . Popu lat ion density ca lcu la t io n and the am ount o f dam age cau sedby b o r e r attack

F o r e v e r y plot o f g round the n u m b e r o f r o w s and the total n u m b e r o f fu r r o w s w e r e de te rm in e d . F u r r o w s w e r e ra n d o m ly s e le c t e d and the p er cent o f in fested s t e m s w as a s ce r ta in e d .

L a r v a e and pupae w e r e r e m o v e d f r o m the in fested s t e m s . B a se d on the n u m b e r o f la rv a e and pupae found in the sa m p le fu r r o w s the density o f in s e c t s w a s d e te rm in e d and the d e v e lo p m e n ta l s tag es w e r e r e c o r d e d .A t the sa m e t im e both the density o f in s e c ts and the s e v e r i ty o f dam age throughout the sa m p le p lots w e r e r e c o r d e d m onthly . E v e r y t im e new fu r r o w s w e r e chosein, w e used the sa m e n u m ber and length o f fu r r o w s as in the f i r s t o b s e rv a t io n .

2 . 3 . M a te r ia ls p r e p a ra t io n and f ie ld r e s e a r c h su p p lies

S M - 102/28 3 1 9

P upae as r e l e a s e m a te r ia l w e r e obta ined f r o m the M ad u k ism o su g ar fa c to r y in cane top s . The r e l e a s e co n ta in e rs c o n s is t e d o f flat earthen d i s c s f ixed on top o f b a m b o o p o le s planted in the f ie ld s . T h e se b a m b o o p o le s w e r e l| m e t r e s lon g and each had two d i s c s f ixed to the top. The upper d is c had a co n e at the bo t tom . Banana le a v e s w e r e h e re sp re a d out to hold the pupae, w hich w e r e then c o v e r e d with s m a l l p ie c e s o f banana l e a v e s . The u pp er d is c w as put on top o f the l o w e r d i s c , which had a flat bottom and w as l a r g e r in s i z e than the u pper . W a te r w as then p ou red on the l o w e r con ta in e r to p reven t ants f r o m h arm in g the pupae and to keep the u pp er con ta in e r hum id . The bo t tom o f the s e c o n d con ta in er w as f ix ed to the top o f the b a m b o o pole .

2 . 4 . L a r v a e r e a r in g and s t e r i l i t y induction

L a r v a e c o l l e c t e d in the f ie ld w e r e la b o r a to r y r e a r e d on su g a r cane until they b e c a m e pupae. T h e se and a l l f i e ld - c o l l e c t e d pupae w e r e e x p o se d to a 60Co s o u r c e . T h is rad ia t ion w as g iven the m o rn in g right b e fo r e r e le a s e .

2 . 5 . S te r i le in s e c t r e l e a s e

. S ter i le pupae w e r e r e le a s e d one o r two days b e f o r e e c lo s io n , the r e l e a s e s be ing c a r r i e d out b e f o r e the r a in y s e a so n . P u p a - r e le a s e is the bes t m ethod , s in c e the b e s t m ating t im e f o r th ese m oths is ju st a fter th e ir m e t a m o r p h o s i s . T h is r e le a s e w as done at defin ite t im e s , the n u m b e r o f in s e c t s to be r e le a s e d be ing m ade equal to the population den s ity o r equ a l ize d with the n u m b e r o f fe m a le m oths e s t im a te d to e m e r g e . On th ree n e igh bou r in g r e l e a s e plots 10 b a m b o o r e l e a s e p i l la r s w e r e p lanted, and the n u m b e r o f pupa put th ere each t im e f o r r e l e a s e depended upon t im e , f r e q u e n c y and d en s ity (Appendix 1).

2 .G . O b s e rv a t io n

The m ethod o f o b s e r v a t io n has a l re a d y been outlined a b o v e . Both c o n t r o l and r e l e a s e p lo ts w e r e kept under o b s e r v a t io n , as w e r e the egg c o lo n i e s and the hatching ra te , which have so fa r y ie ld e d no d e f in it ive data.M onthly o b s e r v a t io n s w e r e c a r r i e d out during r e l e a s e t im e , when the cane w as m o s t su s c e p t ib le to white top b o r e r attack , w h ile final o b s e r v a tions w e r e m a d e b e f o r e h a r v e s t t im e to d e te rm in e w hether the s t e r i l e in se c t r e l e a s e had had any e f fe c t .

3. RESULTS

The tab les and f ig u r e s i l lu s tra te the r e s u l t s o f the e x a m in a tion s .At the s t e r i l e in s e c t r e l e a s e p lo ts (F ig . 1), a d e c r e a s e o f attack

intensity and population density w as n o t ice d , w hile at the c o n t r o l plots (F ig . 2) an in c r e a s e o f both o c c u r r e d .

The f i r s t pupal r e l e a s e took p la ce on 13 S e p te m b e r 1966, in p lots 1,i l , III w hich had a f ixed a re a c o m p a r is o n . T he fo l lo w in g n u m b e rs o f pupae

3 2 0 HATMOSOEWARNO

w e re r e le a s e d in s u c c e s s i o n : 75, 75 and 150, r e s p e c t iv e ly ; s im u lta n e o u s ly the fo l lo w in g dam age w as o b s e r v e d : 9 .4 0 % , 5. 1G% and 6. 28% with population d e n s it ie s o f 8 .5 0 , 4 .4 5 and 5 .7 1 (T a b le I). Not a l l pupae r e le a s e d b e c a m e m o th s ; the fo l lo w in g n u m b e rs o f m oths w e r e o b s e r v e d : 51, 60 and 136. At the se co n d r e l e a s e , on 4 O c t o b e r 1966, the d am age to p lots I, II and III d e c r e a s e d to 4. 35%, 1. 54% and 3. 27%, r e s p e c t iv e ly ,

FIG. 1. D am age and population density at release plots.------------- : D am age ('/Ú)-------------: Population density.

while population d en s it ie s b e c a m e 3 .7 9 , 1 .0 0 and 0 .7 2 . By that t im e an oth er pupal r e l e a s e had been p e r f o r m a l : 54, 54 and 72, r e s p e c t iv e ly , o f which 41, 43 and 66 b e c a m e m oth s . The th ird r e l e a s e , on 15 N o v e m b e r 1966, p ro d u ce d the fo l lo w in g d am age in p lo ts I, II and 111: 2 .8 5 % , 2 .0 4% and 1 .2 5 % , w hile the population density f ig u r e s w e r e 1 .2 4 , 0 .2 5 and 0 ,3 7 . O f the th ird pupal r e l e a s e , o f 45, 45 and 00 pupae, 44 , 45 and

S M - 102/28 321

60 b e c a m e m o th s . Both o b s e r v a t io n and in s e c t r e le a s e w e r e then te r m in a te d , b e c a u se by that t im e the su g ar can e w as no lo n g e r su s c e p t ib le to white top b o r e r attack.

The f ig u r e s on da m ag e and in s e c t population d en s ity g iven be low w e r e obtained f r o m the c o n t r o l p lots (T a b le 11). The f i r s t o b s e r v a t io n on 7 S e p te m b e r 1966 sh ow ed the fo l lo w in g d am age p e r c e n ta g e s : p lo ts i , II

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FIG- 2 . D am age and population density at control plots.------------- : D am age (%)-------------- popu lation density.

and III, 14 .69% , 6. 74% and 8. 54% ,r e s p e c t i v e l y , w hile the density w as12. 50, 5. 76 and 7. 13. The s e c o n d o b s e rv a t io n took p la ce on 27 S ep tem b er 1966, and y i e ld e d t h e fo l low in g da m ag e f ig u r e s : 5 .7 6 % , 4 .7 9 % and 5 .87% with population d e n s it ie s o f 5 .6 5 , 4 .5 0 and 5 .4 6 , r e s p e c t i v e ly . F ig u r e s fo r both dam age and population d en s ity b e c a m e s m a l l e r . On the oth er hand, a s tr ik in g in c r e a s e in these f ig u r e s w as o b s e r v e d in the fo l low in g

2UMARCH

T A B L E I. D A M A G E A N D I N S E C T P O P U L A T I O N D E N S I T Y A T R E L E A S E P L O T S ( P r e l i m i n a r y e x p e r i m e n t o n c a n e f i e l d s [7 ])

. D ate P lot N o .

T o t a l o f pupae T o ta l stem in furrowsD a m a g e

T o ta l (In sa m p le furrow s)

D en sity

R e lea se R em ainderT ota l

nu m berIn fested

stem

№ )L arvae Pupae

I 75 24 234 22 9 .4 0 13 7 8 .5 0

13 S ep t. 1966 .

II 75 15 290 15 5 .1 6 10 3 - 4 .4 3

III 15 0 14 271 П 6 .2 8 9 6 5 .7 1

4 O c t , 1966

I 54. 13 414 18 4 .3 5 12 4 3 .7 9

II 54 11 453 7 1 .5 4 4 1 1 .0 0

III 72 6 367 12 3 .2 7 3 0 . 0 .7 2

I 45 1 801 23 2 .8 5 8 2 1 .2 4

15 N o v .II 45 0 932 19 2 .0 4 2 0 0 .2 5

.1966III 60 0 800 10 1 .2 5 2 1 0 .3 7

322 H

ATM

OSO

EW

AR

NO

T A B L E II. D A M A G E A N D IN S E C T P O P U L A T I O N D E N S I T Y A T C O N T R O L P L O T S ( P r e l i m i n a r y e x p e r i m e n t on c a n e f i e l d s [7 ])

D ate P lo t N o .T o ta l stem in s a m p le furrow s

D a m a g e

(7°)

T o ta l (In sa m p le furrow s)

D en sity

T o t a l num ber In fested stem L arvae Pupae

I 471 69 1 4 .6 9 42 17 1 2 .5 0

7 S e p t.II 346 2 4 6 .7 4 15 5 5 .7 6

1966III 49 2 4 2 . 8 .5 4 26 9 7 .1 3

I 521 30 5 .7 6 18 11 5 .6 527 S e p t.

II 420 ■ 20 4 .7 9 12 • 7 4 .5 01966

III 698 41 5 .8 7 25 13 5 .4 6

I 437 74 1 6 .9 7 40 19 1 3 .4 925 O c t .

1966II 574 59 1 0 .2 2 27 18 7 .8 3

III 812 112 1 3 .7 6 63 34 1 2 .0 0

I 621 107 1 7 .2 5 71 28 1 6 .0 0

1 5 N o v .II 519 65 1 2 .5 0 33 31 1 2 .2 7

1966III 907 126 1 3 .9 2 96 2 4 1 3 .2 0

SM

-102/28 323

T A B L K 111. D A M A G E A N D IN S E C T P O P U L A T IO N D E N S IT Y A T R E L E A S E P L O T S ( L a s t o b s e r v a t i o n b e f o r e h a r v e s t in g )

D a te Plot N o .Rows T o t a l o f s tem T o t a l o f

T o ta l

L etter N o . Furrow T o ta l n u m b er In fested stem L arvae H upae(L a r v a e + pu pae)

14 M a r ch I A 1 4 10 3 42 11 1719 67 5 3 14 84 19 7 2 9

С r> 24 8 4 26 1 3 0 13D 7 34 82 29 12 4 16E g 44 57 18 5 2 7

T o t a l : 4 1 0 1 3 4 . 62D a m a g e (% ) P o p u la t io n d en s ity

3 2 .41 5 .1 2

II F i l 2 9 0 39 14 4 18G 13 32 84 29 9 2 Иil 15 22 66 2 a 11 12

17 .12 78 2Ь 9 3 12J 19 42 83 22 D 0 6

T o t a l : ' 40 1 143 59D a m a g e (°/i>) P o p u la t io n d en s ity

3 5 . К1 4 .7 1

HI К •2 17 89 45 9 14I. 4 07 87 24 6 3 9M 37 59 17 12 13

N R 47 71 2 fi 3 3 6О 10 Г) 7 70 15 5 3 8P 12 G? 78 21 6 1 7

Q 14 77 H7 41 1.4 2 17

1? 16 87 01 35 7 12

T o t a l : 592 224 86D a m a g e (°}¡) P o p u la tio n d en s ity

3 7 .81 4 .5 3

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o b s e r v a t io n that took p la ce on 25 O c t o b e r 1966 - they w e r e h igh er by far than f ig u r e s f r o m the f i r s t and s e c o n d t e s ts : f o r da m ag e 16. 97%, 10. 22% and 13 .76% , r e s p e c t iv e ly , and f o r population d en s ity 1 3 .4 9 , 7 .8 3 and 12 .0 0 . The fourth o b s e r v a t io n took p lace on 15 N o v e m b e r 1966: the am ount o f dam age was 17 .25% , 12 .50% and 13.92%, w hile the population d e n s it ie s w e re 1 6 .0 0 , 12 .27 and 1 3 .2 0 , r e s p e c t iv e ly .

A c c o r d in g to the f ig u r e s obta ined f r o m the ab ove m en tioned o b s e r v a t io n s , c o n c e rn in g both c o n t r o l p lots and r e l e a s e p lo ts , i t ' i s ob v io u s that the se co n d o b s e rv a t io n y ie ld e d d e c r e a s e d f ig u r e s f o r d am age and population density . 'F urther o b s e r v a t io n s y ie ld e d d e c r e a s e d f ig u r e s f o r r e l e a s e p lo ts and in c r e a s e d f ig u r e s f o r c o n t ro l p lots .

B e f o r e cane h arves t in g , o b s e r v a t io n s w e r e c a r r i e d out o n ce m o r e (T a b le 111). O b s e rv a t io n s at the r e le a s e p lots w e r e p e r fo r m e d on 14 M arch 1967 and gave the fo l lo w in g d am age f ig u r e s : 3 2 .4 % , 35.6%) and 3 7 .8 % , r e s p e c t iv e ly , w hile the population d en s ity f ig u r e s w e r e 15. 12, 14 .71 and 14 .5 3 . O b s e rv a t io n was c a r r i e d out at the c o n t ro l p lots on 24 M a rch 1967 and y ie ld e d the fo l lo w in g d am age f ig u r e s : 4 1 .3 2 % , 3 8 .2 1 % and 4 3 .8 6 % , r e s p e c t iv e ly , w hile f o r population density the f ig u r e s w e r e 19. 12, 16 .07 and 19 .27 .

In this la s t o b s e rv a t io n b e fo r e h arves t in g (T a b le IV), both m ethod and o b je c t w e r e the sa m e as f o r the p r e ce d in g o n e s . The am ount o f d am age and the population density at the r e le a s e p lots w e r e seen to be n e a r ly the s a m e as at the c o n t r o l p lo ts . The on ly d i f f e r e n c e w as in the a p pe a ra n ce o f the dam age and it w as o bv iou s that the d am age at the c o n t r o l p lots w as ca u se d by a l in g e r in g , g radua l in fe c t io n , while the d am age at the r e l e a s e plots w as a new in fe c t io n , which , f o r the g r e a te r part , w as cau sed by a new in festation a ff l ic t in g the can e tops in m o s t c a s e s .

4. DISCUSSION

B e c a u s e o f t e ch n ica l d i f f i c u l t i e s , this e x p e r im e n t w as l im ite d to studying the e f fe c t o f s t e r i l e in se c t r e le a s e upon attack intensity and population d en sity . The data obta ined w e r e a m eans to c o m p a r e the o b s e rv a t io n f ig u r e s at both r e l e a s e and c o n t r o l p lo ts .

The d is ta n ce betw een the r e l e a s e p lots and the c o n t r o l p lots was about 5 km to av o id any e f fe c t ca u se d by m ig r a t io n , s in c e the m a x im u m flight ca p a c ity o f the white top b o r e r moth is + 1 km.

If the m o t h s 1 f light ca p a c ity and the r e la t iv e ly s m a l l a re a o f the r e le a s e p lots ( ± 0 . 5 ha) a r e taken into c o n s id e r a t io n , the r e le a s e o f the s t e r i le in s e c t s can be done in one on ly a r e a o f each plot. In o u r e x p e r im e n t the r e l e a s e was done in s e v e r a l a r e a s to en su re that the en tire plot would be c o v e r e d .

Both m a le and fe m a le m oths w e re r e le a s e d to p r o v id e a p o s s ib i l i t y fo r s t e r i l e f e m a le m oths to m ate with f e r t i l e m a le s and thus r e d u c e the p ro b a b il i ty o f f e r t i le m a le s m ating with f e r t i l e f e m a le s .

Not a l l r e l e a s e d pupae b e c a m e m oths . The h ighest death ra te was found a fte r the f i r s t r e l e a s e , pa rt ly b e c a u se o f the p o o r r e l e a s e technique and part ly b e c a u s e o f w eath er d is tu rb a n ce s .

B e c a u s e o f the fac t that th e re w as no constan t supply o f pupae to be r e le a s e d , the n u m b e r o f s t e r i l e pupae did not a lw ays c o r r e s p o n d with the es t im a te d population , as is u su a lly the c a s e with such a r e l e a s e .

T A B L E IV. D A M A G E A N D INSRCT P O P U L A T I O N DENSITY A T C O N T H O L P L O T S ( L a s t o b s e r v a t i o n b e f o r e h a r v e s t in g ) •

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24 M a r ch I A o 12 92 12 4 4 8Ш 7 B 4 22 "2 41 11 18 29

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T o t a l : 455 1RS 87D a m a g e (°¡o) P o p u la tio n d en s ity

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T o t a l : 773 33 9 149D a m a g e (fa ) P o p u la t io n d en s ity

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O b s e rv a t io n s at r e l e a s e p lo ts w e r e on ly p e r f o r m e d fo u r t im e s , but f iv e t im e s at the c o n t r o l p lo t s ; the fourth o b s e r v a t io n at the c o n t r o l p lots w as to a s c e r ta in w hether th e re had been a rep e t it io n o f the d e c r e a s e in both popu lation and attack intensity as had been noted a fte r the s e c o n d o b s e rv a t io n . The c o n t r a r y w a s , in fa c t , o b s e r v e d ; the fourth o b s e rv a t io n y ie lded in c r e a s e d f ig u r e s , h igh er than those f r o m any o f the p r e ce d in g tes ts on both f ie ld s , f o r both attack in tensity and population den sity .The o b s e r v a t io n s w e r e c a r r i e d out at d i f fe re n t t im e s b e c a u se o f the d i f f e r e n c e in the age o f the su g a r cane .

A c o m p le te m a th e m a t ica l ca lcu la t io n cannot be p r e se n te d yet b e ca u se o f the la ck o f re p e a te d e x p e r im e n ts . F o r the t im e being it should su f f ic e to say that w e have c o m p a r a t iv e ly in cr e a s in g f ig u r e s f o r c o n t r o l p lots and d e c r e a s in g f ig u r e s f o r r e l e a s e p lo ts w h e r e both dam age in tensity and population d en s ity a r e c o n c e r n e d .

5. CONCLUSIONS

The r e le a s e o f s t e r i l e insfects ca u se d a d e c r e a s e in both attack intensity and in se c t population d en sity .

The d e c r e a s e o f attack in tensity at the r e l e a s e p lots a fte r the se co n d o b s e rv a t io n w as not s o m a rk e d , but the d e c r e a s e o f the population density w as o b v io u s . T h is w as p a rt ly b e c a u s e o f the fa c t that the in fested plants be long ing to the f i r s t test w e r e in cluded in the s e c o n d , w hile during the se c o n d o b s e rv a t io n th ere w e r e not m any new sh o o ts .

The th ird o b s e rv a t io n at the r e le a s e p lots r e v e a le d a d e c r e a s e in both dam age and population d en s ity , w hile o b s e rv a t io n at the c o n t r o l p lots show ed an ob v io u s and steady in c r e a s e in th ese f ig u r e s .

Since the e x p e r im e n t has on ly been c a r r i e d out o n ce within one g ro w in g p e r io d o f the su g a r ca n e , it w il l be n e c e s s a r y to rep e a t the e x p e r im e n t arid to u se im p r o v e d r e l e a s e apparatus and m o r e intense o b s e rv a t io n m e th o d s . A s e c o n d tes t w i l l s o o n be under way.

It has been p r o v e d that a s t e r i l e in s e c t r e l e a s e can be s im p ly and s u c c e s s f u l ly c a r r i e d out. If the s e c o n d e x p e r im e n t y ie ld s p o s it iv e r e s u l t s , the m ethod can be put into p r a c t i c e soon on su g ar cane plantations .

6. SU M M A R Y

S u c c e s s fu l u se o f the s t e r i l e - m a l e technique in c o n t ro l l in g in se c t populations w as r e p o r t e d in s e v e r a l c o u n tr ie s . C erta in c h a r a c t e r i s t i c s o f a candidate in s e c t a r e im p ortan t f o r the e m p lo y m e n t o f this technique. Since 1965, e x p e r im e n ts have been p e r f o r m e d at the State Sugar A c a d e m y in J o g ja k arta , Indonesia , to e x am in e th ese c h a r a c t e r i s t i c s in the-white top b o r e r (S c irpop h ag a n ive l la F . ) , a d e s t r u c t iv e su g ar can e p es t , and to d e te rm in e the su itab i l i ty o f the s t e r i l e - m a l e m ethod . S e v e r a l im portan t c h a r a c t e r i s t i c s w e r e found su itab le , n a m e ly , copu la t ion a b i l i ty did not d e c r e a s e a f t e r ra d ia t io n , and r e a r in g on an a r t i f i c ia l m e d iu m is p o s s ib le fo r a l l i i f e - c y c l e s ta g e s . R adiation at-the pupal stage induced s t e r i l i ty in the adult.

P r e l im i n a r y e x p e r im e n ts in a su g ar can e plantation w e r e p e r f o r m e d to d e te rm in e the e f fe c t o f r e le a s e d s t e r i l e in s e c ts upon attack in tensity

3 28 HATMOSOEVVARNO

and popu lation d en sity . The n um ber o f r e le a s e d s t e r i l e in s e c ts a p p r o x i m a te ly equ a lled that o f the natural population . R e le a s e w as p e r f o r m e d at a t im e when su g ar cane w as su sc e p t ib le to attack in an a r e a which had p r e v io u s ly r e p o r t e d 10% annual l o s s e s due to the ac t iv ity o f this in se c t .

R e le a s e d s t e r i le in se c ts cau sed a d e c r e a s e in both attack intensity and population density . F r o m the exam ination s c a r r i e d out just b e fo r e h arve s t in g , a fte r the r e le a s e o f the in se c ts had been te rm in a ted , we found that the in se c t population den sity at the c o n t r o l p lots as w e l l as at the r e le a s e p lo ts w as roughly equal. T h is fa c t le d us to the c o n c lu s io n that the r e le a s e o f the s t e r i le in se c ts w il l be e f fe c t iv e in su p p r e ss in g pop u la t ion in c r e a s e i f it is done constan tly . T h e re is another fa c t that su p p orts this op in ion , i . e . although the f ig u re s shown by the c o n t ro l p lots and the r e l e a s e p lots ju st b e fo r e .harvesting a r e a lm o s t equal, the d am age in the r e l e a s e p lots w as to le a v e s only.

T hus , i f we c o n s id e r e d that dam age ca u se d by this pes t to su gar cane 10 m onths old o r o ld e r m a y be ig n o r e d , c o n t r o l cou ld be a c c o m p l i s h e d by r e le a s in g s t e r i l e in se c ts during the se n s i t iv e p e r io d o f su g ar can e grow th . If, h o w e v e r , w e want to d e s tr o y this p e s t c o m p le t e ly , the r e l e a s e o f the s t e r i l e in se c ts m ust be done constan tly during the grow in g p e r io d o f the sugar can e .

Л С. К N O W L E D O E M E N T S

The author w ish e s to e x p r e s s his gratitude to the D ir e c t o r o f the State Sugar A c a d e m y , M r . S oe p ard im e n , w'ho enabled h im to c a r r y out ex te n s iv e r e s e a r c h w o rk in the la b o r a to r y and in the f ie ld and who p r o v id e d a d v ice and guidance during the p r e p a ra t io n o f this p a p e r . Thanks a r e a lso due to the Head o f the A g r o n o m y Section o f the sa m e institution, M r . S oep ra p top o , f o r his aid during the e x p e r im e n t , and to the D ir e c t o r o f M ad u k ism o Sugar F a c t o r y and his s ta ff , who p ro v id e d r e s e a r c h su p p l ies and valuab le a s s i s ta n c e in the su gar cane f ie ld s . The author f ina lly w is h e s to e x p r e s s his a p p re c ia t io n to the Head o f the National A t o m ic E n e r g y A g e n cy A d m in is t ra t io n , Jog jakarta Section , f o r the g e n e r o u s supply o f rad ia tion s o u r c e s .

R E F E R E N C E S

[ 1 ] CHARPENTIER, I ..J . , MATHES, R. , M cCORM ICK, W .J . , SANFORD, J. W. , Injury and Losses Caused by the Sugar Cane Borer in Louisiana, E ntom ology Research D ivision , A gricultural Research S erv ice , USDA (1965).

[ 2 ] CLAUS, W .D . f Radiation B iology and M ed icin e , Addison -W estly Publishing C om pany, Inc. (1958) 107 -0S , 688 -70 0 .

[ 3 ] DEVENTER, W . V an, De d ie tlijk e vijanden van het suikernet en hunne parasieten. H andboek ten dienste van de suikerrietcultuur en de suikerrietfabricage op Java 2 , J .H . DE BUSSY Amsterdam (1 9 1 2 )1 1 4 -2 6 .

[ 4 ] GLASSTONE, S . , Sourcebook on A tom ic Energy, D . Van Nostrand C o . , Inc. , New Jersey, Charles E. Tutle C om pany, T ok yo (1960),

[ 5 ] HATMOSOEWARNO, S . , Rearing dan sexing Scirpophaga n ivella F . , l.aporan tahap pertam a "T ea m penggunaan radiasi atom untuk tanaman tebu" bidang ham a dan pen jak ii. K eidja sama antaraP .G . M adukism o, Fakultas Pertanian IJ. G . M . , Fakultas Biologi U .G .M . . Fakultas Ilmu Pasti dan Alam U. G .M . , Balai Penjelid ikan Perusahaan Perusahaan Gula (B. PM. G. ) Pasuruan dan A kadem i Gula Negara Jogjakarta (1965).

S M - 102/28 329

[6 ] HATMOSOEWARNO, S . , Kemungkinan penggunaan tenaga atom untuk pemberantasán llama penggerek putjuk putih ( Scirpophaga n ive lla F .) pada tanaman tebu. Paper B -10 , Sim posium radioisotop I, Bandung, Indonesia (1966).

[ 7 ] HATMOSOEWARNO, S. , Laporan tahap k e -em p a t "T eam penggunaan radiasi atom untuk tanaman tebu" bidang ham a dan penjakit. Kerdja sama antara P .G . M adukism o, Fakultas Pertanian U .G .M . , Fakultas B io log i U .G .M . , Fakultas Ilm u Pasti dan Alam U .G .M . , Balai Pen jelid ikan Perusahaan Perusahaan Gula (В. P3. G .) Pasuruan dan A kadem i Gula Negara log jakarta (1 966 ).

[ 8 ] HATMOSOEWARNO, S . . A tom dimasa dam ai dan kem ungkinan penggunaan tenaga atom untuk pemberantasan ham a penggerek putjuk putih ( Scirpophaga n ive lla F .) pada tanam an tebu. Pidato Dies Natalis A kadem i Gula N egara Jogjakarta, ke XVI (1966).

[ 9 ] KALSHOVEN, L .G .E . , De plagen van de cultuur gewassen in Indonesia. N .V . U itgeverij w . van h o e v e ’ s gravenhage, Bandung 1 (1960) 4 2 4 -2 9 .

[1 0 ] MARTORELL, L. F . , M ED INA-G AU D , S . , Status o f Important Insect Pests o f Sugar C ane in PuertoRico and Their C on trol, Agricultural Experim ent Station, University o f Puerto R ico , Rio-Piedras, P.R. (1965)

[1 1 ] SANFORD, J .W . , CHARPENTIER, L.J. , MATHES, R . , C h em ica l C ontrol o f the Sugar Cane Borer in Louisiana, E ntom ology Research D ivision , A gricultural Research S erv ice , USDA (1965).

[1 2 ] A rch ief voor de suikerindustrie in Nederland Indie dan M ededelingen van het proefstation voor de Java suikerindustrie (1927 s /d 1934).

[1 3 ] USAEC, Radioisotopes in S c ien ce and Industry, A sp ecia l report o f the United States A tom ic Energy C om m ission (I9 6 0 ) 1 2 -1 4 .

D I S C U S S I O N

M. F R IE D : Could you p le a se ind icate how la r g e the treated plots w e r e , how fa r distant they w e r e f r o m the untreated p lo ts , and what w as the ratio o f s t e r i le in s e c ts to native in s e c ts ?

S. H A T M O SO E W A R N O : The tre a te d p lots w e r e 0 . 5 ha each , and the d is tance betw een them and the untreated plots w as about 2. 5 km.The ratio o f s t e r i l e in s e c ts to native in s e c ts w as brought as c l o s e as p o s s ib le to unity by ca lcu la t io n o f the population density a w eek b e fo r e r e l e a s e o f the s t e r i l e in se c ts .

II. E R D M A N : If you w e r e to e r a d ic a te S. n ive l la would you e x p ect g r e a te r d am age f r o m o th er i n s e c t s ? That i s , on e l im in at ion o f this c om p on en t f r o m the en v iron m en t , w il l an oth er s p e c ie s o f in s e c t pest b e c o m e m o r e d e s t r u c t iv e ? I b e l ie v e that the p o s s ib i l i t y o f s p e c i e s r e p la ce m e n t should be in vestigated .

S. H A T M O SO E W A R N O : I think that o th e r in se c t p e s ts cau s in g d am age to su g a r can e plantations in Indonesia a r e not n e a r ly so im portan t as the white top b o r e r , s o that by c o n t ro l l in g this pest w e m ay re a so n a b ly hope f o r in c r e a s e d can e prod uct ion . It should be b o r n e in m ind , h o w e v e r , that this f i r s t e x p e r im e n t o f m in e w as s im p ly a p r e l im in a r y one c o v e r in g on ly 1. 5 ha, w h e r e a s by now I am w ork in g on a m uch l a r g e r a r e a so that the p o s s ib i l i t y o f another d e s tr u c t iv e p es t assu m in g p r o m in e n c e can soon be thoroughly ex am in ed .

CONTRO L OF A D U LT PESTS BY TIIE IR RA DIA TIO N - O F - MA LE M ETH O D "

R. C. VON BORSTEL BIOLOGY DIVISION,OAK RIDGE NATIONAL LABORATORY,OAK RIDGE, TENN. ( UNITED STATES OF AMERICA

Abstract

CONTROL OF'ADULT PESTS BY THE IRRADIÀTION-OF-MALE METHOD. T h e Grosch e ffect (i. e . , o f radiation interfering with u tilization o f nutrients in insects) b eca m e a p p lica b le to pest control when Galun and Warburg d iscovered that irradiated ticks fed o n ce would seldom i f ever feed again. This l ife lon g sensation o f 'im p le tio n ' does not interfere w ith sexual com petitiveness, but it does render harmless a pest that is a pest during the adult stage o f its l ife c y c le . It w ould seem that the general princip le o f treating adult pests to lim it their feed in g cap acity cou ld be extended to.other pests. When insect pests are- liv in g in stored food products, it is a general rule that desirably low levels o f radiation can be used to eradicate the pest i f steriliz ing doses rather than in sectic id a l.doses are used. With the entire population o f both m ales and fem ales being irradiated, the sterilizing dose is often m uch low er than that w hich w ould ’ b e desirable when the insects are m ass-reared for irradiation and release. W here m ass-rearing techniques are not feasib le , or where the pestiferousness o f adults cannot be o v ercom e by radiation or other treatments, fie ld -irrad ia tion fa c ilities in baited traps.can .be devised. These have som e advantages over sim ilar traps contain ing chem osterilants. W herever it can be used, the method o f m ass-rearing, irradiation, and release is still the m ost desirable w ay to control insect pests.

IN T R O D U C T IO N

The re lia b ility of the irradiation-of-male method of insect control depends upon four effects of radiation t l ] , each of which must be studied for the insect under consideration, and each of which requires precise dosimetry. The first effect is that dominant lethal mutations are induced in the sperm by radiation . A dose of radiation must be used that induces dominant lethal mutations in more than half, and preferably upward of 9 9 % , of the sperm. The second effect is that .. spermatogonia I cells are k illed by radiation . A dose of radiation must be used that is high enough to elim inate repopulation of the testes w ith normal spermatogonia by k illing a ll of the spermatogonial stem c e lls . The third effect is that sperm are inactivated by radiation . A dose must be used that does not inactivate appreciable quantities of the sperm of polygamous species. For monogamous species, it does not matter whether the sperm are inactivated or whether dominant lethal mutations are induced ¡ The fourth effect is that males are weakened by radiation . A dose of radiation must be used that does not deb ilitate the effectiveness o f irradiated males when they are competing with normal males for. the sexual attention o f the females ¿

By d fortunate circum stance, for many insect pests the first two effects of radiation co incide at a dose of radiation below that at which the third and fourth effects become c r it ica l . Further, it is unnecessary to determine any of these or

* Research sponsored by the United States A tom ic Energy Com m ission under contract with the Union Carbide Corporation. 1

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332 VON BORSTEL

any other rad iobio log ical parameters for females if the adult is not also a pest, unless there is discovered the u n like ly insect where the oogonia are less sensitive than the spermatogonia and the females reproduce parthenogenetica lly .

IR RA D IA T IO N A N D RELEASE O F ADULT PESTS

When only the female of a species is a pest, means for separation of the sexes can be invented, and the standard irradiation-of-male procedures can be used . The means of separation that have so ingeniously been devised.comprise the physical (e .g by utiliz ing w eight, size, or osmotic differences), the genetic (e .g ., marking the sex chromosome with a mutant gene for egg co loration), or the tropistic ( e .g . , using sex attractan ts ). If both adults are pests, or the male alone is a pest, the problem takes on new dimensions.

Grosch [2 ] was the first investigator to demonstrate that the life-span was the same in starved and fed insects at doses above 100 kilorads. It could be inferred that in both cases the Habrobracon jug land is (Ash mead) females starved to death after being given large doses of radiation . The Grosch effect ( i . e . , of radiation interfering with utilization of nutrients) became app licab le to insect control when Galun and Warburg [3 ] made the epochal discovery that irradiated ticks fed once would seldom if ever feed again , even at exposures of on ly 4000 R . This lifelong sensation of "im pletion" rendered harmless a pest that is a pest when in the adult stage of its life cyc le . Studies on other insects demonstrate that it is not simple to app ly this princip le un iversa lly . L ittle [4 ] has shown that the earw ig , Chelisoches morio (F .) , and the M editerranean fruit f ly , Ceratitis capitata (W iedem ann), retained normal intestinal epithelium after they had been irradiated with doses of 10 kilorads. Destruction of intestinal cells became obvious at 100 k ilorads. On the other hand, Riemann and F lin t [5 ] showed that when the boll w e e v il, Anthonomus grandis Boheman, is exposed to 4000 R, the male-weakening from destruction of the intestinal tissues proceeds much too rap id ly for the conventional irradiation-of-m ale procedures to be useful.

The exhaustive analyses by Riemann and F lin t, the Grosch e ffec t, and the phenomenon of Galun-W arburg impletion leave no doubt that k illing the cells of the intestine destroys the capac ity to u tilize nutrients. C lark and Rubin [6 ] observed that Habrobracon serinopae (Ram kr.) females that were fed on a d iet of honey and water died much later (92 days) than females given only paralyzed or unparalyzed larvae of their host Ephestia kühniella Z e lle r (40 d ay s ). When radiation was applied at increasing doses, the life-span decreased in both cases. The honey-fed wasps always lived longer than the Ephestia-fed wasps, but the percent decline at each dose was essentially identical . This indicates that nutrient u tilization is declin ing in both cases to the same degree.

Work on mammals [7 ] has shown that rats become ravenous just before dying from radiation-induced intestinal in jury because the blood sugar becomes dep leted . The level of glucose in the blood triggers the sensation of hunger in mammals.That the phenomenon of Galun-W arburg impletion exists argues strongly for the necessity of physiological studies on insects to determine the mechanism of the hunger response. If irradiation of the intestine at doses suitable for insect control does not curb the feeding instincts of an insect as L ittle has shown, or if irradiation k ills the insect at doses too low for insect control as Riemann and Flint have found, then some other treatment with or without radiation might bring on a permanent sensation of impletion; standard irradiation-of-m ale procedures

SM-102/34 333

could then be applied . Understanding the origin and control of appetite and hunger in insects w ill open the way to using the irradiation-of-male method on any insect pest .-1

IR R A D IA T IO N O F ST O RA G E PESTS

When the pests are living in stored food products, it would be preferable if re la tive ly low doses of radiation were used . Radiation induces long-lived free radicals in grain, and the tox ic ity , carc inogen ic ity, and m utagenicity of these free radicals have not yet been adequately eva luated . Since lower doses give fewer free rad icals, the lowest possible doses should be used to elim inate the storage pests. For this reason, steriliz ing doses rather than insecticidal doses should be se lected . Steriliz ing doses genera lly are lower by far than insecticidal doses.

Nature is helpful too. The storage pest most suitable for the irradiation- of-male method would have the following characteristics: The eggs are more sensitive than the sperm. When these sensitive eggs are exhausted and the more resistant oocytes fo llow , then the spermatocytes that are much more sensitive than the sperm have matured to be sperm ready to fertilize the eggs coming from the resistant oocytes. A sterile period ensues for the m ale. By the time the spermatogonia have regenerated, the much more sensitive oogonia have rendered the females completely s te rile . Since many insects conform to a pattern near to the ideal of alternating maxima of radiation sensitivities for the male and female, research with very low doses is worth trying for many insect pests. Further, the combined embryo survival from lacking dominant lethal mutations is a multiple of the sensitivities of the sexes at each stage of gametogenesis.

The principal storage pests that would give d iff icu lty are the Lepidopteran species. As a ru le, the Lepidopteran gametes are quite resistant to radiation [ 1 ] , and the insecticidal doses range beyond 100 kilorads. Minimum steriliz ing doses here w ell may be in the range of 20 kilorads or h igher. A t this le ve l, one may indeed question the desirab ility of de-infesting foodstuffs w ith radiation . Another characteristic of Lepidoptera that in one respect makes it an organism of choice for the irradiation-of-male method is that adults of many species do not contain gonial ce lls , because gametogenesis is terminated during pupation.

The doses of radiation to infested grain can be made lower still if the grain were to be flooded with oxygen at the time of irrad iation . This would enhance the radiation effect on the storage pests, probably without increasing the amount of free radicals in the grain .

IR RA D IA T IO N O F PESTS IN THE FIELD

Where mass-rearing techniques are not feasib le, but where baiting can be easily ach ieved , then fac ilitie s for irradiation in the fie ld can be developed.Such facilities to induce dominant lethal mutations have been developed for a lky la ting agents (chem osterilants). It would seem that radioisotopes could be as e ffective as chemosterilants in these traps. Even with strong B-emitting

1 The m echanism s underlying the phenom enon o f hunger are beginning to be understood in som e insects [ c f . DETHIER, V. G ., GELPERIN, A . , J. exp. Biol. 47 (19G7) 191 -200].

334 VON BORSTEL

90 90isotopes such as strontium through its daughter yttrium , the traps would notneed to have much more radiation shielding than a conventional chemosterilanttrap . W ith y-em itting isotopes, a considerable amount of radiation shieldingwould be required .

One advantage of using radioisotopes in bait traps is that they can be contained upon a surface in such a way that the insect would not carry any of the radioisotope away with him . Another advantage of using radioisotopes is that they are easy to find with ion chambers, whereas chemosterilants are not easily detectable . It is also lik e ly that radiation-warning signs on the traps would be heeded because the public has become educated to the dangers of radiation over the last 20 years .

The disadvantages of such traps are the same as those for any trap or field-applied insectic ide . Mutants of insects that are not attracted to the traps w ill be rapidly selected for in the population, and other possibly beneficial insects may go into the traps.2 The best method is still to mass-rear, irrad iaté, and release

O V E R C O M IN G THE L IM IT A T IO N S O F THE IR R A D IA T IO N -O F-M A LE M ETHO D

When thé irradiation-of-male method is ine ffective , it is usually because the dose given is not high enough to prevent spermatogonial ce ll repopulation of the testes, or because weakening of males occurs at a dose below that required for inducing a high level of dominant le tha lity or gonial 's te r ility .

The first case can be rectified simply by raising the dose of radiation . The second case presents d ifficu lties which are by no m eansinsoluble, but which might demand different solutions for different species.

If dominant lethality is too low when male-weakening becomes c r it ic a l, then protracted (chronic) or fractionated doses of radiation can be g iven . In most insects, dominant lethal mutations in sperm are not repaired between fractionated doses of radiation or during protracted doses, whereas growing or d ividing types of cells are repaired . In this w ay , high frequencies of dominant lethal mutations can be accumulated in the sperm and the animal w ill be stronger than when single acute doses are given .

If the dominant lethal test is madé oñ the basis of hatchab'ility, the criterion can be changed to adult survival since some of the dominant lethal mutations act during the larval and pupal stages. Spermatocytes seem always to be more sensitive than sperm; and by irradiating appropriate pupal stages, a greater sperm dominant le tha lity to male-weakening ratio might be ach ieved .

If the fa ilure of the method lies with weakening of the males before gonial ste rility is ach ieved , then protracted or fractionated radiation probably w ill not be helpful . This is because gonial cells are capable of répair, largely through the effect described by Kimball [8 ] where cells slowed in their rate of division by radiation have more time to repair their chromosomal damage before the mutations are rendered permanent by cell d iv is ion .

Nonetheless, dominant le tha lity can be induced in spermatogonia [9 ] as w ell as oogonia [1 0 ] of certain insects.. Thus, if the ratio of dominant lethal

2 These disadvantages are. avoided .by the m ethod o f capture, irradiation, and release d eveloped by E. Horber (these Proceedings, S M -1 0 2 /3 2 ).

S M -1 0 2 / 3 4 335

mutations to total sperm from a ll matings over the lifetime of the male exceeds 0 .50 , the irradiation-of-male method w ill work despite spermatogonia I regeneration. It is w ell known that radiation given at appropriate times during development w ill enhance the aging processes. It may be possible that irradiation with low Joses at the right time of development could avoid male-weakening, would induce гчзпу dominant lethal mutations, and might enhance aging in a manner such that male-weakening could be established at approximately the same time that gonial ce ll repopulation of the testes takes p la ce . Finding an appropriate "dominant lethal w indow" during development would require a lot of research effort, but it would be worth a lot of effort to develop the irradiation- of-male method to control, for example, the boll w e e v i l .

G re ll [1 1 ] has made an interesting suggestion based upon the observation of Edington [12 ] that the radioprotective substance AET does not protect Drosophila sperm from radiation-induced dominant and recessive le th a lity . G re ll suggested that if the boll w eev il could be induced to feed upon it, AET might protect intestinal tissue from radiation damage without affording protection to the spermatogonia or the cells in different stages of spermatogenesis.

F in a lly , as Knipling and others [1 3 ] have pointed out, if everything else fa ils , partial-body irradiation procedures could be developed and used to avoid the male-weakening problem that occurs with certain insects. Since partia l-cell X-irrad iation procedures have been developed for insect eggs by U lrich and his colleagues [1 4 ,1 5 ], it should not be too large an engineering problem to automate the process for objects thousands of times larger.

Thus it would seem that w ith appropriate developments of myriads of techniques, any insect pest is suitable for selection to be driven v irtua lly to extinction in infested areas by irradiating and releasing the pest; This assertion holds true for a ll but the thelytokous species where females produce female offspring parthenogenetica lly . Possibly even some of the thelytokous species may be controllable by release of irradiated females if the phenomenon of "flushing" described by Monro [1 6 ] operates e ffe c tive ly to drive out the pest simply by the pressure of adult populations demanding " Lebensraum ."

C O N C L U S IO N

The three keys to the widespread ap p licab ility of the irradiation-of-male method of pest control are that it can work [1 7 ,1 8 ,1 9 ], that it can work on any insect that reproduces sexually no matter how many times it mates [20,21 ] , and that there is no lim itation of the method to. larval pests, for some pests that are pests as adults can be released after irradiation without their necessarily causing any damage [ 3 ] . This knowledge makes the irradiation-of-male method justifiable as a research that is a ll at once basic, app lied , and current. It is an ideal type of project in radiation biology for investigators in countries where research funds are lim ited, because it is easy, inexpensive, and app licab le to the often desperate needs of the country. Assistance from the United Nations is a necessity since an insect pays no attention to borders of countries and the w arlike individuals on both sides of them. The individuals in bordering nations must often unite in their efforts to control the insect. As a peaceful use of atomic energy, research on the irradiation-of-male method most nearly fits the appellation of being truly international in scope.

336 VON BORSTEL

R E F E H E N C E S

[ 1] von BORSTEL, R . C . , Radiation and radioisotopes applied to insects of agricultural importance, IA E A , V ienna (1963) 367-385.

[ 2 ] G R O S C H , D . S . , J . Econ . En t. 49 (1956) 629-631 .[ 3 ] G A L U N , R . , W A R BU R G , М . , Use of isotopes and radiation in entomology,

IA E A , V ienna (1968) in press.[ 4 ] LITTLE, H . F . , A . en t. S o c . Am . 60 (1967) 412-414.[ 5 ] R IE M A N N , J . G . , F L IN T , H . М . , A . en t. So c . Am . 60(1967) 298-308.[ 6 ] C L A R K , A . М . , R U B IN , M . A . , Radiation Res. J 5 (1961) 244-253.[ 7 ] L IN D O P , P . J . , personal communication.[ 8 ] K IM B A L L , R . F . , Repair from genetic radiation damage and differential

radiosensitivity in germ cells (SO B EL S , F . H . , E d .) , Pergamon Press,London (1963) 167-178.

[ 9 ] A LEX A N D ER , M . L . , Repair from genetic radiation damage and d ifferential radiosensitivity in germ cells (SO B E L S , F¿ H . , E d . ) , Pergamon Press,London ( 1963) 232.

[10] L a C H A N C E , L . E . , Rad ia t. Res. J J _ (1959) 218-228.[11] G R ELL , E . H . , personal communication.[1 2 ] E D IN G T O N , C . W . , A m . N a t . 92 (1958) 371-374.[1 3 ] von BO RSTEL, R . C . , Technical Reports Series N o . 44, Advances in insect

population control by the sterile-m ale technique, IA E A , V ienna (1965) 71-73.

[1 4 ] U LR IC H , H ., V e rhand l. Deutsch. Z o o l. G e s . in Hamburg 1956, 150-182.[1 5 ] W Ü R G LER , F . E . , U LR IC H , H . , SC H N E ID ER -M IN D ER , A . , Repair from

genetic radiation damage and differential radiosensitivity in germ cells (S O B E L S , F . H . , E d .) , Pergamon Press, London (1963) 101-106.

[1 6 ] M O N R O , J . , Technical Reports Series N o . 44, Advances in insect population control by the sterile-m ale technique, IA E A , V ienna (1965) 22-24.

[1 7 ] B U SH LA N D , R . C . , H O P K IN S , D . E . , J . econ . En t. 46 (1953) 648-656.[1 8 ] B A U M H O V ER , A . H . , G R A H A M , A . J . , BITTER, B . A . , H O P K IN S , D .

E . , N E W , W . D . , D U D LEY , F . H . , B U SH LA N D , R . С . , J . e c o n . Ent.48 (1955) 462-466.

[1 9 ] K N IP L IN G , E . F . , Sc ience 130(1959) 902-904.[2 0 ] von BO RSTEL, R . C . , .S c ie n c e ]31_ ( 1960) 878, 880-882.[2 1 ] von BO RSTEL, R . C . , BU Z Z A T I-T R A V ER SO , A . A . , Radioisotopes and

radiation in entomology, IA E A , V ienna (1962) 273-278.

D I S C U S S I O N

D. S. GROSCH: I thank you fo r y o u r re co g n i t io n o f m y w o rk . P e r haps I should explain why, in o u r w ork , l it t le attention w as g iven to the a p p ro a ch w h e re b y rad ia tion is used to in te r fe r e with the feed ing o f adult in s e c t s . At the t im e o f p u b lica tion o f the r e p o r t in the J ou rn a l o f E c o n o m ic E n to m o lo g y m u ch attention w as be ing f o c u s s e d on c o n t r o l o f s t o r e d gra in p e s t s . H o w e ve r , p u re food and drug law s in the United States con d em n any g ra in o r f lo u r in which p a rt s o f in s e c t s , su ch as le g s , w in gs , t e r g i t e s , s t e rn it e s and so on, a r e found, and t h e r e fo r e this a p p ro a ch w as c o n s id e r e d

SM-102/34 3 37

im p r a c t i c a l , but now that it is p r o p o s e d to ap p ly this technique to t i c k s , its future u se s e e m s p r o m is in g .

R. C. VON B O R S T E L : I 'm p le a s e d that you too a r e o p t im is t i c on thiss c o r e .

V. L A B E Y R IE : T he f e m a le s o f O ry za e p h i lu s s u r in a m e n s is (C o le o p te r a :C u cu jid ae ) o v ip o s i t o v e r a p e r io d o f s e v e r a l m onths; v i r g in f e m a le s do not o v ip o s i t ; the m a le s induce o v ip o s i t in g by mating . F e m a le s mating with n o n -v i r i l e m a le s lay e g gs which a r e s t e r i le , but if , a f t e r 30 days o f n o n -p r o d u c t iv e ov ip o s i t in g , these f e m a le s a re put tog e th er with v ir i le m a le s they a re found to lay fe r t i le e g g s . T h is sh ow s that a s e c o n d mating can c a u s e o v ip o s i t in g o f f e r t i le e g gs when the f i r s t d o s e o f s p e rm a to p h o r e is in capab le o f f e r t i l i z a t io n . It is thus quite fe a s ib le that f e m a le s f i r s t m ated with m a le s w h o se s p e r m has been d e s tr o y e d b y ir ra d ia t io n m a y yet be f e r t i l i z e d by n o r m a l m a le s .

R. C. VON B O R S T E L : Thank you f o r this in fo rm a t io n . D r. G o m e z -jNunez told m e o f his e v id e n ce with Rhodnius ind icat ing that inactivated s p e r m cannot c o m p e te with n o r m a l s p e r m , but it is c e r t a in ly known f r o m w ork on s e v e r a l o th er s p e c i e s that ' le th a l ' s p e r m do c o m p e te .

D. T . NORTH : I w ould l ik e to c o m m e n t on s e v e r a l o f the points youm a d e . F ir s t , when c o n s id e r in g the p o s s ib le use o f a s p e r m ie m a le s to r e d u c e the r e p r o d u c t iv e potent ia l o f a population o f a p o ly g a m o u s s p e c i e s , it m ust be r e m e m b e r e d that these can on ly r e a so n a b ly be e f fe c t iv e w here the density o f population is such that the a v e r a g e n um ber o f m atin gs p e r fe m a le is low , i . e . 1.0 o r b e lo w . In o th e r w o r d s , the e f f e c t iv e n e s s o f a r e le a s e d s t e r i le m a le is dependent on the p r o b a b i l i ty o f his be ing ab le to r e d u c e the r e p ro d u c t iv e potent ia l and this v a r ie s with e a ch type o f population . .

S econ d ly , I shou ld l ike to m en tion that data do e x is t on the p r o b le m o f ra d ia t io n - in d u ce d d e s tru c t io n o f the gut in the b o l l - w e e v i l . When dealing with an in se c t s p e c i e s in which the adult c a u s e s d e s tru c t io n , a s h o r t - l i v e d and e f f e c t iv e s t e r i l e m a le m a y w e l l be an ad vantageous p r o p o s it io n . I sh ou ld a l s o like to point out that y o u r su g g e s t io n on the use o f r a d io p r o te c t iv e agents l ike A E T has been put to the tes t in o u r l a b o r a to r y by Dr. H. M. M int, with no s u c c e s s . He found, fu r t h e r m o r e , that by v ir tu e o f its m od e o f a c t ion a p r o te c t iv e agent m a y w e l l p r o te c t the s p e r m fr o m induction o f dom inant le th a ls , th e re b y lea v in g the p r o b le m u n so lv e d . With r e g a r d to yo u r p r o p o s a l to sh ie ld the gut w hilst ir ra d ia t in g the te s te s I r e f e r you to a pa per by R iem an n and Flint (R iem an n , J. G. and Flint, П. M. , Ir ra d ia t ion e f f e c t s on m idguts and t e s te s o f the adult b o l l w e e v i l , A nthonom us g r a n d i s , d e te rm in e d by h is t o lo g i c a l and sh ie ld in g s tu d ies , Ann. ent. S o c . A m . C>0 (19C7) 2 9 8 -3 08 ) p u b lish ed in 1967, in w hich they d e m o n s tra te d this e f fe c t and sh ow ed that the gut w as in too c l o s e a p r o x im it y to the te s te s f o r the m ethod to be p r a c t i c a b le .

RADIATION E F F E C T STUDIES: GENETIC

(Session VIII, Part 1)

C h a i r m a n : 0 . S T R 0 M N A E S

N O R W A Y

RADIATION SENSITIVITY AND REPAIR OF CHROM OSOMAL DAM AGE

0 . STR0MNAESIN STITU TE OF GENERAL GENETICS,

UNIVERSITY OF OSLO,OSLO, NORWAY

Abstract

RADIATION SENSITIVITY AND REPAIR OF CHROMOSOMAL DAMAGE. If the ste r ile -m a le technique in pest control is used, it is important for irradiated sperm to have a dom inant lethal e ffe c t . In Drosophila, sperm and sperm atogonia are radiation resistant ce lls com pared with spermatids and sperm atocytes. After a m oderate radiation dose, the m ale m ight be fertile for several days and then g o through a sterile period. Fertility is regained when ce lls that were sperm atogonia during the tim e o f irradiation are ava ilab le for fe rt ilization . It is therefore important for the irradiation dose to be lethal to a ll sperm atogonia so that no repopulation o f sperm atogonial ce lls can occu r. It should be possible to find a radiation dose w hich renders insects sterile without im pairing their m ating behaviour and v ita lity . The radiation dose can also be adjusted by changing internal or external environm ents during irradiation.

1. IN TROD UCTION

Our knowledge o f m utagen ic ity has g re a t ly in c r e a s e d in the 40 y e a r s that have e la p se d s in c e M u ller [l] d is c o v e r e d that io n iz in g rad iation in d u ce s mutation. A little is known about how c h e m ic a l m utagens act at the m o le c u la r le v e l , and we have so m e idea how u l t r a -v io le t radiation p r o d u c e s m u ta t io n s . With r e g a rd to ion iz in g radiation , ou r know ledge o f the m o le c u la r ch a n g es that lead to in t e r - and in tragen ic ch a n ges is s t i l l v e r y l im ited . A m o n g the many u n so lved p r o b le m s is , f o r e x a m p le , the m o le c u la r s t ru c tu re o f c h r o m o s o m e s in e u c a r y o te s , w hether they c o n s is t o f s ing le o r m u lt i -s t ra n d e d DNA, and how the n u c le o p r o te in s a r e a r ra n g e d .

2. STRAIN D IF F E R E N C E S IN RADIATIO N SENSITIVITY

Radiation se n s i t iv i ty v a r i e s g r e a t ly betw een o r g a n is m s , s tra in s , c e l l types , and c e l l s in d i f fe re n t s tag es o f d e v e lo p m e n t . Ogaki and Tanaka [2] , who stud ied the s u r v iv a l ra te o f v a r io u s D ro so p h i la s tra in s , found the L D 50 f o r f e m a le s in two wild type s t ra in s to be aoout 150 000 R, while fo r the f e m a le s in two mutant stra in s it w as 120 000 R. The LD50 fo r m a le s was som ew h at lo w e r , 130 000 and 100 000 R, r e s p e c t iv e ly . Radiation r e s is ta n c e w as shown to be dom inant, and m o s t ly c o n t ro l l e d by the third c h r o m o s o m e .

The m u tab il ity o f 13 re la ted s tra in s and 17 unrelated stra in s o f D ro s o p h i la m e la n o g a s te r e x p o se d to X - r a y treatm en t w as in vestigated by Str^mnaes [з]~! He found s ign if ican t d i f f e r e n c e s in m utabil ity between s t ra in s , and it w as su g ges ted that s o m e s tra in s have g en otypes e x t r a se n s i t iv e to ir ra d ia t io n , and that th ere a r e both fa ir ly h o m o g e n e o u s and h e te r o g e n e o u s s t ra in s in th is r e s p e c t . T h e se data a re s t ro n g ly supported by data published by o th er in v e s t ig a to rs [ 4 - 6 ] , F u r th e r te s ts w e re p e r f o r m e d by Str^mnaes [7], who m ade 11 rep e a ts o v e r a p e r io d o f 4 y e a r s at two d i f fe re n t in st itu tes on two d i f fe re n t s t ra in s , I s o -A m h e r s t and O s lo .

341

342 STR(OMNAES

FIG. 1. Sensitivity patterns for two w ild type strains o f Drosophila m elanogaster. T h e upper two curves r e present dom inant lethals (Str»fmnaes[8]), and the lower two curves represent recessive sex -lin ked lethals (Ward and Bird [13]).

Mate age in days

FIG. 2. H atchability o f eggs fertilized by sperm from m ales irradiated at d ifferent ages. T h e lines in d ica tethe general trend given in the data b y -------------- Dempster [ 1 4 3 , ------------Shtfmnaes [151, a n d ---------- Bonnierand Lilning [16].

The h igh er mutation f r e q u e n cy w as o b s e r v e d in the O s lo stra in , and it w a s la te r shown [8] that the two s to ck s a lso have d if fe ren t b ro o d pa ttern s .

That a d i f f e r e n c e in rad ia t ion se n s it iv ity a l s o e x is ts betw een s tra in s o f E s c h e r i c h ia c o l i has been d e m o n stra te d by Hill [9]. She show ed that Strain B s is m uch m o r e se n s i t iv e to X - r a y s than Strain B. Strain B$ was d e r iv e d f r o m Strain В by u . v . treatm en t, and it i s th e r e fo r e m o s t l ik e ly that the d i f f e r e n c e in se n s it iv ity to X - r a y s is cau sed by a u .v . - in d u c e d m utation , in y east , h o m o z y g o s i ty f o r the m a tin g -typ e gene lea d s to h igh er se n s it iv ity to ion iz in g rad ia tion than h e te r o z y g o s i ty [Ю], and th ere a r e is o la te d r a d ia t io n -r e s is ta n t [11] and r a d ia t io n -s e n s i t iv e mutants [12].

S M - 102/35 343

_________ DAYSS p e r m

S p e r m a t i d s

S p e r m a t o c y t e sS p e r m a t o g o n i a

FIG. 3. Sensitivity patterns for different types o f radiation e ffects in sperm ava ila b le for fertilization on various days after irradiation o f D rosophila m elanogaster m ales. D om inant lethals (Liming [21 ]), rfkO fem ales (Savhagen [3 5 ]), non-disjunction fem ales (Savhagen [2 9 ]), recessive lethals (Ives [2 8 ]), translocations (Savhagen [3 5 ]). T h e ordinate is the re la tive frequency o f each radiation e ffe c t norm alized to approxim ately the sam e value at a ll peaks (from Savhagen [104]).

W ard and B ird [13] i r ra d ia te d m a le s o f the sa m e two s t o ck s as Str^mnaes [8] had used , and s c o r e d the rate o f s e x - l in k e d r e c e s s iv e l e thals on days 1, 3, 5, 6, 7, 8, 9 and 11 a f t e r ir ra d ia t io n . T h e ir data strong ly su pported those found by Str^mnaes (F ig . 1). W ard and B ird a lso in v e s t igated c y t o c h r o m e С o x id a s e ac t iv ity and found it to g ive two d i f ferent patterns in the two s t ra in s . T h e ir c o n c lu s io n is that the d i f f e r e n c e in rad ia tion se n s it iv ity betw een the two s tra in s cannot be exp la ined on the b a s is o f a p r o te c t iv e e f fe c t o f the c y t o c h r o m e s y s te m a lo n e . T o explain the en t ire se n s it iv ity pattern o f the two s tra in s , the o p e ra t io n o f two o th er fa c t o r s b e s id e s the e n z y m e m entioned is n e c e s s a r y .

Thus, d i f fe re n t s t ra in s can have d i f fe ren t se n s i t iv i ty to i r ra d ia t ion , which m ay a l s o be r e f le c t e d in th e ir b r o o d pattern . In vestigat ion s by D e m p s te r [14], Str^mnaes [15], and B on n ier and Lüning [16] d em o n stra te that the mutation f r e q u e n cy is dependent on the age o f the m a le at the tim e o f i r ra d ia t io n (F ig . 2 ) . T he sen s it iv ity in c r e a s e s with in c r e a s in g age o f the m a le . A s im i la r e f fe c t w as found when e g g s w e re aged [17]..

B on n ier and Lüning [16] a lso o b s e r v e d a d e c r e a s e in hatching rate with in c r e a s e in t im e f r o m ir ra d ia t io n to fe r t i l i za t io n .

3. RA D IA TIO N SENSITIVITY DURING SP ERM AT O G E N ESIS

H a r r is [18] e m p lo y e d mating p e r io d s o f 4 days and found a d r o p in se x - l in k e d letha ls in the b r o o d c o m in g 12 to 16 days a fte r ir ra d ia t io n .

344 STR)OMNÆS

A u e rb a ch [19] started using constant mating p e r io d s o f 3 days and found the h ighest rate o f s e x - l in k e d r e c e s s iv e letha ls in b ro o d C, which i s in good a g re e m e n t with Liining 's [20] o b s e rv a t io n .

3 . 1 . Dom inant lethal mutations

With the in trodu ct ion o f 2 4 -h o u r mating p e r io d s a much m o r e deta iled p ic tu re w as obtained o f the r a d io s e n s it iv i ty o f the d if fe ren t c e l l s tag es o f s p e r m a to g e n e s is . T he lack o f hatchability o f e g gs fe r t i l i z e d by ir ra d ia te d s p e r m has usually been r e f e r r e d to as dom inant le tha ls . F r o m such stud ies it w as ev ident that f r o m the 5th day a fter ir ra d ia t io n th e re is a m a rk e d in c r e a s e in the fr e q u e n c y o f dom inant letha ls , with a m a x im u m betw een the 6th and 10th day fo l lo w e d by a d e c r e a s e on the 11th day [18,21, 22, 23, 24, 25] (F ig .3 ) .

3 . 2 . R e c e s s iv e lethal mutations

F o r o th er types o f induced genet ic d am age such as s e x - l in k e d letha ls [2 6 -2 8 ] , an in c r e a s e in f r e q u e n c y is o b s e r v e d f r o m the 4th day with the peak r e a c h e d 6 to 7 days a fte r ir ra d ia t ion , f o l lo w e d by a sh arp d e c r e a s e on the 8th day. A s im i la r sen s it iv ity pattern is r e p o r te d f o r a u to so m a l r e c e s s i v e le tha ls [26].

3 . 3 . N on -d is ju n c t io n

Still another m ethod o f studying induced g en et ic d am age is to study l o s s e s o f paterna l s e x c h r o m o s o m e s and n o n -d is ju n ct io n as a s im u ltaneou s l o s s o f paterna l X and Y c h r o m o s o m e s . Savhagen [29] p e r fo r m e d such a study and found n o n -d is ju n ct io n to be h ighest in s p e r m ava i lab le f o r fe r t i l i z a t io n 7 to 10 days a fte r i r ra d ia t io n . She conc lu ded that these c e l l s had been in e a r ly m e io s i s , p r io r to anaphase I, at the tim e o f i r ra d ia t io n .

3 . 4 . H ecom bination

A n oth er w ay o f d e term in in g m e io s i s is by studying the p ro d u c ts o f c r o s s in g o v e r in s u c c e s s iv e b r o o d s . If c r o s s in g o v e r o c c u r s in f i r s t m e io t i c d iv is io n the r e co m b in a n ts should o c c u r singly , but o c c u r in bundles i f they a r e the re su lt o f m itot ic c r o s s in g o v e r . Savhagen [30] found on ly s ing le re co m b in a n ts in sp e rm ready fo r fe r t i l i z a t io n on the 7th and 8th day a fte r i r ra d ia t ion , m o s t ly s ing ly , but o c c a s io n a l ly a lso bundles o f iden t ica l re co m b in a n ts on the 9th to the 11th day, and on la ter days c lu s t e r s o f c r o s s o v e r s w e re frequently o b s e r v e d . T h is m eans that s p e r m ava i lab le f o r fe r t i l i z a t io n f r o m the 12th day on must have been ir ra d ia te d as sp e rm a to g o n ia . The c o n c lu s io n i s in g e n e r a l a g re e m e n t with o b s e r v a t io n s m ade by o th er in v e s t ig a to rs [26, 28, 31, 32, 33, 34].

3 . 5 . T r a n s lo c a t io n s

F o r a u to so m a l t r a n s lo ca t io n s , v e r y m uch the sa m e sen s it iv ity pattern has been found as f o r s e x - l in k e d r e c e s s i v e le tha is . Thus, the peak is r e a c h e d on the 6th day fo l lo w e d by a sh arp d r o p on the 8th day a fte r i r r a d ia tion [27, 35, 36].

S M - 102/35 345

F r o m se n s i t iv ity patterns o f the types m entioned , the c o n c lu s io n can be draw n that s p e rm ava i lab le f o r f e r t i l i z a t io n the f i r s t th ree days a fter i r ra d ia t io n o f m atu re m a le s must have been m ature s p e r m at the t im e o f ir ra d ia t io n . S p erm ava i lab le in the next two days m ust have been i r r a diated as s p e rm a t id s , and the s p e r m ava i lab le f r o m the 6th to the 10th day m ust have been s p e r m a to c y t e s ; s p e r m c o m in g on la ter dates m ust have been ir ra d ia te d as s p e rm a to g o n ia .

T he b ro o d pattern m ay v a r y som ew h at depending on the stra in used, the age o f the m a le s , the mating p r o ce d u r e , and p erhaps the ir ra d ia t io n d o s e [37 ] . B e s id e s , th ere a re v a r ia t io n s betw een individual m a le s ; and the p o s s ib i l i t y e x is t s o f m ix ing o f s p e r m f r o m d if fe ren t stages o f d e v e lo p m ent during ir ra d ia t io n . Thus, s p e r m sa m p led 8 -1 0 days a fte r i r r a d i a t ion m ay have been ir ra d ia te d e i th e r as s p e r m a to c y t e s o r s p e rm a to g o n ia .

3 . 6 . C onclusion

4. SENSITIVITY O F S P E R M A T O C Y T E S

A s p e c ia l study o f induction o f mutations in s p e r m a to c y t e s w as p e r f o r m e d by Chandley [38 ] . She found a high fre q u e n cy o f r e c e s s i v e s e x - l inked lethals on days 5, 6 and 7 a fte r i r ra d ia t io n and then a low f r e q u e n cy on day 8. F o r tr a n s lo ca t io n s the high rate was o b s e r v e d 5 and 6 days a fte r i r ra d ia t io n with a d e c r e a s e on day 7 and a low l e v e l on day 8. The h ighest f r e q u e n c ie s o f dom inant lethals w e re o b s e r v e d on days 6, 7 and 8 a f te r i r ra d ia t ion . The induced f r e q u e n cy o f c r o s s in g o v e r and h yperp io id f e m a le s w e re low on day 5 and in c r e a s e d on la ter dates , rea ch in g peak value on day 8. H er data and in te rp re ta t io n s a r e in g e n e r a l a g re e m e n t with those published by o th er authors, and a lso h er c o n c lu s io n that:"o n e should n e v e r talk l o o s e ly o f a 's e n s i t iv e ' o r ' in s e n s i t iv e ' stage without indicating what type o f mutation is under co n s id e ra t io n . "

5. SPERM ATOGONIAL, SENSITIVITY

A low f r e q u e n cy o f mutation is o b s e r v e d in sp e rm ir ra d ia te d as sp e rm a to g o n ia . Mutations invo lv ing g r o s s c h r o m o s o m a l a b e rr a t io n s w i l l m os t l ik e ly be e l im in ated during m e io s i s . O akberg [39, 40] found the c h ie f radiation e f fe c t to be kill ing o f m ost o f type A sp e rm a to g o n ia and a l l in te rm e d ia te s and type В s p e rm a to g o n ia . O ftedai [41] su ggested that f o r D ro s o p h i la two sp e rm a to g o n ia l c o m pon en ts e x is t that have d if ferent m utabil ity and v iab i l i ty sen s it iv ity in r e g a rd to X - r a y s . The sa l iv a ry g lands o f s u c c e s s i v e b ro o d s o f i r ra d ia te d m a le s w e re studied by S lizynska [42 ] . She lo o k e d f o r ch a n g es such as t r a n s lo ca t io n s , in v e r s io n s , d e f i c i e n c i e s , rep e a ts , dup licat ion , and lo s s o f the 4th c h r o m o s o m e . A l l m a le s s e e m to exhibit the sa m e sen s it iv ity when the s p e rm and sp e rm a t id stages a r e ir ra d ia te d . H ow ever , the sp e rm a to g o n ia l stage d i s c l o s e s a h e t e r o g en eou s population o f m a le s ; s o m e have sp e rm a to g o n ia that a re e x t r a - s e n s i t iv e to X - r a y s , w hile o th e rs have sp e rm a to g o n ia that a re in se n s it iv e . S lizynska exp la ins this v a r ia t ion in sen s it iv ity in sp e rm a to g o n ia by the p r e s e n c e o r a b se n ce o f sp e rm a to g o n ia l m i to s i s .

M cC a rth y and Nafei [43] found no m a le s with ext^ a -sen s it ive s p e r m a togon ia . That s p e rm a to g o n ia in m i c e constitute a h e te ro g e n e o u s popula -

346 STR0MNAES

t ion has been v e r i f i e d by N ebe l , Coulon and M cW hinnie [44]. They found that sp e rm a to g o n ia o f type В a re m o r e se n s it iv e than type A, but within the la tter the stages G i and S a r e e x tr a -se n s i t iv e . T hus , it m a y be m o r e c o r r e c t to ta lk about a population o f c e l l s with sen s it iv e s tag es in a n o rm a l c e l l c y c l e , than to ta lk about a h ete ro ge n e o u s population o f c e l l s .

6. S P E R M SENSITIVITY

6 . 1 . Within m a les

Sp erm ava i lab le f o r fe r t i l i za t io n on the f i r s t two days a fte r i r ra d ia t io n has been studied in te n s ive ly . It has been known fo r s o m e tim e [45] that w hen m a le s 3 - 4 days o ld a r e ir ra d ia te d , the secon d -day s p e rm has a l o w e r mutation f r e q u e n cy than the f i r s t day s p e r m . T h is led to a d is c u s s io n o f w hether d i f fe ren t ia l sen s it iv ity e x is ted , o r i f r e c o v e r y f r o m d am age o f the g en et ic m a te r ia l o c c u r r e d f r o m one day to the next [4 6 -5 0 ] . T he c o n c lu s io n r e a c h e d by N o rd b a ck and A u erb a ch [51] w as that r e c o v e r y o c c u r s f r o m in te rge n ic as w e l l as in tragen ic d am age to the genet ic m a te r ia l .T rau t [52] c o m e s to another co n c lu s io n , n am ely that d i f fe re n t ia l s e n s i t i v ity e x is t s ra th er than r e c o v e r y o f g en et ic dam age f r o m one d a y 's s p e r m to the next. T h is v iew is a l s o held by o th e rs [53, 54], who b e l ie v e that m ix in g o f s p e r m o f d i f fe re n t sen s it iv ity can o c c u r in te s te s a f t e r i r r a d ia tion . M ü lle r [55] su p p orts th is v iew and says that the m ix in g g ro u p s o f s p e r m a r e c h a r a c t e r i z e d by d i f fe ren t d e g r e e s o f oxygenation , an in t e r preta tion that i s c re d i te d to O s te r . M u lle r a lso r e v ie w s pertinent p a p e rs by o th e r s .

In d is c u s s io n s on induced mutation fr e q u e n c y in f i r s t - v e r s u s s e c o n d - day sp e rm , the age o f the m a le at the t im e o f ir ra d ia t io n i s o f the u tm ost im p o r t a n c e . Khishin [56, 57] d e m o n stra te d that the f i r s t m o t i le s p e r m in m a le s o f the O r e g o n - K s to ck w as o b s e r v e d 7 ± 1 h ours a f te r e c l o s io n . Str^mnaes and Kvelland [58] studied in m o r e d e ta il the sex u a l ac t iv ity o f D ro s o p h i la m a le s , and found that o ld e r m a le s a re cap a b le o f m ating m o r e t im e s than you n ger m a le s in a tw e lv e -h o u r p e r io d . The o ld e r m a le s a re a b le to p ro d u ce m o r e o f f s p r in g than the yo u n ge r o n es as w e l l as a la r g e r to ta l per fe r t i le m ating. F u r t h e r m o r e , a 1 2 -h o u r c e l ib a c y p e r io d is su f f ic ie n t to b r in g the am ount o f s p e rm ava i lab le f o r fe r t i l i z a t io n up to the am ount ava i lab le in m a le s o f the sa m e age but p r e v io u s ly not m ated. ( F o r m o r e d eta ils see a lso R e fs [59 -63 ] ). That se co n d -d a y s p e r m m ay have a d i f fe ren t se n s i t iv ity to i r ra d ia t io n than f i r s t -d a y s p e r m s e e m s to be brought out by the data published. H o w ever , when it c o m e s to m a le s w ithheld f r o m mating f o r one day, m ix in g o f f i r s t - and s e c o n d -d a y s p e r m shou ld be m o r e pronou n ced in young than in o ld m a le s .

If s ing le e ja cu la te s a r e studied, m uch m o r e deta iled in fo rm a t io n about m utation se n s it iv ity in m atu re and n e a r ly m atu re s p e r m should be obta ined . M o s s ig e [64] p e r fo r m e d an e x p e r im e n t w h e re mating w as o b s e r v e d and the fr e q u e n c y o f mutation in s u c c e s s iv e e ja cu la te s studied . She found that

i the f r e q u e n c y o f s e x - l in k e d le tha ls d e c r e a s e d in c o n s e cu t iv e m atin gs f o r \ a p e r io d up to about 24 h ours when 0 - 4 - h o u r - o W m a le s w e re ir ra d ia te d .

F o r 2 - d a y -o ld m a le s th ere w as a d e c r e a s e in fr e q u e n c y with time^p~~to .9 h ou rs . In 7 -d a y -o ld m a le s th ere w as a d e c r e a s e f r o m f i r s t to s econd

I e ja cu la te and rïo~fürtïïër d e c r e a s e . M o s s ig e ir ra d ia te d the m a le s irTair,

S M -102/35 347

ox yg en and n itrogen , and f r o m th ese data she co n c lu d e d that the d e c r e a s e o b s e r v e d is due tó a d i f f e r e n c e in ox yg en sen s it iv ity and to r e c o v e r y with t im e f o r m a tu re s p e r m f r o m m a le s up to 2 days o ld . Str^mnaes and K velland [65, 66] studied the f r e q u e n c y o f induced M inutes, s e x - l in k e d le tha ls , and t r a n s lo ca t io n s in s u c c e s s iv e e ja cu la te s during two tw e lv e - hour p e r io d s sep a ra te d by a t w e lv e -h o u r m a le c e l ib a c y p e r io d . The m a le s at the t im e o f ir ra d ia t io n w e r e 0 -2 , 12, 24, and 72 h ou rs o ld . F o r s e x - l in k e d le tha ls and tr a n s lo ca t io n s th ere w e r e s ign if ican t d i f f e r e n c e s in mutation fr e q u e n c y betw een m a le age g ro u p s in the f i r s t 1 2 -h ou r m ating p er iod a f t e r i r ra d ia t io n , w hile the v a r ia t io n betw een age g ro u p s in the se co n d mating p e r io d w as in s ign if ica n t . F u r t h e r m o r e , by test in g s u c c e s s iv e s p e rm e ja cu la te s , it w as p o s s ib le to d e te rm in e the t im e when s p e r m o f l e s s se n s it iv ity b e c a m e a v a i la b le . T h is w as found to be a v a i la b le e a r l i e r in v e r y young m a le s than in o ld e r m a le s . S im ila r r e s u l t s w e re obtained by LUning [67] who found that a change in mutation f r e q u e n cy o c c u r r e d a fte r 4 h ou rs f o r m a le s i r ra d ia te d when 0 -2 4 h ours old , while no change in mutation f r e q u e n c y w as found the f i r s t 12 h ou rs a f t e r i r r a diation o f 3 - 4 - d a y - o l d m a le s (s e e a l s o R e f . [64] ).

D i f fe re n t ia l m u tab il ity and r e c o v e r y f r o m ir ra d ia t io n d am age in the d i f fe re n t m ale g e r m c e l l s tag es and indiv idual e ja cu la te s have a lso been d i s c u s s e d by L e fe v r e and J o n ss o n [68] and L e fe v r e [69 ] .

W hether d i f fe r e n t ia l se n s i t iv i ty a n d /o r r e c o v e r y with t im e op era te in m ature o r a lm o s t m atu re s p e r m is d i f f i cu lt to d e te rm in e , s in ce e ja c u late seq u en ce is not independent o f t im e . H ow ever , we have now a s u b stantial am ount o f data which a re be ing te s te d by pa rt ia l c o r r e la t i o n analy s is , and we hope the r e su lt w i l l shed m o r e light on this question .

If r e c o v e r y is go ing on in m a le g e r m c e l l s and is c o n t r o l l e d by a p rote in , lo w e r in g the m e t a b o l i s m should a f fe c t the induced mutation rate . T h is p r o b le m w as studied by W edv ik and S tr^ m n æ s [70] and Sollunn and Str^mnaes [71 ] . T h ey found c e r t a in ch a n g e s in the b r o o d pattern fo r 3 - d a y - o ld m a le s . Thus, the f r e q u e n c y o f dom inant letha ls is h igh er in the seven f i r s t b r o o d s f o r the m a le s i r ra d ia te d at 7° С than f o r th ose i r r a diated at 22° C, and no s ta t is t ica l ly s ign if ican t d i f f e r e n c e s w e r e o b s e r v e d f o r la ter b r o o d s .

When ir ra d ia t io n is p e r fo r m e d at 0° С and 22° C, b r o o d c o m p a r is o n s f o r the th ree f i r s t b r o o d s a lw ays r e v e a l a h igh er f r e q u e n c y o f dom inant le tha ls and r e c e s s i v e s e x - l in k e d le tha ls at 0° С than at 22° С . Varia tion betw een f i r s t and se co n d b ro o d i s s ign if ican t f o r s e x - l in k e d letha ls at both te m p e r a tu r e s and on ly at 22° С f o r dom inant le tha ls . T h e s e r e su lt s a re co m p a t ib le with a r e p a i r en zym e s y s te m that is o p e ra t iv e in s p e r m and s p e rm a t id s at r o o m te m p e ra tu re but is inactivated at 0 °C . App arently , the en zym e a c ts on the break a ge rest i tu t ion p r o c e s s (dominant letha ls) , and d o e s not a f fe c t n o t ice a b ly r e c e s s i v e s e x - l in k e d le tha ls .

6 . 2 . Within f e m a le s

B on n ier and Liining [72] w e r e the f i r s t to o b s e r v e that s p e r m i rra d ia ted in f e m a le s i s m o r e se n s i t iv e to i r ra d ia t io n than s p e r m trea ted in the m a le . Data supporting this finding w e re published by A b ra h a m so n and T e l f e r [ 7 3 ] , T rau t [74] found that ir ra d ia t io n o f in sem in ated f e m a le s gave no h igher mutation fr e q u e n c y in the s p e r m than w as found a fter i r ra d ia t in g sp e rm in the m a le , and L e fe v r e [75] c a m e to the c o n c lu s io n that s p e r m ir ra d ia te d

348 STR0MNÆS

in the m ale exh ib its a d e te c ta b iy h igher mutation fre q u e n cy than that i r r a diated in the fe m a le . It was b e l ie v e d [76] that when s p e rm is ir ra d ia te d in the m a le , p r e -e x i s t in g d e tr im e n ta ls m ay act s y n e r g is t i c a l ly with in d uced o n es to g ive c o m p le t e ly lethal c h r o m o s o m e s , while this e f fe c t is absen t with s p e r m ir ra d ia te d in the fe m a le . H ow ever , m o r e r e ce n t e x p e r im e n ts by L e fe v r e [77] show that th ere is a m o r e h ighly s ign if ican t re d u ct ion in mutation f r e q u e n cy in the fe m a le g en om e when both pa terna l and m a tern a l g e n o m e s a re ir ra d ia te d than i f only the fe m a le g e n o m e is i r ra d ia te d . T h is is va lid f o r mutations d i s c o v e r e d in e g gs la id the f i r s t f o u r d ays a fte r i r ra d ia t ion , but not f o r e g g s laid la ter , w hich in d ica te s that in re la t iv e ly m atu re g e r m c e l l s there is p r e fe re n t ia l e l im in at ion o f c h r o m o s o m a l a b e rr a t io n s by an in te ra c t io n o f the two ir ra d ia te d g e n o m e s .

L e fe v r e [78] re in v e st ig a te d the e f fe c t o f ir ra d ia t io n o f fu lly m ature s p e r m in the m a le and in the fe m a le . He found a red u ced f r e q u e n c y o f h yp erp io id m a le s and s e x - l in k e d r e c e s s iv e letha ls when in sem in ated f e m a le s rath er than m a le s w e r e ir ra d ia te d . L e fe v r e fa v o u rs the id ea that the d i f fe r e n c e in o b s e r v e d mutation f r e q u e n cy is ca u se d by e l im in at ion o f s o m e zy g o te s when both g e n o m e s are ir ra d ia te d , and s e e s no r e a s o n why the s p e r m should have a d i f fe ren t sen s it iv ity in the two e n v iro n m e n ts o r should be p r e fe re n t ia l ly r e p a ir e d when ir ra d ia te d in the fe m a le .

7. A L T E R A T IO N O F THE EN VIRO N M EN T

Ila d iosen s it iv ity can be in flu enced by a l te ra t ion s in e x tern a l and in te r n a l en v iron m en t b e fo r e , during o r a fter i r ra d ia t io n . A l te ra t io n s o f th is type a lso in flu ence the m e c h a n ism o f r e p a ir f r o m g en et ic dam age . S e v e r a l r e v ie w p a p ers and book s on the su b ject have been published lately [7 9 -8 2 ] . R e p a ir and rad io sen s it iv ity phenom ena in D ro s o p h i la w e re studied in c o n s id e r a b le deta il by Sobe ls and c o w o r k e r s (see r e v ie w s by Sob e ls [83-85] ). One o f the in te re s t in g findings i s that when D ro s o p h i la m a le s a re e x p o se d to a high d o s e - r a t e o f X - r a y s under anoxia , a reduction in the f r e q u e n cy o f r e c e s s i v e lethals and tr a n s lo ca t io n s is found in s p e r m a to z o a when the p o s t - t r e a tm e n t is N.,, but when the p o s t - tre a tm e n t is 0.> the re d u ce d fr e q u e n c y is o b s e r v e d in e a r ly s p e rm a t id s . The sa m e re su lt is obta ined when sp e rm a to z o a a re ir ra d ia ted in f e m a le s o r sp e rm a t id s in 2 4 - h o u r - o ld pupae. It i s su ggested that the p o s t -ra d ia t io n e f fe c t s in both ty p es o f c e l l s a r is e f r o m en zym at ic r e p a i r o f potential l e s io n s . Sobels thinks that in s p e r m both g ly c o ly t i c e n z y m e s and RNA o r p rote in syn th es is a r e in vo lved in the r e p a i r p r o c e s s . F o r r e p a ir to o c c u r in e a r ly s p e r m a t ids , oxygen is needed , and a re d u ct ion in mutation f r e q u e n cy is o b s e r v e d when inhibit ion o f RNA a n d /o r prote in sy n th es is o c c u r s .

D ick e r m a n [86] studied the induction o f dom inant letha is by ir ra d ia t in g D r o s o p h i la v i r i l i s o o c y t e s in d if fe ren t g a s e s . The g a se s used w e re a ir , a rg o n , he lium , methane, ca r b o n m on ox ide , and oxygen . He found that the f r e q u e n c y o f dom inant lethals induced in Stage 7 and Stage 14 o o c y t e s in c r e a s e d with in c r e a s e d oxygen ten sion during ir ra d ia t io n . T he data in d ica ted a p r o te c t iv e e f fe c t o f argon , methane, and he lium . Since methane i s a lip id so lub le gas , it is su ggested that the oxygen site is a lip id f ra c t io n o f the c e l l . D o se c f fe c t c u r v e s show ed that Stage 7 o o c y t e s a r e m uch m o r e re s is ta n t than Stage 14 o o c y t e s . The explanation g iven f o r this is that Stage 7 o o c y t e s a re m e ta b o l i c a i ly ac t ive and ab le to r e p a ir c h r o m o s o m a l

S M - 102/35 3 4 9

dam age in a sh ort t im e , while Stage 14 o o c y t e s a re l e s s a c t iv e , but when f e r t i l i z e d , go ra p id ly through m e i o s i s w h e re b y c h r o m o s o m e m o v e m e n ts m ake rest i tu t ion l e s s l ik e ly .

Induced dom inant lethality in o o c y t e s has a l s o been studied by R inehart [87 ] . H is c o n c lu s io n is that in Stage 7 o o c y t e s th ere is an o x y g en -d ep en d en t r e p a i r sy s te m . The r e p a i r s y s te m is d eg ra d ed in the a b s e n c e o f oxygen , but r e g e n e r a te s again when oxygen is a d m in is te re d . R inehart thinks this r e p a i r s y s te m is in o p e ra t ive in Stage 14 o o c y t e s until a fte r fe r t i l i z a t io n .T h is , tog e th er with c h r o m o s o m e m o v e m e n ts , is part o f the explanation why sen s it iv ity is g re a t ly in c r e a s e d in Stage 14 o o c y t e s as c o m p a r e d with Stage 7 o o c y t e s .

8 . TH E BASIC LESION AND ITS R E P A IR

In form ation about rad iation sen s it iv ity and r e p a ir can a l s o be obtained by em p lo y in g d o s e frac t ion at ion . One might e x p e ct that the mutation f r e qu en cy would be the sa m e f o r the sa m e tota l d o se no m atter how the d o se is d e l iv e r e d . H o w ever , Sax [88, 89] show ed that the y ie ld o f c h r o m o s o m e a b e rr a t io n s f o r low I ,E T g iven in two equal f r a c t io n s depen ds on the t im e in te r v a l betw een the fr a c t io n s . The usual explanation is that rad ia t io n - induced b r e a k s have a c e r ta in l i fe t im e within w hich they can in terac t and p ro d u ce e x ch a n g e s . If th ere is not a se co n d b r e a k within r e jo in in g d i s tance the bre a k m a y rest i tu te . Thus, a s the t im e between radiation f r a c t ion s is extended f e w e r b re a k s w i l l be ava i lab le f o r the fo rm a t io n o f e x ch a n g es .

Savage [90] d is t in gu ish es betw een 3 typ es o f s i te s which can have 2,1 o r no exch an ge l e s io n s . E xchange can take p la ce at s i te s with 2 le s io n s , w hile s i te s with one le s io n m ay undergo restitu tion . T h is w il l lead to d i m in ish ed in te ra c t io n w hich can a lso be a ch ie ve d by in activat ion o f s i t e s . Savage found that the y ie ld o f c h r o m o s o m e ex ch a n ge s is l e s s than s im p le ad ditiv ity o f the y ie ld o f the two d o s e s g iven independently , when the secon d d o s e i s g iven at a t im e when ex ch a n ge s with le s io n s p ro d u ce d by the f i r s t d o s e a r e m in im a l . T h is i s con s is te n t with the idea o f r e p a ir o r r e s t i tution o f potential exch an ge le s io n s , but the data cannot ex c lu d e the p o s s i b i l ity o f d o s e -d e p e n d e n t inactivat ion o f s i te s .

Evans [91] p r e se n ts the h ypothes is that rad iation do e s not p ro d u ce a d i r e c t b re a k a ge o f c h r o m o s o m e s , but induces le s io n s that a re in it ia lly unstab le . T h e se le s io n s b e c o m e stab il ized , and, s o m e h ours a fte r i r r a diation , undergo r e p a i r . The r e p a ir m ay be analogous to that found in c e r ta in m i c r o - o r g a n i s m s e x p o se d to u l t r a -v io le t light o r alkylating agents and m a y in vo lve the e x c i s i o n o f the dam aged zone and the re s v n th e s is and p o ly m e r iz a t io n o f new c h r o m o s o m e m a te r ia l s . C h r o m o s o m e a b e rr a t io n s a r e th e r e fo r e su ggested to be a c o n s e q u e n ce o f m i s - r e p a i r ( c r o s s p o ly m e r iza t io n ) invo lv ing two le s io n s . Savage su ggested that r e g io n s that have not undergone r e p a ir in in terph ase ap pear at m i to s i s as n on -sta in ing gaps in the c h r o m a t id s . E v id en ce i s p re se n te d that s ing le o r pa ired gaps show ing l in ea r and d o s e - s q u a r e d k in etics a r e re p a ir a b le le s io n s , and that the m a jo r i ty o f gaps a re r e p a ir e d as a c o n s e q u e n c e o f DNA syn th es is .

The bes t ev id e n ce published so fa r that ir ra d ia t io n b r e a k s c h r o m o s o m e s by break ing the DNA has been published by von B o r s t e l , M il le r and C a r r i e r [92, 93]. T h ey ir ra d ia te d am phibian lam p bru sh c h r o m o s o m e s in

350 STRlDMNAES

in tact o o c y t e s , in is o la te d n u c le i , and ir ra d ia te d the is o la te d c h r o m o s o m e se ts . In this way, they w e re able to d em o n stra te that c h r o m o s o m e b reak s a r e induced by d ir e c t act ion o f X -r a d ia t io n ra th er than v ia so m e c e l l s t ru c tu re .

K irb y -S m ith and Randolph [94] su ggested that X - r a y - in d u c e d dam age is in the prote in part o f c h r o m o s o m e s , while u . v . dam age is in the DNA. F u r t h e r m o r e , data p re se n te d by Chu [95] a lso in d icate that prote in sy n th e s is is n e c e s s a r y in r e p a ir o f c h r o m o s o m e dam age . R e v e l l [96] advanced the id ea that the c h r o m o s o m e s get act ivated sp ots , which , when lo c a te d c l o s e enough to o th er such spots , b re a k and r e jo in to fo r m e x ch a n g e s . W o l f f [97] d i s a g r e e s with R e v e l l on the point o f a ct iva ted sp ots , and b e l ie v e s an actual b re a k i s p rod u ced at the t im e o f ir ra d ia t io n . With r e g a r d to restitution , W o l f f [98] finds that f rac t ion at ion o f d o se with a p ro te in syn th es is inh ib itor p re se n t w i l l inhibit the restitu tion o f c h r o m o s o m e bre ak s in the in terva l between rad iation d o s e s , FUdR, w hich in h ib its DNA syn th es is , has no e f fe c t on the rest itu tion p r o c e s s . W o lf f [9 9 ] sa y s that r e p a ir o c c u r s in th ose parts o f the c e l l c y c l e w here no DNA sy n th e s is takes p la ce , and that the bon ds f o r m e d in c h r o m o s o m e r e jo in in g a r e p r o te in a c e o u s . Thus, th ere s e e m s to be substantial ev id e n ce that rest i tu t ion o f b re a k s is dependent on p rote in sy n th es is . It has not been d e m o n stra te d that DNA syn th es is is go ing on at the sa m e t im e , but this c a n not be c o m p le t e ly exc lu d ed .

Since restitu tion i s dependent on p ro te in syn th es is , it i s l ik e ly that d e f e c t s in p ro te in s m ay lead to c h r o m o s o m e bre a k s . It i s known that the m e t a b o l i c inh ib itor para fluoroph enyla lan in e (p F P A ) can be built into p r o te in s . A r e ce n t study by Str^mnaes [100] o f the m utagenic e f fe c t o f p F P A in S a c c h a r o m y c e s show s that p F P A in d u ces c r o s s in g o v e r , t ra n s lo ca t io n , in v e r s io n and m o n o s o m ie s . The e f fe c t o f p F P A is b e l ie v e d to be through in co r p o r a t io n in p ro te in s n e c e s s a r y fo r c h r o m o s o m e in tegrity and d i s t r ib u tion o r DNA duplicat ion .

Som e in terest in g p r e l im in a r y data on the e f fe c t o f d i f fe ren t d o s e s o f i r ra d ia t io n on the su rv iv a l o f y east c e l l s have been obta ined by Str^mnaes and M o r t im e r (unpublished). T h e se data show that f o r low d o s a g e s th ere i s a d r o p in su r v iv a l fre q u e n cy , while h igh er d o s a g e s g ive a h igh er s u r v iv a l f r e q u e n c y until such d o s a g e s w h ere the su r v iv a l f r e q u e n c ie s again d e c r e a s e . T he explanation m ay be that a c e r ta in ir ra d ia t io n d o se i s n e c e s s a r y to a ct iva te a r a d ia t io n -p r o te c t iv e e n zym e sy s te m . Thus, l i n e a r i ty f o r low d o s e s would not be e x p e cte d . C a lk ins [ 101] o p e r a t e s with two ty p e s o f r e c o v e r y s y s t e m s o f which one, the. T sy s te m , d o e s not o p e ra te a f t e r low d o s e s , but d o e s o p e ra te at h igh er d o s e s , and m ay thus be s im i la r to what we b e l ie v e to have found. C a lk ins sa ys that act iva tion o f the T s y s t e m r e q u ir e s rad ia tion d am age o r so m e equivalent st im u lu s . If a ra d ia t io n d o s e o f a c e r ta in s i z e i s n e c e s s a r y to st im ulate a r e p a ir e n zy m e sy s te m , this m a y be the explanation why Shiom i [102] finds no s ig n if ica n t d i f f e r e n c e betw een f i r s t - and s e c o n d -d a y s p e r m trea ted with 1000 R, but o b s e r v e s a s ign if ican t d i f f e r e n c e a f t e r treatm en t with 2000 R, 3000 R, and 4000 R . He studied s e x - l in k e d le tha ls and t r a n s lo ca t io n s . Sankaranarayanan [103] found no s ign if ican t d i f f e r e n c e betw een n itrogen and oxygen p o s t - t r e a tm e n t in the fre q u e n cy o f dom inant letha ls induced by 4000 R o f X - r a y s under anox ia . Sankaranarayanan thinks on ly a sm a l l f r a c t io n o f the dom inant lethal dam age is cap a b le o f be ing r e p a ir e d , while the m a jo r p or t ion is i r r e p a r a b le .

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[82] Radiation Research. Proc. Third Int. Cong. Rad. R es.,Cortina d'Ampezzo, Italy, 1966.(SILINI, G . , Ed.) North-Holland Publ. C o . , Amsterdam (1967).

[83] SOBELS, F . H . , Genetics Today, (GEERTS, S. J. , Ed.)Proc. XI Int. Congress Genetics, Hague 1963 - Pergamon Press, London (1965) 235.

[84] SOBELS, H. F . , in ref. [79] 179.

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354 STRlOMNAES

[85] SOBELS, F. H. , MICHAEL, B. , MUKHERJEE, R ., OLIVIERI, G . , SANKARANARAYANAN, K. , WATSON, W . A . F . , in reír. [82] 502.

[86[87

[88

[89

[90

[91[92

[93

[94

[95

[96

[97

[98

[99

[100[101

[102[103

DICKERMAN, R . C . , Genetics 48 (1963) 311.

RINEHART, R. R. , Genetics 49 (1964) 855.

SAX, K ., Proc. Nat. Acad. Sci. 25 (1939) 225.

SAX, K . , Cold Spring Harbor Symp. Quant. Biol. 9 (1941) 93. SAVAGE, J . R. K. , inrefr. [80].

EVANS, H.J. , inrefr. [80] 31.MILLER, O.L. Jr. , CARRIER, R . F . , von BORSTEL, R. C . , Nature 206 (1965) 905.

von BORSTEL, R . C . , MILLER, O . L . , CARRIER, R. F. , Chromosomes Today (DARLINGTON, C.D. , LEWIS, K . R . , Eds.) Oliver and Boyd, Edinburgh Vol 1 (1966) 141.

KIRBY-SMITH, J.S. , RANDOLPH, M . L . , J. Cell, comp Physiol., Supplement 58 (1960) 1.

CHU, E.H. Y . , Mutation Res. 2 (1965) 75.

REVELL, S . H. , Radiation Induced Chromosome Aberrations. (WOLFF, S ., Ed. ) Columbia University Press, N.Y. (1963) 41.

WOLFF, S ., in refer, [80] 1.WOLFF, S ., Amer. Nat. 94 (1960) 85.

WOLFF, S . , Mechanisms of the Dose Rate Effect of Radiation at the Genetic and Cellular Levels. Supplement J. Genetics 40 (1965) 38.STR0MNÆS, 0 . , Hereditas (1968) In press.

CALKINS, J . , Int. J. Rad. Biol. 12 (1967) 297.

SHIOMI, T ., Mutation Res. 4 (1967) 323.

SANKARANARAYANAN, K. , Mutation Res. 4 (1967) 641.

DI SCUS SI ON

R:C. VON BORSTEL: It would appear that meiosis proceeds more slowly in the radiation-sensitive strain. Do you believe that there may be a correlation between these two characteristics?

O. STR0MNAES: Yes, developmental rate may well be partially responsible for the difference in radiation sensitivity.

E F F E T S COM PARES D'IRRADIATIONS A U X RAYONS GAM M A E T A U X NEUTRONS SUR LES ORGANES REPRODUCTEURS DE LA MOUCHE M EDITERRANEENNE DES FRUITS, C eratitis capitata W ied. (D IPTERA: TR YPE TID A E)

R. CAUSSE ET M . FERONINRA, STATION DE ZOOLOGIE AGRICOLE,CENTRE DE RECHERCHES AGRONOMIQUES DU SU D -EST,

MONTFAVET

ETP. PEREAU-LEROYCEA, CENTRE D'ETUDES NUCLEAIRES DE CADARACHE,

FRANCE


COMPARATIVE STUDY OF THE EFFECT OF G AM M A AND NEUTRON IRRADIATION ON THE REPRODUCTIVE ORGANS OF THE MEDITERRANEAN FRUIT FLY Ceratitis Capitata W ied . (DIPTERA-.TRYPETIDAE). Plant radiob io logy studies have dem onstrated that plants grown from grains exposed to neutron irradiation show, for a g iven survival rate, greater sterility than those grown from grains exposed to gam m a rays.It therefore seem ed o f interest to determ ine whether the sam e phenom enon occurs with insects, with a view to making use o f it for pest con tro l. i

Treatm ent with gam m a rays at steriliza tion doses (m ore than 3 krad) inhibits ovogenesis; at sub- sterilization doses a variable num ber o f ovarioles m ay show norm al d evelopm en t. In the m ale, at higher doses (8 -1 0 krad), the sperm atozoids form ed rem ain norm al and the testes retain their usual appearance, but sperm atogenesis is greatly reduced.

Irradiation with neutrons by exposing pupae in a reactor co re causes quite different e ffe c ts . Relatively high doses o f the order o f 1000 rad inhibit the developm en t o f the gonads and lea v e the m ale with a residual fertility w hich is very w eak or n on -existen t, the testes being atrophied by progressive reduction o f the region containing sperm atocytes and sperm atogonia. In the fem ales, the ovary appears as an undifferentiated mass. At low er doses, the fem ales show a typ ica l developm ent o f certain ovarioles, resulting in the form ation o f m orp h olog ica lly abnorm al eggs; h isto log ica l studies show an aberrant disposition o f the v ite llin e ce lls , and various abnorm alities.

EFFETS COMPARES D ’IRRADIATIONS AUX RAYONS G AM M A ET AUX NEUTRONS SUR LES ORGANES REPRODUCTEURS DE LA MOUCHE MEDITERRANEENNE DES FRUITS C eratitis capitata W ied. (DIPTERA: TRYPETIDAE). Des études de ra d iob io log ie végéta le ont m ontré que les plantes issues de graines irradiées aux neutrons présentent, à survie ég a le , une plus forte stérilité que ce lle s provenant de graines ayant reçu des rayons gam m a. Il paraissait donc intéréssant de rechercher si le m êm e phénom ène se produit ch ez les insectes, en vue de son app lication à la lutte contre les ennem is des cultures.

Le traitem ent aux rayons gam m a à des doses stérilisantes (plus de 3 krad) provoque ch ez la fe m e lle un b lo ca g e de l 'ov o g é n è se ; aux doses substérilisantes un nom bre variable d 'ovario les peut montrer un développem en t norm al. C hez le m aie, â des doses plus é lev ées (8 à 10 krad) les sperm atozoïdes formés restent normaux, m ais la sperm atogénèse est très fortem ent ralentie.

L 'irradiation aux neutrons par exposition des pupes dans une p ile a des effets assez différents. Des doses relativem ent fortes, de l 'ordre de 1000 rad, induisent un b lo ca g e du d éveloppem en t des gonades et laissent ch ez le m âle une fertilité résiduelle très fa ib le ou nulle : les testicu les s ’atrophient par réduction progressive de la zone à sperm atocytes et à sperm atogonies; ch ez la fe m e lle l 'o v a ire apparaît com m e une masse in d ifféren ciée . A v ec des doses plus fa ib les les fem elles présentent un développem en t atypique de certaines ovarioles aboutissant à la form ation d 'œ ufs m orphologiquem ent anorm aux; l 'é tu d e h istologique m ontre une disposition aberrante des ce llu les vite llin es et diverses anom alies.

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356 CAUSSE et al.


L 'e f f ic a c i t é b io lo g iq u e re la t iv e d e s n eu tron s su r d e s in s e c te s a été m e s u r é e par p lu s ie u rs au teu rs . L es ré su lta ts , fon d és so it su r d es m e s u r e s de D L50, so it su r d es m e s u r e s de taux de m utation , donnent d es v a le u r s allant le plus souvent de 1, 76 à 4 e n v iron [ l - б ] . On d oit donc s 'a tten d re à ce que l 'a c t io n d e s n eu tron s so it plus e f f ic a c e que c e l le d es ra y o n s gam m a pour o b te n ir la s té r i lis a t io n d 'in s e c te s ; ou e n co r e que, p ou r obtenir- le m êm e taux de s t é r i l ité , i l fa i lle e m p lo y e r une d o se m oin s fo r te d 'ir ra d ia t io n aux n eu tron s qu 'au x ra y on s gam m a.

N ous sa von s par a i l le u r s que, ch e z le s végétau x, l 'a c t io n c o m p a ré e d e s ra y on s et d es n eu tron s m on tre , à taux de su rv ie ég a l, une s t é r i l ité b ea u cou p plus é le v é e ch e z le s p lantes proven an t de g ra in e s tr a ité e s aux n eu tron s [7 -9 ]. L 'a c t io n d e s n eu tron s su r le s p lantes e st d 'a u tre part a s s e z r é g u liè r e p ou r p e rm e ttre la m ise au point d 'un e tech n iqu e de d o s i - m é tr ie b io lo g iq u e ; c e l l e - c i e s t fon dée su r la m e su re de la p r e m iè r e fe u ille d es p lantu les is s u e s de g ra in e s d 'o r g e ir r a d ié e s s è c h e s [1 0 -1 2 ] ,Etant donné la d iff icu lté de m e s u r e r d ire c te m e n t la d o se d 'ir ra d ia t io n en n eu tron s re çu e dans un ré a c te u r p a r du m a té r ie l b io lo g iq u e , i l p a r a is sa it in té re ssa n t de c o m p a r e r dans c e s co n d itio n s l 'a c t io n b io lo g iq u e su r Les g ra in s d 'o r g e et su r un in se c te , i c i la m ou che m éd iterra n éen n e d es fr u it s C e r a t it is cap ita ta W ied .

E nfin , i l nous p a ra is s a it ég a lem en t in té re ssa n t de c o m p a r e r , su rC. cap ita ta , l 'a c t io n d e s ra y o n s gam m a, d é jà p a rt ie lle m e n t é tu d iée [13] , et d es n eu tron s su r le d év e lop p em en t et la stru ctu re d es g on a d es . C 'e s t d 'a i l le u r s su r c e d e r n ie r point du p ro g ra m m e en c o u r s que le s ré su lta ts le s p lus n ets ont été ju s q u 'à p ré se n t e n r e g is t r é s .

2. TECH NIQ UES

L e s pupes de C. cap itata d e s tin é e s à ê tr e ir r a d ié e s p rov ien n en t d 'un é le v a g e perm an en t de la Station de z o o lo g ie de M ontfavet.

L e s ir ra d ia t io n s son t p ra tiq u ées , p ou r le s ra y on s gam m a dans un ir ra d ia te u r au c o b a lt -6 0 de 1200 Ci donnant un débit de d o se de 1 0 0 0 ra d /m in , et p ou r le s n eutron s dans un ré a c te u r de type p is c in e , la p ile PEG G Y, de fa ib le p u issa n ce (in fé r ie u re à 1 kVV), le co m b u st ib le étant de l'u ra n iu m e n r ic h i à 90%. C es in sta lla tio n s se trou ven t au C en tre d 'é tu d e s n u c lé a ire s de C a d a ra ch e .

L e s éch a n tillon s b io lo g iq u e s p réa la b lem en t e m b a llé s dans d es sa ch ets de polyéth y lèn e sou d és sont p lon g és dans l 'e a u du r é a c te u r , au con tact de l 'é c r a n de p ro te c t io n du coeu r (dans le p r e m ie r ce n t im è tre d 'eau le flu x n eutron iqu e e st de 5* 1 0 9 n c m -2 s -1 quand la pu issan ce du ré a c te u r e s t de 300 W ).

L es pupes ou le s g ra in e s d 'o r g e sont é ta lé e s su r d es p la qu es pour que l 'é p a is s e u r de l 'e n s e m b le ne d ép a sse pas 1 c m . Dans c e s co n d itio n s , la d o s im é tr ie b io log iq u e a v ec l 'o r g e a m on tré qu 'une d o se de 750 rad en v iron éta it attein te en 12 m in d 'ir ra d ia t io n .

L e s pupes ir r a d ié e s sont en su ite m is e s en é c lo s io n à la Station de z o o lo g ie de M ontfavet. L es in s e c te s é c lo s sont ex a m in és quant à le u r v ita lité , le u r fé co n d ité et le u r fe r t i l i t é ; leu r d is s e c t io n et l 'e x a m e n h is - to lo g iq u e d es gon ades p erm et de c o m p a r e r l 'a c t io n d e s ra y on s gam m a et d e s n eu tron s.

S M - 10 j¡/3íi 357

3. H ISTOLO G IE DES O RG AN ES G EN ITA U X

3 .1 . O rg a n es génitaux m â les

L e s o rg a n e s gén itau x du m âle sont fo r m é s p a r deux te s t ic u le s g l o bu leux, de fo r m e o v a la ire , de 470 ц m de lon gu eu r su r 180 Mm de d ia m è tre en m o y e n n e .

C hez l'a d u lte m â le à l 'é m e r g e n c e chaque g lande gén ita le contient de n om b reu x c y s te s à sp e rm a to g o n ie s et à sp e rm a to cy te s a in s i que de n o m bre u x fa is ce a u x de sp e rm a tid e s et de s p e rm a to z o ïd e s .

Au c o u r s d e s t r o is p r e m ie r s jo u r s de la v ie im a g ín a le la p a rtie in fé r ie u r e du te s t ic u le va se d if fé r e n c ie r p r o g r e s s iv e m e n t en une zon e c o n tenant uniquem ent d es sp e rm a to z o ïd e s in d iv id u a lisé s . C ette so r te de p och e r e p ré s e n te a lo r s la m o it ié e n v iron de la ta ille de l 'o rg a n e g én ita l e n tie r , ce d e r n ie r ayant atteint sa s tru ctu re d é fin it iv e .

3 .2 . O rg an es génitaux fe m e lle s

L e s o rg a n e s génitaux de la fe m e lle à l 'é m e r g e n c e sont r e p ré s e n té s par deux o v a ir e s m é ro i's t iq u e s p o ly tro p h iq u e s de 270 ium de lon gu eu r en v iro n , qui p résen ten t la s tru ctu re h is to lo g iq u e su ivan te : d 'u n e p art le g e r - m a riu m con stitu é par la p artie a p ica le d es o v a r io le s contenant le s o v o g o n ie s et le s c e l lu le s fo l l i c u la ir e s ; d 'a u tre p art le v ite lla r iu m où s 'e ffe c tu e n t la d iv is io n et la d if fé r e n c ia t io n d e s o v o g o n ie s en p r é o v o c y te s et c e l lu le s v ite ll in e s pou r fo r m e r le s fo l l i c u le s ou c y to b la s te s . A c e stade, chaque o v a r io le con tien t deux et q u e lq u e fo is t r o is fo l l i c u le s à la su ite le s uns d es a u tre s et de ta ille c r o is s a n te .

De 24 à 48 h a p rè s l 'é m e r g e n c e on c o m m e n c e à n o te r une lé g è re au gm en tation de v o lu m e d es o v a ir e s et à p a rt ir du d eu x ièm e jo u r la c r o i s sa n ce se d é c le n ch e de fa ço n t r è s ra p id e . C hez le s im a g o s de 3 j le s o v a ir e s atteignent une lon gu eu r de 650 д ш en m oyenne et contienn en t d es o v o c y te s à tou s le s sta d es de m atu ration , dont t r o is ou qu atre en fin de d é v e lop p em en t. Au q u a tr ièm e jo u r de v ie im a g ín a le c e n om b re devien t p lu s im portan t et à p a rt ir du c in q u iè m e jo u r la m atu rité se x u e lle des fe m e lle s e st a ttein te ; p ra tiqu em en t chaque o v a r io le con tien t un o v o cy te co m p lè te m e n t d év e lop p é et p rêt à ê tre pondu au stade d 'o v o c y te I. A ce m om en t le s o v a ir e s o ccu p en t p resq u e la to ta lité de la ca v ité abdom in a le d es fe m e l le s a v ec une lon gu eu r de 800 à 1000 jum.

4. IR R A D IA TIO N DES PU P E S AU X RAYO N S G AM M A

4 .1 . A ctio n su r la v ita lité et la r e p ro d u ctio n

L 'a c t io n d es ray on s gam m a su r C . cap ita ta a d é jà été étu d iée par p lu s ie u rs au teu rs su r le plan de la v ita lité , la fé co n d ité et la fe r t ilité [1 3 -1 5 ] .

La d u rée de v ie d es in s e c te s ir r a d ié s à 10 krad e st sou vent lé g è r e m ent su p é r ie u re à c e l le d e s té m o in s .

L 'ir r a d ia t io n d es pupes de 8 j (à la v e il le de i 'é c lo s io n ) ju sq u 'à la d ose de 15 krad ne d im inue pas le taux d 'é c lo s io n par ra p p ort aux té m o in s .

L 'ir r a d ia t io n d es pupes à 1 krad a b a is se la fe r t ili té d e s adultes à 50% e n v iron pou r le s fe m e lle s et 30% p ou r le s m â le s . L 'ir r a d ia t io n

3 5 8 CAUSSE et al.

à plus de 3 krad p rovoq u e la s té r i lité to ta le d e s fe m e lle s p a r b lo ca g e du d év e lop p em en t o v a r ie n . Une d o se de 10 krad e st n é c e s s a ir e pou r ob te n ir une s t é r i l ité p resq u e to ta le d es m â le s ; dans c e c a s le s s p e rm a to z o ïd e s fo r m é s peuvent fé c o n d e r l'o e u f m a is sont p o r te u rs de m utations lé ta le s d om in a n tes ; la fo rm a tio n de nouveaux s p e rm a to z o ïd e s e st b loq u ée .

4 .2 . A ctio n su r le s gon a d es m â les

L 'é tu d e h isto log iq u e d e s o rg a n e s génitaux m â le s a é té fa ite su r d es in d iv id u s â g é s g é n é ra le m e n t de 4, 9 et 16 j a p rè s ir ra d ia tio n de pupes à 2, 3, 4, 5 et 9 krad .

C hez d es in d iv idu s â g é s de 4 j le s te s t ic u le s c o n s e rv e n t une ta ille n o rm a le q u e lle que so it la d ose d 'ir ra d ia t io n . La p a rtie b a sa le de c e s o r g a n e s p ré se n te un a s p e c t n o rm a l et c o m p re n d une zone à sp e rm a to z o ïd e s in d iv id u a lisé s a in s i que de t r è s n om breu x s p e rm a to d e s m e s . On ne v o it p lu s de sp e rm a tid e s q u e lle que so it la d o s e . L a p a rtie a p ica le p a r co n tre e s t d é s o r g a n is é e . A 2 et 3 krad on peut e n co r e a p e r c e v o ir q u e lq u es c y s te s à s p e rm a to g o n ie s ; c e s d e r n ie rs d is p a ra is se n t to ta lem en t aux d o s e s su p é r ie u r e s . A in s i, à. 4 et 5 krad toute la p a rtie a p ica le e s t fo r m é e par p lu s ie u rs am as de noyaux p a ra issa n t c o r r e s p o n d r e à d e s c y s te s dont le s c e l lu le s au ra ien t perdu le u r m em bran e cy to p la sm iq u e .

P o u r le s in d iv id u s p lus â g é s (7 à 9 j ) la zon e à s p e rm a to z o ïd e s in d iv id u a lisé s e st to u jo u rs bien d év e lop p ée et la d if fé r e n c e p orte su rtou t su r le n o m b re de s p e rm a to d e s m e s , qui e s t beaucou p plus réd u it. L e s sp e rm a tid e s sont to u jo u rs a b se n te s . La p artie a p ica le , p a rt icu liè re m e n t à la d o se s té r ilis a n te de 9 krad , prend un a s p e c t c a r a c té r is t iq u e p r é s e n tant le s am as tro u v é s à 4 j , m a is i c i a r r o n d is , bien in d iv id u a lisé s et r e m p lis de noyaux ou tout au tre c o rp u s c u le fo r te m e n t c o lo r é par l ’h é m a - to x y lin e . C hez le s in d iv id u s â g é s de 16 j a p rè s ir ra d ia tio n à m oin s de9 krad le te s t icu le ga rd e sa ta ille n o rm a le . L e s s p e rm a to z o ïd e s o ccu p en t p re sq u e tout l ’o rg a n e ; on ne v o it p lus que qu elqu es s p e rm a to d e s m e s .A p r è s ir ra d ia tio n à 2 et 3 krad on a p e rç o it e n c o r e qu e lq u es c y s te s à sp e rm a to c y te s , m a is la zon e a p ica le , com p orta n t e n co r e qu e lq u es am as c e l lu la ir e s in d if fé r e n c ié s , a p resq u e d isp a ru . A p r è s ir ra d ia tio n à 9 krad on ne v o it p lus de c y s te s et le te s t ic u le p ré se n te une ta ille réd u ite . C hez un m â le âgé de 1 m o is a p rè s ir ra d ia t io n à 9 krad le s te s t ic u le s sont n e tte m ent a tro p h ié s et réd u its à l 'é ta t de deux s a c s ne contenant que d e s s p e r m a t o z o ïd e s in d iv id u a lisé s .

C e s in d ica tio n s sem b len t s ig n if ie r d 'un e part que, q u e lle que so it la d o s e , le s sp e rm a tid e s se d iffé re n c ie n t rap id em en t en sp e rm a to z o ïd e s . D 'a u tre part, aux d o s e s fa ib le s , la zon e à s p e rm a to c y te s ap para ît p a r t ie lle m e n t d é s o r g a n is é e , p a rt ie lle m e n t b loqu ée dans son d év e lop p em en t, m a is ca p a b le e n co r e p ou r une part d 'u n e év o lu tion n o rm a le . Enfin , à 9 krad i l y a d é s o rg a n is a t io n puis d é g é n é r e s c e n c e de c e tte zon e a b ou tissan t à une a trop h ie de l 'o r g a n e .

4 .3 . A ction su r le s gon ad es fe m e lle s

C on tra irem en t aux gon a d es m â les le s o rg a n e s génitaux fe m e lle s sont t r è s peu d é v e lop p és ch e z le s pupes â g é e s de 8 j au m om ent de l ' i r r a d ia tion ; ce fa it p ou rra it p e rm e ttre d 'e x p liq u e r l 'a c t io n beaucou p plus fo r te d es ra y o n s gam m a à d ose é g a le su r le s fe m e l le s . L es co u p e s h isto lo g iq u e s

S M - 102/36 3 5 9

de fe m e lle s i r r a d ié e s à 2, 3, 4, 5 et 9 krad ont été e ffe c tu é e s su r le s in s e c te s â g é s de 16 j . D é jà pou r 2 krad le s o v a ir e s sont de ta ille rédu ite m a is p résen ten t une o v o g é n è se n o rm a le a v ec tou s le s sta d es de d é v e lo p pem ent d e s fo l l i c u le s ; c e tte d im in ution de ta ille p rov ien t su rtou t d ’un n o m b re p lu s réd u it d 'o v o c y t e s venus à m a tu rité .

A la d o se de 3 krad, a u -d e là de la q u e lle la s té r i lité e st co m p lè te , nous trou von s une a ction beau cou p plus m a rq u ée et c a r a c té r is t iq u e des ra y o n s gam m a. L e s o v a ir e s , to u jo u rs pour le s fe m e lle s de 16 j , sont de ta ille rédu ite m a is peuvent p r é se n te r d es s tru c tu re s v a r ié e s . On peut t r o u v e r un ce r ta in n om b re d 'o v o c y te s n orm a lem en t d é v e lo p p é s .On peut a u ss i en tr o u v e r un n o m b re t r è s rédu it, p a r fo is m êm e un seu l.On con sta te par c o n tre un n om b re plus ou m oin s im portan t de fo l l i c u le s a typ iq u es : o v o cy te s p lu s ou m oin s id e n tifia b le s , c e l lu le s n o u r r ic iè r e s ir r é g u l iè r e s , p a r fo is con fon d u es en une m a ss e in d if fé r e n c ié e , p r é se n ce de n om breu x petits noyaux re sse m b la n t à ceu x d es c e llu le s fo l l i c u la ir e s et r é p a r t is au m ilie u d e s c e llu le s n o u r r ic iè r e s .

A p a rt ir de 4 krad le s deux o v a ir e s de ta ille identique à c e l le q u 'ils ava ien t au m om en t de l 'ir r a d ia t io n ne su b issen t aucun d év e lop p em en t.A la p a rtie a p ica le le s o v a r io le s contienn en t un m élan ge fo r m é par de p etits fo l l i c u le s de s tru ctu re a b e rra n te , de n om b reu x noyaux de c e llu le s fo l l i c u la ir e s , d e s m a s s e s a r r o n d ie s contenant de n om breu x petits noyaux a in s i que d es c e l lu le s v ite ll in e s i s o lé e s . La p a rtie b a sa le n 'e s t fo r m é e que par le s p a ro is d es o v a r io le s .

A 9 krad ce tte a trop h ie d es o v a ir e s e st m a x im a le et c e s d e r n ie rs ne sont p lus fo r m é s que par le s o v a r io le s v id e s dont la p a ro i c e llu la ir e e s t p lus é p a is s e dans la p a rtie b a sa le d e s gonades.

5. IR R A D IA TIO N DES P U P E S A U X NEUTRONS

5 .1 . A ction su r la v ita lité et la re p ro d u ctio n

L e s in s e c te s ont été i r r a d ié s aux n eu tron s au stade pupe âgée de 8 j , par e x p o s it io n plus ou m oin s longue au flu x de la p ile PE G G Y , g é n é ra le m e n t pendant d es tem p s de 5, 10, 15 et 20 m in.

L e s taux d 'é m e r g e n c e de c e s pupes ir r a d ié e s son t a b so lu m en t iden tiqu es à c e u x o b s e r v é s ch e z le s in s e c te s té m o in s et d ép a ssen t dans tou s le s c a s 95%. (D ans la d é term in a tion de c e taux d 'é m e r g e n c e ne sont p r is en c o n s id é ra t io n que le s im a g o s e n tiè re m e n t d é b a r r a s s é s de le u r puparium et non ce u x qui se trou ven t à m o it ié ou p re sq u e s o r t is de le u r en ve lop p e n ym pha le , b ien q u 'i ls p u issen t r e s t e r a in s i v iva n ts pendant qu elque te m p s . )

La lon g év ité d e s ad u ltes a é té c o n t r ô lé e dans tou tes le s e x p é r ie n c e s pendant une d u ré e de 20 j a p rè s le u r é m e r g e n c e . D e s te m p s d 'ir ra d ia t io n de 5 à 15 m in ne sem b len t pas a f fe c te r la lo n g é v ité d e s in s e c te s en c o m p a ra iso n e c e l le d 'in s e c te s n orm au x , tout au m oin s ju sq u 'a u v in g tièm e jo u r , oïi la m o r ta lité e st en m oyen ne de 10%. Seuls d es te m p s d 'ir ra d ia t io n de 20 m in d im in uen t lé g è re m e n t la lo n g é v ité d e s fe m e l le s et p lus f o r t e m ent c e l le d e s m â le s (3 5% de m o r ta lité p ou r c e s d e r n ie r s ) (f ig . 1).

En c e qui c o n c e rn e l 'é tu d e de la fé co n d ité et de la fe r t i l i t é , le s r é su lta ts ob ten u s sont a s s e z v a r ia b le s ; c e c i e s t p e u t-ê tr e dû au fa it que le s d o s e s r e ç u e s pendant un m êm e te m p s d 'e x p o s it io n sont e l le s -m ê m e s v a r ia b le s d 'u n e sé a n ce d 'ir r a d ia t io n à l 'a u tr e . Cependant le s e ffe ts de c e s i r r a d ia -

360 CAUSSE et al.

% de mortalité en 20 jours lOOl

75

50

25

e maies o femelles

5312

10625

15937

20 Temps d'irradiation(mn) 1250 Dose évaluée (rad)

FIG. 1. Pourcentage de m orta lité tota le pour des Lots d 'in sectes (1000 individus environ pour chaque lot) exposés aux neutrons pendant des temps variés.

% de fertilité 50

30

10

5 10 15 20 Temps d'irradiation(mn)312 625 9 3 ? 1250 Dose évoluée (rad)

FIG. 2. Fertilité résultant de croisem ents $ normales x c?issus de pupes de 8 j irradiées aux neutrons.

S M - 1 02/36 361

t io n s sont d é jà t r è s s e n s ib le s pou r d e s tem p s d 'e x p o s it io n de 2 à 5 m in et augm entent a v e c d e s tem p s plus é le v é s .

En c e qui c o n c e rn e le s m â le s , d es c r o is e m e n ts e ffe c tu é s en tre m â le s i r r a d ié s et fe m e lle s n o rm a le s donnent une fe r t ili té m oyen ne de l 'o r d r e de 20% pou r d e s e x p o s it io n s de 5 m in, et de 7 à 8% pou r d e s e x p o s it io n s de10 m in . L e s d o s e s s té r i l is a n te s ne sont atte in tes q u 'à p a r t ir de 15 m in, où su b s is te e n co r e une fe r t i l i té r é s id u e lle de 1, 7%, c e tte fe r t i l i t é n 'é tan t p lu s que de 0, 3% à 20 m in (95% étant la fe r t i l i té m oyen ne ch e z le s té m o in s ) ( f ig .2 ) .

M ais le fa it le p lus in té re ssa n t e st la d if fé r e n c e d 'a c t io n qui se m b le e x is t e r en tre le s ir ra d ia t io n s aux ra y o n s gam m a et aux n eu tron s ch ez le s fe m e l le s .

R a p p elon s que le s d o s e s s u b s té r ilis a n te s de ra y on s gam m a perm etten t la fo rm a t io n d 'un n o m b re rédu it d 'œ u fs , m a is que c e u x - c i son t m o r p h o lo g iq u e m ent n orm au x . D ans le c a s de n os e x p o s it io n s aux n eu tron s nous avon s obtenu une s t é r i l ité to ta le d es fe m e l le s à p a r t ir de 15 m in d 'e x p o s it io n .P a r co n tre , p ou r 10 m in d 'e x p o s it io n nous avon s o b s e r v é la fo rm a tio n et la ponte d 'œ u fs m o rp h o lo g iq u e m e n t an orm au x ; c e u x - c i a p p a ra isse n t d é fo r m é s , p a r fo is r a c c o u r c is ju s q u 'à d e v e n ir g lobu leu x , et p résen ten t sou vent une s tru ctu re in tern e h é té ro g è n e v is ib le à t r a v e r s le ch o r io n .

5 .2 . A ctio n su r le s g on a d es m â les

T o u te s le s o b s e rv a t io n s portant su r d es in s e c te s is s u s de pupes ir r a d ié e s aux n eu tron s pendant d e s tem p s de 5, 10, 15 et 20 m in ont été e ffe c tu é e s su r d e s in d iv id u s â g é s de 25 j .

C hez le s m â le s ir r a d ié s pendant 5 m in, d o se qui d im in ue c o n s id é r a b le m ent la fe r t i l i t é , n ous ne co n sta to n s aucune m o d ifica t io n au n iveau d es g on a d es , qui r e s te n t de ta ille et de s tru ctu re an a logu es à c e l le s d 'o r g a n e s génitaux de m â le s n orm au x .

L 'a c t io n e s t bea u cou p plus nette et ap para ît de fa ço n bru ta le ch e z d e s in s e c te s i r r a d ié s pendant 10 m in . En e ffe t , ch e z c e s in d iv id u s le s te s t ic u le s peuvent ê tr e tout deux t r è s a tro p h ié s et réd u its à d es s o r te s de p o ch e s a llo n g é e s ne contenant que d e s s p e rm a to z o ïd e s in d iv id u a lisé s a v e c p a r fo is qu e lq u es c y s te s à sp e rm a to g o n ie s . M ais , dans le c a s le p lu s g é n é ra l, c e s m â le s ne p résen ten t qu 'un se u l d es o rg a n e s génitaux a in s i a trop h ié , a lo r s que l 'a u tr e c o n s e r v e une stru ctu re n o rm a le présen tan t tou s le s sta d es de la sp e rm a to g é n è se , sau f p e u t-ê tre qu elqu es m o d ifica tio n s aux n iveaux d es sp e rm a to g o n ie s et s p e rm a to c y te s .

P ou r d e s tem p s d 'ir r a d ia t io n de 15 et 20 m in i l y a to u jo u rs b lo ca g e co m p le t de la sp e rm a to g é n è se , et le s o rg a n e s génitaux sont tou s deux ré d u its à d e s s o r te s de s a c s à p a ro is plus é p a is s e s ne contenant que des sp e rm a to z o ïd e s a v e c qu e lq u es r a r e s sp e rm a to d e s m e s à 15 m in.

5 .3 . A ction su r le s gon a des fe m e lle s

C om m e dans le c a s d e s ra y on s gam m a l 'ir r a d ia t io n aux n eutron s, m êm e à fa ib le d o s e , p rovoq u e d 'im p o r ta n te s m o d ifica tio n s au n iveau d es c e l lu le s r e p r o d u c tr ic e s .

D é jà pou r d e s tem p s d 'ir ra d ia t io n de 5 m in le s o rg a n e s génitaux sont à la fo is de ta ille rédu ite (de m o itié e n v iron par ra p p o rt à la n orm a le ) et l 'o v o g é n è s e e st p lus ou m oin s p e r tu rb é e . P a rm i le s fo l l i c u le s se

362 CAUSSE et al.

développan t n orm a le m e n t se trou ven t q u e lq u es fo l l i c u le s a typ iqu es ayant l 'a s p e c t de m a s s e s a r r o n d ie s de d iffé re n te s fo r m e s com p orta n t, so it en to ta lité , so it s im p lem en t en b o rd u re , une m ultitude de p etits noyaux.L e s d if fé r e n c e s in d iv id u e lle s sont t r è s im p o rta n te s d 'un e fe m e lle à l 'a u tr e ; dans c e r ta in s c a s l 'o v a ir e ne con tien t qu 'un ou deux fo l l i c u le s a b e rra n ts , dans d 'a u tre s au c o n tra ire i l e st en tiè re m e n t fo r m é par c e s m a s s e s a r r o n d ie s où se trou ven t e n co r e qu elqu es fo l l i c u le s n orm au x .

A la su ite d 'u n e ir ra d ia tio n de 10 m in, le s gon a des sont g é n é ra le m e n t de ta ille e n co r e p lus rédu ite , m a is le s d if fé r e n c e s in d iv id u e lle s su bs is ten t to u jo u r s . On peut n o te r dans tou s le s c a s un n om b re plus ou m oin s im portant de c e s m a s s e s t is s u la ir e s a m o rp h e s r e m p lie s de p etits noyaux, qu i p a ra isse n t ê tr e c a r a c té r is t iq u e s d es ir ra d ia t io n s aux n eu tron s.C e s « f o l l i c u l e s » a typ iqu es de fo r m e s i r r é g u l iè r e s , qui peuvent m êm e fu s io n n e r en tre eux, dénotent une ce rta in e p r o lifé r a t io n c e l lu la ir e . Sur c e r ta in e s co u p e s h is to lo g iq u e s on peut n o te r l 'a n a lo g ie ex istan t en tre c e s n oyau x de p r o lifé r a t io n c e llu la ir e et ceu x d e s c e l lu le s fo l l i c u la ir e s qui su b s is te n t en b o rd u re par e n d ro its .

Aux d o s e s s t é r i lis a n te s a p rè s ir ra d ia tio n de 15 ou 20 m in, le s r é s u lta ts son t p lus h om og èn es et nous o b s e rv o n s une a ction c o m p a ra b le à c e l le p rod u ite par le s ra y o n s gam m a aux d o s e s su p é r ie u re s à 4 krad . L 'o v o g é n è s e e s t in h ibée par b lo ca g e d e s d iv is io n s c e l lu la ir e s , et le s o v a ir e s e n tiè re m e n t a tro p h ié s , de ta ille co m p a ra b le à ceu x de fe m e lle s im m a tu re s , ne contienn en t aucun fo l l ic u le (du m oin s en c e qui c o n c e rn e d e s in d iv idu s fe m e l le s â g é s de 25 j ) . C es gon a d es ne sont fo r m é e s que par une s é r ie d 'o v a r io le s v id e s p lus fo r te m e n t c o lo r é e s à le u r p a rtie b a sa le , c o m m e n ous a v io n s vu dans le c a s d 'ir r a d ia t io n s aux ra y on s gam m a à p a r t ir de 4 krad .

6. CONCLUSION

L a c o m p a ra iso n de l 'a c t io n d e s ray on s gam m a et d e s n eu tron s su rC . cap itata a é té fa ite en partant de l 'h y p o th è se d 'un e e f f ic a c it é b io lo g iq u e r e la t iv e d e s n eu tron s su p é r ie u re k c e l le d es ra y o n s gam m a. La d i f f i c u l té de m e s u r e r d ire c te m e n t la d o s e d 'ir ra d ia t io n aux n eu tron s dans le r é a c te u r nous a fa it u t i l is e r un éta lon b io lo g iq u e connu, la c r o is s a n c e de p la n tu les is s u e s de g ra in e s d 'o r g e ir r a d ié e s . D ans c e s co n d itio n s , d es te m p s d 'e x p o s it io n aux n eu tron s de 5, 10, 15 et 20 m inu tes c o r r e s p o n d a ient à d e s d o s e s c a lc u lé e s de 310, 620, .940 et 1250 rad .

L e s ir ra d ia tio n s de pupes â g é e s ju sq u 'a u x d o s e s s té r i lis a n te s de ra y o n s gam m a ou de n eu tron s ne dim inuent pas le s taux d 'é c lo s io n . P a r c o n tre la lon gév ité d es ad u ltes, non a ffe c té e par le s ray on s gam m a ju s q u 'à 10 krad , e st d im in u ée a p rè s ir ra d ia t io n aux n eu tron s à 1250 rad .

L 'e f f i c a c i t é b io log iq u e re la t iv e é le v é e d e s n eutron s appara ît lo r s q u 'o n exa m in e le s t a u x de fé co n d ité et de fe r t ili té . D ans le s co n d itio n s de nos e x p é r ie n c e s , on peut en d éd u ire que l 'e f f i c a c i t é b io log iq u e d e s n eu tron s a été e n v iron de 7 fo is c e l le d e s ra y on s gam m a.

L 'a c t io n d es ra y on s gam m a et d es n eu tron s su r le s gon a des m â les p a ra ît a s s e z se m b la b le . Aux d o s e s fa ib le s le s te s t ic u le s resten t de d im e n s io n s n o rm a le s , m a is l 'e x a m e n h isto log iq u e m on tre que la s p e rm a - to g é n è se a été a r r ê té e . Aux d o s e s é le v é e s , d ites s té r i lis a n te s , on c o n s tate que le s gon ad es sont ré d u ite s k d e s s o r te s de sa cs contenant u n iqu em ent le s s p e rm a to z o ïd e s .

S M - 102/36 363

L 'a c t io n d e s ra y on s gam m a et d es n eu tron s su r le s g on a d es fe m e lle s aux d o s e s s té r i lis a n te s p rovoq u e l 'a tro p h ie co m p lè te d es o v a ir e s .

P a r co n tre , aux d o s e s su b s té r ilisa n te s ap p ara ît une d if fé r e n c e d 'a c t io n d 'autant p lus m a rq u ée que l 'o n a p p roch e de la d o se s té r i lis a n te . A p rè s ir ra d ia t io n aux ra y on s gam m a on co n s ta te la ponte d 'un n o m b re réduit d 'oeu fs , c e u x -c i p résen ta n t un a s p e c t n o rm a l. A p rè s ir ra d ia t io n aux n eu tr o n s on con sta te a u ss i la ponte d 'un n om b re rédu it d 'œ u fs m a is une p artie de c e u x - c i ont un a s p e c t an orm a l, d é fo rm é , sou vent g lobu leu x ; c e s œ u fs son t s t é r i le s . L 'e x a m e n h is to lo g iq u e c o n fir m e dans le s o v a ir e s la p r é s e n c e de fo l l i c u le s a b e rra n ts co n s titu é s de m a s s e s t i s s u la ir e s a m o rp h e s .

Il appara ît d on c que l 'e f f i c a c i t é b io lo g iq u e r e la t iv e d e s n eu tron s est su p é r ie u re à c e l le d e s ra y on s gam m a, m a is qu 'en m êm e tem p s le m ode d 'a c t io n des n eu tron s su r le s o rg a n e s r e p ro d u c te u rs n 'e s t pas ex actem en t se m b la b le à c e lu i d e s ra y o n s gam m a.

R E F E R E N C E S

[1 ] ABELEVA, E .A . , LAPKIN, YU . A . , R adiob iologiya 2 2 (1962) 29 3 -9 7 .[2] ABELEVA, E .A . , LAPKIN, YU . A . , R adiob iologiya 3 3 (1 9 6 3 )4 2 0 -2 1 .[3 ] BICKER, A .E ., Thesis, Kansas U n iv ., Lawrence, T ID -15962 (1 9 6 2 ) 52 p .И ] MURAKAMI, A . , KONDO, S ., Rep. natn. Inst. G e n e t ., M isim a 14 (1963) 106-07 ,'15] MURAKAMI, A . , KONDO, S . , Jap. J. G enet. (Idengaku Zasshi) 39 (1964 ) 102 -14 .[6] MURAKAMI, A . , KONDO, S . , T A Z IM A , Y . , Jap. J. G enet. (Idengaku Zasshi) 40 (1965 ) 113 -12 4 .[7] ERENBERG, L . , SEALAND, E ., J. n u cl. Energy 1 (1964) 150 -69 .[8] M AC -K EY, J ., Hereditas 37 (1951 ) 4 2 1 -6 4 .[9] PEREAU-LEROY, P ., Bull. Inf. S c i. T e ch . C .E .A . 93 (1965 ) 19 -2 6 .

[10] AHNSTRON, G .. GUERRIERI, G ., GIACONELU, М ., DI PALO, L ., Lab. A p p lic . A grie . C .S .N . C asaccia , Public, n" 8 6 (1 9 6 4 ).

[11 ] ARNAUD, Y . . Bull. Inf. S c i. T e ch . C .E .A . 32 (1964) 5 8 -6 2 .[12] KONZAC, C . F .. Radiat. Res. 1 (1954 ) 220.[13] FERON, М ., Annls Epiphyt. 17 2 (1966 ) 2 2 9 -3 9 .[14] ARROYO, М ., JIMENEZ, A . , MELLADO, L ., CABALLERO, F ., Boln. Patol. veg . Ent. agrie. 2 8 (196 5 )

2 5 7 -9 8 . _[15] KATIYAR, K .P . , VALERIO, J .. Turrialba 14 (1964) 2 1 1 -2 1 .

D I S C U S S I O N

W .F . BALD W IN : It is u su a lly im p o s s ib le to obtain g a m m a -fr e e neutron ir ra d ia tio n s fr o m r e a c to r s . D id th is p resen t any p ro b le m fo r you?

P . P E L E G R IN : P erh a p s I m ight a n sw er th is on D r. C a u s s e ' sb eh a lf. The neutron rad ia tion ca m e fro m the P EG G Y s w im m in g -p o o l r e a c to r at C a d arach e , in the tank o f w hich fast n eu tron s a r e g en era ted at the sa m e tim e as gam m a ra y s and th e rm a l n eu tron s. The neutron sp ectru m is not c le a r ly known, but it is known that the gam m a flu x is low . A w o rk e r in F ra n ce w ish in g to in v estig a te fu rth e r cou ld use the 'b io lo g ic a l c a v ity ' o f the E L 3 r e a c to r at S aclay w h ere , by m ean s o f a u ran iu m c o n v e rte r , a high flux o f fa s t n eu tron s is m ade a v a ila b le with a lm o st to ta l a b sen ce o f gam m a ra y s .

W .F . BALDW IN : In the one o r two stu d ies a v a ila b le fo r c o m p a r is o n it has been show n that fa s t n eutron s (14 M eV) g ive an in c r e a s e in e ffe c t o v e r that o f gam m a rad ia tion o f about 1 . 4 (o r a p p ro x im a te ly 4U%), w h erea s

364 CAUSSE et a l .

in you r w o rk you found a ¡seven -fo ld in c r e a s e . I should be in te re ste d to lea rn o f the m ethod w h ereby you co m p a re d the neutron and gam m a e f fe c t s .

P . PE L E G R IN : A s stated in the paper, the re la t iv e b io lo g ic a l e f f e c t iv e n e s s o f the n eutron s, as co m p a re d with that o f the gam m a rad ia tion , has been d eterm in ed by D r. P . P e r e a u -L e r o y , at both S aclay and C a da - ra ch e , with d ry ir ra d ia te d b a r le y g ra in s , and h is f ig u re s a re co m p a ra b le to th ose g iven h e re .

W .J . LE QUESNE: W ould it be p o s s ib le to m ake use o f neutron s o u r c e s o f the type co n s is t in g o f a m ixtu re o f an a lp h a -e m itte r and b e r y l liu m in th is w ork , and w ould th is m ake fo r a lo w e r gam m a d osa g e than that obta in ed using a r e a c to r ? I understand that e m is s io n s o f up to 10s n e u tr o n s /s can be obtained , and w on der w hether th is le v e l w ould be su ffic ie n t ly high.

R . CAUSSE: I think it is quite fe a s ib le to use o th er n eutron s o u r c e s in o r d e r to cut out the gam m a rad ia tion . One cou ld even e lim in a te it c o m p le te ly by a c a r e fu l c h o ic e o f is o to p e , but in th is c a s e it w ould c e r ta in ly not be p o s s ib le to obtain such a high neutron flux.

INCREASED YIELD OF G A M M A - INDUCED E YE COLOUR MUTATIONS FROM CHRONIC VERSUS ACU TE EXPOSURES IN Dahlbominus

W .F . BALDWINDIVISION OF BIOLOGY AND HEALTH PHYSICS,CHALK RIVER NUCLEAR LABORATORIES,ATOMIC ENERGY OF CANADA LIM ITED ,CHALK RIVER, O N T ., CANADA

Abstract

INCREASED YIELD OF GAM M A-INDUCED EYE COLOUR MUTATIONS FROM CHRONIC VERSUS ACUTE EXPOSURES IN D ahlbom inus. The interpretation o f apparent d ifferences in the m utagenic e ffects o f chronic and acute irradiations is often com p lica ted because the exposed ce lls may not have rem ained in com parable stages o f the m itotic c y c le in the two circum stances and because it is usually im possible to test for d ifferential k illing follow ing the two treatments. Thus, in the first case , the early part o f a chronic exposure could have the e ffe c t o f slowing down the rate at w hich ce lls proceed through the subsequent normal sequence o f changes in radiosensitivity . A method o f avoiding this source o f bias in studies o f the eye co lou r mutations o f Dahlbom inus was sought, and it has been found that susceptibility to induced mutation reaches a plateau in aging fem ales about nine days after they b ecom e adult, and that over the follow ing four days the mutation rate maintains a com paratively constant frequency. A ccurate records o f the number o f cocoon s parasitized by Dahlbom inus and the number o f progeny per fem ale have also m ade it possible to test for d ifferential killing by ascertaining whether decreased fecundity is associated with a depression o f m utagenic rates.

Studies o f the e ffects o f 500 R o f gam m a radiation delivered ch ron ica lly over the period o f com parable sensitivity, i . e . over 100 hours from 9 days to 13 days after the pupal stage, and o f the sam e dose as acute exposures at different tim es in this period are now com p le te , and are com plem en ted by sim ilar studies with 250 R. The results with 500 R indicate an approxim ately 20% greater m utagenic e ffe c t o f the chronic as com pared with the acute exposures. At the present t im e , an interpretation in terms o f a possibly greater lethal e ffe c t o f the acute irradiations on ce lls that are potentia lly more susceptible to mutagenesis cannot be ruled out. H ow ever, any contribution from this source should diminish with low er, less lethal doses.The results at 250 R show that the d ifferen ce in m utation frequency with acute as com pared with chronic doses is less at this low er exposure, im plying that the d ifferen ce in e ffe c t is probably the result o f a disproportionately greater k illing by the acute irradiation o f ce lls which would have produced mutations. Records o f parasitization and the number o f progeny per fem ale follow ing acute and chronic exposures at 500 and 250 R g ive con flic tin g results, indicating that d ifferentia l k illing m ay not be a factor in determ ining m utagenesis. The results are based on 923 e y e co lou r mutations in a total o f 346 171 flies scored.

1 . IN T R O D U C T IO N

E a r ly w ork with D ah lbom inus on the e ffe c ts o f acute and c h r o n ic d ose ra te s fa ile d to d em on stra te d if fe r e n c e s in the fr e q u e n c ie s o f in du ced eye c o lo u r m utations in ir ra d ia te d o o g o n ia e x p o se d to s im ila r to ta l d o s e s [1 ] .A fu rth e r e x ten sion o f the s tu d ies , h o w e v e r , did in d ica te a s m a ll but s ta t is t ica lly s ig n ifica n t fa l l in m utation rate with c h r o n ic , as c o m p a re d with acute, e x p o su re s o f oS gon ia [2 ] , an e f fe c t w hich su p p orted r e su lts with o th e r o r g a n is m s su ch as m ice [3] and s ilk w o rm s [4 ] , w h ere m utation ra te s w e re lik e w is e h igh er with acute d o s e s . T he e f fe c t w as m ost apparent in m ic e , and R u s s e ll w as able to sh ow in sp e rm a to g o n ia that

365

366 BALDWIN

e x p o su re r a te s o f 0 .009 R /m in gave on ly o n e -q u a r te r as m any s p e c i f i c lo c u s m u tation s as co m p a re d with a ra te o f 90 R /m in . T h e se r e s u lts , and th ose fr o m oth er , s im ila r s tu d ies , have led R u ss e ll to postu late that so m e step in the ra d ia tio n -in d u ce d m utation p r o c e s s m ay be cap a b le o f r e p a ir , a ph enom enon w hich w ould have im portan t im p lica t io n s in the e st im a tio n o f g en etic h a za rd s in m an. In re a ch in g h is c o n c lu s io n s ,R u s s e ll u sed data fr o m fe m a le m ice , w hich sh ow ed a m uch g re a te r d o s e -r a te e f fe c t than had been o b s e rv e d fo r sp e rm a to g o n ia . In fe m a le m ic e , g e rm c e l l s r e m a in in the d ictya te stage o f the p r im a ry o o cy te f r o m sh o r t ly a fte r b irth until ovu la tion ; thus, the d o s e -r a te e f fe c t in fe m a le s o c c u r r e d in a n on -d iv id in g c e l l popu lation [5 ] . R u s s e ll a lso a tta ch es so m e s ig n ifica n ce to the fa ct that o o c y te s in la te f o l l i c le s ta g e s a re re s is ta n t to k illin g by rad ia tion , a con d ition w hich w ould le s s e n the p o s s ib il it y o f a m utation ra te d if fe r e n c e as a re su lt o f d iffe r e n t ia l c e l l su rv iv a l fo llo w in g acute and ch ro n ic ir ra d ia t io n s .

In the D ah lbom inus w o rk m en tion ed above [2 ] , in w hich a s ig n ifica n t d if fe r e n c e in m utation w as found with the tw o d iffe re n t tre a tm e n ts , fe m a le s ir ra d ia te d as la rv a e w e re a llow ed to d e p o s it e g g s on h ost c o c o o n s fr o m the age o f fo u r days until d ea th ; the p ro g e n y thus in clu d ed in d iv id u a ls fr o m a ll s ta g e s o f oO gen es is . A s show n e a r l ie r [6 ] , m utation ra te s in fe m a le s in c r e a s e sh a rp ly w ith age, and in adults ir ra d ia te d at d iffe re n t ag es the n u m ber o f m utants at fo u r days is a p p ro x im a te ly f iv e t im e s g re a te r than in pupae, p re su m a b ly the re su lt o f an in c r e a s e in the n u m ber o f h igh ly s e n s it iv e o o c y te s . In the p re se n t e x p e r im e n ts , it w as d is c o v e r e d that D ah lbom in us fe m a le s ir ra d ia te d at 9, 11, and 13 d a y s , w hen on ly m a tu re o S cy te s w e re p re se n t in the o v a r io le s , gave co m p a ra b le m utation fr e q u e n c ie s . Thus, c h r o n ic e x p o su re s cou ld be a d m in iste re d o v e r th is p e r io d f o r c o m p a r is o n w ith acute d o s e s . U n der th ese co n d itio n s , the fr e q u e n cy o f m utation w as found to be lo w e r with acute e x p o su re s than with c h r o n ic , a r e su lt w hich w as the r e v e r s e o f the e f fe c t show n by R u s s e ll in m ic e .

2. M A T E R IA L S AND M ETH ODS

An advantage o f D ah lbom in us as a su b je c t f o r g e n e t ica l s tu d ies is that a ll m u tation s in du ced in fe m a le .u n fe r t il iz e d g e rm p la sm w ill ap pear in f ir s t gen era tion pa rth en ogen etic h ap lo id m a le s . In addition , the s c o r in g o f eye c o lo u r m utants in th is s p e c ie s is c o m p a ra t iv e ly e a sy , the c o lo u r s ap pearin g as m utant ph enotypes r u s s e t (ru ), chestnut ( c s ) , ca rm in e (c ) and c la r e t (c t ) , c o n tra stin g sh a rp ly with the a lm o st b la ck w ild type eye c o lo u r . A s d e s c r ib e d re ce n t ly , th ese ph enotypes can a r is e at a m in im u m o f 8 lo c i , d em on stra tin g tw o lin k age g rou p s and tw o a l le l ic s e r ie s o f th ree a l le le s each [7 ] . A d e s c r ip t io n o f the b io lo g y o f D ah lbom in us and the d iffe re n t eye c o lo u r s has been pu b lish ed e ls e w h e r e [8 ] .

Ir ra d ia tio n o f e x p e r im e n ta l fe m a le s w as done d a ily in .s m a ll p la s t ic c o n ta in e rs at about 200 fe m a le s p e r day . T re a tm e n ts at both acute and c h r o n ic ra te s w ere continu ed until s u ffic ie n t unm ated fe m a le s had been p la ce d with c o c o o n s to p ro d u ce at le a s t 35 000 m a le s . The acute e x p o su re s w e re done with a G am m abeam 150 (C o m m e r c ia l P ro d u c ts , A E C L , Ottawa) con ta in in g a p p ro x im a te ly 700 Ci o f 60C o ; f o r c h r o n ic ir ra d ia t io n s , la stin g o v e r fo u r days at each e x p o su re , a s m a lle r s o u r c e o f about 100 m C i o f

S M - 1 0 2 / 3 7 367

60C o w as p la ce d in a ch a m b e r w h ere te m p e ra tu re and h u m id ity cou ld be m aintained at r e a r in g r o o m le v e ls . T he in s e c ts w e re p o s it io n e d at a p p rop r ia te d is ta n ce s to g ive the lo w e st p o s s ib le c h ro n ic e x p o su re fo r a fo u r -d a y p e r io d . The d ose ra te s f o r the 500 and 250 R to ta l e x p o s u re s in th ese te s ts w e re g iven at an acute ra te o f 100 R /m in , w hile the c h r o n ic d o se ra te s am ounted to 0 .082 and 0.041 R /m in , r e s p e c t iv e ly . The to ta l d o s e s and d ose ra te s w e re m e a su re d by m ean s o f a c a lib ra te d E . I . L . p o r ta b le e le c t r o m e t e r (M o d e l 37A ).

3. O BSE RVA TIO N S

3 .1 . M utation fr e q u e n c ie s in b r o o d s o f ad vancin g ages

S ince p r io r r e s u lts had show n that age w as a d e te rm in in g fa c to r in the in c r e a s e in m utation se n s it iv ity o f D ah lbom in u s, the f i r s t e x p e r im e n t o f th is s e r ie s w as d es ign ed to c o m p a re the m utation fr e q u e n c y in e g g s d e p o s ite d by unm ated fe m a le s at on e , tw o, th re e and fo u r days a fter ir ra d ia t io n . D a ily e x p o s u re s o f s u c c e s s iv e g rou p s o f unm ated fe m a le s at fo u r days o f age w e re continu ed (e x p o su re 1000 R at 100 R /m in ) until a su ffic ie n t n u m ber to p ro d u ce at le a s t 3500 m a le s had b een p la ce d with h o s t c o c o o n s . T h e c o c o o n s in e a ch g rou p w e re r e p la c e d ea ch day o v e r a fo u r -d a y p e r io d , p ro d u c in g se p a ra te b r o o d s fo r e a ch o f the fo u r days (F ig . 1). The m utation fre q u e n cy , o r ig in a lly at 600 (X 1 0 "5) on the f ir s t day, f e l l ra p id ly to a le v e l at fo u r da ys , a p p rox im a tin g the fr e q u e n c ie s show n p r e v io u s ly f o r o o g o n ia (50 X 1 0 '5) (B a ldw in [1 ] ) . T h is fin d in g p ro v e d that v a r ia b le s e n s it iv ity in v a r io u s s ta g e s o f o ô g e n e s is w ould con stitu te a s o u r c e o f s ig n ifica n t e r r o r in m utation fr e q u e n c y te s ts , e s p e c ia lly i f adult fe m a le s w e re le ft with c o c o o n s fo r s e v e r a l days a fte r ir ra d ia tio n . T h us, in th is w ork , a ll fe m a le s w e re re m o v e d fr o m c o c o o n s a fte r 24 h o u rs . T he sam e fa c to r w ould be im p ortan t with c h ro n ic trea tm en ts exten d in g o v e r s e v e r a l days, e s p e c ia l ly i f the e a r ly part o f the e x p o su re s low ed dev e lop m en t, and a study o f eg g p ro d u ctio n in unm ated fe m a le s w e re undertaken to d e te rm in e when ir ra d ia t io n s cou ld be con fin ed to a s in g le stage fo r both the acute and ch r o n ic tre a tm e n ts .

3 .2 . O flgen esis in o v a r io le s

Unm ated fe m a le in s e c ts w e re d is s e c te d at the a g es o f 60, 132 and 252 h ou rs a fte r e m e r g e n c e fr o m the pupal sta g e . T he re p ro d u c tiv e sy s te m s w ere te a se d aw ay fr o m su rrou n d in g abdom in a l t is su e and d isp la yed on a s lid e in a sm a ll d rop o f w a ter . In F ig . 2(a) it can be se e n that each o v a r y i s c o m p o s e d o f s e v e n o v a r io le s each em p ty in g in to a com m on ov id u ct . N o rm a lly , the e g g s w ould be fe r t i l iz e d sifter le a v in g the o v id u ct.In th ese e x p e r im e n ts , h o w e v e r , a ll m a le s w e re d is ca rd e d as pupae m aking it im p o s s ib le fo r fe r t iliz a t io n to o c c u r . At 60 h ou rs o f age (F ig . 2 (b ) ) the g e rm a r iu m con ta in ed n u m erou s oO gonia, w hile im m a tu re o S cy te s (w ith n u rse c e l ls ) and m atu re oO cytes , re a d y to be d is ch a rg e d , w e re p resen t in the o v a r io le . In the o v a r io le s fr o m 1 3 2 -h ou r fe m a le s (F ig . 3 (a)), a ll t r a c e s o f oO gonia had d isa p p e a re d , and on ly im m a tu re and m ature o 6 cy te s w e re v is ib le . In the la s t illu s tra t io n (F ig . 3 (b )) , at 252 h o u rs , on ly m atu re o ficy te s w e re p re se n t, a r e su lt w hich in d ica ted that fe m a le s

368 BALDWIN

800 -

700 -

-ï 600 r X (38/6184)

BROOD I BROOD 0 BR O O D Ш BROOD 12

FIG. 1. D ecreasing m utagenesis w ith increasing age in irradiated D ahlbom inus fem ales.

ir ra d ia te d at th is age w ould p rod u ce on ly m atu re oS cy te s , at le a s t f o r the f ir s t 24 h o u rs . In th ese o ld e r f l ie s , d is in teg ra tin g oC cytes (s e e F ig . 3 (b )) w e re found in m any in d iv id u a ls .

F r o m th ese d is s e c t io n s , it ap peared that the p rod u ction o f new eg gs c e a s e d f iv e days a fter e m e r g e n c e : by ten days, a ll e v id e n ce o f oO gonia and im m a tu re o ô c y te s had d isa p p ea red , p ro v in g that on ly m atu re oO cytes w ould be p re se n t fo r ir ra d ia tio n at th ese a g e s .

T o substantiate th ese fin d in gs fu rth er , coun ts o f m atu re oO cytes in the o v a r ie s w ere m ade o v e r the p e r io d fr o m e m e rg e n ce to 20 days o f age. In F ig u re 4, the a v era g e n u m bers o f m a tu re oO cytes fr o m eight fe m a le s at d iffe re n t ages show that the to ta ls r o s e to a p lateau at f iv e d a ys ; fr o m h e re the n u m bers p re se n t rem a in ed constan t to about ten days o f age, w h ere a gradu a l d e c r e a s e began , the re su lt o f the r e -a b s o r p t io n o f o ld e r oO cy tes . T hus, it w as co n c lu d cd that the n u m ber o f h igh ly m utable m a tu re oO cytes re m a in s con stan t o v e r a c o n s id e r a b le p e r io d , and that it w ould be p o s s ib le to a d m in is te r c h ro n ic d o s e s during a p e r io d when se n s it iv ity to m utation w ould rem a in c o m p a ra t iv e ly con stan t.

3 .3 . M utation fr e q u e n c ie s at d iffe re n t ages

T o co m p a re the m utation fr e q u e n c ie s o v e r the p e r io d when co m p a ra b le n u m b ers o f o figon ia w ere found in the o v a r io le s , fe m a le s w ere ir ra d ia te d (500 R at 100 R /m in ) at 7, 9, 11 and 13 days a fte r e m e r g e n c e . The r e s u lts , in T ab le I, sh ow that the fr e q u e n c ie s at th ese ages w e re , in fa c t .

(a)

S M - 1 0 2 / 3 7 369

S - SPERM A TH ECA AG - A CCESSO RY GLAND G -• GLAND ULAR T IS S U E

( b )

- OVIDUCT

NURSEC EL LS

FIG. 2 . (a) S ch em atic drawing o f ovary o f Dahlbom inus fem a le , showing 7 ovarioles on each side.(b) O variole at 60 hours o f age, illustrating presence o f oogon ia , im m ature oocy tes , and m ature oocytes.

(a)N URSE C E L L S

IMMATURE OOCYTESHRINK ING FROM O VA R IO LE W ALL

FIG. 3. (a) O variole at 132 hours, showing absence o f oogon ia l ce lls . (b) O variole at 252 hours (10 days) showing absence o f a ll stages e x cep t m ature oocytes.

3 7 0 BALDWIN

AGE IN HOURS (DAYS) AFTER EMERGENCE

FIG. 4. Increasing numbers o f m ature oocytes in aging fem ales w ith plateau at tí to 10 clays.

T A B L E I. M U T A T IO N F R E Q U E N C IE S O V E R T H E P E R IO D F R O M 7 T O 13 D A Y S A F T E R E M E R G E N C E O F A D U L T F E M A L E S (5 0 0 R at 100 R /m i n ) .

A g e (days) O ffsprin g M utants M ean freq u en cy ( x 1 0 s)

7 57 9S4 160 276 ( 3 1 1 - 240)

9 46 831 140 298 ( 3 4 0 - 253)

11 3 5 1 3 6 129 3G7 ( 4 2 6 - 307)

13 4 У 987 176 3 5 2 ( 3 7 4 - 310)

C on tro l 42 048 8 19 ( 2 9 - 8 )

a lm o st co m p a ra b le in m agn itude. T h e le v e l at 11 days w as h ig h er than the 13 -d a y value ow in g to a c lu s te r o f seven c la r e t eye c o lo u r m utants in th is g rou p ; when the c lu s te r w as c o n s id e r e d as one m utation , the ca lcu la te d fr e q u e n cy w as e x a c t ly equ al to the value at 13 days (350 X 1 0 '5 ).

S M - 1 0 2 / 3 7 371

AGE OF ADU LT F E M A LE S

FIG. 5. M utation frequencies fo llow in g irradiation o f fem ales o f d ifferent ages at 500 R (100 R /m in ).

T h e plateau fr o m 9 to 13 days (F ig . 5) con stitu ted a fo u r -d a y p e r io d when ch ro n ic e x p o su re s cou ld be a d m in iste re d f o r c o m p a r is o n with com b in ed v a lu es fr o m the acute ir ra d ia t io n s at 9, 11 and 13 da ys .

3 .4 . M utation fr e q u e n c ie s with acute and c h r o n ic d o se ra te s

E x p e r im e n ts to c o m p a re the e f fe c t s o f acute and c h r o n ic rad ia tion w e re co m p le te d at two d ose le v e ls , 500 R and 250 R . At the h ig h er d o se , the acute value w as d e r iv e d by co m b in in g the data fr o m 9, 11 and 13 days show n in T a b le I and F ig . 5. At 25Ó K, fe m a le s at 11 days o f age w ere u sed fo r acute ir r a d ia t io n s . C h ron ic e x p o s u re s fo r both d o s e s exten ded fr o m 9 to 13 days a fte r e m e r g e n c e , at 0.08 R /m in fo r the 500 R d o se and at 0 .04 fo r the 250 R e x p o s u re .

As show n in T a b le II, the m utation fr e q u e n c ie s fo r the c h ro n ic ir ra d ia t io n s a re h ig h er at both d o se le v e ls than in the acute e x p o s u r e s .At 500 R, the d if fe r e n c e is s ig n ifica n t ( P < 0. 002); at the 250 R e x p o su re the l im its o v e r la p . In F ig . 6, an a lm o s t e x a ct l in e a r re la t io n s h ip e x is ts betw een the v a lu e s at 0, 250 and 500 R; a lso , the d if fe r e n c e in e f fe c t at the tw o d ose le v e ls o f ch r o n ic ir ra d ia t io n is ap parent in the f ig u r e .

3 .5 . F ecu n d ity o f fe m a le s fo llo w in g acute and ch r o n ic e x p o s u re s

In th e se s tu d ies with D ah lbom in u s, a te s t f o r b ia s in v o lv in g d iffe r e n t ia l k illin g o f p o ten tia lly m utant e g g s is a v a ila b le f r o m re a r in g data. In the d o se ra te e x p e r im e n ts , a ccu ra te r e c o r d s o f the n u m ber o f c o c o o n s p a ra s it iz e d by the fe m a le s and the n u m ber o f h ap lo id m a le p rog en y p rod u ced by the fe m a le s w e re m a in ta ined . T he r e s u lts (T a b le П1) show f ir s t that the p e rce n ta g e o f fe m a le s p a ra s it iz in g c o c o o n s and the n um ber o f v ia b le e g g s p e r fe m a le w e re both re d u ce d in the ir ra d ia te d g ro u p s .

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T A B L E II. D A T A F R O M T E S T S O F A C U T E A N D C H R O N IC IR R A D IA T IO N S A T 500 A N D 250 R

Exposure (R) R /m in O ffsprin g M utants M ean freq u en cy (x 1 0 s )

C lusters

500 100 131 954 455 377 ( 3 6 3 - 311) 4 ( 7 , 2 , 2 . 2 ){a )

500 0 .0 8 62 966 270 428 ( 4 7 1 - 3S5) 1 ( 2 )

250 100 57 780 99 171 ( 1 9 9 - 143) 2 ( 2 ,4 )

250 0 .0 4 51 423 101 196 ( 2 7 7 - 165) 1 ( 2 )

C on tro l - 42 048 g 1 9 ( 2 9 - 3 ) -

(a )Eye c o lo u r o f a ll in d iv id u a ls in a l l clusters was c la re t

FIG. 8. M utation frequencies fo llow in g acute and chron ic irradiations at 500, 250 and 0 R.

At 500 R, the c h ro n ic ir ra d ia t io n gave le s s p a ra s it iza t io n and fe w e r o f fs p r in g p e r fe m a le , w hile at 250 R, th ese tw o f ig u r e s w ere r e v e r s e d , w ith ch r o n ic e x p o su re s g iv in g s lig h tly m o r e in both c a s e s . T h u s, it d o e s not s e e m that d if fe r e n t ia l k ill in g in the eg g stag e cou ld p o s s ib ly be im p orta n t in d e te rm in in g the re la t iv e m utation fr e q u e n c ie s fo llo w in g acute and c h r o n ic e x p o s u r e s .

T A B L E III. N U M B E R O F P A R A S IT IZ E D H O S T C O C O O N S A N D N U M B E R O F P R O G E N Y P E R F E M A L E F O L L O W IN G A C U T E A N D C H R O N IC E X P O S U R E S A T 500 A N D 250 R

Exposure Exposure ra te

N um ber o f c o c o o n s

N um berparasir ized % P

N um ber o f fem a les

N um ber o f m ales

M ea n n u m ber o f m a les P

500 A c u te (a ) 2532 2 0 3 3 (b ) SO. 4 2038 3 5 1 3 6 1 7 .4

500 C h ron ic S225 406 6 6 0 .3 < 0 .0 0 1 49 6U 62 966 1 2 .3 < 0 .0 0 1

250 A cu te 5167 301 6 5 8 .3 3 01 6 . 57 780 1 6 .1

250 C h ron ic ' 4321 2S23 65 . 3 < 0 .0 0 1 2823 51 423 1 8 .6 < 0 .0 0 1

C o n tro l - 1305 1100 8 4 .2 < 0 .0 0 1 11ÜÜ 42 043 3 8 .2 < 0.U01

(a ) D ata from 11 days o f a g e o n ly used in this ta b le .(b ) N ot s ig n if ic a n t ly d iffe re n t from co n tro ls (P > 0 .0 5 ) .

SM

-102/37 373

374 BALDWIN

T h e m ost im p ortan t fa c t to e m e r g e fr o m th is r e s e a r c h w as that m utation fr e q u e n c ie s fo llo w in g acute e x p o s u re s w e re not s o h igh as th o se f r o m c h r o n ic d o s e s in m atu re o ficy te s at one stage o f d ev e lop m en t and at tw o d iffe re n t d ose le v e ls . The e x p e r im e n ta l c o n d itio n s in D ah lbom in u s w e re unique, s in ce adult fe m a le s w e re held fo r ir ra d ia t io n until 9 to 13 da ys , when on ly m atu re o flcy te s w e re p re se n t in the o v a r io le s . T h us acute and ch r o n ic d o s e s cou ld be a d m in is te re d o v e r a p e r io d of fo u r days w hile the g e rm c e l l s rem a in ed in the sa m e stag e , a situ ation w hich has not b een a ch iev ed in o th er o r g a n is m s , with the p o s s ib le e x ce p t io n o f d ictya te oO cytes in the m o u se . In D ro so p h ila , P u rd o m [9] s ta te s that the v a r io u s s ta g e s o f s p e rm a to g e n e s is v a ry c o n s id e r a b ly in th e ir s e n s it iv ity to ir ra d ia t io n , and s t r ic t ly c o m p a ra b le sa m p lin g fr o m s u c c e s s iv e s ta g e s o f the d e v e lop m en t o f the f ly is im p o s s ib le w hen d iffe re n t p e r io d s a re u se d . "He d o e s co n c lu d e , h o w e v e r , that a d o s e ra te e f fe c t c o m p a ra b le to that in the m ou se d o e s not o c c u r in the m o r e m a tu re sp e rm a to g o n ia l c e l l s o f D ro s o p h ila . T a z im a [10] b e l ie v e s that tw o ty p es o f d o s e -r a te d ep en d en ce o c c u r in young g on ia l s ta g e s o f the s i lk w o rm , one co n s is t in g o f a lo w e r m u tagen ic e ffe c t iv e n e s s in c h ro n ic ra th e r than in acute ir ra d ia tio n , and anoth er in w hich c h r o n ic e ffe c ts a re h ig h er than fo r acute tre a tm e n ts . H ere again , h o w e v e r , th is author s t r e s s e s the co m p le x ity in in te rp re tin g d ose ra te data due to d ev e lop m en ta l sta g e . In D ro so p h ila , O s te r [11] sta tes that despite,, the la r g e - s c a le w ork a lre a d y c a r r ie d out, th e re is s t i l l no c le a r - c u t b a s is f o r a d ose ra te e ffe c t in the p ro d u ctio n o f point ch an ges in im m a tu re fe m a le c e l ls , and the w ork h as been co m p lica te d by "d if fe re n t ia l r a d io s e n s it iv ity m ask in g re p a ir m e c h a n is m s " . T hus, R u s s e ll ’ s r e su lts sh ow in g s ig n ifica n t m u tagen ic d if fe r e n c e s with d ose ra te , and h is c o n c lu s io n s co n ce rn in g p rem u ta tion a l r e p a ir in m ou se c e l ls stand a lone, se e m in g ly u nsupp orted by c o n c re te e v id e n ce fr o m o th er o r g a n is m s .

In c o n s id e r in g the r e s p o n s e s to m utation at the tw o d ose le v e ls in D ah lbom in u s, it w as in te re s t in g that the d if fe r e n c e betw een the acute and c h r o n ic trea tm en ts w as g re a te r at 500 than at 250 R. A s the lo w e r d ose can be p re su m e d to be le s s le th a l than the h igh er one, th is r e su lt w ould be e x p e cte d i f the acute ir ra d ia t io n s e le c t iv e ly k illed c e l ls w hich w ould h ave p ro d u ce d eye c o lo u r m u ta tion s. The p a ra s it iza tion and p rog en y r e c o r d s re v e a l, h ow ev er , that m o re c o c o o n s w e re p a ra s it iz e d and m o re p ro g e n y w e re p rod u ced fo llo w in g acute ra th e r than ch ro n ic e x p o su re s at 500 R, and that th ese r e su lts w ere r e v e r s e d at 250 R. Thus, it s e e m s im p o s s ib le that s e le c t iv e k ill in g o r s e le c t iv e su rv iv a l o f p oten tia lly m u table c e l ls cou ld accou n t fo r the s m a lle r d if fe r e n c e in m utation rate at a lo w e r , le s s leth a l d o s e . F u rth er, the n u m ber o f p rogen y fr o m ir ra d ia te d fe m a le s in th is w o rk w as le s s than h a lf the n um ber p ro d u ce d in c o n t r o ls . T hus, e x ten siv e k illin g o c c u r r e d in m atu re oO cytes, an e ffe c t w hich did not s e e m to p ro d u ce d iffe re n t ia l m o r ta lity in p o ten tia lly m utable c e l l s .


T he author w ish es to e x p r e s s h is s in c e r e ap p rec ia tion to D r . H .B . N ew com be fo r h is continu ing in te r e s t in the w ork , to

4 . D ISC U SSIO N

S M - 1 0 2 / 3 7 3 7 5

M e s s r s P . and A . Knight fo r th e ir e x ce lle n t te ch n ica l a s s is ta n c e , and to M iss R . T an n er, who, as a su m m e r student, stud ied the r e p ro d u c t iv e p r o c e s s e s in D ah lbom in us r e p o r te d h e re .

R E F E R E N C E S

[1 ] BALDWIN, W .F . . R adial. Res. 21 (1962) 121.[2] BALDWIN, W .F . . M utation Res. 2 (1965) 55.[3] RUSSELL, W .L . et a l, S cien ce 128 (1958) 1546.[4] T A Z IM A , Y .S . et a l, G enetics4Ô "(1961) 1335.[5] RUSSELL, W -.L ., P roc. C on f. on M ech . o f the D ose Rate E ffect o f Radiation, O iso, N ov. 4 - 1 1964,

128.[6] BALDWIN, W .F ., M utation Res. 2 (1965) 530.[1] BALDWIN, W .F . et a l. C an . J. G enet. C y to l. 2 (1966) 245.[8] BALDWIN, W .F ., et a l, C an. ] . G enet. C y to l. 4 (1964) 453 .[9] PURDOM, C .E . , M cSllEEHY, T .W . , Int. J. Radiât. B iol. J7 (1963) 265.

[10] T A Z IM A , Y . , Proc. C on f. on M ech . o f the D ose Rate E ffect o f Radiation, O iso , N o v .2 -1 1964, 6S.[11] OSTER. 1 .1 ., Proc. C on f. on M ech . o f the Dose Rate E ffect o f Radiation, O iso, N o v .2 -7 1964, S3.

D I S C U S S I O N

R . C . VON B O R S T E L : I sh ou ld lik e to poin t out that D r. Baldw in is one o f the few in v e s t ig a to rs who is in the p r o c e s s o f p ro v id in g m utation data w h ere m utation fr e q u e n c ie s can actu a lly be an a lysed at fr e q u e n c ie s be low the spontan eous m utation le v e l . By p lo ttin g the data on a lo g - lo g p lo t o f m utation fr e q u e n cy v e rs u s d o s e , and by su b tra ctin g the sp ontaneous m utation fr e q u e n c ie s fr o m the in du ced m utation fr e q u e n c ie s , it can be d e term in ed w heth er lin e a r ity is m ain ta ined , even at the v e r y low d o s e s w here the spontan eous m utation data and the in du ced m utation data appear to m e r g e .

REPROD UCTIVE PER FO R M AN C E OF F E M A L E BRACONIDS COM PARED A F T E R (A) BRIEF AND (B) P R O TR A CTED EXPOSURES TO IONIZING RADIATIONS

D .S . GROSCH GENETICS DEPARTMENT,NORTH CAROLINA STATE UNIVERSITY,RALEIGH, N. C . , UNITED STATES OF AMERICA

Abstract

REPRODUCTIVE PERFORMANCE OF FEMALE BRA CO NIDS COMPARED AFTER (A) BRIEF AND (B) PROTRACTED EXPOSURES T O IONIZING RADIATIONS. 85Sr sources are installed in the in itia l US b iosatellites to provide ca lcu la b le dose levels during three-d ay orbita l space flights. Such protracted exposures are longer than those used custom arily in insect rad iob io logy and shorter than those o f e co lo g ica l studies. This paper concerns results from the ground controls o f ill - fa te d US B iosatellite A and com pares them with results from other dose rates and types o f radiation.

Males are packaged separately and used for m utational studies to be reported elsew here. T o com pare the vulnerability o f c e ll types in the ov a r io le sequence, nearly 1000 virgin fem a les from a vigorous ou t- cross are used, h a lf for ground controls and half for the sate llite launched. Sam ples o f 2 0 -2 5 wasps are packed in each o f two screw -capped capsules inserted into housings in p lastic m odules w hich also incorporate thermisters and radiodosim eters. These packages are fixed in shielded positions as w ell as in p laces w hich re ce iv e one o f four leve ls o f gam m a rays from the 85Sr source. Each treatm ent thus consists o f 4 0 -5 0 virgins; a sim ilar number receives a sensitizing preflight exposure to 2000 R o f X -rays. A fter the flight period da ily egg production is scored to d etect resorption fo llow in g gross chrom osom al dam age, em bryon ic deaths to re vea l m ore subtle dam age, and maternal l i fe span as a measure o f som atic fitness.

In most insects bundles o f numerous ovarioles confound interpretation relating c e ll status during e x posure to eggs deposited subsequently. H abrobracon*s four synchronized ovarioles provide a uniquely suitab le system for studying radiosensitivity o f a sequence com p le te from sp ec ia lized oocytes through o o c y te - trophocyte units to prim itive interphase ce lls . F o llow in g a series o f doses, the fa m ily o f oviposition curves reflects the vulnerability o f d ifferentiating units in a v a lley w hich deepens and broadens w ith increased dose. At high dose rates, low est egg production occurs on day 7. The pattern, w ell established for X -rays and :,2P 6 -rays, has now been dem onstrated for y -rays from the Raleigh 60C o G a m m a cell and the Woods H ole 137Cs source, although 1 .4 tim es the X -ra y dose is required to produce the sam e am ount o f dam age. When the gam m a exposure was spread over three days the v a lley did not appear until the eighth day. Fem ales tested sim ultaneously and treated id en tica lly except that their X -ra y exposure was brie f provided the usual 7th-day low . Previously v a lley deferm ent was observed on ly when studying ingested radioisotopes where a necessary prestarvation obscured the sign ifican ce . Another unpredicted response to the protracted gam m a dose was a control lev e l productivity o f eggs derived from exposed oogon ia . R ecovery m echanism s w ere evidently ab le to keep p a ce w ith radiation dam age. On the other hand, em bryon ic leth ality was related to dose during all periods studied and dose rate was not important except for the m ost m ature oocytes. As scored by von Borstel categories, stage I deaths decreased and stage III types increased w ith age o f the mothers until the latter predom inated. This change is a ge-re la ted and not dose-dependen t. B ioch em ica lly , predom inance o f stage III death can b e induced, even in eggs from young mothers, by feed in g either RNA or protein inhibitors, but not by interfering with DNA synthesis. W e postulate ph ysio log ica l involvem en t o f nurse ce lls and fat body or their interrelations. M ean l ife span ranged from 19.52 ± 1 .3 4 days after 4320 R to 22.35 ± 0 .9 1 for controls.

B iosatellite B, the second effort, was recovered successfully. A fter two days in orbit the reproductive perform an ce o f fem ales d iffered strikingly from that repeated in ground controls. Most significant was egg deposit in a nearly contourless pattern rem iniscent o f a com pensatory response to m ito tic inhibition.

377

3 7 8 GROSCH

The release of "sterile" females is often a feature of experimental insect control. They may serve as an infertile siphon for sperm from fertile males or they may be a convenient way to avoid sexing a.large sample. For an understanding of the sterilization of female insects it is important to distinguish between the agent's effects upon egg production and its effects upon the hatching of any eggs laid. These types of responses to radiation depend upon differences in the vulnerability of a variety of cell types.

The greatest variety of cell types occurs in the polytrophic ovariole which is the functional unit typical of the ovaries in holometabolous orders and a few others as well. In the polytrophic

type of egg-forming tube, specialized nurse cells accompany each oocyte and both the differentiation and the functioning of the entire follicle- enclosed unit are important. An investigation merely concerned with scoring viable offspring provides no basis for analysis of the cytological action of the agent employed.

Even when egg production is recorded, the pattern of response may be obscured by an unsynchronized ovariole performance. This is likely to occur in species characterized by large bundles of egg tubes when they engage in continuous oviposition. A favorable system for studying radiosensitivity of the ovariole cell sequence is provided by a small number of synchronized ovarioles per ovary. The parasitoid wasp Bracon hebetor=Habrobracon juglandis offers such an advantage with.only two synchronous ovarioles per ovary.

This year we had the opportunity to use braconids in experiments with Sr85 gamma ray sources which provided dose rates and exposure times differing from those usually used in insect radiobiology or ecological studies. The radiation sources were installed in U. S. biosatellites A and В and duplicate sources were employed for ground controls. In addition to 2 and 3 day gamma.ray exposures a brief preflight exposure to X-rays was given to a group of wasps. This paper compares reproductive performance for biosatellite experiments with results for experiments using other dose rates and types of radiation. Criteria of damage scored include the pattern of egg production, egg hatchability, and the stage of development■achieved before embryonic death. Since records are obtained from youth into senility, present results throw light upon the influence of aging on reproductive performance. Space flight introduces additional experimental conditions to which, biologists have given little attention (mechanical vibration) and for which there has not been study opportunity (low gravity) .

Of broad interest in bioastronautics is the general question of the performance during space flight of progenitive tissue with a stem cell component. The insect ovariole with its single series of units including all stages from fully differentiated products (the eggs) to interphase

oogonia seems ideally suited for the studies. Eggs are dispensed in order and those deposited at any designated time can be traced back to their cytological condition during the exposure period. Quantitative modifications in numbers of eggs or hatchability may thus be correlated with the cellular state and critical periods of sensitivity identified.

MATERIALS AND METHODS

A vigorous wild type Bracon stock collected in Raleigh, North Carolina provided females used in the first biosatellite attempt

SM -102/38

(December 14-17, 1966). Phenotypically wild type females heterozygous for three linked marker genes provided females for the second experiment (September 7-9, 1967). The genetic background came from a wild type stock collected in Lumberton, North Carolina.

Newly isolated virgin females were packed in screw-capped plastic capsules incorporating glass rod dosimeters. In turn the capsules were inserted into housings in plastic modules containing thermistors. The completed packages were fixed within the Experiment Capsule in positions aft of a radiation shield and a fore group arranged concentrically around the Sr85 source. Positioning was such that four different radiation doses were experienced by samples of wasps in the fore group of experiments.

Each position held two samples of 20-25 wasps each so that 40-50 virgins experienced each treatment. Included in shielded position were females which had been X-rayed with 2,000 R. In analysis of data, homogeneity was ascertained before pooling the replicated data for presentation of results. Ground controls duplicated the shielded and exposed position for samples of virgins. Only the ground controls were available for study when Bios A failed to respond to retro-rocket firing command. Both ground control and flight animals were obtained from the second attempt, Bios B.

During the experiments females were maintained at 19-21° C.After recovery they were provided with a constant supply of host caterpillars and incubated at 30 C. During the first day, transfers were made every two hours for a study of genetic damage to metaphase and prophase oocytes by R. C. Von Borstel and R. H. Smith of Oak Ridge. Subsequently, the collection of eggs and provisioning with a fresh host caterpillar per female followed a daily schedule. Routinely eggs were immersed in mineral oil for determining their hatchability. This is an empirical method which not only minimizes variability but also expedites scoring inviable embryos by Von Borstel [1] categories. The stage of development achieved in each unhatched egg was determined. Life span was recorded for each of the ovipositing females in control and experimental groups.

RESULTS

EGG PRODUCTION

Bios A Ground Controls

After irradiation the more sensitive cells deteriorate and debris is resorbed. Subsequently, egg deposit is decreased on the day for which the resorbed cells would have produced eggs. Typically the ovariole response to an acute dose of ionizing radiation has been in the form of a trough or valley wherein lowest egg production was achieved by the seventh day of oviposition. In the Bios A experiment this response was demonstrated for the 2,000 R X-ray exposure given before packaging of a group of females (Fig.l, upper).

A consistent difference in oviposition pattern resulted from spreading doses of Sr85 gamma rays over three days of continuous exposure. As shown In Flg.l (lower), the valleys characteristic of radiation damage developed their low points on the eighth day of oviposition. The entire family of curves shows this displacement or deferment in the pattern of response. In descending order 3-day gamma ray doses were 4,320 R, 2,410 R, 1,320 R, and 650 R obtained by position

3 8 0 GROSCH

D A Y SFIG. 1. Mean daily egg production p lotted for sam ples o f 40 braconid wasps. T h e upper graph gives the pattern o f response to an acute dose o f 2000 R o f X -rays. T h e low er one plots data from unirradiated controls and from four doses o f 8SSr gam m a rays delivered over a three-day period. T h e fam ily o f curves showing an eighth day dip was obtained after G50, 1340, 2410, and 4320 R, respectively .

GAMMA

______ 1______ i i______ I______ i______ i i______ i______ I______ I______ ;______ t i______ I______ j______ I______ I______ I______ I I

5 10 15 20D A Y S

FIG. 2. M ean daily egg production for sam ples o f fem a le braconids subsequent to rem oval from the ground experim ents v e h ic le (upper graph), and subsequent to recovery from B iosatellite В (low er graph). The latter wasps had experienced 30 orbits 170 m iles in space. In each case curves have been drawn for un irradiated controls, for wasps rece iv in g an acute preflight dose o f 2000 R o f X -rays, and for wasps rece iv in g 2667 R in a gam m a exposure spread over two days.

S M - 102/38 381

of the package in respect to the source. The top solid line gives the control record. Except for displacement of the low point, the curve shape appears to be essentially unchanged. During the last ten days of study egg production approached or exceeded control values.

Bios J5 Ground Controls and Flight Wasps

Oviposition records from females packaged but held on the ground at Cape Kennedy again demonstrated deferred low egg production for wasps receiving protracted doses of gamma rays. The-valley did not reach its low point until the ninth day. Fig.2 (upper) contrasts the daily egg production pattern for females receiving the highest gamma dose of 2,667 R in a two-day exposure with data obtained from a brief exposure to 2,000 R of X-rays. Data from controls are also plotted.

Wasps recovered after a two-day space flight show a strikingly different pattern of oviposition when their gamma ray exposure was spread over most of the two days in 170 mile high orbit. No true valley developed. Instead, after a sloping plateau egg production rose in a fashion tending to compensate for relatively low oviposition during the first week. Fig.2 (lower) shows this pattern along with plots of data from females receiving 2,000 R of X-rays before the flight. Data for unirradiated control wasps shows more than the usual variability when plotted.

A detail of the curve shape in Bios A results which is not typical of Bios В results is a slight second day drop resulting from transporting the females from Cape Kennedy, Florida to Raleigh, North Carolina by plane at winter temperature.

EGG HATCHABILITY

Bios A Ground Controls

In contrast to egg production records, embryonic lethality was not modified significantly by a low dose rate for gamma rays. These results are presented as bar graphs on Figure 3. In order to summarize on thebasis of cell types exposed, egg hatchability data are pooled in groupsorganized with respect to the days of deposit. Metaphase eggs and oocytes in late prophase of first maturation division give rise to ova deposited on the first day. Eggs laid on days 2-5 were prophase oocytesduring the exposure period. Differentiating cells of the transitionalperiod give' rise to eggs laid on days 6-10- Eggs deposited during subsequent periods were derived from cells which were oogonia when treated but we recognize that days 16-20 correspond to a period of maternal senility. On that basis we distinguish it from the 11-15 day period.

The solid bars show how control hatchability decreases with the age of the mother. The diagonally shaded bars represent results from the X-rayed females. Through the important two weeks of the experiment from day 2 through day 15, hatchability after the acute 2,000 R did not differ significantly from the protracted dose results after 2,410 R of gamma rays. The 2,410 R results are represented by the open bar to the left of the shaded bar. Other open bars demonstrate the dose dependent pattern of egg hatching. In each grouping by days the dose decreases from left to right.

382 GROSCH

FIG. 3. Bar graphs sum m arizing hatchability for the eggs o f Fig. 1. A key to dosages in R is g iven at the upper right: -4 = 4320, 2 = 2410 gam m a and 2000 X-rays, 1 = 1340, . S = fi50, and С = con tro l. The shaded bar identifies results after 2000 R o f X -rays. As explained in the text, data are grouped into periods w hich re flect the cy to lo g ica l stage at the start o f exposure.

Bios _B

Analysis of hatchability of eggs laid by Bios В females is still in progress at the time of writing this preprint document. Nevertheless, the excellent hatchability evident in inspection of the raw data provides no indication of synergistic damage from space flight conditions in combination with radiation.

Stage of Embryonic Death

Immersion in clear oil and the thin (2ц) transparent chorion of the braconid egg make it possible by microscopic examination to determine the stage of development achieved by unemerged embryos. Von Borstel's laboratory Г1] has classified stages of embryonic death into five categories. In our scoring of unhatched eggs, stages 1> 3 and 4 accounted for nearly all deaths in Bios A eggs (see Table I). Stage 1 deaths occur before blastoderm formation. State 3 shows that yolk has changed from white to yellow and is becoming localized. Stage 4 has developed to the point where tracheal elements and urate cells are visible.

S M - 102/38 383

Stage 1 deaths decreased and stage 3 types increased with age of the mothers until the latter predominated. Stage 1 showed the dose dependent trend except during the period of maternal senility (Days 14-20). On the other hand stage 3 proved to be primarily age related. Figure 4 provides an impressive demonstration of the increased predominance of

stage 3 deaths, represented by the open circles. The triangles indicate the percentage of unhatched eggs which failed to progress beyond stage 1. Interestingly enough, the fifth type of lethal syndrome in which braconid embryos die late in development and which has been recognized as an enhancement of aging processes [1] did not contribute significantly to lethality in present results.

The statistical analysis of results from Bios В is not yet complete, but again age dependent stage 3 types predominate in lethality records after the first week.

LIFE SPAN

Table II presents a summary of life span for the Bios A females. Evidently conditions of the experiment including handling and packaging do not shorten life span appreciably.

With Bios В females 1007» survival was obtained in both ground controls and flight groups. Furthermore with rare exception all these females survived more than 20 days of study. Final calculation of mean life span for these wasps must be delayed until all of them are dead.

DISCUSSION

The characteristic shape of the egg production curve for irradiated mothers is two hills separated by a valley which broadens and deepens with

increased dose. The hills respectively represent the eggs derived from cells which were differentiated oocytes with nurse cells and from cells which were interphase oogonia at the time of irradiation. Both conditions provide a measure of resistance to the cell destructive action of ionizing radiation but mutational damage is another matter entirely.

Complete sterilization of a female with polytrophic ovarioles requires a radiation dose adequate to guarantee destruction of all oogonia and to induce at least one dominant lethal change in all oocytes. Although oogonial destruction after 5,000 R of X-rays [2] results in a cessation of egg production during middle and old age, the more mature oocytes can develop into eggs after any irradiation short of massive doses in the "knock down" range. Sterilization can be accomplished at moderate doses only because 10,000 R is adequate to induce dominant lethality in all oocytes 13]. These lethal mutations are expressed as embryonic death.

The valley reflects the vulnerability of differentiating units.Five successive mitotic divisions are required to produce the 32-cell oocyte-trophocyte unit from a single oogonium. In addition endomitosis must be accomplished in nurse cells to provide the high polyploidy of the functional state. Both mitosis and endomitosis are vulnerable to radiation damage. However, changes in shape and position of the valley obtained from differing dose rates imply a certain resilience in the cell physiology concerned with normalizing chromosome activity after disruptive ionizations from irradiation.

сосоrfb

T A B L E I. ST A G E S O F D E V E L O P M E N T A C H IE V E D B Y B ra co n E M B R Y O S IN TH E U N IIATCH ED EGGS O F TH E BIOS A E X P E R IM E N T . P E R C E N T A G E S O F TH E T O T A I, D EAD .

Days 2-5 6-10 11-15 16-20

Stages 1 3 4 1 3 4 . 1 3 4 1 3 4

Control 30.0 40.0 30.0 19.0 73.0 7.9 4.6 82.3 13.1 1.1 94.5 4.4

3 day gamma

650 R 45.1 40.8 14.1 8.6 81.5 9.9 4.2 84.2 11.7 3.5 84.8 11.6

1340 R 44.0 54.8 1.2 11.1 84.6 4.3 7.2 84.4 8.3 1.6 90.5 7.9

2410 R 53.7 42.0 3.3 21.6 76.7 1.7 7.5 82.0 10.6 1.3 86.3 12.5

4320 R 56.1 38.6 5.3 23.8 72.1 4.1 13.6 81.9 4.5 1.8 92.1 6.1

Acute X-ray

2000 R 39.1 51.1 9.8 1.2 91.7 7.1 4.5 80.5 15.0 1.7 91.3 7.0

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FIG. 4. T h e re la tive contribution to unhatched eggs by two stages o f em bryon ic death in Bios A ground experim ents. The percentage o f deaths in stage 1 (triangles) and in stage 3 (circles ) are plotted against dose and organized w ith respect tu periods o f deposit. Note that stage ] deaths representing c lea v a g e d ifficu lties show a pronounced dosc-depen den t slope particularly for eggs from days 2 -5 . In contrast is the nearly horizontal distribution o f stage 3 deaths w hich predom inate later deposits.

Fractional dose experiments such as those used for studying chromosomal rejoining mechanisms in plants have provided some insight. By altering the length of the interfraction period [4] or the temperature during interfraction period [5] or by feeding inhibitors of protein synthesis £6] the derivation of eggs from oogonia was much altered.These treatments flattened the post-valley hill of the oviposition curve but did not shift the valley in time. Similarly although egg production changed quantitatively when Co^O doses required a day for their delivery[7] valley position was not shifted. Subsequently a variety of gamma sources including the Woods Hole Csl37 device has been used but no changes in valley position were obtained. The displaced valley reported herein resulted from spreading the gamma ray exposure over three days.This is nearly half of the important first week for insects which have an active life of little more than three weeks. Females tested simultaneously and treated identically except that their exposure was to X-rays and brief, provided the usual low at the end of the first week.

25

T A B L E II. T H E M E AM L IF E S P A N O F F E M A L E B R A C O NIDS F R O M B I O S A T E L L I T E E X P E R IM E N T S

T re a tm e n tD e ce m b e r 196(5

GroundS e p te m b e r 1967

G round F lig h t

C on tro l 2 2 .4 i 0 . 9 2 3 .3 ± 0 . 9 2 5 .1 è 0 .73 2 .1 i 2 . 5 ( a)

P r e -X -r a y e d 200 0 R 2 0 .2 ± 0 . 9 2 1 . 9 ± 1 . 9 2 4 . 2 ± 1. 5

3 - d a y /-r a y s 43 2 0 R 19. 5 ± 1. 3241 0 R 2 2 . 8 ± 0. 91340 R ' 2 1 .8 ± 1. 1

650 R 2 0 .8 ± 1. 5

2 -d a y y-rays 2 3 5 0 R 2 4 . 9 ± 1. 5 3 4 .1 è 4. 4 (a )

1200 R 23 . 8 ± 1 . 9 2 4 . 3 i 1 . 4700 R 17 . 2 ± 2 . 7

2 1 . 4 ± 2 . 32 4 . 5 ± 0 . 5

3 4 o R 2 1 . 6 ± 1. 8 2 3 . 3 i 1 . 4

(a) R e c e iv e d s p e c ia l c a r e d u rin g the last w e e k o f l i f e .

386 G

RO

SCH

SM -102/38 387

Until now we had observed deferment of the valley only when studying females fed radioisotopes I8]. Here too the radiation dose accumulated over a period of days, but the significance of the observation was obscured because of the necessary prestarvation in preparation for ingestion experiments.

That there are efficient recovery mechanisms was revealed by control level production of eggs derived from oogonia of Sr®5 exposed females. Presumably recovery mechanisms can keep pace even during irradiation and, if the dose rate is low enough, presumptive lesions can be corrected before the irrevocable biochemical events develop.

Cells differing in stage may be expected to differ in the efficiency of their recovery mechanisms. Also unless the system is completely'at rest, certain cells may progress to a stage of lower vulnerability while others have entered a stage of high vulnerability before a critical dose has accumulated. The S stage of braconid interphase oogoniá has never' been localized. Perhaps its occurrence is related temporally with the delayed valley.

In these considerations it is important to appreciate that cells do not necessarily die in the stage irradiated. They die when faced with an insurmountable cytological crisis such as the mitotic period and the endomitotic period necessary to differentiate the polytrophic contents of the ovariole. We are not concerned about the follicle because its cells come from a different germ layer and have proved relatively radioresistant.

In contrast to the radiation response a different type of pattern for the oviposition curve was obtained from females irradiated while in space flight. The curve most nearly resembles compensating ovipositional records obtained from feeding mitotic inhibitors [9]. This type of response did not appear either with centrifuged or vibrated females [10]. Therefore, we postulate an effect of low gravity on dividing and differentiating cellular units of the ovariole. A well established way to moderate radiation damage to chromosomes is the use of a mitotic inhibitor such as colchicine [11] . Good egg production and hatchability can be explained by low gravity contributing a similar influence.

Most of this discussion has centered on egg production. This is important not merely as the limiting factor to insect fertility but because it provides the same type of curves seen in vertebrate peripheral hematology [12] and in sperm studies of domestic animals [13]. Now we shall briefly consider matters which become evident after eggs are deposited.

Considerable information is already available on egg hatchability especially for eggs laid during the first week [1]. An additional contribution to our growing understanding of embryonic survival is demonstration of the predominance of stage three deaths when mothers have been ovipositing for a week or more. The change is age-related and not dose-dependent. Depletion of the abdominal fat body and accumulation of urate deposits characterize actively ovipositing braconids. Under such circumstances, although the abdomen's important somatic tissues can sustain life for a normal span, their efficiency in supplying nurse cells with the materials needed for oocytes may decline after a week or more of peak operation. This could mean that compensatory oviposition in flight animals would jeopardize eggs to age defects

3 8 8 GROSCH

by causing more eggs to be deposited late in the mother's life. Even before oviposition began stress on tissue reserves was experienced by both ground controls and flight wasps since they were packaged without a food supply.

Stage three deaths indicate that the embryo was able to accomplish cleavage and form the blastoderm but lacked either the information or the materials to proceed in development. Nuclear imbalance has characterized genetically contrived stage three lethals but we are inclined toward cytosomal defects in explaining the prominance of the syndrome in Bios experiments. Radiation injury need not enter the explanation. A. R. Whiting [14] has demonstrated that cytoplasmic injury does not kill eggs at the doses employed here. Results with chemical agents have strengthened our position. A predominance of stage three deaths can be induced even in young mothers by administering inhibitors of RNA as well as by agents which cause DNA cross linkage [15]. Perhaps cell division and differentiation is proceeding at a faster rate in the ovarioles than the somatic tissues of an aging insect can manufacture and transfer materials to the ovary.

ACKNOWLEDGMENTS

This work has been supported by N.A.S.A. Research Grant NsG 678.It could not have been performed without the cooperation of Dr. R. C. Von Borstel and Dr. R. H. Smith of Oak Ridge National Laboratory nor without assistance from Mrs. Ruth Ann Carpenter, A. C. Hoffman, L. R. Valcovic and many others from the Raleigh campus.

SUMMARY

Three criteria of damage can be employed in radiation experiments with female braconids. Egg production records provide a measure of the gross chromosomal and nuclear damage to a variety of cell types.Egg hatchability reveals more subtle damage, both genic and age related. Life span records reflect the ability of somatic tissues to function for general body maintenance.

The shape of the egg production curve is discussed from the standpoint of appreciating the cytology of the insect ovariole. Hatchability data are analyzed with respect to the stage of embryonic development achieved in unhatched eggs. Large scale experiments performed in conjunction with the first and second U. S. biosatellite experiments provided striking evidence of

(1) a deferred low period of egg production from protracted gamma ray doses delivered in ground experiments,

(2) no pronounced low for females recovered from space flight,

(3) both dose-dependent and age-influenced components of embryonic lethality,

R E F E R E N C E S

[1 ] VON BORSTEL, R. C . , REKEMEYER, M. L . , G enetics 44 (1959) 1053;VON BORSTEL, R. C . , St. AM AND, W . , Repair from G enetic Radiation (SOBELS, F. H . , Ed. ) Pergam on, Oxford (1963) 8.

S M - 102/38 3 8 9

[2 ] GROSCH, D .S . , SULLIVAN, R. L . , Radiat. Res. 1 (1954) 294.[31 HEIDENTHAL, G . , G enetics 30 (1945) 197.[4] GROSCH, D .S . , p. 83 Annual Review o f Entom ology (STEINHAUS, E. Л . , SMITH, R .F . , Eds)

Annual Reviews I n c . , Palo A lto, C aliforn ia (19G2).[5] GROSCI I, D .S . , B io log ica l Effects o f Radiations, B laisdell/G inn, W altham , Mass. (1 % 5 ) 174.[51 GROSCH, D .S . , Radiat. Res. 14 (1961) 470.[7 ] GROSCH, D. S . , Radiat. Res. 9 (1958) 123.[8] GROSCII, D .S . et a l . , Radiat.Res. 5 (1956) 281.[9] GROSCH, D .S . , Ann. ent. S oc. A m . 52 (1959) 294.

[101 GROSCII, D .S . , 3rd. Int. Congr. Radiat. Res. (1966) Abstract 390.[ I l l LEA, D. E ., p. 220 Actions o f Radiations on Living Cells, C am bridge Univ. Press (1947).[12] JACOBSON, L. O . , H em a tolog ic Effects o f Ion izing Radiation, Ch. 16: Radiation B iology

(IIOLLAENDER, A . , E d .), M cG raw -H ill, New York.[131 GILLETTE, E .L . et a l . , Radiat. Res. 22 (1904) 264.[14] WHITING, A. R . , Radiat. Res. 2 (1955) 71.[15] SMITH, R. H . , P h .D . Thesis, N. C. State University (1965) R aleigh, N. C . , USA.

D I S C U S S I O N •

K. K. NAIR: C ou ld you g iv e so m e d eta ils on the le v e ls o f c o s m icrad ia tion in sid e the sp a ce ca p su le in y ou r e x p e r im e n ts and explain w hether you o b s e rv e d any in c r e a s e in the fre q u e n cy o f r e c e s s iv e m u ta tion s?

R. C . VON B O R S T E L : If I m ay a n sw er the f ir s t part o f th is qu estion ,the o rb it o f B io sa te llite II w as b e low the Van A lle n Beit, and h en ce no r a d ia tion fr o m that s o u r c e con trib u ted s ig n ifica n tly to the e f fe c t . In fact, rad ia tion fr o m heavy c o s m ic e le m e n ts , as m e a su re d d o s im e tr ic a l ly on the s p a c e c r a ft , w as n e g lig ib le . The r e c e s s iv e le th a l m utation data a re s t i l l under a n a ly s is .

D. S. GROSCH : W ith the p ostpon em en t o f B io sa të llite II untilS ep tem b er, the 2 0 -d a y o v ip o s it io n e x p e r im e n ts w ere b a r e ly co m p le te d b e fo r e the dead lin e se t fo r su b m is s io n o f p a p e rs to th is S y m p osiu m . A n a ly s is o f r e c e s s iv e le th a ls n e ce s s ita te s study o f g e n e ra t io n s subsequent to the F i and so th is w ork is as yet in co m p le te .

GEN ETIC AND C YTO G EN ETIC BASIS OF R ADIATIO N-IN DUCED STE R IL IT Y IN THE AD U LT M A LE CABBAG E LO O PER Trichoplusia ni

D .T . NORTH AND G .G . HOLTMETABOLISM AND RADIATION RESEARCH LABORATORY, ENTOMOLOGY RESEARCH D IVISIO N ; ARS,UNITED STA TES DEPARTMENT OF AGRICULTURE,FARGO, N. D A K ., UNITED STA TES OF AMERICA

Abstract

GENETIC AND CYTOGENETIC BASIS OF RADIATION-INDUCED STERILITY IN THE ADULT MALE CABBAGE LOOPER T richlop lusia n i. The relationship o f egg hatch as a function o f radiation dose is o f a two*.hit nature when irradiated adult m ales are m ated to non-irradiated virgin fem ales. Sterility as a function o f dose is usually linear in insects. H ow ever, other species o f moths respond sim ilarly to irradiation, so it is ind ica tive .that the m echanism involved in producing sterility in Lepidoptera is basically different from that in other insects. The sign ifican ce o f the tw o-h it kinetics is discussed in relation to the chrom osom e structure and possible m echanism s for the induction o f sterility in Lepidoptera. Many workers using Lepidoptera have reported that fem ales mated to irradiated m ales oviposit substantially fewer eggs than norm ally . This response has been correlated to a lack o f sperm transfer by irradiated m ales, even though they pass a sperm atophore. The phenom enon is dose-dependen t. Although Lepidoptera are far m ore radioresistant than other insect species when measured by the induction o f sterility in the m ale , there appears to be very lit t le d ifferen ce when longevity is used as the criterion . The radiation dose required to reduce the lifespan o f a new ly em erged cabba ge looper m ale by 50a/o was found to be approxim ately the sam e as that for the house fly . High doses o f radiation have no im m ediate e ffe c t on the mating behaviour o f the irradiated m a le . With a recessive e y e -co lo u r mutant as a sperm m arker, it was determ ined in tests u tiliz in g double matings that the second m ating is the e ffe c t iv e m atin g. Sperm m ixing is not prevalent; rather it appears to be a *sperm flushing' phenom enon in that sperm from the first m ating are displaced by sperm from the second m ating. Radiation studies with the cabba ge looper have demonstrated that the progeny o f a cross where the m ale parent receives a sub-sterilizing dose o f gam m a radiation are often serni-sterile when mated to non-irradiated individuals. The am ount o f inherited sterility is d irectly dependent on the amount o f radiation g iven the orig ina l parent. A dose o f 10 krad to a Pj m a le , for exam p le , on ly induces 15 to 20% sterility. H ow ever, o f the surviving progeny as many as 50% w ill be sern i-sterile with 20% being com p le te ly sterile when irated to non-irradiated individuals. The cy togen etic im p lication s o f this are discussed; nam ely , the e ffe c t o f diffuse centrom eres, and the induction o f reciproca l translocations. D ata are presented on induced translocation frequencies by various doses o f radiation and the F2 - bred behaviour o f these individuals. This approach possibly affords a more e ffe c t iv e too l in insect con tro l.


The s u c c e s s o f the s t e r i le -m a le technique in e ra d ica t in g o r c o n tro ll in g the s c r e w -w o r m fly , C o ch lio m y ia h o m in iv o ra x (C o q u e r e l) , [1, 2] has led en to m o lo g is ts to in c r e a s e th e ir e f fo r ts to apply th is tech n iqu e to m any oth er in s e c t s . Such r e le a s e s o f s t e r i le m a le s into n atu ral popu lation s have been e ffe c t iv e with m any s p e c ie s o f D ip tera [3 -7 J, but to date have not had g rea t s u c c e s s with s p e c ie s o f L e p id o p tc ra . One d ifficu lty is that L e p id o p te ra are h igh ly re s is ta n t to ir ra d ia tio n when the c r it e r io n is in du ced s te r i l ity , e . g . 3 0 -4 0 krad a re re q u ire d to s t e r i l iz e the co d lin g

391

3 9 2 NORTH and HOLT

m oth C a rp o ca p sa p o m o n e lla L . |8 J, the E u ropean c o r n b o r e r O str in ia n u b ila lis (H tlbner) |9l, the cabb age lo o p e r T r ich o p lu s ia ni (H ilb n e r )[ I 0 1, the to b a c co bu dw orm I-Ieliothis v ir e s c e n s l1'. [ 11] , and the s u g a r -ca n e b o r e r D ia tra ea s a c c h a r a lis IT. (12 I, and 100 krad a re needed fo r the In d ia n -m ea l rnoth P lo d ia in te rp u n ctc lla (H ü bn er), and the A n gou m ois gra in m oth S itog rog a c e r e a le l la (O liv ie r ) [ 13] . About a tenth as m uch is re q u ire d to in du ce m a le s t e r i l ity in D ipteran s p e c ie s , and m o s t re q u ire on ly 4 -5 krad a d m in iste re d to the adult, e . g . the s c r e w - w o rm [ 14 J, the M exican fru it f ly A n astreph a ludens (L oew ) ( 15) , and the h ou se f ly M usca d o m e stica L.

In se c ts that re q u ire 30 krad o r m o re to in du ce s te r i lity are m o re lik e ly to in cu r s e v e r e p h y s io lo g ic a l and so m a tic dam age than are m o re ra d io s e n s it iv e s p e c ie s . In p a rt icu la r , the h igh er d ose m akes ir ra d ia te d m a le s m uch le s s co m p e tit iv e than the unir radiated m a le s in a w ild popu la tion . S u c ce s s fu l ap p lica tion of the s t e r i le m a le technique depen ds on the p rodu ction o f s t e r i le m a le s that a re co m p e tit iv e enough to d e c r e a s e the r e p ro d u c tiv e potentia l o f the popu lation in to w hich th e y 'a re r e le a s e d . T h u s, r e a liz a t io n o f th is o b je c t iv e depends on studying and u nderstan d in g the e f fe c t s o f ir ra d ia t io n on the r e p ro d u c t iv e b io lo g y o f the in s e c ts . With L ep id op teran s p e c ie s it is a d is t in c tly m o re d iff icu lt p rob lem than with D ipteran s p e c ie s , w here s t e r i le m a le s a re u su a lly co m p e tit iv e . O nly a fte r the v a r io u s fa c to r s in flu en c in g co m p e tit iv e n e ss in L ep id o p te ra are known can the fa c to r s r e s p o n s ib le fo r n o n -c o m p e t it iv e n e ss be o v e r c o m e . The stu d ies re p o rte d h e re a re th e r e fo r e an attem pt by the au th ors to evalu ate the fa c to r s in flu en c in g the s t e r i l ity and c o m p e tit iv e n e ss o f the cabb age lo o p e r and to su g g est so lu tio n s to p ro b le m s en cou n tered with this and o th er L ep id op teran s p e c ie s .

2. M A T E R IA L S ANO M ETHODS

T he cabbage lo o p e r stra in used in th ese e x p e rim e n ts was o r ig in a lly obta in ed fro m the la b o r a to r y o f the F ru it and V egeta b le In sects Tiranch o f the US D epartm ent o f A g r icu ltu re at R iv e rs id e , C a lifo rn ia ; it has s in ce been m aintained at F argo through m o r e than 30 g en era tion s . A ll in s e c ts w e re re a r e d in d iv id u a lly in 1 -o z p la s t ic je l ly cu ps on a se m i-s y n th e t ic m ed iu m f 16 J. The adults w ere fed a 10% so lu tion o f s u c r o s e and m ain ta ined at 27.5 ± 2°C with a 12-h p h otop eriod and a re la tiv e hum id ity o f about 7 5%. M ales ir ra d ia te d as 2 - to 4 -d a y -o ld adults w ere used in s in g le -p a ir c r o s s e s with u n irra d ia ted v irg in fe m a le s . The h old in g c a g e s w e re m e s h -s c r e e n c y lin d e r s ( 7 . 5 X 17.5 c m ). A fte r one night, the m a le s w ere re m o v e d and the fe m a le s w ere a llow ed to o v ip o s it on wax p a p e r that w as p la ced around the ou tsid e o f the ca g e s each day. The next day, any dam aged eg gs w e re r e m o v e d , and the o th ers w e re gently bru sh ed fr o m the p a p er and p laced in row s on dam p, s t e r i le b la ck m u slin o r on b la ck f i lt e r p a p er in s t e r i le p e tr i d ish e s . The petri d ish e s w ere than p la ced in se a le d p la s t ic bags , with a wet paper tow e l around e v e r y two d ish es to p reven t d e s ic c a t io n , and incubated fo r th ree days at 28 .5°C . A fte r incubation , the p e tr i d ish es w ere exam in ed under a d is s e c t in g m ic r o s c o p e , and the p e rce n ta g e o f hatched eg gs w as d e te rm in e d . The re su ltin g la rv a e w ere tr a n s fe r r e d to la rv a l d iet cu ps if the p rog en y w e re to be r e c o v e r e d .

S M - 1 0 2 / 3 9 393

T e s ts o f c o m p e tit iv e n e ss w e re m ade with one ir ra d ia te d m a le , one u n irrad ia ted m a le and one v irg in fe m a le . O ther co n d itio n s w e re id e n tica l to th ose d e s c r ib e d ex ce p t that the m a le s w ere not re m o v e d fro m the ca g e s until the end o f the 5 - to 7 -d a y te s t . In th ese e x p e r im e n ts , the 2 - to 4 -d a y -o ld adult u nanaesthetized m a le s w ere ir ra d ia te d with gam m a ra y s fr o m a co b a lt -6 0 s o u r c e at a d ose rate o f 7440 r a d /m in . S ince on ly adult m a le s w e re ir ra d ia te d , m atu re sp erm w as the on ly stage te s te d .

A ll fe m a le s w e re d is s e c te d a fte r the study and the n u m ber o f sp e rm a to p h o re s in the b u rsa co p u la tr ix w e re r e c o r d e d , as w as the p r e se n ce o r a b se n ce o f sp e rm in the sp e rm a th e ca e (d e te rm in e d by squash ing the sp e rm a th e ca e into p h y s io lo g ic a l sa lin e so lu tion and by m ic r o s c o p e ex am in a tion ). A ll c y to lo g ic a l o b s e rv a t io n s w e re m ade by squash in g the te s te s o f la te - in s ta r la rv a e and sta in in g with a c e to -o r c e in .

3. R A D IA T IO N -IN D U C E D M A L E S T E R IL IT Y

T h irty krad o f gam m a rad ia tion c a u se s n e a r ly c o m p le te s te r i lity in the adult m a le cabb age lo o p e r (T a b le I). The d o s e -r e s p o n s e cu rv e f o r in du ced m a le s t e r i l i ty is n o n -lin e a r (F ig . 1) in stead o f l in e a r as in m ost in se c t D ip teran s p e c ie s (E ig .2 ) . No s ig n ifica n t s t e r i l ity o c c u r s in the cabb age lo o p e r until a d ose o f o v e r 10 krad is a d m in iste re d , but s t e r i l ity thus in c r e a s e s ra p id ly until lit t le h atch ing o c c u r s . Thus d o s e - r e sp o n se c u rv e s fo r m a le s te r i lity in the cabb age lo o p e r and oth er L ep id o p te ra a re d is t in c tly d iffe re n t than f o r D ip tera . The cabb age lo o p e r is ap paren tly m o r e re s is ta n t to ra d ia tio n -in d u ce d s t e r i l ity than the house fly , and the o r ig in and nature o f the s t e r i l ity a re a ls o d iffe re n t .

The d o s e -r e s p o n s e cu rv e o f the cabb age lo o p e r , and p ro b a b ly that o f a ll L ep id op tera , is b a s ic to the u se o f th ese s p e c ie s in s t e r i le m a le r e le a s e s . S ince la rg e d o s e s o f ir ra d ia tio n are re q u ire d to in du ce s te r i lity , the lo w e st p o s s ib le d o se m u st be s e le c t e d . F o r ex a m p le , i f a h ou se fly is o v e rd o s e d by as m u ch as 20%, on ly an add itiona l thousand rad o r le s s are a d m in iste re d . Tf the cabb age lo o p e r is o v e rd o s e d by the sa m e p e rce n ta g e , an ad d ition a l 8000 rad o f rad ia tion a re a b so rb e d and so la rg e a d ose m ay cau se s e r io u s p h y s io lo g ic a l dam age in clu d in g the lo s s of c o m p e tit iv e n e ss in the s t e r i le m a le . In addition , as P ig . 1 sh ow s, ad m in istra tion o f 40 in stead o f 30 krad d o e s not in c r e a s e the am ount of in du ced s te r i lity a p p re c ia b ly but re p re s e n ts a 25% o v e r d o s e .

4 . E F F E C T OF RA D IA TIO N ON LO N G E V ITY

Although the cabb age lo o p e r is fa r m o re re s is ta n t to induced s te r i lity than the house fly , the red u ction o f lon g ev ity ca u sed by ir r a d i ation o f both s p e c ie s with 50 krad w as about the sa m e (F ig . 3). S ince 50 krad is on ly 20% h igh er than the s t e r i l iz in g d ose fo r the cabb age lo o p e r and s in ce the lon g ev ity o f the house f ly is litt le a ffe cted at the 4 -k ra d s t e r i liz in g d o se , the cabb age lo o p e r is c o m p a ra t iv e ly r a d io r e s is ta n t in te r m s o f s t e r i l ity but not in te r m s o f lon gev ity .

Reduced lo n g e v ity h'as v a r ia b le e ffe c ts on the e f f ic ie n c y o f s t e r i le m a le s (a deta iled d is cu s s io n is beyon d the s c o p e o f th is p a p er), but in po ly g am ou s s p e c ie s such as L e p id o p te ra it has an o b v io u s e f fe c t . If

394 NORTH and HOLT

T A B L E I. T H E R E L A T IO N O F G A M M A IR R A D IA T IO N D O SE T O IN D U C E D S T E R IL IT Y IN 3 -D A Y -O L D A D U L T C A B B A G E L O O P E R S AS D E T E R M IN E D B Y IN D IV ID U A L P A IR M A T IN G S (T R E A T E D M A L E S X U N T R E A T E D F E M A L E S )

D ose X 1 0 3 radN um ber o f

eggs

N um ber o f eggs hatch ed

Per ce n t hatch ¡ S . Ü .

0 10 261 9287 90 . 5 i 5 .9

1. 2S96 2662 9 1 .9 (a )

2 4577 4163 9 0 .0 ± 3 .2

3 2550 2279 8 9 .4 (a )

4 52S5 4620 8 7 .0 i 5 .0

5 6277 5227 8 3 .4 i 1 . 5

6 3403 2919 85 . / ± 1 . 5

7 3122 25S3 82 . 7 ± 2 .9

S 3589 3055 8 5 .1 i 0 .9 2

9 1712 1250 7 3 .0 i 5 .3

10 8442 5006 5 9 .3 ■ 9 .8

12 2522 1557 6 1 .7 - 0 . 3 5

15 2921 1590 5 4 .5 i 0 .8 0

18 1324 291 2 2 .0 (a )

20 175 26 1 4 .9 : 3 .5

25 2136 132 6 .2 ! 4 . 6

30 3713 131 3 . 5 i 2 .1

35 2403 107 4 . 3 ■■ 0 .7 0

40 1137 0 0 (a )

50 54-1 0 0 (a )

(a)O n ly o n e re p lic a t io n .

s t e r i le m a le s on ly liv ed lon g enough to m ate o n ce , then e ith e r a la rg e n u m ber o f m a les w ould have to be r e le a s e d o r frequ ent r e le a s e s w ould have to be m ade.

5. E F F E C T O F RADIATIO N ON THE A B IL IT Y OF THE M ALES T O M ATE

T he e ffe c t o f red u ced lon g ev ity on the m ating ab ility o f s t e r i le m a les w as stud ied to d e term in e its im p o rta n ce . The 3 -d a y -o ld m a le cabbage

S M - 1 0 2 / 3 9 3 9 5

DOSE X I03 rads

FIG. 1. The relationship o f radiation gam m a dose to induced m a le sterility (2 - to 4 -d a y -o ld irradiated m ales crossed individually with non-irradiated virgin fem ales).

DOSE X Ю2 R

FIG. 2. The relationship o f X -ra y dose to the frequency o f induced dom inant lethal m utations in 3 -d a y -o ld adult m a le house flies.

lo o p e r s w e re ir ra d ia te d (0 -5 0 krad) and p la ce d in d iv idu a lly with v irg in ■ fe m a le s fo r one night; m atin g w as d e term in ed by d is s e c t in g the fe m a le s the fo llo w in g day to d e te rm in e the p r e s e n ce o f a sp e rm a to p h o re . The a b ility o f the m a le to m ate w as not in it ia lly im p a ire d , even a fte r 50 krad o f rad ia tion (T a b le II). H o w e v e r , the data do not m e a su re the m ating a b ility o f the ir ra d ia te d m a le o v e r a lo n g e r p e r io d o f t im e , and to b a c co budw orm m a le s that r e c e iv e d 40 krad w ere 20% le s s e ffe c t iv e in m ating than the c o n tro ls o v e r the life t im e o f the m a le s [ 11] .

396 NORTH and HOLT

DOSE X I03 rads

FIG. 3. T h e e ffe c t o f l ife shortening by gam m a radiation for 1 -d a y -o ld adult cabba ge loopers and house ily m ales.

6. OVIPOSITTOX E L IC IT E D B Y IR R A D IA T E D M ALES

A co m m o n o b se rv a t io n b y in v e s t ig a to rs o f L ep id o p te ra has been the fa ilu re o f the ir ra d ia te d m a le to e l ic i t n o rm a l o v ip o s it io n in m atin gs with u n irra d ia ted fe m a le s . F o r ex a m p le , u n irra d ia ted cabbage lo o p e r fe m a le s m ated to m a le s that had r e c e iv e d a s t e r i l iz in g d ose o f 30 krad o v ip o s ite d 50% fe w e r eg gs than the c o n tr o ls (T a b le III). T h is red u ced o v ip o s it io n a ffe c ts the evalu ation o f the co m p e tit iv e n e ss o f s t e r i le m a le s , tf m ating co m p e tit iv e n e ss is equal, that is , i f o n e -h a lf the fe m a le s m ate with s t e r i le m a le s , the e g g hatch w ill be 66%, not 50% o f n o rm a l (b a se d on 100% fo r the n o rm a l eg g hatch o f an u n irra d ia ted m a le m ated with an u n irra d ia ted fe m a le and 0% fo r the hatch re su ltin g fr o m the c r o s s o f a s t e r i le m a le and an u n irrad ia ted fe m a le ). T h e re fo r e , the s t e r i le m ale m ay be m ating co m p e tit iv e ly though he is unable to red u ce e g g hatch e f fe c t iv e ly . U n less th is fa c t is e sta b lish e d , la ck o f c o m p e tit iv e n e ss m igh t be attributed to a la ck o f m ating a b ility w hich w ould be unfounded.

7. S P E R M U T IL IZA T IO N

Since the cabbage lo o p e r is a p o ly g am ou s s p e c ie s , we exam in ed the s p e rm -u tiliz a t io n pattern o f fe m a le s m ated with both s t e r i le and u n irra d ia ted m a le s . Such in form a tion is n eeded to evaluate the e f fe c t o f s t e r i le m a le r e le a s e into a popu lation on the red u ction o f that p op u la tion 's r e p ro d u c tiv e p oten tia l. T he e ffe c t the s t e r i le m a le has on the natural popu lation depends on w hether sp e rm m ix in the sp e rm th e ca e a fter m u ltip le m atin gs o r a re u sed p re fe re n t ia lly . T hus eg g hatch depen ds on w hether sp e rm are used ran d om ly in fe r t i l iz in g eg gs o r w hether sp erm fro m on ly one m ating a re u sed .

S p erm u tiliza tion ih the cabb age lo o p e r w as th e r e fo re in vestig a ted by u sin g a r e c e s s iv e y e llo w -e y e d mutant as a sp e rm m a rk e r . The

S M - 102/39 3 97

T A B L E II. T H E E F F E C T O F G A M M A R A D IA T IO N ON T H E M A T IN G R E S P O N S E O F 3 -D A Y -O L D A D U L T M A L E C A B B A G E L O O P E R S AS D E T E R M IN E D B Y IN D IV ID U A L P A IR M A T IN G S (T R E A T E D M A L E S X U N T R E A T E D F E M A L E S ) F O R O N E P E R IO D O F 24 h

D ose X 1 0 3 rad Per cen t m ales m ated

0 5 0 .0 0

1 6 6 .6 7

2 6 2 .5 0

3 C 3 .1 6

4 6 3 .6 4

5 5 5 .8 3

6 6 9 .2 3

7 5 4 .5 5

S 4 6 .6 7

9 6 1 .5 4

10 6 9 .2 3

12 6 4 .2 8

15 5 6 .2 5

18 6 6 .6 7

20 4 0 .0 0

25 6 6 .6 6

30 6 5 . 52

35 4 6 .4 3

40 7 7 .2 7

50 6 4 .2 S

h om ozy gou s y e llo w -e y e d fe m a le s w ere a ltern a te ly m ated fo r one night each to n o r m a l-e y e d m a le s and y e llo w -e y e d m a le s , and the p rogen y w ere r e c o v e r e d . O nly the p rogen y fro m fe m a le s having tw o sp e rm a to p h o re s and sp e rm in the sp e rm a th e ca e w e re c o lle c te d . S ince cabb age lo o p e r s r a r e ly m ate tw ice the sa m e night, the u se o f sp e rm a to p h o re coun ts to d e term in e the n u m ber o f m atin gs a p p ears v a lid . S ince the mutant is r e c e s s iv e , any sp e rm b ea rin g the m utant phenotype p rod u ced a y e l lo w e y ed in d iv id u a l. S perm p o s s e s s in g the w ild -ty p e a lle le (n o rm a l eye) p rod u ced p ro g e n y having n o rm a l ey e c o lo u r .

When a n o rm a l m a le w as the f ir s t m ate and the se co n d w as a y e llo w eyed m a le , about 85% o f the re su lt in g p rogen y had y e llo w e y e s . If the f ir s t m ating w as with a y e llo w -e y e d m a le and the secon d with a n o rm a l-


T A B L E III. R A D IO S E N S IT IV IT Y O F 2 - T O 3 -D A Y -O L D M A L E C A B B A G E L O O P E R S D E F IN E D B Y D E P R E S S E D F E C U N D IT Y AS D E T E R M IN E D B Y IN D IV ID U A L P A IR M A T IN G S (T R E A T E D M A L E S X U N T R E A T E D F E M A L E S )

D ose X 103 rad

N um ber o f fem a les

N um ber o f

eggs

N um ber o f e g g s /fe m a le

A v g . e g g s / ov ip o s it io n

C on tro l 22 10 261 46G .4 1 4 8 .1

1 5 2896 5 7 9 .2 1 4 4 .8

о 10 ■ 457 7 4 5 7 .7 1 4 3 .0

3 5 2550 5 1 0 .0 1 2 7 .5

4 14 5285 3 7 7 .5 1 0 1 .6

5 IS 627 / 3 4 8 .7 1 2 5 .5

6 9 3404 3 7 8 .0 8 5 .0 8

7 11 3122 2 8 3 .8 1 2 0 .1

8 7 3589 5 1 2 .7 1 4 3 .6

9 8 1712 2 1 4 .0 8 5 . 6

10 27 3442 3 1 2 .7 9 9 .3

12 10 2522 2 5 2 .2 9 3 .4

15 16 2921 1 8 2 .6 1 0 4 .3

IS 5 1324 2 6 4 .8 8 8 .3

20 4 17 5 4 3 .8 2 1 .9

25 3 213 G 267 .Ü 4 6 .4

30 19 3713 1 9 5 .4 7 4 .3

35 13 2463 1 8 9 .5 61 . G

40 22 1137 5 1 .7 2 4 .7

50 14 .544 3 8 .9 2 0 .1

V irg in 159 2230 1 4 .0 1 0 .0

ey ed m a le , 98% o f the re su ltin g p rogen y had n o rm a l eye c o lo u r ( F i g .4 ); that is , the la s t m ating w as the e ffe c t iv e m ating.

8 . SP E R M TR A N SFE R B Y IR R A D IA T E D M A LE S

If the c o n c lu s io n s co n ce rn in g polygam y are va lid , the ir ra d ia te d m a le in a tes t o f m ating c o m p e tit iv e n e ss m ust con trib u te a p h y s io lo g ic a lly n o rm a l (a ls o adequate in n u m bers) co m p le m e n t o f sp e rm at each m atin g . '

SM- 1 0 2 /3 9 3 9 9

SPERM UTILIZATION TEST I---- 1NORMAL

% OF TOTAL OFFSPRING

FIG. 4. T h e utilization o f sperm from two d ifferent matings by fem a le cabba ge loopers (a ll w ere m ated to y e /y e and + /+ m ales. T h e resulting progeny are indicated by phenotype indicating the source o f the sperm used).

100

9 0

8 O1

70

E 6o(оосUJ 50Q.

40

30

20

10□ — % OF F E MALES MATED

О — % OF F E M A L E S INSEMINATED

8 10 15DOSE X Ю3 rad

20 25

FIG. S. The relationship o f m ating to insem ination o f fem ales by irradiated m a le cabba ge loopers.

In A tteva p u n cte lla (C r a m e r ) , the p r im a ry stim u lu s f o r o v ip o s it io n is the s u c c e s s fu l t r a n s fe r o f an adequate supply o f sp e rm [ 17] . T h is w as tru e fo r the cabbage lo o p e r ; h o w e v e r , ir ra d ia te d m a le s m ay tr a n s fe r on ly sp e rm a to p h o re s at m ating, and they often fa ile d to supply an adequate am ount o f sp e rm o r any sp e rm s in ce th is t r a n s fe r is d o s e - dependent (P ig . 5 ). M ale cabb age lo o p e r s ir ra d ia te d with 0 -2 5 krad m ated as w e ll as the u n irra d ia ted c o n tro ls , but as the d ose in cr e a s e d , the p e rce n ta g e o f m a le s that t r a n s fe r r e d sp e rm ca p a b le o f re a ch in g the sp e rm a th e ca e o f the fe m a le d e c r e a s e d rapid ly '. At 20 krad , le s s than 30% o f the m a le s s u c c e s s fu lly t r a n s fe r r e d sp e rm to the fe m a le co m p a re d


with 63% o f the u n irrad ia ted m a le s . T h is fa ilu re to in sem in ate adequ ately ex p la in s why ir ra d ia te d m a le s fa i l to e l ic it n o rm a l o v ip o s it io n from u n irra d ia ted fe m a le s . Thus the lo w e st ir ra d ia tio n that w ill s t e r i l iz e the m a le m ust be u sed if a c o m p e titiv e s t e r i le m a le is to be p ro d u ced .

Stud ies by o th er w o rk e r s with trap c o lle c t io n s have shown that ca b b a g e lo o p e r fe m a le s in w ild popu lation s m ate an a v era g e 2.5 t im e s [18] , as d e te rm in e d b y sp e rm a to p h o re cou n ts . T hus, if the s t e r i le m a le co n tr ib u te s a n o rm a l co m p le m e n t o f sp e rm at e a ch m ating, p o ly g a m y w ill not be a p ro b le m and shou ld not d e tra ct fr o m the e f fe c t iv e n e s s o r e f f ic ie n c y o f a s t e r i le m a le r e le a s e .

9. S T E R IL IT Y IN THE F j G EN E RATIO N

The c h r o m o s o m e s o f L e p id o p te ra have d iffu se c e n t r o m e r e s [ 19 ] .Such c h r o m o s o m e s w ould im p a rt a high d e g re e o f r a d io r e s is ta n c e to the in du ction o f s t e r i l ity [ 10] , b e ca u se the lo s s o f c h r o m o s o m a l m a te r ia l at c e l l d iv is io n due to c h r o m o s o m a l fra g m en ts w ould not be e x p e cte d and th e r e fo r e shou ld not lea d to dom inant le th a lity . C on seq u en tly , the ty p ica l c h r o m o s o m a l a n o m a lie s that lead to dom inant leth a l m u tation s in s p e c ie s that have m o n o c e n tr ic c h r o m o s o m e s p ro b a b ly do not ca u se dom inant le th a l m utations in L e p id o p te ra .

H ow ever , the d iffu se c e n tro m e re a lso a llow s a s o u r ce o f s t e r i l ity not av a ila b le in m any o th er o r d e r s o f in s e c ts . The b a s is f o r th is unique tr a n s m is s ib le s t e r i lity is g iven b e low .

In addition to the lo s s o r re ten tion o f c h r o m o s o m e fra g m e n ts , we m u st c o n s id e r the co n s e q u e n ce s o f r e jo in in g o f the brok en c h r o m o s o m e s and its e f fe c t on in se c t s t e r i l ity . B rok en c h r o m o s o m e s can reu n ite in m any w ays [ 20] . One type o f c h r o m o s o m e re a rra n g e m e n t in v o lv in g n o n -h o m o lo g o u s c h r o m o s o m e s y ie ld s one o r tw o a ce n tr ic fra g m e n ts (w ithout c e n tr o m e r e s ) and c h r o m o s o m e s with tw o c e n t r o m e r e s (d ic e n tr ic ) . At anaphase o f the next so m a t ic d iv is io n , a d ic e n tr ic c h r o m o s o m e p ro d u ce s a ch r o m o s o m e b r id g e w hich , when brok en , r e su lts in daughter c e l ls that a re d e fic ie n t fo r so m e p o rtio n o f the g cn e tic m a te r ia l and p o s s e s s so m e du p lica ted c h r o m o s o m a l se g m e n ts . T h e re su lt is c e l l death — a cy to g e n e tic b a s is f o r the le th a l action o f dom inant leth a l m u ta tion s. In L e p id o p te ra , d ic e n tr ic s o f th is o r ig in p o s s ib ly do not e x is t b e ca u se e v e r y such exch an ge is e s s e n t ia lly a r e c ip r o c a l tra n s lo ca tio n .M ost r e c ip r o c a l tra n s lo ca t io n s do not in v o lv e any a p p re c ia b le lo s s o f c h r o m o s o m a l m a te r ia l and a re th e r e fo r e tra n sm itted to the o ffs p r in g .T hus a ll o f th ese ty p es of re a rra n g e m e n ts a re tra n sm itte d to the p ro g e n y w hich can d ev e lop n o rm a lly . H ow ever , at m e io s is in the p rog en y , the in h er ited r e c ip r o c a l tra n s lo ca t io n cou ld r e su lt in the fo rm a tio n o f g e rm c e l ls that contain du p lica ted and d e fic ie n t am ounts o f g en etic m a te r ia l [ 21] . When th ese s p e rm a re u sed in fe r t iliz a t io n , th ey a re le th a l to the e m b ry o f o r the sa m e r e a so n s that sp e rm con ta in in g a dom inant le th a l m utation im p a rt death to the d e v e lo p in g e m b ry o in s p e c ie s w ithout d iffu se c e n t r o m e r e s . The p e rce n ta g e o f s t e r i l ity in the gen era tion ca u sed by su ch a re a rra n g e m e n t i s then dependent on the n u m b er o f c h r o m o s o m e s in v o lv e d in the e x ch a n g e s . If parts o f tw o d iffe re n t c h r o m o s o m e s are exch an ged , 50% o f the sp e rm o r ova p rod u ced by an adult w ill be in v ia b le .

S M - 102/39 4 0 1

A tra n s lo ca tio n in v o lv in g p a rts o f th ree d iffe re n t c h r o m o s o m e s th e o r e t ic a lly w ill ca u sc 75% s te r i lity .

T h us, i f s e v e r a l r e c ip r o c a l tr a n s lo ca t io n s w e re in du ced in e a ch sp e rm o f the ir ra d ia te d m a le p aren t, h is p ro g e n y w ould be p a rt ia lly to fu lly s t e r i l ç , and in deed , th is o c c u r s in the cabbage lo o p e r [ 1 0 j . L a rg e n u m bers o f t r a n s lo ca t io n s w o re o b s e rv e d in the d e v e lo p in g m e io t ic g e rm c e l ls o f F1 m a le s w hose m a le p a ren ts r e c e iv e d a s u b -s t e r i l i z in g d o se o f gam m a ir ra d ia t io n . (T h e c h r o m o s o m a l co n fig u ra tio n fo r m e d by su ch a r e c ip r o c a l tra n s lo ca t io n can be e a s ily d e tected c y to lo g ic a lly in the te s te s o f la rv a e . ) The la rg e n u m b ers o f h e te ro zy g o u s r e c ip r o c a l t r a n s lo ca tion s obtained with su b -s te r iliz in g d o s e s then exp la in the s t e r i lity o b s e rv e d in the Fj g en era tion . S ince no s iz e a b le n u m ber o f dom inant leth a l m utations are in du ced by even r e la t iv e ly la rg e d o s e s o f rad ia tion , dom inant le th a l m utation in L e p id o p te ra cannot lim it the r e c o v e r y o f the in du ced r e c ip r o c a l t r a n s lo ca t io n s as they do in D íp tera o r in s p e c ie s h av ing c h r o m o s o m e s w'ith lo c a liz e d c e n t r o m e r e s . In D ipteran s p e c ie s , f o r ex a m p le , the in du ction o f dom inant le th a l m u tation s in 100% o f the sp e rm at r e la t iv e ly low d o s e s (b e lo w 10 krad) lim its the n u m ber o f r e c ip r o c a l tra n s lo ca t io n s that can be r e c o v e r e d .

T he d iffu se (n o n - lo c a l iz e d ) c e n t r o m e r e s o f the cabb age lo o p e r o f fe r the p o s s ib ility o f u sin g the in du ced r e c ip r o c a l tr a n s lo ca t io n s as a s o u r ce o f Fj s t e r i l ity and as an ad d ition a l and som ew h at unique to o l to im p ro v e the e f f ic ie n c y o f a s t e r i le m a le r e le a s e p ro g ra m m e . A lo w e r d o se o f rad ia tion f o r adult m a le s that a re to be u sed in fie ld r e le a s e s w ould p ro b a b ly a llow th ese m a le s to be m o r e co m p e tit iv e . The c r o s s betw een a m a le that had r e c e iv e d 20 krad and a n o rm a l u n irra d ia ted fe m a le would p rod u ce an eg g hatch o f 15-20% (T a b le IV ). H ow ever , the p ro g e n y that su rv iv ed to adulthood w ould be s t e r i le . F u rth e rm o re , th ose p rogen y that did su rv iv e w ould be a com pon en t in the w ild popu la tion o f the next gen era tion and w ould p rod u ce a popu lation with inbu ilt s t e r i l ity . S u cce s s o f such an a p p lica tion d epen ds on m any fa c to r s , not a ll o f w hich are in te r re la te d . F o r e x a m p le , it has yet to be p roved that the s t e r i le Fj m a le is co m p e tit iv e with u n irra d ia ted m a le s . A lso , m eth od s o f in c r e a s in g

T A B L E IV . IN H E R IT E D S T E R I L I T Y O F P R O G E N Y O F A M A L E C A B B A G E L O O P E R T H A T R E C E IV E D A S U B -S T E R IL IZ IN G D O SE O F R A D IA T IO N C R O S S E D W IT H A N U N IR R A D IA T E D V IR G IN F E M A L E (Fj. E G G S O B T A IN E D B Y C R O S SIN G F 2 IN D IV ID U A L S T O U N IR R A D IA T E D V IR G IN S O F T H E O P P O S IT E S E X )

D ose r e ce iv e d by Pj m a le (krad)

N um ber o f

Pi eggs

°Io h atch o f

Pi eggs

N um ber o f

Pi eggs

% h a tch o f

P i eggs

C on tro l 213 3 8 5 . G 2964 8 3 .4

1Û 2338 4 6 .0 8226 3 0 .7

15 2068 3 9 . 5 5629 1 1 .5

20 2345 1 5 .7 1317 3 .8


the in du ction o f t r a n s lo ca t io n s with agents o th e r than gam m a irra d ia tio n a re cu rre n tly being ex a m in ed . W ith in cr e a s e d in form a tion about the p rod u ction o f F j s te r i lity , a p r a c t ic a l and e ffic ie n t m ethod o f u sin g th is p r o ce d u r e can be d ev e lop ed that w ill ben e fit any s t e r i le m a le r e le a s e p ro g ra m m e fo r the c o n tro l o f L e p id o p te ra and f o r o th er in s e c ts h aving c h r o m o s o m e s p o s s e s s in g n o n -lo c a l iz e d c e n t r o m e r e s .

A C K N O W L E D G E M E N T

The au th ors g ra te fu lly a ck n ow led ge the w ork p e r fo rm e d by the fo llo w in g students fr o m N orth D akota State U n iv e rs ity du rin g the stu d ies re p o rte d h e re : Jane A rm s tro n g , Cathy F itz g e r a ld , C o leen K arpen k o, C a r o l M on dor, and S h ir le y S ch erb en sk e .

R E F E R E N C E S

[1 ] KNIPLING, E .F ., J. e co n . Ent. 48 (1955) 4 5 9 -6 2 .[2 ] KNIPLING. E .F ., S cien ce 130 (1959) 902.[3] STEINER. L .F . , MITCHELL. W .C . , BAUMHOVER, A .H . , Int. J. app l. Radiat. Isotopes Ki (1962)

4 2 7 -3 4 .[4 ] STEINER, L .F . , HARRIS, E .J . , MITCHELL, W .C . , FUJIMOTO, M .S . , CHRISTENSON, L .D . ,

J. e co n . Ent. 58 (1965) 519.[5] CHRISTENSON, L . D . , Insect Population C ontrol by the S ter ile -M a le T ech n iqu e, T ech n ica l Reports

Series N o .21 , IAEA, Vienna (1963) 31 .[6] STEINER, L .F . , Advances in Insect Population C ontrol by the S ter ile -M a le T ech n iqu e, T ech n ica l

Reports Series N o. 44, IAEA, Vienna (1965) 28 .[1] K ATIYAR, K .P . , Ib id ., 20 .[8] PROVERBS, M .D . , NEWTON. J .R . , Can. Ent. _94 (1963) 11 6 2 -7 0 .[9] WALKER, J .R . , BRINDLEY, T . A . , J. e co n . Enr. _5fi ( 1903) 522- 25.

[10] NORTH, D .T . , HOLT, G .G . , J. e co n . E n t., in the press.[11 ] FLINT, H .M . , KRESSIN, E .L . , J. e co n . E n t., in the press.[12] WALKER, D .W . , Puerto R ico N uclear Center Rep. J (1 9 6 5 ).[13] COGBURN, R .R ., TILTON, E .W . , BURKHOLDER, W .E ., J. e co n . Ent. 5 9 (1 9 6 6 ) 6 8 2 -8 5 .[14] BUSHLAND, R .C . . HOPKINS, D .E ., J. e co n . Ent. 4-1 ( 1951) 7 2 5 -3 1 .[15] RHODE, R .H ., LOPEZ, D . , EQUISA, F. , TELICli. G . , J. e co n . Ent. _54 (1961) 202 -03 .[16] 1GNOFFO, C . M . , Ann. ent. S oc . Am ._5£>(1963) 17 8 -8 2 .[17] TAYLOR, O .R . , I r . , Ann. ent. S oc . A m . _60 (1967) 58 3 -9 0 .[18] GENTRY, R . , personal com m u n ica tion (O c t . 1967).[19 ] VIRKK1, N .. J. A grie . U niv. Puerto R ico XLVII (1963) 10 2 -3 7 .[20] LEA, D .E ., Actions o f Radiation on Living C ells, C am bridge Univ. Press, London (1 946 ).[21] PAINTER, T . S . , MULLER, H .J . , J. H ered. 20 (192Э) 28 7 -9 S .

D I S C U S S I O N

C . F . CU RTIS: I did not fu lly understand T ab le IV o f y o u r p a per, sh ow in g the fe r t il i ty o f the Pj and Fj g en era tion s a fte r ir ra d ia t io n . If, as I understand it, the re su lt f o r the F j, r e fe r r e d to e g g s p rod u ced by the son s and daughters o f the ir ra d ia te d in s e c ts , I do not se e why th ese eg gs sh ou ld not have exh ib ited the m in im u m le v e l o f fe r t ility , s in ce the e ffe c ts o f tra n s lo ca tio n w ould have show n th e m s e lv e s at th is stag e .

D. T . NO RTH : One m u st b e a r in m ind that in the c a s é o f s t e r i l i ty in the P i gen era tion one is sp eak in g o f la r g e ly p h y s io lo g ic a l dam age tog e th er

S M - 102/39 403

with a ce rta in am ount o f gen etic dam age, and in the Fj g en era tion one is sp eak in g o f m a in ly g en etic dam age. In a sen se the dominant, le th a ls , as th ey o c c u r in s p e c ie s such as D ip tera , a re not fu lly r e c o v e r e d until the se co n d gen era tion in L e p id o p te ra .

A . B . B O R K O V E C : I w as in te re s te d in the statem en t that a ra d ia tio n - s t e r i l iz e d m a le can re d u ce the fecu n d ity o f the fe m a le o f the s p e c ie s .T h e re is an an alogy h e re w ith the o b s e rv e d e ffe c t s o f m atin g c h e m o s te r i l iz e d m a le s o f the azuki bean w e e v il ( C o llo so b ru ch u s ch in e n s is ) to u ntreated fe m a le s . At f ir s t we thought that so m e o f the s te r ila n t w'as t r a n s fe r r e d fr o m the m a le to the fe m a le du rin g cop u la tion and that the d im in ish ed fecu n d ity w'as a d ir e c t e f fe c t o f the c h e m ica l, but fo r v a r io u s r e a so n s th is exp lan ation is inadequate and I w ould lik e to know w hether you have any data in d ica tin g that the d rop in fecu n d ity o c c u r r e d on ly when th ere w as no adequate sp e rm tr a n s fe r o r w hether it o c c u r r e d a lso when tr a n s fe r w as adequate.

D . T . N O RTH : T o the b e s t o f ou r kn ow ledge, when a fu ll co m p lem en t o f sp e rm is t r a n s fe r r e d and r e a c h e s the sp e rm a th e ca e o f the fe m a le , she o v ip o s its a n o rm a l n u m ber o f e g g s . It is on ly when she r e c e iv e s an inadequate am ount o f sp e rm o r se m in a l flu id that h e r o v ip o s it io n re sp o n se is lo w e re d .

V. L A B E Y R IE : Y o u r h yp oth es is on the p o s s ib le e f fe c t s o f e lim in ation o f c e rta in s e c r e t io n s n o rm a lly p re se n t in the se m in a l flu id c o r r e s p o n d s to o b s e rv a t io n s m ade on fe m a le s m ated with m a le s w hich had a lrea d y p e r fo rm e d a c e r ta in n u m ber o f cop u la tion s , o r w h ere cop u la tion was a r t i f ic ia l ly in terru p ted . A ll th ese e x p e r im e n ts show ed s u c c e s s iv e tr a n s fe r o f v a r io u s su b sta n ces du rin g the p e r io d o f fo rm a tio n o f the sp erm a top h ore w ithin the b u rsa co p u la tr ix o f the fe m a le . In A ca n th o sce lid e s obtectu s the fin a l su b sta n ces t r a n s fe r r e d by the m a le a re th ose w hich act to stim u la te m ig ra tio n o f sp e rm a to z o a tow a rd s the sp e rm a th e ca ; one o r m o re m ay w e ll be p ro te in s a ffe ctin g o v o g e n e s is .

D. T . N O RTH : I w as not aw are o f the situ ation you d e s c r ib e fo r A . o b te ctu s , and find it m o s t in te re s t in g . A w ord o f cau tion shou ld be e x p re s s e d , h o w e v e r , in that at the p re se n t tim e w e are not su re w hether the sp e rm a to p h o re con ta in s any sp e rm at a ll and, i f so , w hether it conta in s on ly in a ctiv e sp e rm o r i f not, w hether it con ta in s a c c e s s o r y flu id . T h is is an im p ortan t point w hich we hope to e lu cid a te in the n e a r fu tu re.

G

ISOTOPE A P P LIC A TIO N S: GEN ETIC

(Session VIII, P art 2)

C h a irm a n : ф . ST R £)M N A E S

N O R W A Y

LA SYNTHESE D 'A D N DANS ' LES NO YAUX GEANTS DES INSECTES -

M O DALITES DE CONTROLE ET STRUCTURES OBSERVEES DANS LA GLANDE SERICIGENE DE Bombyx m ori

V. NIGON, J . DAILLIE, S. CILLOT,M. M. NEULAT-PORTIER ET M. de TURENNE

FACULTE DES SCIENCES,UNIVERSITE DE LYON,

FRANCE


DNA SYNTHESIS IN THE GIANT NUCLEI OF INSECTS - CONTROL MACHINERY AND STRUCTURES OBSERVED IN THE SILK-PRODUCING GLAND OF Bom byx m orí. T h e ex isten ce in m any insect organs o f giant n u cle i w ithout v is ib le chrom osom es raises the question o f possible h om olog ies betw een the chrom atin structures o f these n u cle i and those o f p o lyten e n u cle i or com m on euploid ce lls . Studies have been m ade o f the n ucle i in the s ilk -produ cin g gland o f Bom byx m ori. T h e DNA synthesis is c y c l i c . During the third stage there are three successive synthesis cy c le s , w hich appear to be re la tive ly autonom ous in the in d ividual n u cle i. For m ore than 24 hours after m oulting, how ever, synthesis is greatly reduced ; m oulting factors thus cause synchronization o f a ll the n u cle i. This leads to the con clu sion that the triggering o f a synthesis c y c le is con trolled by general factors external to the c e l l . At the end o f larval developm ent, DNA synthesis is suspended at the m om ent w hen la rg e -sca le secretion o f silk begins. E valu ation 'o f the p oo l o f endogen ic precursors o f DNA shows that it is considerably reduced at the end o f the DNA synthesis period. T h e hypothesis proposed is that la rg e -sca le synthesis o f fibroin requires polariza tion o f the m e ta bolism , h en ce the d ep letion o f the n u cleotid e p o o l and the end o f D NA synthesis. DNA synthesis w ithin a single nucleus is to som e extent asynchronic. In particu lar, a w e ll-d e fin e d , delayed-synthesis structure v isib le on ly in the fem a le seem s to be a possible h om olog u e o f a sex ch rom osom e. Other asynchronisms are also apparent, though less c lea rly . Functional studies thus allow the supposition that in the giant nucleus rep lication units retain an individuality com parab le to that o f a polyten e ch rom osom e. These observations together lead to the conclu sion that a nucleus in the silk -produ cin g gland has p h ysio log ica l and structural characteristics sim ilar to those o f a polyten e nucleus, d ifferin g from it essentially in the lesser degree o f condensation o f its structures.

LA SYNTHESE D ’ ADN DANS LES NOYAU X GEANTS DES INSECTES - MODALITES DE CONTROLE ET STRUCTURES OBSERVEES DANS LA GLANDE SERICIGENE DE Bom byx m ori. L 'ex isten ce , dans beaucoup d'organes des insectes, d e noyaux géants, sans chrom osom es visibles» pose la question des h om olog ies possibles entre les structures chrom atiniennes de ces noyaux et ce lle s d e noyaux polytènes ou d e ce llu les euploides banales. Des études ont porté sur les noyaux d e la glande sêric igèn e d e Bom byx m ori. La synthèse d 'AD N s’ e ffe c tu e d e façon cy c liq u e . Au cours du 3èm e âge, on dénom bre trois cy c le s successifs de synthèse. D 'un noyau à l 'au tre , les cy c le s d e synthèse paraissent se dérouler d e fa çon rela tivem ent auton om e. Cependant après la m ue, et durant plus d e 24 heures, la synthèse est très réduite, les facteurs d e la m ue déterm inant ainsi une m ise à l ’ unisson d e l 'e n se m b le des noyaux. Ces observations ob ligen t à c o n clu re que l e d éclen ch em en t d 'u n c y c le de synthèse est con trô lé par des facteurs généraux extérieurs à la ce llu le . A la fin du dévelop p em en t larvaire, la synthèse d ’ADN est suspendue au m om en t oîi co m m e n ce la sécrétion m assive d e so ie . L ’ évaluation du poo l des précurseurs endogènes d e l 'A D N m ontre que c e lu i - c i est considérablem ent réduit à la fin de la p ériode d e synthèse de l ’ ADN. L 'hypothèse avan cée est que la synthèse m assive d e fibroïne e x ig e une polarisation du m étabo lism e d ’où résulteraient l 'appauvrissem ent du p o o l des nucléotides et l ’ arrêt de la synthèse d 'AD N .

407

4 0 8 NIGON et al.

L 'e x is te n ce , chez le s In se c te s , d 'organ es formés de c e l lu le s géantes pourvues de noyaux endopolyp loïdes pose de nombreux problèm es. Les conséquences de ce mode p a r t ic u l ie r de la d i f fé r e n c ia t io n c e l lu la i r e resten t encore inconnues pour la p lu p a rt . En revanche, nous commençons à e n tre v o ir une p a rt ie des mécanismes responsables de c e t te é v o lu t io n . Dans le s noyaux p o ly - tèn es , la r é p lic a t io n des chromatides p a ra ît correspondre aux stru ctu res et aux processus correspondants d'une c e l lu le norm ale, tand is qu 'on t disparu le s év o lu tion s ca ra c té r is t iq u e s de la prophase a in s i que le s éléments c in é tiq u e s de la m itose. Cependant, dans la p lupart des noyaux géan ts, tou te tra ce de stru ctu re chromosomique est apparemment absente. De so r te que l 'o n peut se demander s i la synthèse d'ADN et sa ré g u la t io n , sa com position et le s stru ctu res auxquelles i l e s t a tta ch é , ne répondent pas a lo rs à une forme plus dégradée encore des c y c le s n u c léa ires h a b itu e ls . C 'e s t à ces questions que nous avons ten té d 'a p p orter quelques éléments de réponse.

La fig u re 1 ra p p e lle un ce rta in nombre de su bd iv is ion s que l 'o n d is t in gue habituellem ent dans le s processus qui gouvernent la synthèse d'ADN. Au sein de chacune de ces d iv is io n s in terviennent des éléments rég u la teu rs . Et c 'e s t évidemment au niveau de ces éléments q u ' i l faut chercher la cause du gigantism e c e l lu la i r e . Dans une c e l lu le d ip lo ïd e , ces processus conduisent généralement à la r é p lic a t io n conforme d'un ADN de com position moyenne in changée. Et l 'o n peut se demander s i le s noyaux en dopolyp loïdes o b é issen t à c e t te r è g le ou s i le u r stru ctu re p a r t ic u liè r e a u to r is e ra it ce rta in e s déviat io n s .

Les données que nous avons rassem blées concernent le s noyaux de la glande sé r ic ig è n e de Bombyx mûri. En passant, nous rapp elleron s quelques données com paratives t i r é e s de la l i t t é r a t u r e en nous ré fé ra n t le p lu s souvent p o s s ib le à des p u b lica tio n s comportant une revue b ib liogra p h iq u e étendue .

F a c t e u r s g é n é r a u x d a n s le mi l i eu i nt é r i eu r cé l é m e n t s t r o p h i q u e s . i ons m i n é r a u x h o r m o n e s . . . . )

1S y n t h e s e d e s

p r é c u r s e u r s d i r e c t s d e l ' A D N

/ n t n e s e d e s

p r o t é i n e si

F a c t e u r s p r o p r e s a c h a q u e t y p e c e l l u l a i r e

S t r u c t u r e d e I A D N m o d è l e a v e c s es p r o t é i n e s a s s o c i é e s

S y n t h e s e d ' A D N

d A T P d T T P d G T P d C T P

A D N-Poly m é r a s e

FIG. 1. Schém a sim p lifié des principaux processus susceptibles d 'in terven ir dans la régulation d e la synthèse d 'A D N .

S M - 102/40 4 0 9

1. LA SYNTHESE DE L'ADN DANS SES RAPPORTS AVEC LA PHYSIOLOGIE DE L'ORGANISME

De nombreuses ob serv a tion s , fa i t e s chez le s V ertébrés , ont montré qu'une m u lt ip lica t io n c e l lu la i r e lo c a l i s é e peut ê tre provoquée par des fa cteu rs d 'o rd re g én éra l, hormonal ou autres [ l , 2 } . Pour le s In se c te s , on c i t e r a plus p articu lièrem en t deux ca té g o r ie s d 'o b se rv a tio n s :

D'une part, HADORN (3} constate que les cellules des disques imaginaux de Drosophile se multiplient indéfiniment lorsque ceux-ci sont implantés dans l'abdomen d'un imago. Au contraire, leur multiplication s'arrête pour faire place à la différenciation si les mêmes disques sont greffés à la pupe.

D'autre part, KRISKNAKUMARAH et coll. (U} décrivent, dans certains organes des larves de Saturnides, une synchronisation de la synthèse d'ADN par rapport à la mue. Des abdomens de nymphes isolés n'effectuent pas de synthèse d'ADN au niveau de l'hypoderme ; celle-ci reprend lorsqu'on leur greffe des glandes prothoraciques actives. Ces auteurs en concluent que la fourniture d'ecdysone déclenche la synthèse d'ADN dans un certain nombre de tissus.

Dans la glande séricigène de Bombyx mori, les noyaux grandissent, sans se diviser et sans jamais former de chromosomes distincts, jusqu'à donner naissance à des structures géantes multilobées, qui peuvent contenir une quantité d'ADN 1 0ц fois supérieure à celle d'un noyau diploïde.

Chez des animaux d'âges croissants, le dosage de la quantité totaled'ADN montre que la synthèse répond à un temps de doublement régulier d'environ U3 heures, coupé par des phases de synthèse ralentie, durant 2k à U8 heures, au voisinage de chaque mue (fig. 2) (5). Les résultats de ces analyses sont confirmés par l'étude cytologique. Si on injecte, à un animal, de la thymidine tritiée puis, après un certain intervalle de temps, de la thymidine ***С, on pourra distinguer, parmi les noyaux marqués, ceux qui le sont par le tritium ou par le carbone ou par les deux marqueurs. Lorsqu'on fait varier l'intervalle de temps qui sépare les deux injections, on constate une variation corrélative dans la proportion des marquages mixtes(fig. З). Sur ce graphique, l'intervalle entre deux maxima représente ladurée qui sépare deux phases de synthèse d'ADN au sein d'un même noyau.

FIC. 2. Evolution d e la quantité d 'AD N dans la glande séricigène d e Vers à soie d 'âges croissants. Les temps sont exprim és en jours n partir du prem ier repas. Les quantités d 'AD N sont exprim ées en ng (= 10 “ 6 m g).

4 1 0 NIGON et al.

Tem ps en jours

FIG. 3. Evolution du pourcentage d e noyaux m anifestant un m arquage m ix te en fonction d e l 'in te rv a lle entre les deux in jections d e thym idine 3II et 14C. Vers du troisièm e âge. La prem ière in jection est pratiquée à la sortie d e m ue.

Appliquée systématiquement aux vers du 3ème âge, c e t te méthode a démontré l 'e x is t e n c e de t r o is c y c le s de synthèse : l e premier dure 60 heures, tand isque le s deux suivants durent moins de !+8 heures. Au moment de la mue, l e nombre de noyaux marqués par une seu le in je c t io n tombe aux a lentours de 25$ a lo rs q u ' i l peut dépasser 75% un jo u r p lus ta rd .

Ces observation s conduisent à penser que, après la mue, le s noyaux mènent à le u r f in le s synthèses déjà commencées mais suspendent l 'e n t r é e dans un nouveau c y c le : dans c e t te hypothèse, la durée prolongée du premierc y c le se r a it la conséquence d'un retard dans l e déclenchement de la synthèse.

L 'e x is te n ce de c e t te d is co n tin u ité présente l 'a v a n ta ge évident d 'a ssu rer une homogénéisation entre le s d ivers noyaux de la glande : en l 'a b se n cede ce p rocessu s, en e f f e t , la v a r ia b i l i t é in d iv id u e lle des d iv ers noyaux pou rra it in trod u ire des d iffé r e n c e s cro issa n te s dans leu rs développements r e s p e c t i f s . I l p a ra ît évident que c e t te d is co n tin u ité trouve son o r ig in e dans un fa c te u r e x té r ie u r à la g lande. Les observation s de KRISHNAKUMARAN in c ite r a ie n t à en chercher la cause dans un changement de la s itu a t io n end ocrin e . Cependant, b ien d 'a u tres fa cte u rs peuvent ê tre invoqués, comme des m od ifica tion s au niveau de m étabolites e s s e n t ie ls , t e l s q u 'i l s peuvent ré su lte r des processus de 3a mue associés au jeûne que subit l 'a n im a l à ce moment.

2. LA REGULATION DE LA SYNTHESE D'ADN AU NIVEAU DU METABOLISME.CELLULAIRE

Lorsque p lu sieu rs noyaux se trouvent dans une même enveloppe c e l lu la i r e , on constate en général que le u r comportement e st étroitem ent synchronisé . L'une des p lus in téressan tes expériences e ffe c tu é e s en ce sens a comporté l 'u t i l i s a t i o n de noyaux p ré levés dans l e t is s u nerveux d'un Xenopus adulte e t donc présumés incapables de se d iv is e r : l o r s q u 'i l s sont in je c té s à des o e u fs , ces noyaux e ffe c tu e n t une synthèse d'ADN ( б ) . Cette homogénéité du comportement n u cléa ire ne peut ê tre due qu 'à des fa cteu rs in tr a c e llu la i r e s .

Parmi le s éléments su sce p tib le s d 'in te r v e n ir à ce n iveau, p lu sieu rs auteurs ont noté l 'e x is t e n c e d 'une c o r r é la t io n entre l'im p orta n ce de la synthèse d'ADN e t certa in es v a r ia tio n s au sein du système producteur des n u c leo tid es précurseurs de l'ADN ( 7 ) . Dans la glande sé r ic ig è n e de Bombyx

S M -102/40 4 1 1

nous avons pu con sta ter une év o lu tion du p oo l des précurseurs th ym id iliqu es qui p a ra ît en rapport avec l ' i n t e n s i t é de la synthèse d'ADN. Ce p o o l des précurseurs th ym id iliqu es représente une quantité extrêmement f a ib le , en viron m ille f o i s p lu s p e t it e que la quantité de thymine présente dans l'ADN de la glande. Son évalu ation n 'a pu ê tre f a i t e que par des méthodes in d irectes fondées sur l 'é tu d e c in é tiq u e des con d ition s de com pétition entre la thymidine ra d io a ct iv e exogène e t l e s précurseurs endogènes, dans des glandes explantées in v i t r o ( 8 ) .

L'ensemble des mesures e ffe c tu é e s conduit aux r é su lta ts du tableau I .

T A B L E A U I. SYNTH ESE DE L 'A D N E T NIVEAU DES PRECU RSEU RS ENDOGENES (L e s c h if fr e s sont donnés en quantité de thym ine (juM) p ou r 100 m g d 'A D N )

In corporation h ora ire Quantité présenteAge.de la glande étudiée dans la dans le

synthèse d'ADN pool endogène

Itème jo u r du 5ème âge 20 à 1*0 10 à 20

6ème jo u r du 5ème âge 5 . 1

L 'e x is te n ce de ces v a r ia t io n s au niveau du p oo l des précurseurs est in téressan te à p lu sieu rs t i t r e s :

1 . E lle conduit à con s id érer avec prudence tou tes le s évalu ation s qu an tita tives de la synthèse d'ADN fondées sur des mesures d 'in co rp o ra tio n d'un précurseur r a d io a c t i f . Chaque f o i s que des évalu ation s comparatives portent sur des c e l lu le s d if fé r e n te s ou sur des stades é lo ig n é s de la v ie d'un animal, e l le s doivent prendre en con s id éra tion une p o s s ib le v a r ia tio n au sein du p oo l des précurseurs endogènes avec le sq u e ls l e précurseur rad io a c t i f en tre en com pétition .

2 . Les rapports entre ces v a r ia t io n s e t le s changements dans la synthèse d'ADN peuvent ê tre conçus de deux façons au moins. Ou b ien on supposera que la rédu ction du p o o l des précurseurs e st une conséquence in d ire cte d 'une synthèse d'ADN diminuée ; ou bien on admettra que la synthèse réd u ite d'ADN ré su lte de l'appauvrissem ent en p récu rseu rs.

En l 'a b se n ce d 'in d ic a t io n s p r é c is e s , la première hypothèse conserve un caractère s p é c u la t if év id en t. Au co n tra ire , de nombreuses observation s démontrent que s i , par l 'e m p lo i de moyens génétiques ou p h ysio log iqu es approp r ié s , on lim ite la form ation des précu rseu rs , la synthèse d'ADN se trouve trè s rapidement b loqu ée . Dans la glande sé r ic ig è n e de Bombyx m ori, on peut fo r t b ien supposer que la m o b ilisa tio n des ap titudes m étaboliques de la c e l lu le vers la synthèse massive de f ib r o ïn e entraîne des d é fic ie n ce s au niveau d 'a u tres secteu rs comme c e lu i qui in té re sse la genèse des précurseurs th ym id iliqu es . On touche p e u t-ê tre a lo rs un aspect p a r t ic u l ie r de ce problème trè s général d 'un ce rta in degré de co n tra d ic tio n en tre la m u lt ip lica tion e t la d i f fé r e n c ia t io n c e l lu la i r e .

4 1 2 NIGON et al.

3. LE CONTROLE DE LA SYNTHESE D'ADN AU NIVEAU DES STRUCTURES CHROMOSOMIQUES

On sait aujourd'hui que l'ADN d'un noyau est formé par un certain nombre d'unités indépendantes de réplication. Entre ces diverses imités, la synthèse se déroule selon un ordre et avec des durées relatives qui semblent caractéristiques (9, l o ) . L'étude des chromosomes polytènes des Diptères est, à cet égard, particulièrement intéressante car elle autorise un pouvoir de résolution très élevé (il, 12). C'est ainsi que PLAUT et NASH (1 3} reconnaissent, dans un seul chromosome géant de Drosophile, au minimum 50 unités de réplication distinctes.

Dans les noyaux endopolyploïdes sans structures chromosomiques visibles, l'observation de synthèses asynchrones d'ADN est quasiment inexistante. Une exception remarquable est représentée par le macronucleus du Cilié Euplotee où deux ondes de synthèse progressent depuis les extrémités vers le centre du noyau'(lit, 1 5 , lé}.

Chez Bombyx mori, le marquage obtenu après incorporation de thymidine radioactive est en général également réparti sur l'ensemble du noyau. Cependant, des marquages limités a certaines parties du noyau ont pu être observés (1 7}. Ces marquages localisés appartiennent à deux types différents. Les uns présentent la forme d'une tache circulaire dans laquelle la synthèse d'ADN est retardée par rapport au reste du noyau (fig. h). D'autres au contraire correspondent à des structures épaisses de forme allongée, (fig. 5). La tache circulaire est aisément identifiable et semble correspondre à une structure de la chromatine plus dense que le reste de la chromatine nucléaire. Cette tache est généralement unique : sa taille s'accroît lorsque l'animal grandit ; elle se rencontre seulement chez les individus de sexe femelle. L'ensemble de ces caractères milite en faveur de l'identification de cette structure avec l'hétérochromatine sexuelle : on sait, eneffet, que chez le Ver à soie le sexe femelle est hétérogamétique.

• - ---------------------

FIG. 4 . Marquages nucléaires montrant la tache à incorporation tardive. Sur la figure de gauche, on vo it aussi un noyau intégralem ent m arqué. Vers du troisièm e. Jge.

Les stru ctu res de forme a llo n g é e , en revanche, peuvent ê tre présentes en p lu sieu rs exem plaires au sein d'un même noyau. Mais leu rs cara ctères morphologiques sont trop peu d i f fé r e n c ié s pour perm ettre d 'é t a b l i r des homolog ie s p ré c ise s d'un noyau à un au tre . I l semble d i f f i c i l e d 'échapper à l ’ hypothèse que ces stru ctu res représentent d 'authentiques chromosomes géants.

S M - 102/40 413

En d é f in i t iv e , l 'e m p lo i de marquages r a d io a c t ifs permet de recon n a ître au se in des noyaux de la glande sé r ic ig è n e des u n ités de r é p lic a t io n qui présentent ce rta in s caractères de stru ctu res chromosomiques g igantesques. Si l 'o n admet c e t te in te rp ré ta t io n , on e s t conduit à supposer que l'en sem b le du noyau p ou rra it ê tr e formé par l 'a p p o s it io n de t e l l e s stru ctu res dont la cont ig u ït é in te r d it normalement de d iscern er le s l im ite s . La forme m u ltilobée du noyau p ou rra it ê tre une conséquence de l 'a s p e c t a llon gé de ces stru ctu res étroitem ent a c c o llé e s entre e l l e s . Le nombre de ces éléments co rre sp o n d -il au nombre des chromosomes ou à un c h i f f r e d if fé r e n t ? Les données a c tu e lle s sont encore in su ffis a n te s pour répondre à c e tte qu estion . On notera seu le ment qu'une éva lu ation du volume n u cléa ire e t du volume de ces stru ctu res montre que le noyau s e r a it su sce p tib le de con ten ir entre 20 e t 60 éléments de ce genre, tand is que la garn iture haploïde du Ver à so ie e st formée de 28 chromosomes.

1*. EXISTE-T-IL 1ШЕ SYNTHESE DIFFERENTIELLE D'ADN ?

Malgré l 'e x is t e n c e de r é p lic a t io n s asynchrones au se in d 'un même noyau, on admet le p lus souvent que le c y c le complet de synthèse corresponde à un doublement rigoureusement conforme de l'en sem b le de l'ADN p ré e x is ta n t . Cependant, des observation s relativem ent is o lé e s semblent in v ite r à l a prudence. D'une p a rt , chez la D rosoph ile , p lu sieu rs auteurs remarquent que la quantité d'ADN hétérochrom atique e st relativem ent réd u ite dans le s chromosomes polytèn es par rapport à sa p rop ortion dans le s c e l lu le s eu p lo ïd e s . D 'autre p a rt , des zones de synthèses ex cessiv es d'ADN (ju sq u 'à 15 f o i s la synthèse normale) ont é té notées dans le s chromosomes de Saîaea e t Rhynoho- scia ra . La question se p o s a it donc de sa v o ir s i la s tru ctu re endopolyploïde n 'e s t pas su sce p t ib le de fa v o r is e r la synthèse p r é fé r e n t ie l le de certa in s secteu rs du génome, conduisant éventuellem ent à un changement dans la compos i t io n moyenne de l'ADN. Pour v é r i f i e r ce p o in t , nous avons c h o is i de comparer l'ADN d'embryons à c e lu i e x tr a it de la glande s é r ic ig è n e . La méthode d 'an a lyse employée a co n s is té à i s o l e r , dans l'ADN, le s d iv ers o lig o n u c lé o - t id e s pyrim idiques de même longueur, ou i e o p l i th e s , e t à analyser c e u x -c i en quantité e t en com position ( l ô ) . Cette méthode fo u r n it , pour un même ADN, un ensemble de ré su lta ts qui peut comprendre p lu s de 15 paramètres d 'id e n t i f i c a t io n indépendants. Les ré su lta ts obtenus montrent que, au degré de sens i b i l i t é extrême a in s i obtenu, i l n 'e x is t e aucune d if fé r e n c e s i g n i f i c a t iv e entre la com position moyenne de l'ADN dans la glande sé r ic ig è n e e t la comp o s it io n de l'ADN d'embryon. A in s i, une d i f fé r e n c ia t io n c e l lu la i r e extrême ne p a ra ît pas, dans ce ca s , com porter un changement v is ib le de la composit io n de l'ADN.

CONCLUSIONS

En d é f in it iv e , l'ensem ble des analyses que nous avons e f fe c tu é e s tend, à démontrer que la synthèse de l'ADN o b é i t , dans le s noyaux endopolyp loïdes de la glande s é r ic ig è n e , à la p lupart des rè g le s généralement observées dans le s c e l lu le s d ip lo ïd e s .

Dans chaque noyau, la synthèse s 'o p è re selon un processus cy c liq u e dont chaque élément correspond sans doute à un doublement. Au se in d'un c y c le de synthèse, on découvre des asynchronismes mettant en évidence l 'e x is t e n c e d 'u n ité s de r é p lic a t io n qui présentent b ien des ca ra ctères de chromosomes gigantesques. Au cours de la synthèse, l a com position de l'ADN semble exactement conservée. E n fin , l e déterminisme de la synthèse o b é it à d ivers fa cteu rs dont le s uns sont e x tér ieu rs à la c e l lu le tand is que d 'a u tres lu i sont in tern es .

FIG. 5. M arquages nucléaires loca lisés au niveau d e form ations a llon gées. Trois coupes successives d e la m ê m e g lande perm ettent d e reconstituer la form e des structures m arquées. Les zones c la ires représentent les sections d e noyaux.

414 N

IGO

N.etal.

S M -102/40 4 1 5

L'acquisition la plus importante paraît concerner la nature et la forme des unités de réplication. Bien qu'il soit encore difficile d'exprimer une opinion définitive sur le nombre de ces unités, les éléments recueillis semblent indiquer que les unités observées pourraient correspondre au nombre des chromosomes. Cette situation rappelle fortement les dispositions bien connues au niveau des noyaux polytènes. Elles tendraient à faire penser que, dans le cas de la glande séricigène comme dans celui des noyaux polytenes, les éléments chromosomiques homologues restent associés pour former des unités d'ordre supérieur à comportement synchronisé. Cette situation correspond vraisemblablement à l'absence du processus de condensation caractéristique de la prophase mitotique.

L'une des questions qui se posent est de savoir si cette structure n'est réalisée que dans les noyaux endopolyploïdes de la glande séricigène ou si elle peut exister également dans d'autres organes et dans d'autres espèces. S'il en était ainsi, on trouverait là un élément d'unification entre les processus de différenciation cellulaire particuliers aux Insectes.

On notera, seulement, pour terminer, que la prudence apparaît nécessaire avant une généralisation trop étendue. Si l'on considère, en effet, les noyaux polyploïdes des Protozoaires, il apparaît que les observations faites chez Euplotes justifieraient peut-être une extension des hypothèses précédentes. Au contraire, les études concernant la régénération du macronucleus chez Paramecium ou la division nucléaire chez le Radiolaire Aulacantha, conduisent à admettre l'existence de sous-noyaux distincts formés chacun d'un assortiment chromosomique complet. De sorte qu'un ensemble d'observations conduirait à supposer l'existence d'une série d'étapes intermédiaires entre la mitose classique et le processus de multiplication que révèlent les chromosomes polytenes. L'étude comparative de ces divers processus est certainement de nature à nous apporter des éclaircissements importants concernant les relations entre les diverses composantes de.la division cellulaire.

R E F E R E N C E S

flj TEIR, H., RYTOMAA, T., Control of cellular growth in adult organisms. Ácad. Press (19 6 7).

(2) LOCKWOOD, D .H ., VOYTOVICH, A .E . , STOCKDALE, F .E . , TOPPER, Y .J . , In su lin dependent DNA polym erase and DNA s y n th e s is in mammary e p i t h e l i a l c e l l s in v i t r o . Proa. Nat. Aoad. Soi. _58 (19 6 7 ) 658.

(3) HADORN, E., "Dynamics of determination", in Major problems in developmental biology (LOCKE, М., ed.) Acad. Press (1966) 85.

(1+] KRISHNAKUMARAN, A., BERRY, S.J., OBERLANDER, H., SCHNEIDERMAN, H.A., Nucleic acid synthesis during Insect development. II, Control of DNA synthesis in the Cecropia silkworm and other Saturniid moths, J. Insect

Physiol. 13 (196 7) 1.(5] GILLOT, S., DAILLIE, J., Rapport entre la mue et la synthèse d'ADN dans

la glande séricigène du Ver à soie, C.R. Acad. Sc. (196 7) sous presse.(6} GURDON, J.B., On the origin and persistance of a cytoplasmic state in

ducing nuclear DNA synthesis in frog's eggs, Proc. Nat. Acad. Sci. _58

(1967) 51+5 •(7 ] DUSPIVA,F., "Enzymatische Aspekte der Mitose" in Probleme der biologis-

chen Reduplikation (SITTE, P., ed.) Springer, Berlin (1966) 120.(8] DAILLIE, J., Métabolisme de la thymidine dans la glande séricigène du

Ver à soie. I, Les principales voies suivies par le précurseur dans la glande incubée in vitro. II, Utilisation des nucléotides radioactifs pour la synthèse de l'ADN. III, Incorporation dans la glande séricigène prélevée au 6ème jour. IV, Etudes sur la glande in situ. Ann. Biol. anim. Bioch. Biophys. 7. (1967) 115 ; 227 ; sous presse.

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(9] HSU, T.С., SCHMID, W. , STUBBLEFIELD, E., "DNA replication sequences in higher animals" in The role of chromosomes in development (LOCKE M.ed.) Acad. Press (1961+) 8 3.

(lo) BARIGOZZI,C., DOLFINI, S., FRACCARO, M.,HALFER,C.,REZZ0NIC0 RAIMONDI G. TIEPOLO,L., A first approach to the somatic cell genetics of Drosophila melanogaster : studies on DNA replication, Atti Ase. Genet. I tal. 1£ (I967) 291.

Ill SWIFT, H., "Molecular morphology of chromosome", In vitro 1 (1965) 26.12] KEYL, H.G., "Lokale DNS-Replikationen in Riesenchromosomen" in Probleme

der biologischen Reduplikation (SITTE P., ed) Springer Berlin (1966) 55

(1 3) PLAUT, W., NASH, D . , "Localized DNA synthesis in polytene chromosomes and its implications" in the role of chromosomes in development (LOCKE, М., ed. ) Acad. Press (196I+) 113-

(lit) FAURE-FREMIET, E., ROUILLER, С., GAUCHERY, М., La reorganisation macromoléculaire chez les Euplotee. Exp. Cell Res. 12 (1957) 135.

(15) GALL, J.G. ,Macronuclear duplication in the ciliated protozoon Eicplotee, J. bioph. biooh. Cytol. ¿ (1959) 295.

(lé) RINGERTZ, N.R., ERICSON, J.L.E., NILSSON, 0., Macronuclear chromatin structures in Euplotee. Exp. Cell Res. (1967) 97-

(1 7 } GILLOT, S., Hétérogénéités fonctionnelles dans l'ADN de noyaux géants. Etude autoradiographique sur la glande séricigène de Bombyx mori, Exp. Cell res. (sous presse).

(l8) NEULAT, M.M., Comparaison de différents DNA animaux par .l'analyse de leurs séquences pyrimidiques, Biooh. Biophys. Aota ll*9 ( 196 7) 1*22.

SYMPOSIUM ON THE USE OF ISOTOPES AND RADIATION IN ENTOM OLOGY

HELD IN VIENNA, 4 - 8 DECEM BER 1967

С1Ш 1Ш КМ OF SESSIONS

SESSIONS 1 AND II

SESSIOXS IIT AND IV

SESSION V

SKSSIOXS VI AND VII

SESSION VIU

П. M ARCH ART

II. NOTHEL

L .K . CUTKOM P

E .M . SIIUMAKOY

(p. STROMNAES

Chana

F ed era l Republic of G erm any

United States of A m erics

USSR

X orw av

SECK ETA RIAT OF THE SYMPOSIUM

S cien tific S e cre ta ry : I-I. ERDMAN

A dm in istra tive S e cre ta ry : II. H. STORIIAUG

Editor

R ecord s O ffice r

P r e s s O ff ic e r s :

C on feren ce O ff ic e r :

E. DOYLE

E ileen PEMBR.1DG

P . A P R IE TO S. WHITE

C la ire BURKE

Joint FAO/IAEA Division o f A tom ic Energy :n Food and A gricu lture

D ivision o f Scientific and T echn ica l Inform ation,

IAEA

D ivision o f Scientific and T echn ica l inform ation,

IAEA

D ivision o f Languages,IAEA

D ivision o f Public in form ation , IAEA

D ivision o f C onference and G eneral S erv ices ,

IAEA

4 1 7

LIST OF PARTICIPANTS

A U S T R IA

Beran, F.

K alil, E.

Russ, K.

S ch m u tzen h ofer, H.

S p erlich , D,

B E LG IU M

P cleren ts, C .A .

R iga, A .

C A N A D A

B aldw in, W .F .

N air, K .K .

C Z E C H O S L O V A K

B elák ov á , A d e la

H alanda , S.

Bundesanstalt fur P flanzenschutz,Trunnerstrasse 5 , 1020 V ien n a ,Postfach 154

Bundesanstalt für P flanzenschutz,Trunnerscrasse 5 , 1020 V ienna,Postfach 154

Bundesanstalt fur P flanzenschutz,Trunnerscrasse 5 , 1020 V ienna ,Postfach 154

Institut für Forstschutz, A b te ilu n g E n tom olog ieund a m tlich e M ittelprU fung an der forstlich en Bundesversuchsanstalt,Schonbrunn, O ber T iro lerga rten , 1131 V ienna

Institut für a llg e m e in e B io lo g ie ,Schw arzspanierstrasse 17 , 1090 Vienna

F a cu lté des S c ie n ce s A gron om iqu es de l ’ Etat, Ghent

C en tre de R echerches A gron om iques de l'E ta t, .Station de C h im ie et de Physique a g r ico le s , G em b lou x

B iology and H ealth Physics D iv is ion ,C h alk River N uclear l aboratories,A to m ic Energy o f C anada L t d . ,C h alk River, O ntario

D ept, o f B io lo g ica l S c ie n ce s , P esto logy C en ter,S im on Fraser U niversity , Burnaby 2 , B .C .

SOC IA L IS T R E P U B L 1С

H ept. o f E n tom ology , Nature Facu lty,U niversity o f C om en iu s , Safarikovo nám 12 , Bratislava

Institute o f E xperim enta l P h ytop ath ology and E n tom ology o f the S lovak A ca d e m y o f S c ien ces ,Ivanka pri Dunaji

4 1 9

4 2 0 LIST OF PARTICIPANTS

liu sá rová , Anna

K m e to v á , Sona

K orb e l, !..

b te p a n o v ico v á , O lga

V ese lovsk y . J.

E T H IO P IA

L em m a , A .

F R A N C ГС

B luzat, R.

B on avita , Annie

C ausse, R.

D urgeat, L . - A .

H urpin, B.

L a beyrie , V .

L e c o m te , J,

Le M asn e, G .

L eroi, B .R.

Dv'pi. o f lv;iU):noii.n.:v, / .o o lo g v r 'a c i l lv ,V n ivcrsitv o f C on u 'iiiu .', Safanl\OV;¡ nám V 2 , Bratislava

D ept, o f A n im a l P h ysio logy ,U niversity o f C om en iu s , M oskovská 2 , Bratislava

D ept, o f E n tom olog y , Z o o lo g y F a cu lty ,U niversity o f C om en iu s , S a farik ovo nám 12 , Bratislava

D ept, o f E n tom olog y , Nature F a cu lty ,U niversity o f C om en iu s , S a fa r ik ov o nám 12 , Bratislava

D ept, o f A n im a l P h y s io log y ,U niversity o f C om en iu s , M oskovská 2 , Bratislava

H aile S e llass ie I U niversity , P .O . Box 1176 , A ddis A baba

Laboratoire de z o o lo g ie de la fa cu lté des sc ien ces d ’ O rsay, U niversité de Paris,Orsay 91

Institut de n eu rop h ysio log ie et p sy ch op h y s io log ie ,D épt. de p sy ch op h ysio log ie c o m p a r é e ,C .N . R . S . - I ,N . P . 7 ,31 ch e m in J osep h -A ig u ier , M arse ille 9^

Station de z o o lo g ie , I . N . R . A . , C .N .R .A . du Sud-Est,84 M on tfa vet (V au clu se )

Institut tech n iqu e français de la betterave in du strie lle ,6 c it é M onth iers, Paris 9e

Institut n a tion a l de la re ch erch e a gron om iqu e ,Station de rech erch es de lutte b io log iq u e et de b io co e n o t iq u e , La M in ière par V ersailles

Laboratoire d * é co lo g ie et b io co e n o t iq u e expérim en ta les, C . S . U . , Parc G randm ont, 37 Tours

Institut n a tion a l de la re ch erch e a gron om iqu e ,91 Bures sur Y v ette

Institut de n eu rop h ysio log ie et p sy ch op h y s io log ie , D épt. de p sy ch op h ysio log ie c o m p a ré e ,C . N . R. S . - I . N. P. 7 , 31 ch e m in J osep h -A igu ier ,M arse ille 9e

Laboratoire d ’ é c o lo g ie et b io co e n o t iq u e ex p ér im en ta les , C . S . U . , Parc G randm ont, 37 Tours

LIST OF PARTICIPANTS 4 2 1

P é leg r in , P.

R age, P.

S im e o n , C .G .

T orossian , C .

G E R M A N Y ,

A d o lp h i, I-l.

H aisch , A .

K lo ft , W .J.

M ayer, K.

N Cthel, II,

GH ANA

M archart, H.

H U N G A R Y

Farkas, J.

N agy , B.

Nigon, V. U niversité de L yon , F a cu lté des sc ien ces de Lyon, S ection de b io lo g ie gén éra le et a p p liq u ée ,43 bd du 1 ] N ovem bre 191S , 69 V illeu rban n e

D épartem en t de b io lo g ie , S erv ice de ra d ioa g ron om ie , GEN C a d a ra ch e , B . P . l ,St. Paul le z D urance 13

Institut de n eu rop h y s io log ie et p sy ch op h y s io log ie ,C . N .R .S . - I . N .P .7 , 31 ch e m in J o sep h -A ig u irer ,13 M a rseille 9e

C om m issariat à l 'é n e r g ie a tom iq u e , rue C a p ita in e S co tt , Paris

F a cu lté des s c ie n ce s d e T ou lou se ,L aboratoire d * e n to m o lo g ie ,1 ] 8 Route de N arbon n e, 31 T ou lou se

F E D E R A L R E P U B L IC OK

B adische A n ilin und Soda Fabrik, L u dw igshafen /R h.

B ayerische Lanciesanstalt fur B odenkultur,P flanzenbau und P ila n /e n sch u tz ,M enzingerstrasse 5 4 , 8000 M unich 19

Institut fiir angew and te Z o o lo g ie , U n iv e rs ité Bonn,An der lm m en b u rg 1, 5300 Bonn

B io log isch e Bundesanstalt für Land- und Forstw irtschaft, K O nigin -L u ise-S trasse 19 , 1000 Berlin 33

Institut für C c n e t ik der Frcien Univcrsittft Berl in, R u deloffw eg 9 , 1000 Berlin 33 , Dahlern

C o c o a R esearch Institu te, Ghana A ca d e m y o f S c ien ces , T a fo , P .O . Box 8 ‘

C en tra l Food R esearch Institute, H erm ann O ttó ut 15 , Budapest II

R esearch Institute for Plant P rotection , H erm ann O tto ut П , Budapest II


INDIA

M ehrotra , K .N .

Rahalkar, G . W.

IN D O N ESIA

H a tm osoew a m o, S.

IS R A E L

G alu n , R ach el

IT A L Y

C ir io , О.

F im ia n i, P.

G u errieri, G .

K E N Y A

O d h ia m b o , T .R .

K O R E A

L ee , K .-B .

N E T H E R L A N D S

d e K ort, C . A . D.

N IG E R IA

D iv is ion o f E n tom ology ,Indian A g ricu ltu ra l Research Institute,New D elh i 12

B io logy D iv is ion , Bhabha A to m ic Research C en tre , M odular L aboratories, T rom b a y , Bom bay 74

State Sugar A ca d e m y , Jogjakarta

Israel Institute for B io lo g ica l R esearch , Ness Z io n a

C o m ita te N a z ion a le Energía N u c le a r c , Laboratorio A g rico ltu ra , C a s a c c ia , Rom e

L aboratorio di E n tom olog ía Agraria 'F . Silvestri*, P o r t ic i , N aples

C o m ita to N azion a le Energía N u clea re ,C . S . N . C a s a cc ia , Via A nguillarese , Rome

D epr. o f / .o o lo g y , University C o l le g e , P .O . Box 3 01 97 , N airobi

A to m ic Energy Research Institute, P .O . Box 7 , C h y u n g -ry a n g -r i, Seoul

Laboratory o f E n tom ology , A gricu ltu ra l U niversity, B inncnhavcn 7 , W ageningen

Baldry, D . A . T . N igerian Inst, for Trypanosom iasis R esearch, Private M a il Bag 2 0 7 7 , K aduua,North C entral State

LIST OF PARTICIPANTS 423

Str<Smnaes, Ф.

N O RW AY

Institute o f G enera l G e n e tics , U niversity o f O slo , O slo 3

P A K IS T A N

Anw ar, M . Pakistan A to m ic Energy C om m iss ion , P .O . Box З П 2 , K arachi 29

P E R U

S im ón , J.E . E stación E xperim ental A g rico la de la M o lin a , C a s illa 2 7 9 1 , l.im a

R O M A N IA

C a trin a , I.

M ih a la ch e , G.

Institut de r e ch erch e fo restiè re . Sos. P ipera N o. 4 6 , Bucharest

institut d e r e ch erch e fo restiè re , Sos. P ipera N o. 4 6 , Bucharest

SW E D E N

E idm ann , H. H. R oyal C o l le g e o f Forestry, S tock h olm 50

S W IT Z E R L A N D

H orber, E. Station féd éra le d 'essa is a g r ico le s , Z u r ich -O e r lik o n

T U R K E Y

Kansu, 1 .Л . F a cu lty o f A g ricu ltu re , U niversity o f A nkara, Ankara

UG AND A

H a rley , J .M .B . East A frica n T rypanosom iasis Research O rga n iza tion , P .O . Box 9 6 , T ororo

U S S R

S hu m akov , E .M . Institute for Plant P ro tect ion , M ertzen Str. 4 2 , Leningrad


U N IT E D A R A B КК1Ч.ГВ1Л.С

Reda El H ag, M .A . F acu lty o f B io lo g y , M oscow State U niversity,M o sco w , USSR and I-lin Shams U niversity , C a iro

U N IT E D K IN G D O M

C o h e n , M .

C u rtis, C .F .

Le Q uesne, W .J .

M inistry o í A g ricu ltu re , F isheries and F ood ,Plant P a th o logy Laboratory,H atch ing G reen , H arpenden, Herts.

Tsetse Research Laboratory,D ept, o f V eterinary M e d ic in e , U niversity o f Bristol, Langford H ouse,Langford, Bristol

U K A EA , R a d ioch em ica l C en tre ,W hite l.ion R oad , A m ersham , Bucks

P h illip s , F .T , M inistry o f O verseas D ev e lop m en t, Rotham sted E xperim enta l S tation , I la rp en den , Herts

T reh ern e , J .E . D ept, o f Z o o lo g y , U niversity o f C a m b rid g e , D ow ning S treet, C a m b ridge

U N ITED S T A T E S O F A M E R IC A

B o ïk o v e c , A .B .

C u tk om p , L .K .

G rosch , D .S .

K atiyar, K .P .

N orth, D .T .

S onen sh in e, D .E .

E n tom ology Research D iv is ion , ARS,US D ep t, o f A g ricu ltu re , B e ltsv ille , M d .20705

U niversity o f M in nesota , St. P au l, M in n . 551.01

North C a ro lin a State U niversity , G en etics D e p t ., Raleigh* N .C . 27607

M eta b o lism and Radiation R esearch Laboratory , ARS, US D ept, o f A g ricu ltu re , Fargo, N . D akota

M etabo lism and Radiation R esearch Laboratory, ARS, US D ept, o f A g ricu ltu re , Fargo, N . Dakota

B io logy D e p t ., Old D om in ion C o l le g e ,N orfo lk , V a .23508

von B orstel, R. C , B io log y D iv is ion , Oak Ridge N ationa l Laboratory, O ak R idge, Term . 37630

LIST OF PARTICIPANTS 4 2 5

ORGANIZA TIONS

E U R A T O M

C a v a llo r o , R.

SchrOder, J .II .

C en tre com m u n de re ch erch e n u c lé a ire , S erv ice de b io lo g ie , S e c t , e n to m o lo g ie , lspra (V a rese ), Italy

G esellschaft für S trahlenforschung M ü n ch en , Ingolstadter Landstrasse 1 , 8042 N euherberg, F ederal R ep u blic o f G erm any

F A O

D e lu c c h i, V, V ia d e lle T erm e di C a ra ca lla , H om e, Ita ly

IA E A

F ried , M .

L angley , P. A.

Lindquist, D.

R oiva in en , 0 .

T /.anakak is , M .E .

Voso., P.

Joint ГЛО/IA E A D iv ision o f A to m ic Energy in Food and A g ricu ltu re , K arntnerring 1 1 -1 3 , А Ю 10 V ienna I , Austria

D iv ision o f Research and Laboratories,KSrntnerring 1 1 -J 3 , A 1 0 ]0 V ienna I , Austria

Joint FA O /IA F A D iv ision o f A to m ic Energy in Food and A gricu ltu re , KJirntnerring 1 1 -1 3 , A 10']0 V ienna I, Austria

C o co a R esearch Institute, P .O . Box 8 , T a fo -A k im , Ghana'

lA l’ A Laboratory,Sc i horsci о г t, Л tM-M N iec lcros terrc ict i, Austria

Joint. F A O /IA K A D ivision o f A to m ic Energy in f-'ooc and A g ricu ltu re , K am tnerring 1 1-1П, A 10'iG V ienna I, Austria

0.1 Ы ’>

T ic lie lc r , J .H .G . Insiiiur. voor :']niiten/.tei<teni<undig O nderiioek , I3iniienhavc.n 1 - , W a g en m g cn , Xctherlancis

W HO

G o m e z -N u R e s , J .C . V ector B io logy and C on tro l, W H O , G en ev a ,Sw itzerland

AUTHOR INDEX

(including participants in discussions)

N um erals underlined re fer to the first p a g e o f a paper b y the author c o n ce rn e d . Further num erals d en ote

com m en ts and questions in the discussions.

A n w a r , М . : 10 9 , 120, 139,147, 286

B a c o y a n n is , A . : 209 B a ld r y , D . A . T . : 246 B a ld w in , W .F . : 52 , 84, 363 ,

365B e r a n , F . : 207B lu za t , R . : H )3, 107 , 108,

121

B o r k o v e c , A . B . : 2 0 1 , 205 ,20 6 , 20 7 , 20 8 , 21 5 , 403

B o r s t e l , v o n , R . C . : 147,20 7 , 208 , 24 5 , 3 3 1 , 337 , 354, 375 , 389

C a u s s e , R . : 15 5 , 35 5 , 364 C a v a llo r o , R . : 28 , 107, 147 C oh en , М . : 21 , 120, 174, 208 C u r t is , C .F . - : 120, 174, 206 ,

233 , 24 5 , 24 6 , 402 C u tk om p , L . K . : 179, 184, 185 D a i l l ie , J. : 407 D e lu c c h i , V . : 207 E h r h a rd t , P . : 23 E id m a n n , H . H . : 75 , 84 E r d m a n , H . : 120 , 154, 208,

329F e r o n , M . : 335 F e r r e r , F . : 165 F r a n ç o i s , E . : J53 F r ie d , M . : 329 F y t iz a s , E . : 209 G a lu n , R a c h e l : 29 , 30 , 52,

20 6 , 207 , 23 2 , 2 4 9 , 257 , 258

G illo t , S . : 407 G ir is h , G .K . : 123 G r o s c h , D . S . : 51, 52, 336 ,

3 7 7 , 389 H a tm o s o e w a r n o : 317, 329

H ill , W .G . : 233 H olt , G . G . : 39J_H o r b e r , E . : 21, 205, 206, 2 5 9 ,

270, 271 H u rp in , B . : 3 0 1 , 315 H w an g, U . K . : 273 Im p e n s , R . : J53K a n su , I. A . : 147, 154, 184, 286 K a t iy a r , K . P . : 165 , 173, 174,

175K lo ft , W . J . : 14, 23 , 29 , 30, 68,

137 , 162, 2 5 7 , 27 1 , 299 , 300 K u n k e l, H . : 23L a b e y r ie , V . : 73 , 74 , 107, 148,

175, 245 , 3 3 7 , 403 L a n g le y , P . A . : 185, 258 L e c o m t e , J. : 2Z» 20 , 21 L e e , К . B . : 273 , 286 L e e , K . S . : 273L e M a sn e , G . : 21 , 107, 137, 164,

315L e m m a , A . : 208L e Q u e sn e , W . J . : 15, 30 , 198,

364L e r o i , B . R . : 147 L in d q u is t , D . : 14 M a r c h a r t , H . : 3, 14, 15, 20 , 30,

51 , 67 , 185, 257 M e h r o tr a , K . N . : 30 , 187, 197,

198N a g y , B . : 148, 175, 270 N a ir , K .K . : 147, 149, 257 , 389 N e u la t -P o r t ie r , М . M . : 407 N ig o n , V . : 25 7 , 407 N o rth , D . T . : 174, 20 7 , 24 6 , 337,

39 1 , 4 0 2 , 403 N& thel, H . : 85_O d h ia m b o , T . R . : 206 P é le g r in , P . : 74 , 137 , 185, 363,

364

427

4 2 8 AU TH O R IN D E X

P e le r e n t s , C .A . : 21 , 148, 205 P e r e a u - L e r o y , P . : 355 P e s s o n , P . : 123 , 137 P h i l l ip s , F . T . : 30 , 184 P o u v r e a u , A . : 17 P r a s a d , Y . : 187 R a h a lk a r , G .W . : 69 , 73, 74 ,

149 , 154, 285 R a m a k r is h n a n , V . : J39 R ig a , A . : 53, 67 , 68 S eth i, G . R . : 187

S h u m a k ov , E .M . (Ш у м а к о в , E . M .): 15, 173, 219, 232

S im ó n , J . E . : 121, 173, 20 8 , 287, 2 9 9 , 300

S o n e n sh in e , D . E . : 3J., 51, 52 S tr^ m n a e s , p . : 3 4 1 , 354 T i c h e l e r , J . H . G . : 163, 286 T o r o s s ia n , C . : 155, 163, 164 T r e h e r n e , J . E . : 197, 198 T u re n n e , d e , M . : 407 T z a n a k a k is , A . B . : 215 W a rb u r g , M . : 249

I A E A S A L E S A G E N T S

O rd e rs for A g e n c y p u b l i c a t i o n s can be p l a c e d with your b o o k s e l l e r or any o f our s a l e s

a g e n t s l i s t e d b e l o w :

A R С К N T I N AC o m i s i ó n N a c i o n a l de E n e rg ía A t ó m ic a A v e n id a d e l L i b e r t a d o r G en era l San Martin 8 2 5 0 B u e n o s A i r e s - S u e . 29

A U S T H A L IAblunter P u b l i c a t i o n s ,23 M c K i l l o p Street M e lb o u r n e , C . l

A U S T H 1 AG e o rg l 'r om m e & C o .S p e n g e r g a s s e 39 A-JüfíO, V ie n n a V

B E L G I U MO f f i c e i n te rn a t io n a l de l ib ra ir ie 30, a v e n u e Marnix B r u s s e l s Г>

В R A Z I LL ivra r ia K o s m o s Ed itora Hua d o R o s a r i o , 13 5 -1 3 7 R i o de J a n e iro

A g e n c i a E x p o e n t e O s c a r M. S i lv a Rua X a v i e r de T o l e d o , 1 4 0 - 1 ° An dar ( C a i x a P o s t a l N o . ñ .6 1 4 ) .S a o l ‘ a u lo

B Y K L O RUSSIAN S O V I E T S O C I A L I S T R E P U B L I C

See und er USSR

C A N A D AT h e Q u e e n ' s P r in ter O tta w a , O ntar io

С H I N A ( T a i w a n )H ook s and S c ien t i f i c : S u p p l i e s S e r v i c e , L t d . ,P . O . B o x 83 T a i p e i

C Z E C H O S L O V A K S O C I A L I S T R E P U B L I CS . N . T . L .Spolr.-na M Mo v e Mo s t o Pr a g u e 1

D E N M A R КE jn ar M u n ksga ard Ltd .6 N o rre g a d e C o p e n h a g e n К

F’ I N L A N DA k a t e e m in e n K ir jak au p p aK e s k u s k a t u 2H e l s i n k i

1-’ R A N С E

O f f i c e i n te rn a t io n a l de d o c u m e n t a t i o n e t l ib ra ir ie 48 , rue G a y » L u s s a c E - 7 5 , P a r i s 5 t:

G E R M A N S , F e d e r a l R e p u b l i c of R. O ld e n b o u r g R o s e n h e i m e r S t r a s s e 145 8 M unich 8

H U N С A Í I Y KulturaH ungarian T r a d in g C o . for B o o k s and N e w s p a p e r s P . O . B . ] 49 B u d a p e s t 62

1 S R A E LH e i l i g e r and C o .3 Nathan S t r a u s s S treet J e r u s a le m

I T A L YA g e n z i a E d i t o r i a le I n t e m a z i o n a l e O r g a n i z z a z i o n i U n iv e r s a l i ( A . E . I . O . U . ) Via M erav i g l i 16 Milan

J A P A NMaruzen C om p a n y L td .6 , T o r i N ic l iom e N ih o n b a s h i

■ ( P . O . B o x 6 0 5 )T o k y o C en tra l

M E X I С ОL ib r e ri a Inter nació n a 1 A v , Son ora 206 M e x i c o 1 1 , D . F .

N E T II E H L A N D SN .V . Mar ’.inus N i j h o f f L a n g e V o o r h o u t 9 T h e H ague

N E W Z E A L A N DW h itcom b e & T o m b s , Ltd.G . P . O . B o x 189 4 W e l l in g t o n , C . l

N О K W A YJohan Grundt Tanum K ar l J o h a n s gate 43 O s l o

P A K I S T A NK a r a c h i E d u c a t io n S o c i o t y H aroon C h am bers South N a p i e r Hoad ( P . O . B ox N o . 4 8 6 6 )K a r a c h i 2

P O L A N DO s r o d e k R o z p o w s z e c h n i a n a W y d a w n i c tw N a u k o w y c h P o l s k a A k a d e m ia Nauk Pa-fac Kultury i N auki W arsaw

R O M A N I A C a rt im exHue A . Briand 11-18 B u c a r e s t

S O U T H A F R I C AVan S c h a i k ' s B o o k s t o r e ( P t y ) L td . L ib r i B u ild in g C h u rch S treet ( P . O . Box 724 )P re to r ia

S P Л I NI -i b re n a B o s c hH onda de la U n iv e rs id a d ПB a f c c I o n a

S W t: D E NC . E , K r i t z e s K u n g l . I I o v b o k h a n d e I K re d sg a ta n 2 S t o c k h o l m 16

S WI T Z E R L A N D L ib r a i r ie P a y o t Rue G ren us 6 1211 G en e vu 1 1

1' L7 R K E Y

L ib ra i r ie H a ch et te 4 6 9 , I s t ik l â l C a d d e s i B e y o g l u , Is tanbu l

U K R A IN IA N S O V I E T S O C I A L I S T R E P U B L I C

S ee und er USSR

UNION OK S O V I E T S O C I A L I S T R E P U B L I C S

M e z h d u n a ro d n a y a Kniga S m o l e n s k a y a - S e n n a y a 3 2 -3 4 M o s c o w G -2 0 0

U N I T E D K ING DO M O F G R E A T B R IT A IN A N D N O R T H E R N I R E L A N D

Her M a j e s t y ’ s Stat ionery' O f f i c e P . O . Box 569 L o n d o n , S . E . l

U N I T E D S T A T E S O K A M E R I C A N a t io n a l A g e n c y for In tern a t ion a l P u b l i c a t i o n s , In c .317 E a s t 34th Street N ew Y o r k , N .Y . 10016

V E N E Z U E L ASr. B r a u l io G a b r ie l C h a c a r e s G o b e r n a d o r a C a n d iU to 37 Santa R o s a l i a ( A p a r i a d o P o s t a l 8 09 2 )Caracas l ) , F .

Y U G O S L A V I AJ u g o s l o v e n s k a K n jig a T e r a z i j e 27 Be Igra de

I A E A p u b l i c a t i o n s can a l s o be p u r c h a s e d re ta i l at the U nited N a t io n s B o o k s h o p at U n it e d N a t io n s H e a d q u a rte r s , N e w Y o r k , at the n e w s - s t a n d at the A g e n c y ' s H e a d q u a r te r s , V i e n n a , and at m o s t c o n f e r e n c e s , s y m p o s i a and s e m in a r s o r g a n i z e d by the A g e n c y .

In order to f a c i l i t a t e the d i s t r i b u t i o n of its p u b l i c a t i o n s , the A g e n c y i s p re p a re d to a c c e p t p a y m e n t in U N E S C O c o u p o n s or in l o c a l c u r r e n c i e s . •

O rd e rs and in q u ir ie s from c o u n t r ie s w h ere s a l e s a g e n t s hav'e not y e t b e e n a p p o in t e d may be s e n t to :

D is t r ib u t io n and S a l e s G rou p , In tern a t ion a l A t o m i c Energy A g e n c y ,K a m tn e r R in g 11, A - 1 0 1 0 , V ie n n a I, A u str ia

INTERNATIONAL ATOMIC EN ER G Y A G EN C Y V IE N N A , 1968

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