"STUDIES ON THE CHROMOSOM'ES OF C·ERTAIN INSECTS WITH SPECIAL REFERENCE

TO THE EFFECT OF DRUGS'"

A THESIS FOR THE PH. D. DEGREE CF'

BHOPAL UNIVERSITY, 8HClPAL 1975

BY

MISS PUS UP A SURlY ASTAV

JR. RESEARCH FELLOW, U. G. C.

MOTIJ.AL VIGYAN MAHAVIDYALAYA

BHOPAL INDIA

I I I I

! ,

I

i i

f

Department of Zoology Motilal Vigyan Mahavidyalaya

Bhopal (M.P.)

Oertified that Miss Pushpa Shrivastav, Junior

Research Fellow, U.G.C., has worked under my guidance on

the "Studies on the chromosomes of certain insects with

special reference to the effect of drugs" for the Ph.D. ,

Degree of Bhopal Unlversity. She worked in the researoh

laboratory of the Department of Zoology, Motllal Vigyan

Mahavidyalaya, Bhopal, for more than 200 days. This

thesis represents her original findings and has not been

submitted either partly or fully, for any other degree

in any other University.

Miss Shrivastav joined the leoturership in

Zoology, Oollegiate Branoh of Eduoation Department,

Government of M.P., late in 1974 and is submitting the

thesis while in the same oadre. The thesis is in a

presentable form and is ready for examination. It ful­

fils the requirements presoribed by the Bhopal University,

Bhopal.

( P. Jaitly )

Assistant Prof. of Zoology

Motilal Vigyan Mahavidyalaya,

Bhopal (M.P.).

ACKNOWLEDGEMENTS

I gratefully aoknowledge the oompetent

supervision of Dr.(Miss) P.Jaitly. who has been a

oonstant souroe of inspiration to me. Her keen

interest in my progress rejuvenated me in my days of

desperation. I am thankful to Dr.R.C.Chourasia, the

Head of the Department of Zoology and the then

Prinoipal, Dr.S,N,Kaveeshwar for permitting me to

work in the Department. The author is also thankful

to Dr.K. C.Mehra, Head of the Anatomy' Department,

Medioal College, Bhopal, for permitting photography

in his Department.

The author is also thankful to the members

of the staff of the Zoology Department, Motilal Soience

College, for their cooperation, and to DR.G.P.Mukerji.,

now the Head of the Zoology Department, Government

Narbada College, Hoshangabad, M.P., for his suggestions

in the rearing of the grasshoppers. Thanks are also due

to my research oolleague, Mias Shail Saxena, for her

help during the oompilation of the thesis.

The author is especially thankful to

Prof. S.P.Ray-Chaudhuri for his suggestion to inolude

the banding pattern in the oaryologioal study. His

student Dr. Lalji Singh helped too by lending me the

literature on banding pattern and giving his valuable

suggestions from time to time. The study of banding

pattern was carried out at the Cytogenetios Laboratary

of Benaras Hindu University, for which the permission.

was kindly given by Dr.T.Sharma and friendly help

extended by his research students. All this is

gratefully acknowledged.

Thanks are due to the British Museum ot

Natural History for getting the specimens identified.

I am much beholden to my father, Prof.P.N.Shr1vastav,

who has taken keen interest in my academic career and

has always been a source of inspiration to me.

Lastly, the financial assistance of

University Grants Commission, New Delhi, is grateful17"

acknowledged for awarding the Junior Researoh Fellowship

to me.

( P.SHRIVASTAV )

CONTENTS

I Introduction.

11 Literature Survey.

II! Material.

IV Experimental Procedure and Methods.

A. Germinal Tissue (testes).

B. Somatic Tissue (gastrio caeca).

C. Drugs.

D. Division-Cycle and Drug-treatment.

E. Banding Pattern.

V Observations.

A. Caryologioal study :

1. Trilophidia annulata Thunberg.

a. Meiosis.

b. Abnormalities in the

Natural Population.

(i) Extra fragment.

(ii) Polyploid oells.

(iii) Anaphase bridge.

(iv) Chromosome breakage

o. Mitosis.

2. ChrotogonuB trachypterus Blanchard.

a. Meiosis.

b. Abnormalities in the Natural Population.

(i) 'Supernumerary Chromosomes.

(ii) Polyploidy.

1 - 6.

1 - 23.

24 - 28.

29 - 34.

29

29 - 30.

30 - 31.

31 - 33.

33 - 34.

35 - 93.

35 - 67.

35 - 43.

35 - 4;).

41 - 42.

43.

43 - 53.

43 - 44.

44 - 53.

(iii) Non-disjunction.

(iv) Understained regions.

(v) Chromatid breakage.

(vi) Extra fragment.

c. Mitosis.

3. Leva indica Bo1iver.

a. Meiosis.

b. Abnormalities in the Natura.l Population.

(i) Extra. fragment.

(ii) Polyploid cells.

(iii) Abnormal spindle.

(1v) Breakage.

c. Mitosis.

4. Attraotomorpha crenulate. 'abrlcius.

a. Abnorme.lities in the Natural Population.

(i) Chromatid breakage.

(1i) Chromatid separation.

b. Mitosis.

5. Poekilocerus ~ictus Fabrioius.

a. Mitosis

b. Banding pattern

B. Effeot of Drugs :

1. Quantitative Study: Caffeine.

2. Qualitative Study: Caffeine.

a. Control Series.

b. Treated Series

53.

53 - 58.

55.

55 - 57.

57 - 58.

53 - 61.

60 - 61.

61.

61 - 67.

62 - 63.

63 - 67.

67 - 92.

67 -78.

78 - 82.

79.

79 - 82.

(i) Mitotio ohanges. 79.

(ii) Ohanges in the Meiotic prophase stages. 79 - 80.

1) High condensation.

2) Lesions.

(iii) Changes at metaphase I ill. 80 - 82.

1) Polyploidy.

2) Olumping.

3) Spindle abnormality.

(iv) Changes at first and second post-metaphase stages.

1) Breakage.

3. Qualitative study : Enteromyoetin.

(i) Mitotio ohanges.

(ii) Changes in the meiotio prophase stages.

1) Breakage.

2) Polyploidy.

82.

82 - 86.

82 - 83.

(iii) Ohanges at metaphase I ·1 11. 83 - 84.

1) Fragmentation.

(iv) Ohanges at first and second post-metaphase stages.

1) Stiokiness.

2) Spindle abnormality.

4. Qualitative Study: Formalin.

(i) Mitotio ohanges.

(ii) Changes in the meiotio prophase stages.

84 - 66.

86 - 90.

86 - 87.

1) Deohromatization and breakage.

2} Interbivalent oonneotion.

3) Breakage.

4) Clumping.

(iii) Changes at metaphase I ~ 11.

1) Inter-bivalent oonneotion.

2) Destruotion of morphology.

3) Lesions.

4) stretohed oentromsre and dechromatization.

5) star-shaped oonfigurations.

(iv) Changes at first and second post-metaphase stages.

5. Qualitative study: Niootine

(i) Mitotio changes.

1) Fragmentation.

(ii) Changes in the meiotio prophase stages.

1) Breakage.

2) Clumping.

(iii) Changes at metaphase I & 11

(iv) Changes at first and seoond post-metaphase stages.

VI Results and Discussion.

A. Meiosis and Mitosis.

B. Banding Pattern of P. Eictus.

C. Abnormalities in the ~atural Population.

D. Effect of Drugs.

87 - 90.

90.

90 - 92.

90.

91.

92.

92.

93 - 125.

93 - 104.

104 - 108.

108 - 117.

117 - 125.

VII Summary. 126 - 132.

A. Caryological study 126 129.

B. Abnormalities in the Natural Population. 129 - 130.

c. Banding Pattern of P. ,Eiotus. 130 - 131

D. Effeot of Drugs. 131 - 132.

VIII Bibliography. 133 - 163.

IX Explanation of Figures. 164 - 132.

X Abbreviations. 183 - 1135.

I

INTRODUCTION

-1-

INTRODUCTION

The chromosomes of grasshoppers have always

been popular with the nuolear cytologists. They enjoy the

topmost position with respeot to the suitability of mate­

rial among the invertebrates. They have large germ and

somatio cells with correspondingly large chromosomes.

Moreover, the extreme olarity of preparation and readily

available divisional stages in their testes. make the

grasshoppers a olassical material for cytological studies.

The faot that India is very rioh in grasshopper

fauna is a boon to our oytologists. Considerable work has

been done on this fauna by various Indian soholars. still,

the studies in India oannot be regarded as very extensive

when we take into consideration other parts of the world,

especially U.S.A., Japan and Australia.

A survey of studies on grasshopper chromosomes

reveals that research in morphological aspeots of chromo­

somes is waning beoause of the current interest in mole­

cular cyto-chemistry. The latter branch has become rich

with information in the last deoade. still, the morpholo­

gical stUdies cannot be regarded as orthodox owing to

their relation to the modern systematios.

In faot, the modern systematists regard the

cytogenetioal information as an essential feature because

the speoies are considered to be the objective reality of

some particular genetic continuity. In a good many cases,

where the species could not be distinguished on purely

morphological grounds, help was sought from the fields of

cytology, genetice, ecology, embryology and biochemistry.

The works of Hughes Schrader (1948), Patterson and stone

(1952), White (1954),Darlington (1956), Makino (1956),

Swanson (1958), Lewio and John (1963), Hew1tt (1964), John

and Lewis (1965), Manna and Mazumder (1961,s), Hsu and

Benirsohke (1967, 1968), John and Hewitt (1968), White

(1968, 1970) and others, advocate the usefulness of cytology

in the taxonomy of plants and animals. The various cytolo­

gical 'toolst, with the help of which systematists establish

homology and analogy between allied species and groups, are

- analysis of caryotype, diploid number, metrical data of

metaphase chromosomes, existence of specialized chromosomes,

behaviour and qrrangement of chromosomes during cell division,

speciality of some oytoplasmic and nuclear bodies and hybri­

dization results, etc. (Manna, 1969). Thus a nuclear cyto­

logist provides an independent set of data for establishing

the inter-relationships of different groups. And the study

of cBryotypes is not limited merely to the counting of

chromosome number and morphology. but also helps in under­

standing the cytogenetic basis of speciation.

The cytological data of Indian grasshoppers are

very limited. In many cases the in~ormation is incomplete

and unevenly scored. The most neglected aspect is the somatic

oaryologioal st~dies. It was, therefore, the author's

earnest desire to take a plunge in this indifferently

oharted ooean. The work includes the morphological studies

-3-

of both germiD!:ll Dnd s01llc1.tic chromosomes.

An attempt is here made to study the effect of

a number of chemicals also, viz., caffeine, nicotine,

formaline and enterornycetln. The importance of testing for

mutagenic effects of substances that are used as preserva­

tives or are coneti tuents of hump...ll food/medicine has been

stressed by Bald.une (1956). All the four chemicals are

important from this point of view. Caffeine is used for

both cualitetive a.nd quantitc.tive studies, while formaline,

enteromycetin a.nd nicotine are used for qualitative studies

only.

The study of chromosomal p.berra.tions by chemi­

cals has a special sienificapce and immense possibilities.

First, chromosome fragmentation followed by translocation

of some fragments may give rise to a new pattern of ohromo­

some segments re-su.1ting in heritable phenotypio difference.

The latter is oov1ously i,ll.portant in the evolution of

new speoies and desirable varieties. Seoondly, the study

of causes and consequenoes of aberrations is fundamental

to our understanding of many important biological problems.

Chromosome breakage is perhaps the most important cause

of the death of cancer cells following irradiation. The

chromosome-breaking property of chemicals has an important

bearing on the ohemotherapy of cancer too. Here it is

pertinent to mention that the effects of not all the

chemicals used in the present work have been reported

in higher vertebrates. However, there is a basic similarity

in the mechanism of replication of the genetic material in

-4-

all forms of life. The same faot allows one to draw para­

llel conolusions to a great extent, irrespective of the

animal type experimented upon. Lastly, the study of

chromosomal aberrations by means of chemioals also helps

in bringing about the differential nature of ohromosome

segments.

The above-mentioned aspeots need a thorough and

systematio effort in investigation. A survey of the availa­

ble literature makes it quite apparent that though a host

of workers have been practising the induction of ohromoso­

mal aberrations by chemical agents, very few tried to do a

thorough and systematic investigation. The author tried

her beat, therefore, to state the facts as systematically

as possible and hopes to bridge a few gaps in the archi­

tecture of relating information.

Though the study on the morphology is being made

and effect of ohemicals Observed still with enthusiasm,

another trend has been set in during the more reoent years.

Cytologists have been trying to identify single ohromosomes

in animals and plants. Their eff'orts resulted in a variety

of chromosome identification methods and their applications.

Unfortunately the investigators could not keep in touch

with each others' works due to the delay in publioation.

This brought about a lag in the organization of further

research and also often duplication of work. To overcome

these difficulties the various investigators gathered toge­

ther in the 23rd Nobel symposium held on 25-27 September,

1912. The proceedings of this meeting are beautifully compile~

-5-

by Caspersson and Zeoh (1973) in their book -

"Chromosome Identifioation : Teohnique and Applioations

in Biology and Medicine".

In this new era, the various workers oonoentrated

on human ohromosome studies with the intention of linking ~)

speoific dideases with distinct sites on chromosomes.

Entire populations were screened through automatic maDns to

detect or prediot inherited abnormalities. The honour of

first outstanding discovery goes to Caspersson !1 al (1968)

who observed a peculiar pattern of fluofescence in the

chromosollles of Vici~ faba and Trillium erectum following

quinacrine mustard staining. The chromosomes when viewed

under ultraviolet light, fluoresced or glowed in alternate

light and dark bands, named as Q-bands. Later on, the

same method was applied to human and animal chromosomes

as well. The 'banding pattern' studies have now become a

'vogue' in nuclear oytology and have triggered a rush on ch­

romatid firle structure analysis all over the world.

Now a greater range of methods is available

( Hsu, 1972 ). A number of chemicals, other than quinacri­

ne and its derivatives, oan also produce bands. These bands

are either identioal with or different from the fluore-

scent ones and are named as - C-, G-, wld R-bands.

The banding pattern study has been undertaken

in grasshopper chromosomes also but the reports are very

scanty. An attempt is made to contribute in this field

with whatever means available. This work, thus, touches

-6-

a variety of fields, viz., morphological studies of

germinal and somatio ohro:nosomes, effect of ohemicals

and the banding pattern.

The unlirr;ited hardships un.dergone to complete

the work will be rewarded. if it is found to have some

value to the disoerning.

))J))J)))J

)3°

11

LITERATURE SURVEY

-7-

LITERATURE SURVEY

The Orthoptera are frequently quoted as one of

the classic examples of karyotype stability. Cytologically

they have been extensively studied and each major group

exhibits a chracteristic pattern. Amongst the two

taxonomical groupings of Acrididae, Cryptosaoci have typi­

oally 2n=23 and Chasmosaooi 2n=19 aorooentric chromosomes.

Cyto-taxonomical oharacterizations of the different

subfamilies or genera of shorthorned grasshoppers have

great limitations beoause of the orthodox ohromosome

number and meiotio pattern.

A good number of scientists have reported on

grasshopper chromosomes from different corners of the world.

The available literature relating to this work is surveyed

by the author under a number of headings, namely, general

( orthodox ) oaryological study; unorthodox ohromosome

behaviour including (a) structural heterozygosity for

unequal and asymmetrical bivalents, (b) supernumerary

chromosomes, (0) heterochromatization leading to polymor­

phism, (d) polymorphism due to change in the charaoteristic

spermatogonial number, (e) neo-XY sex chromosomes, and

(t) spontaneous meiotic aberrations; parthenogenesis;

ohemioally induoed aberrations; telocentricity and finally,

the banding pattern.

The orthodox pattern in the oaryology of Aori­

doidea has been reported by a host of workers. The Indian

fauna was paid attention to by - Asana (1934), who published

,/

-8-

a series of papers on the chromosomes of IndiBn grassho­

ppers; Rao (1937) oompared the oaryo1ogy of eight genera

of Pyrgomorphini; Asana ~ 81 ( 1938, 1939 ) made a

chromosomal survey of Indian speoies belonging to Pami1y

Aorididae. A oomparative study was undertaken by Ray­

Chaudhuri and Dutt (1947) in three genera. A cytologioal

ingestigation of Ph10eba sp., Tristis Eu1vinat~ and

Chrotogonus sp. was done by Dutt (1948 s,b; 1950). Manna

gave a oomprehensive aocount of meiosis in a number of

oriental speoies ( Manna, 1954, 1967 a, 1969 ). Srivastava

( 1954, 1956 ) reported the ohromosome behaviour in

Chroto~onus inoertus and Dissosteira oarolina. Sharma!!!±

,I (1963e) reported the variation in the mean ohiasma --' frequenoy per nuoleus in the wild populations of Chrotogonus

traohyPterus. Ohatterjee (1971) reported the orthodox

meiotic pattern of 0ela uvarovi and Xenocatant09s humi1is

humi1~s and again of Euprepocnemis roseus, Ceracris

def1orata, Aulacobothrus 1uteioeps and Hieroglyphus banian

(Ohatterjee et aI, 1971). --Orthodox pattern has been reported in Aoridoidea

from other parts of the world also. McClung (1902, 1905,

1914 ) conoentrated on the behaviour of the spermatocyte

ohromosomes, but his aocount was somewhat unsatisfactory

in the se~se that he oould not state the multiple sex

ohromosome meohanism distinotly. Metrioal ?~alysis of

ohromosomes, chiasma frequency study, etc. ( Ooleman,

1943 ) yielded supportive evidences which helped in

-9-

establishing the relationship of some species. A detailed

account of the cytology of Oxalis disper was reported by

Marks (1957). A comparative study of male and female

meiosis in the grasshopper Stethophyma grossum showed that

both the frequency and distribution of chiasmata in the

oocytes were Quite different from the male situation (Perry

and Jones, 1974). Besides these individual efforts, groups

of scientists scanned the populations of particular regions.

The British population was through1y investigated by Hewitt

(1964, 1965), John and Hewitt (1966, 1910) and Hewitt and

John (1968, 1972). The grasshopper fauna of Japan was

studied by Asana, Makino and Niyama (1938, 1939), Hareyama

(1941), Makino and Momma (1950) and Makino (1956). White

and co-workers paid attention to the African, American

and Australian populations and released a series of exce­

llent pob1ioations ( White, 1940, 1941b, 1953, 1954, 1956,

1957 a,b,o, 1961, 1963, 1965, 1966, 1968, 1970; White, Mesa

and Mesa, 1967; ~~ite, Cheyney and Key, 1963; White, Carson

and Cheyney, 1964; White and Cheyney, 1966; White and Key

1957d; "~ite and Andrew, 1960, 1962; White, Lewontin and

Andrew, 1963; White, B1aokith, Blackith and Cheyney, 1967,

and Atchley, 1974).

The various studies were facilitated by an

improvement in the methods of handling the hereditary

material ( Darlington, 1960, Sharma and Sharma, 1972 ) ."

and an understanding of the mode of evolution of chromo-

) somes in the Orthoptera ( White, 1941 b, 1954; Ray-............... , ..

Choudhuri and Manna, 1951; John and Hewitt, 1968).

-10-

Most of these reports oentred around morphome­

trio data, oomparative study, behaviour of autosomes and

sex ohromosomes. meiosis and ohiasma formation, eto.

Recently an interesting report had been given

by Nur (1973) in his work on the random arrangement of

ohromosomes in the radial metaphase oonfiguration of

Melanoplus femurrubrum. The report offers several advan­

tages for studying ohromosome assooiations. Any treatment

whioh ohanges the position of the chromosomes, is also

expected to destroy the radial oonfiguration. Thus, it is

possible to ohoose for study only unaffeoted oells.

In the animal world, natural heterozygotes have

always been attraoting the attention of oytologists. They

play a major role in oaryotypic evolution. The structural

rearrangements, whioh are oonsidered as ohromosomal mutation

are generally detectable oytologically. A l::;rge fraction

of them remains unaocounted because they are likely to

produce inviability and sterility. Natural seleotion plays

its part in seleoting the superior adaptive ones from the

structural heterozygotes and gives rise to a polymorphio

population. Chromosomal polymorphisma h:_ve been studied

with regard to para- and peri-oentrio invession. translo­

oation, deletion, duplioation and non-disjunotion. Such

studies mainly oentred around the salivary gland ohromoso­

mes of dipterans ( Drosophila and Rhynohosoiara ) in the

beginning. But, since the germinal ohromosoH.es of

Drosophila are unsuit8ble for meiotic study, attention was

-11-

diverted later to the germinal chromosomes of grasshoppers.

The nutural heterozygotes in grasshoppers have been very

efficiently reviewed by White ( 1954, 1956. 1951 a,b,o,

1961, 1963, 1966; White et aI, 1960, 1962, 1963, 1966, 1961).

Several instances of unorthodox ohromosome behaviour have

been reported in about forty speoies of grasshoppers by

Manna (1969).

The oocurrence of supernumerary chromoso~es was

reported in Aiolopus sp. ( Ray-Chaudhuri and Manna, 1951;

Ray-Chaudhuri and Guha, 1955 ). in Tagarta indica (Manna,

1954 ), in Myrmeleotettix maoulatus ( John and Hewitt,

1965 a,b; Barkar, 1966; Hewitt and John. 1967 ). in

Chrotogonus traohypterus ( Sharma,. Parshad and Gupta, 1965)

in Loousts migratoria ( Rahiman and Rajasekarasetty, 1967a)

in Chorthippus paral1e1u~ ( Hewitt and John, 1968 ), in

Aorotylus humbertianu6 ( Gururaj and Rajasekarasetty, 1971)

and in Aorida lata and Oedaleus infernalis ( Takizawa and

Narasawa, 1971 ). It was established earlier ( John and

Hewitt, 1965a and b) that British populations of the mottled

grasshopper Myrme1eotettix maculatus were polymorphic with

respeot to the presence of B-ohromosomes. In one population

10-70 ~ of the individuals possesed 1-3 supernumerary

elements while none in the other population. This condition

results in the occurrenoe of four caryotypes, namely,

S ( standard diploid oomplement ), 5+1, S+2 and S+3. The

cytogenetio systems of grasshoppers and loousts were

reviewed by John (1913), giving speoial referenoe tothe

-12-

origin and evolution of supernamerary segments. The namber

of B-chromosomes varied from 0-7 in the Japanese population

of Attractomorpha bedeli ( Sannomiya, 1973 ). The effect I

of supernamerary heterochromatilU'iwas observed in three I

species by Schroeter and Godfrey (1974).

The deviation in the characteristio spermatogo­

nial number towards the lower ones due to the centric

fusion of two aorocentrio elements had been enoountered by

a number of workers. Besides, studies on the sex chromosome

mechanisms of the Aoridoidea revealed the existenoe of

many speoies in whioh the original XO : XX mechanism has

been replaoed by an XY IXX one, beoause of a centrio fusion

between the original X and an acrocentric autosome ( V'I'hi te,

1940, 1941b; King, 1950; RaY-Ch+udhuri and Guha, 1952; I

Manna and Chatterjee, 1963; White and Cheyney, 1966;

Manna, 1967a; White, Mesa and Mesa, 1961; Mesa and Mesa,

1961; Hewitt and John, 1972).

Newly arisen XI meohanisms are of considerable

genetic interest. One may expect to find in them a series

of genetio transformations, namely, evolutionary hetero­

chromatization of ohromosome regions, acquisition of new

differential segment and perhaps, development of new

mechanisms of dosage compensation. It is evident from the

available report that the newly arisen XI mechanisms are

not a long :1term evolutionary success. Instead, they tend

to oocur in isolated species or small groups of closely

re la ted speoies. There is not a single genus in which 8.11

/'

-13-

the members have neo-XY sex chromosome mechanisms.

The X1X2Y : XI X1X2X2 systems have also been

reported in a few short-horned grasshoppers ( King and

Beams, 1938; White, 1940, 1941b, 1953; White and Cheyney,

1966; Mesa and Mesa, 1967 ). This system is an advanoe

over the XI : XX one and has arisen thro~gh an additional

Y-autosome fusion. It is also s short-lived failure from

the evolutionary view-point. On the other hand, the

X1X2Y system of praying mantids. is a oonspicuous evolu­

tionary suooess, and has arisen through a mutual translo­

oation in an XO form without passing through a neo-XY stage

( White, 1940, 1941a, 1965; Hughes-Sohrader, 1950, 1953 )

Among the Tettigonoides, five speoies have so

far been reported to possess the multiple sex chromosome

mechanism. One is the "Mormon Cricket" - Anabrus simplex

( Decticinae ), reported by McClung ( 1902, 1905, 1914 ).

The other two are an unidentified species of Isopsera --( Phaneropterinae ) with XY males and Letana atomifera

( Tettigoninae ) with X1X2Y males. These two belong to

India and were reported by Dave ( 1965 ). Another two,

Theodoria me1anocnemis ( Phaneropterinae ) and Yorkiella

piots ( Listrosoelinae ) were reported by White, Mesa and

Mesa (1966).

-----Polyploidy in the natural oondition is not an

infrequent phenomenon. Meiosis of the diploid and

tetraploid spermatooytes was studied by Ray-Chaudhuri

and Bose (1948) in Attractomorpha speoies. John and

-14-

Henderson ( 1962 ) reported polyploidy in ~sto~erca

Earane~.Sharma, Prasad and Gupta ( 1965 ) found

scattered polyploid cells in four individuals of

ghroto~onus trachg,pterus. Tetraploidy was reported in

PyreomorEha bispinosa by Nirmala and Rajasekarasetty (1971) ,

while the chromosome complement of Plrgomorpha kraussi

was found to be 22-ploid ( Lewis and John, 1959 ).

Parthenogenesis, as is apparent from the acanty literature,

is not as frequent a phenomenon. White, Cheyney and Key

( 1963 ) reported a case with complex structural hetero­

zygosity.

Literature on spontaneous meiotic aberrations

in grasshoppers is quite rich. The association between

two nonhornologous chromosomes in Gesonia punctifrons

was reported by Ray-Choudhuri and Manna ( 1950 ). In

the Bryodema species White ( 1954a ) noticed an extreme

form of chiasma localization. Anomalous asoooiations and

the behaviour of pseudo-bivalents was studied in the

meiosis of two inter - specific hybrids of Bromus

( WaIters, 1954 ), where the chromosomes seemed to be held

together merely by chromosome matrix connections. White

( 1951c ), while releasing a series of papers on the

cytogenetics of Moraba scurra, reported heterozygosity

for "elastic constrictions" in that species. Srivastava

( 1959 ) came aoross Et number of spontaneous meiotio

aberrations while studying the spermatogenesis of

Poeki1ocera Eiate. Kayano and Nakamura (1960) in their

-15-

ohiasma study, reported a heterozygote for El translooa­

tion in Acridt! lata. An inversion heterozygote was obser­

ved by Lewontin and White ( 1960 ) in Moraba sourra.

Calliptamus palaestinensis possessed an unequal bivalent

( Nur, 1961 ). Ray-Choudhuri and Sherma , 1962 } observed

dioentric bridge and cleavage in certain grasshoppers. A

large number of aberrations were noted by Sharma, Pr6sad

and Bedi ( 1962, 1963 ) in their study on the populations

of Chrotogonus traohypterus. They reported suppression of

paChyten~airing, anaphase bridges, anaphase laggards,

polyploidy, chromosomal variability, unequal bivalents and

translooation heterozygotes. and discussed various aspects

of the breakdown of mitotic and meiotic stability. Adap­

tive properties of inversion were reviewed in Moraba

sourra ( White, Lewontin and Andrew, 1963 ). A spontane­

ous interchange was marked in Chorthippus brunneus with

extensive ohiasma formation in an interstitial segment

( John and Hewitt, 1963 ). A spontaneus chromosome brea­

kage was reported in a grasshcpper of British population,

and in an undescribed species of Morsbinee; and its meio­

tio oonsequences were studied by Lewis and John ( 1966 )

and White ( 1966 ) respeotively. l!;volutionary implioations

of pericentric inversions in Austrioce-tes interioris were

disoussed by Nrulkivell ( 1967 ). In Metrioptera brachyptera

a prolonged non-homologous association resulted in the

formation of a pseudomultiple ( Southern, 1961a ). A -'-' survey of 'vie,tiQa' group of morabine grasshoppers revealed

-16-

the occurrence of a number of spontaneous meiotic aherra­

tions ( White, Blackith, Blackith and Oheyney, 1967 ). A

male grasshopper, Oamnula pellucida, was found to be

heterozygous for a paracentric inversion ( Nur, 1968 ).

Nirmala and Rajasekarasetty ( 1971 ) reported a number of

chromosome abnormalities in the wild populations of

Plrgomorpha bispinosa.

All the above referred casea of polymorphism

have been found to be playing a key role in the evolution

of caryotypes in animal speciation. Their role in the

cellular adaptability has also been proved to be extremely

important for the preparation of in vitro cell lines as

well as with regard to prognosis of cancer.

Since the frequency of spontaneous aberrations

is very low, the meohanism of structural roarrangements

and chromosomal evolution is understood better from the

indu.ced aberration studies. ;tIuch of our information regar­

ding the nature, mechanism and extent of induced changes

in the chromosomes has co~e from the study of pl~~t

material. It h1=Js now gradually become apparent that there

are certain important differences betWeen the reactions

of the plant and animal chromosomes, a-t least so far as

ohemical treatments are concerned. For example, very many

different chemicals can induce ohromosome breakages in the

former ( Sharma and Sha~~a, 1960 ) whereas there are only

a few chemicals which call produce trlle ohromosome breaka­

ges in animal chromosomes as distinct from stickiness and

its consequences. A part of' this dif'f'srence in behaviour

i I f

-17-

may be due to metabolic differences in beheviour in the

cell and nuolear membranes of anima.l and plants, but

nothing definite is known. Therefore it seems desirable

to have more data of a quantitative kind on the induced

chromosome changes seen in animal cell divisions immedia-

tely following treatment.

The neoessary impetus for research on the effects

of outside agencies on chromosomes was provided by the

discovery of X-ray induced mutation in Drosophila by

Muller ( 1921 ), In the meantime, the polyploidising

action of oolchicine and its effective application on

plants had been disclosed through the works of Blakeslee

and Avery ( 1937 land their collaborators. The result of

this Ilenthusiasm led to the disoovery of mutagenic action

of nitrogen mustard in Drosophila,. Since then various che­

ffiicals and drugs have been reviewed from time to time.

Work on grasshopper chromosomes was very limited in the

sixties ( Gaulden and Carlson, 1951; Dutta. Gupta and

DivakEr8n, 1959; Rao; 1960 and others ). With the ad~ent ,

of the. seventies, the literature gradually became recher

and richer with the contributions of Manna and co-workers, (\.,

and few others - Carlson and Gaulden ( 1964 ), Manna and

Mazumder ( 1964 ), Manna and Roy ( 1964 ), Manna and

Purida ( 1965 a,b ), Desai, Sirsi, Shankarf;lppa and Kasturi

Bai ( 1966 ); Manna and Lahiri ( 1966 ); Manna and

Mukherjee ( 1966 ); Jain and Singh ( 1967 ); Manna (1967a); -'---Manna and Parida ( 1967 ); Wilson and Friedkin ( 1967 );

-18-

r;~anna and Perida ( 1968 ) ; Klassen, Chang and Eide (1969);

!VIanna and Parida ( 1970 ) ; Bhunya and Manna ( 1971 ) ;

Manna Fmd Bhunya ( 1971 ) ; Manna and Parida ( 1971 ) ;

Mue11er, Gaulden and Drane ( 1911 ); Manna and Bhunya

( 1972 ); Bhattacharya ( 1973 ); Bhunya and Acharya (1973);

Bhunya and Das ( 1973 ); Nirmala and Rajasekarasetty

( 1973 ); Pati and Bhunya ( 19'73 ); Saha ( 1973 ) ;

Saha and Chatterjee ( 1973 ); Bhattacharya and Dab

( 1914 ); Bhunya, Farida and Ghosh ( 1974 ); Saha and

Chatterjee ( 1914 ); and Saha and Khudabakeh ( 1974 ).

The varipus chemicals tested by these soientists

were - (1) antibiotios - erythromycin, oh1oram.pheniool,

bTiSeofu1vin, novobiocin, st~eptomyoin, tetracyoline eto.;

(2) hormones - corline, insulin, ovocyo1ine, testoveron

eto.; (3) phenols - dihydroxy-oha1oone, gallio aoid,

parabenzoquinone eto.; (4) ohelating agents - oupferron,

EDTA, versene etc.; (5) a1oohos - ethyl aloohol, methyl

aloohol, t-butyl a1oohol; (6) nuoleic aoid analogues -

BUDR, IUDR, maleio hydrazide; ('7) metal1io salts -,

aluminium oh1oride, oaloium chloride, merourio iodide,

merourio nitrate, potassium cyanide; (8) alkaloids -

oolohioine, a.trophine; (9) tranquiliser - largaoti1;

(10) formaline; (11) arginine; (12) semioarbazide;

(13) apho1ate; (14) urethane, etc.

A survey of the findings obtained after using

the above-mentioned ohemioals revealed an interesting

oolleotion of generalizations - (1) feeding method of

-19-

administration was found to be quantitatively more effective

than the injection method, as evidenced from the comparative

aberration frequency study. Qualitatively, however, the

effects were the same in both cases and they were (a) in the

spindle appara.tus - manifested by fusion of cells, polyploi­

dy, failure of spindle formation or the destruction of the

spindle, and (b) in the chromosome - manifested by failure

of synapsis, reduction of chiasma, C-meiotic metaphase, star

or ball configurations, lagging chromosomes, failure of ori­

entation, sticky effect, etc. (2) The meiotic chromosomes of

grasshoppers did not have equal responses to some particular

chemicals. (3) Some chemicals affected the X-chromosome more

than the autosomes, while no preferential effect had been

observed with other chemicals. (4) The effect of most of the

chemicals on the chromosomes of different stages of division

appeared to be non-specific while corline had been found to

be stage sensitive since it damaged more specifically the

anaphase chromosomes. (5) The breaking point in the X-chro­

mosome followed more clearly at diplotene-diakinesis stages

indicated that breaks were more common to the 2/5 and 4/5

segments. (6) There is inter-specific sensitivity with re~a­

rd to X-rays as well as chemicals. (7) The aberration fre­

quency data of the X-chromosome indicated some cumulative

effect when the specimens were treated with a mixture of

maleic hydrazide and aluminium chloride and the results were

compared with those of separately treated ones. The results

sug;ested thDt they might be locus specific in action.

-20-

(8) Some chemicals tested for the dosage-frequency relation­

ship did not yield positive result. (9) The time of first

appearance of aberration va.ried considerable. This is

expected since the mode of action of various chemicals is

different.

The above stu.dies have yielded u.seful information

on the architecture and the mode of action of the genes,

chromosomes and cells. Chromosome breakages perhaps lie

at the basis of the chain of reactions which follow whole

body irr'adiation leading to the death of experimental

animals. In the present age, when we intend to exploit

atomic energy -are ,essential for an intelligent planning

for the protection of the hereditary material from the

hazards of radiation ( Ray-Choudhuri, 1961 ).

There has always been a confusion regarding the

position of the centromere in grasshopper chromosomes.

The strongly preva.iling view was that chromosomes with

strictly terminaloontromeres could not survive in nature.

Probably the first person to suggest and adopt this view

was Ne.vashin (1916). After him this remained the generally

accepted view. The school of thought which rejected the

concept of telocentricity argued that all stable rod

chromosomes were acrooentrics. It was believed that the

minute heads situated at the centric ends of rod chromo­

somes clearly demonstrated the existence of short arms

( White, 1935; Coleman, 1943). Thus,the occasional formation

of ditactic biva.lents ( Dgrlington, 1936 ) could then be

-21-

explained only by the association of these very small arms.

It was also pointed out that when telocentrios either

arose spontaneously ( Upcott, 1937 ) or following X-irradia­

tion ( Rhoades, 1936 ) through the fraoture of an inter­

stitial centromere, their unstable behaviour usually led to

their rapid elimination.

However, as the techniques of chromosome analysis

improved over the years, there has been a gradual rise in

the data which supported another school of thought. The

latter considered that not all rod chromosomes are

acrocentrics. Works of Schrader (1939), Ostergren (1947)

and Lima-De-Faria (1956) made it clear with some degree

of certainty that for many rod chromosomes the so-called

"small arms" represent fused centromeric chromosomes.

There are a number of exmples of derived telocentrics in , I

plants ( ~arlington and La Court 1950; Marks, 1957a;

Strid, 1968 ). The various confusions regarding the

nomenclature of centromeric position were skilfully dealt

with by Levant Fredga and Sandberg (1964 ). John and

Hewitt ( 1966 ) suggested that the production of ditac­

tic bivalents could be the result of a chiasma actually

forming within the terminal centromeres of homologous

rods.

White and some of his co-workers claim that in

animals the prevailing rod choromosome is an acrocentric.

They are of the opinion that misdivision leading to the

establishment of stable telocentrics h.as not contributed

-22-

in any significant way to the evolution of animal chro-

mosomes. John and colleagues, however, repeatedly ques-

tione4 such beliefs, because their own studies on

Orthopteran species have clearly revealed the existence

of telocentrics ( Southern, 19618.; John and Hewitt. 1966,

1968 ). Nur ( 1968 ) also reported stable telocentric

chromosomes in Myrmeleotettix maculatus. The only

debatable point concerns their origin. Unlike White,

these workers have not ruled out centric fission as a

possible pathway.

still there are scholars who are unwilling to

8.ccept that stable telocentrics may exist in the normal

chromosome complement in certain species. The probable

reason for this attitude is that it would demand a

radical change in their outlook concerning caryotype

evolution. It would particularly necessitate a reapp­

I'a.isal of the role that centric fission may have had in

this process.

Lastly, coming to the banding pattern, its

discovery not only gave new impetus to research but has

actually rendered all previous findings obsolete. The

present developments in the chromosome technology make

the old preparations look like a palaeolithic research

tool.

The techniques are immensely important for the

fundamental approach to the organization of the ;gene

systems and on its mode of work in cell differenciation

-23- •

and oell funotion.

The banding teohniques not only make an invaluable

oontribution to the fields of medicine and biology, but

also to evolution ( Grouohy !!!!!.' 1913, and Pearson, 1973).

The information available so far mainly centres around

mammalian subjects and has been compiled, as previously

stated, by Caspersson and Zeoh ( 1973 ). One paper,

however, deals with the banding pattern of heterochromatio

B-chromosomes of M,p:meleotettix maculetus ( Galaghar,

Hewitt and Gibson, 1973 ). Lateral subunits in the C-bands

of unreplioated ohromosomes of Dis:;;,osteira carolina were

studied by Bregman ( 1913 ). The location, structure and

behaviour of C-heterochromatin was reported in Dichroplus

silveira~idoi (Cardoso et aI, 1914 ). An unusual

behaviour of the heteroohromatio blocks looated in the

so-oalled synaptio region of the sex bivalent ( Neo Y-

Neo X ) was found to remain paired from early prophase

through metaphase I. Brown and Wilmore ( 1974 ) reported

the looation of repetitious DNA in the chromosomes of the

desert looust Schistooeroa gregaria using a modified giemsa

staining teohnique. Klaste.rska., ~atarajan and Ramel (1974)

utilized Q-banding and C-banding techniques to olarify

the relationship between heteroohromatin, centromere and

ohiasma localization in Bryodema tuberculata. The author

has also made a hesitant attempt to step in this territory

and hopes that the present study will add a little bit to

the existing knowledge on the subject.

III

MATERIAL

· M/M : rmmnn !

19 .1

M/M i rmmnn ~

F1g.2

"nTmmn j

Fi g . 3

-24-

MATERIAL

A. SPECIES

The following species of short-horned grasshoppers

belonging to the family Acrididae ( Orthoptera : Acridoidea)

constitute the material for the present study:

(l) Tril0;Ehidia annulate Thunberg.

(2) Chrotos:onus trachypterus Blanchard.

( 3) Leva indica Boliver.

(4) AttraotomorEha crenulata Fabr1oius.

(5) Poekilooerus Eictus Fabric1us.

TriloEhid1a annulata I

( Fig. 1 ) The specimen belongs to the sub -

family Oedipodinae. In winter, the adults invariably gather

around the carrot plants, while the nymphs conceal them­

selves in between the branohes. In summer and rainy

eeasons they are found scattered in the grass. Adults are

capable of short flights.

Chrotogonu6 trachypterus I

( Fig. 2 ) Commonly oalled sand-hopper, this

specimen belongs to the sub - family pyrgomorphinae.

The males can easily be distinguished from the muoh stouter

females. The males can flyover short distances but the

females only hop because of their heavy bodies.

Leva indica z

( Fig. 3 ) This specimen belongs to the sub­

family Gomphocerinae. Both the male and female adults are

fast fliers. The nymphs are aotive too. The adult males

f'"

-25-

make a oharaoteristic 'ohirping' sound with their hind

legs.

Attraotomorpha orenulata :

( Fig. 4 ) This slender grasshopper belongs to

the sub-family Pyrgomorphinae. The adults are not very

aotive and oan hop over short distanoes. A oharaoteristio

oolour ohange is observed in this speoimen. During the

hotter months it aquires a dull, buff - brown oolour,

while during the oold and moist weather it has a fresh

green oolour. Suoh oolour polymorphism is not a rare

phenomenon in the inseot world ruld is directly related to

temperature changes.

Poekilooerus piotus :

( Fig. 5 ) This large speoimen measuring about

c' 5-7 mm. belongs to the sub-family pyrgomorphinae. The

pigment is red, yellow and green in the nymphal stages

and turns to prominent green patches on yellow baokground

in the adults. When irritated, the insect seoretes an ill­

smelling liquid.

B. COLLECTION

All the speoimens were oollected from the bota­

nical garden of Motilal Scienoe College,Bhopal except

Poekilocerus piotus. The latter was oollected from the

Gwalior University oampus. One population of Chrotogonus

traohypterus was also available at the Arera Colony locality

of Bhopal from where individuals were ocoasionally oolleoted

for examination and oomparison with the Science College

population. Both the populations are widely separated.

M/M r

fTlllllTTTl ~

Fig. 4

\

\

/

~ o 10 10 ]0

Fig. 5

-26-

Since the activity of the grasshoppers varies

directly with the temperature, collecting was easiest

when the day warmed enough so that they could easily be

flushed out of the grass, but when it was hot they began

taking flight too quickly to be caught with a net. Nymphs

could easily be caught by hand more easily than by net.

Poekilocerus pictus was available from May to

August. Rest of the grasshoppers were available through­

out the year.

C. REARING

The grasshopper Chrotogonus traohypterus was

reared by the ~ethod of Carlson and Gaulden (1964). No

long - term rearicg was required for the rest of the looal

grasshoppers. Adults and last instar nymphs were colleoted

directly from the field and kept in the cages until requi­

red for examination. As Poekilocerus piotus ({id not belong

to the local inseot - fauna, it had to be reared from the

early nymphal inste,rs. Every possible attempt was made

to provide natural surroundings to the insects.

D. C.flGES

The cages used were similar to the type descri­

bed by Carlson and Gau1den ( 1964 ), me2suring 23 om. high

x 18 om. wide x 15 cm. deep ( Fig.6 ). For Poekiloc~

pictus larger oages were provided, measuring 15 om. high

x )0 om. wide x 25 cm. deep. (lig.7). About 10 - 15

adults were plaoed in one oage.

F1g.6

Fig. 7

-21-

E. FOOD

The nymphs and adults of Poekilocerus pictus

were fed with the leaves of the lutex - seoreting plant

Calotropis of the family Asclepidiaceae. Other grassho­

ppers were given a variety of food, including, wheat

seedling, carrot tops and fresh grass soaked in water.

The grass was placed inside the cage in a ata.ining jar

filled with water. The grasshoppers were often seen to

ingest drops of water from the grass.

F. TISSUE

A review of' ava.ila.ble literature makes it

obvious that much attention has been paid to the study

of ohromosomes of the germinal tissue, and that too of

the male grasshoppers. The caryology of the somatic tisDue

has been largely ignored. However, about a deoade ago,

an excellent study was undertaken by Carlson and Gaulden

( 1964 ). They utilized to good advantage the large

neuroblasts in an early embryonic stage of the grazshopper

Chrotophaga viridifasoiata. There are, however, oertain

limitations of the neuroblast teohnique. In order to have

a oontinuous supply of young embryos throughout the year,

it is neoessary to maintain a healthy stock of individuals

in oages under controlled conditions. Besides, the oulture

teohnique itself is very elaborate and involves the use of

various delicate and expensive equ1pments.

Fox ( 1969, 1970 ) used abdominal fit body and

-28-

malpighian tubules in his report on the DNA va~ues in

somatic fiscues. These tissues, are, however, extremely r""

delicate and need to be handled by leaving it attached to

the abdominal sternite and the gut, respectively, during

fixation and staining.

In the present study gastric caeca huve been

used for SOIru:itic caryologioal study. Though a very useful

tissue, its use seems to be rather limited. Nankivell

( 196'1 ) used the gastric caeca of the females of

Austriocetes interioris to locate polymorphism for

perioentric inversions in the sex chromosomes. Nanda

( unpublished ) used the same tissue in her study on the

somatic chromosomea of a cockroach. The author used the

tissue in her report on the caryology of three speoies of

short horned grasshoppera ( ShrivastCi.v, 1975 ).

The testes of either lest instar n~ph or young

adul t were taken for the study of meiosis and bfUldirig

pattern.

.)-0-0-0-0-0-0 0-0-0

o

IV

EXPERIMENTAL PROCEDURE

AND METHODS

-29-

EXPERINTENTAL PROCEDURE A;':D M.F~THODS

A. Germinal Tissue (teste~) :

Testes of the pr-e-hdul t and adult male,:;rasGho­

ppers were dissected out in invertebrate saline and kept

in 1% Sodium citrate for 15 - 20 minutes. Aceto-alcohol

( 1 : 3 ) was used as fixative. Staining was done accor­

ding to the schedule followed by Mangalangi and su~ra.manium

( 1963 ), using 0.5% aqueous Heidenhain's haematoxylin.

Temporary slides were sealed with vcseline for observation.

The schedule was sli~htly modified for making the slides

permanent. After removing the seal with xylene, the slides

were inverted in a large petri - dish containing absolute

butanol and a piece of glass rod. The whole set-up was

covered with another petri - dish of lar5er diameter.

After the separation of the cover - ;~lass ( which required

15 minutes to 24 hours ), mounting was done in D.P.X.

B. Somatic Tissue ( gastric caeca ) :

The orthodox squash techni<1ue proved inadequate

for the routine analysis of the somatic tissue and, there­

fore, an air - drying technique ( Crozier, 1970 ) was

adopted after some alterations. The technique may be

summarised as follows :-

1. Actively feeding nymphs were injected with J.2 ml. of

0.1% colchicine prepared in Ringer Solution 'A'.

2. The nymphs ware dissected in 1% Sodium citrate after

4 hours. The gastric caeca were removed while still

................ -----------------------

-JJ-

attached to a portion of the gut and were kept in fresh

hypotonic solution for 10 - 20 minutes.

3. The tissue was fixed in aceta-methanol ( 1 : 3 ) for

30 minutes.

4. A small piece of tissue was transferred to a drop of

60% acetic acid ( aqueous ) on a clean warmed slide. This

brought about a rapid dissociation of the cell.

5. A drop of aceta - methanol was added to the preparation

and the slide was tilted in HII directions to achieve a

maximum spread of the li(~uid.

6. The slide was stained in 2% aceto-c8rmine, mounted, and

placed in a covered container inside an oven. It was left

for 3 hours or until microscopic81 exmination revealed a

satisfactory staining.

7. The slides were placed vertically in acetic-ethanol

to loosen the cover-glasses.

8. After dehydration in 95% and absolute ethanol, and

clearing in xylene, the slides were mounted in D.P.X.

Originally devised for ants ( Order Hymenoptera ),

this method reC'luires very small amount of lnateriCiI. A regular

and complete fla.ttening of a dispersion of cells is obt&ined

without the use of mechanical means. The slides can be

m~1de permanent with rninimel cell loss.

c. Dru'Is:

Following concentratioi:Js were prepared in

invertebrate saline fit the time of treatment :-

1. Caffeine :-:).1%, :).2-', :).3%, 0.4%, ·).5fo, ).61', J. 7~t

-31-

J.d>b t ;).9%, and 1. (w/v).

2. Formaline:- 3.0,0 (v/v).

3. Enteroll2l.oetin ( ohlorbmphellicol cl3psules ) :- O.25}b (w/v).

4. Niootine:- J.5% ( v/V).

The drugs were introduced into the body of the

insect by injection. The needle was inserted through the

coxal cavity of the meta-thorasic leg, arId ,).2 ml. solution

was allowed to escape the syringe.

D. Division - Cycle and Drug - treatment :

To seoure the effect of chemicals on the chromo­

somes, various artificial means are adopted to secure

maximum availability of metaphases - t~ stage at whioh the

detection of aberrations is most convenient. The alkaloid

~olchicine, e.g., and its derivative colcemid are commonly

used to arrest the division at metaphase. Ap(~trt from the

advan tages, hO'Never, these agen ta can induoe a number of

aberrations either independently or in combination with

the chemical, the effect of which is to be investigated.

It is difficult, if not impossible, to deiect and differe­

ntiate the 'independent' effect from the 'combination'

effect. Moreover, the use of such means would complicate

the quantitative analysis of the effect of the ohemical.

No metaphase - arresting agent was therefore, used. along

with the chemic21s.

In the present investigation, the effect was

studied on the IDcle germ cells of Chrotogonus trachl~terus

because sperme.togenesis in the testes tubules of this

-32-

species oocurs in an orderly menner. The large chr'omosomes

Hre also well - suited for analysis, The approximate

duration of various divisional Gtages was determined in the

schedule of spermatogenesis ( Fig. a ) according to the

instrnctions given by Subramanium ( 1971 ). This schedule

allowed tentatively to deduce the time during the pre -

meiotic cell - stage when the chrom080meu were exposed to

the chemical.

Thirty - five days old gras.:.::;hoppers were chosen

for experiment. In the first lot, a sst of five grl.:isshoppers

each, were treated with the ten concentrations of ca.ffeine.

Controls were maintained for eaoh set. This experiment helped

to seleot that concentration which was most effective. This

effective concentration of caffeine was then used in the

second lot to study its rospo.ase to the increase of tre::: t­

ment time. For this experiment, the testes were fixed at

4, Id, 48 !lnd 72 hours and 6, 7, 8, 14 and 21 dbYs.

In the third, fourth and fifth lots, the selected

concentrations of formaline, rticotine and enteromycetin

were uc'ied Eind the testes were fixed at the above - mentioned

:timings. The tisGue was proceeDed &5 described e'· rlier for

rsermint·d tissue. bU.t the hypotonic tre8.tment was given

for one hour.

Chromosome figures were se:lected for analysis

under low ma~lification ( 10 X objective J.Only those

figures v.ere chosen that could be reliably analysod by

using oil immersion. The cell stages analysed were diakinesis.

-- --26 DAYS------

i PI?IMORDIAL MI SPERMATlDS

I SPERMS

I SPERMATOGONIA PREMEIOTIC PROPHASE I! SPERMATO

I SPERMATOGON IAL GENES:JS DIVISIONS

lE 8DAYS I DAY ·9DAYS

SCHEQULE OF SPERMATOGENSIS IN

Fig.8

DIAGRAMMATIC REPRESENTATION OF THE METHOD OF COUNTING METAPHASES IN THE SQUASH OF

A TESTIS-FOLLICLE

Stages other than metaphase.

Metaphases.

Path follo",e d by the ocular micr·;meter (by moving the stage of the microscooe).

1. Slide. 2. Cover - glass.

3. Area covered by the checKered ocular micrometer at a pertlcular focus.

4. Initial position of ocular micrometer. 5. Ultimate position of ocular m!crQmeter~

Fig. 9

-33-

metaphl:;sc I, and anaphGse 1. Chromosomal abnormalities

observed in spermc.togonial metaprtases are not reported

pecBuse of uncertainty about the exact stage of the mitotic

cell - cycle during which treatment occurred. The spermato­

gonial mitoses require only 1-2 days hod the srunpling in

this investigation was too infrequent to give a reliable

sensitivity of ohromosomes throughout the mitotic cycle.

The meiotic divisions, on the other hand, take only a

short time ( 1 to 2 days ) to complete. Therefore, the data

from these stages of' meiosis were pooled and the two daughter

oells of enaphase I were counted as one cell in the tabula­

tion.

The cells were oounted with the help of' a cheque­

red ocular miorometer n.nd the slide wa.s thorouehly scanned

by a method represented diagrammatically in Fig. 9.

E. Banding Pattern

The testes of Pocki1ocerus pictus were fixed in

acetic-ethanol ( 1 I 3 ) for 1 hour. The tissue was then

transferred to 60% acetic acid for About 15 - 20 minutes.

This brought about breaking down of the tissue. Aggregates

of cells settled at the bottom of the tube. The tissue was

spun at 1)00 r.p.m. for 5 minutes. Afterwards the super­

natant was drained out with the help of a pipette. Freshly

prepared fixative was added to the tube and again the tissue

was spun at 100) r.p.m. for 5 minutes. The supernatant was

removed and the air- dried prepp,rations were made with the

rem2ining fluid.

-34-

The slides were stored for 24 hours and then the

procedure followed wee according to Sumnerts (1912)

technique with a few altera.tions :-

(i) hydrolysis in 0.2 NHol was carried out for 30 minutes

instead of an hour;

(ii) the time of treatment with 5% barium hydroxide ranged

from 3 to 7 minutes, instead of 5 to 15 minutes;

(iii) incubation in 2xSSC was carried out for 2 hours

instead of one.

The camera luoida diagrams were made using an

oil - immersion objective and a 15 x eye-piece. The

photomicrographs were taken using an oil-immersion or 40 x c

obje.tive and an 8 x eye-piece. Thus the final magnifications ,. of the photographs are x 800 and x 320, respectively.

*=*=*=*=""=*=*

V

OBSERVATIONS

-35-

OBSERVATIONS

A. Oaryo1ogice1 Study

The caryo1ogy of Trilophidia annulate is reported in

detail. As far as the author is aware, this species has

not been reported in the past. Two populations of Chroto­

gonus traohYEterus were scanned by Sharma and oo-workers

in a series of papers ( 1962, 1963, 1964, 1965 and 1967 ).

Their findings are compared and oontrasted with those of

the two local popu1ations of the same speoies. Also the

details of the morphometrio analysis, which were not

available in the above-mentioned papers, are inoluded in

this work.

A brief report of the meiosis and mitosis of

Leva indioa is given. The meiosis of the diploid and te~ra­

ploid spermatocytes of Attraotomorpha speoies has already

been reported by Ray-Chaudhuri and Bose ( 1948 ) and,

therefore, only morphometric data and mitotic study is

undertaken here. The spermatogenesis of Peekilocera picts

was worked out by Srivastsva (1959) and hence in this case

too, only mitotic oaryological study is reported, along

with the banding pattern.

1. !ri1ophidia annulata Thunberg.

a. Meiosis I The diploid number of ohromosomes in the

male germ cells of T. annulata is 23 ( Fig. 10 ). Measure­

ment of chromosomes was made from twenty clear well-plaoed

metaphase plates ( Table 1 ). The chromosomes oan be

• \

•

• o

• • •

Fig.ID

2 3 4 5 6

(\~ nn 1\0 n" 1\1\ 1\/\ (X'~

7 8 9 10 11 'x'

f\A I\A AA AI\ " .. A , IO? j

Fi g.ll

-36-

classified into three long, seven medium and two short

chromosomes ( Fig. 11 ). The sex chromosome ( X ) is the

third in order of length. The minute second arm is not

visible in ~y chromosome. The sex chromosome shows slight

heteropyonosis at the spermatogonial metaphase and can

be easily demarcated from the autosomes ( Fig. 12 ). The

general course of meiosis is of the classical type.

In the prophase stage, the pachytene nuclei

reveal the X as a strongly heteropycnotic body (Fig. 13 t 14).

in tne pachytene nualei. The eleven autosomal bivalents

show a clear sp11 t down the middle '( Fig. 14 ), which the

later stages show to be reductional, i.e. between the two

synapsed chromosomes, rather than between the chromatids

of a single chromosome. There is an indication of the

presence of small arms ( marked by arrow ) in one of the

bivalents ( Fig. 14 }.There is a distinot tendency to

heteropycnosis at one end of several of the autosomal

bivalents. The heteropycnotio end is probably the proximal ,

one, as believed by White ( 196i'). Small blocks of hetero-'

chromatin are present interstitially in the bivalents

( Fig. 15 ).

At diplotene, the bivalents show a greater affi­

nity for stain and exhibit the lampbrush fibres so chara­

cteristic of grasshopper spermatocytes. This characteristic

can be seen at pachytene also ( Fig. 14 ). A single split

can be observed separating the two homologous chromosomes

of which the bivalent is composed. The split is thus

-37-

Table 1 : Mean length in miora of the spermatogonial

metaphase ohromosomes.

I Chromosome No. Length ~ S.D.

1 1.38 + 0.0101

2 6.16 + 0.0226 -3 5.87 .:!: 0.0001 I

I

4 5.41 + 0.0001 -5 4.75 + 0.0001 -6 4.56 + J.OOOl

-7 4.03 .:!: 0.0001

8 3.62 + 0.0002 -9 2.89 + 0.0002 -

10 1.91 :!: 0.0001

11 1.37! O.lJOO

• X' 6.34 + 0.0001 -

. ''''-"

, :1. ,

. "" ,.-., .. ~ I t , ,-

\ .i.i~ . ;

.

h.b _. __ ..

'\l

~\J " .. " l.'-X

)"> •

"

"'\' ~ A

Fi g .l:-2

$ ' h.4b

~ 1,.-- ' ., .. ' " ' .. "

~ -.. "

Fig.l3

h.b--:---

Fig.14

-, ,-"

~ ,

'. ,

. ~ .

t. . J

"0

I' • ~

Fig.15

.'

F1g.16

F1g.17

- ..... \ -' I 0( .. . . ,.

-38-

Table 2 : Chiasma frequency at different stages of

meiosis

-Number or Stages of Total Mean chiasma frequenoy

Nuo1ei Meiosis Chiasmata per Nuo1eus

10 Diplotene 160 16.00 + 0.02 -15 Diakinesis 172 11.46 + 1.24 -20 Metaphase I 227 11.35 + 1.26 -

reductional and not an equational one. In this stage the

X chromosome shows a pronounoed negative heteropycnosis.

The latter is more evidect in the understained nuolei. Where

the stain is dark, the X chromosome looks just like the

8utosomae bivalents ( Fig. 17 ). Each of the two long

autosomal bivalents invariably contains at least two chias­

mata, and sometimes even three ( Fig. 11 ). The number of d

chiasmata never excees two in the medium bivalents and one 1\

in the small bivalents. Mean ohiasmatfrequenoy per nucleus

is 16 + 0.02 ( ~able 2 ). -In the diakinesis stage, the 'woolly' appearanoe

is lost to a great extent and the bivalents aquire an

almost smooth outline. A variety of shapes of the bivalents

is found in this stage ( Fig. 31 ). The two long bivalents

-39-

exhibit either 'diamond' or 'ring' shapes, the former

when the interstitial chiamata are at the end point of

terminalization, and the latter when the terminalization

is complete. Three or four of the medium sized bivalents I ~

r

r

look 'oross' - shaped owing to the presenoe of one

interstitial ohiasma, which has not been terminalized. Rest

of the medium bivalents look either ·v· or'rodt shaped j'.,-

because of the terminalization of the interstitial ohias-

mata. Eaoh of the two small bivalents looks like two

adjaoent dots. Mean ohi8sma fre~uency per nacleus is

11.46 .:t 1.24.

Maximum condensation oCourrs at metaphase I and

no heteroohromatio ohromosome or chromosome region can be

Observed. In the polar view ( Fig. 16 ) the eleven

bivalents are seen to form an irregular oircle. The large

and medium bivalents ocouPY the peripheral position while

the small ones remain in the centre. The X ohromosome can

easily be distinguished from the others because it always

lingers outside the circle, either 'above' or 'below' the

equatorial plate ( Fig. 18 ). The proximal ends of the

chromosomes are distinctly pulled apart at first metaphase,

but only over a short distance. It may 'be taken as an indi­

cation of a 'premetaphase-atretch' during spindle formation,

but this is nothing like as strongly developed as it is in

many mantids and phssmids ( Schrader. 1944; Hughes-Schrader,

1947 ). Mean chiasma freQuency per nucleus is 11.35 + 1.26. -Thus the loss of chiasmata is more between diplotene and

1\ / .

[ 1

• • .. ......

", --x 4' . , "

If,. + J .," J , .~. r'- ~ ·

-"" ..... - ' ­. ' "

_________________ ~~ _______ !~I1J~~~\~~~ __ ._J

I ~

Fig.18

Fig.19

Fig. 20

J \

-40-

diakinesis than between diakinesis and Metaphase I.

Anaphase I oonsists simply in pulling asunder of

the two ohromoBomes of eaoh bivalent ( Fig. 20 ). Sometimes,

the bivalents exhibit the 'pseudo-ohiasmata' ( Fig. 19 ),

when the terminal points of the separating bivalents stiok

together for a oonsiderable time. A,distinot split is

visible between the ohromatids of whioh each of the

separating chromosome is composed. Thus in early anaphase

I the daughter aorooentrios appear 'V'-shaped as is normal

in the ohiasmate meiosis. The univalent X ohromosome also

shows a split between its ohromatids in the late ( Fig. 21 )

if not in the early anaphase ( Fig. 22 ). In the normal

oourse no thread-like oonneotion is seen to conneot the

separating daughter ohromosomes.

At telophase If the spindle fibres conneoting the

separating daughter ohromosomes stain quite deeply and

are clearly visible ( Fig. 25 ). Deoondens.ation sets in

at late telophase and the daughter nuclei look like loosely

spun balls of chromatin fibres.

The seoond meiotio division follows the usual

oourse. At metaphase 11 the chromosomes look rounded and

condensed ( Fig. 23 and 24 ), but are smaller in size and

closer together as oompared to metaphase I chromosomes.

At anaphase two, the ohromosomes divide equationally. The

_ J 'rod' shaped 'chromatids' or the daughter chromosomes move ! I'" t'- towards the opposite poles ( Fig. 23 and 24 ). The 'rods'

assume a star-like appearanoe at the end of telophase 11

( Fig. 26 ).

-~

-?fr-

~ .,.

'I

... " . "

"'~

... ,'" , l".

.. .. 't. ."

. j,;

Fig. 21

~ 'X'-( "," ,V

", i • ... t". . 11 , ., "" o 7 '

Fig. 22

/),

J ' 6 ? f (. ..

:/' I... •

I, '"

~

. .. . '{i ~

.~

~

Fig.23

\ ) /~ \,

',,~ ~'. ~

!i' .......

-:-.. , - ( ~,

•

, .. ~~:. .-,. ·11 ~. _ d", .'

~ J£ . ~ ~ :

• -::.t

• •

Fi g . 24

~- -:.;'

"liW .. .

Fi g . 25

Fig. 26

-

ii' .;,' '.

, ~

•

., .' ~.

•

. J~

1'. I

-41-

b. Abnormalities in the Natural Population :-

The frequency of individuals showing aberrations

in the natural population is very low ( T8ble 3 ). One

hundred and six individuals were scored in all. The aberra-

tions can be analyzed under the following headings :-

(i) Extra frasment : In a single individual, three

metaphase I cells out of 35 and one anaphase r cell out of

50, have been found withhan extra element. The latter is i

round in shape and lies free ( Fig. 21 and 28 ). The

fragment is smaller than the smallest autosomal bivalent

and does not show negative heteropycnosis.

(ii) Polyploid cells: In a single follicle of an

individual, 13 cells in diakinesis are found to be

polyploid ( Fig. 29, 30 and 31 ). All the polyploid cells

observed are tetraploid in nature. The two XS have never

been found to pair, though they may lie close to each

other. No multivalents are found in the cells. In some of

the other follicles of the same individual, a few scattered

cells are found.

(iii) Anaphase bridge: Only one out of a total of 99

cells in anaphase It is found to possess s.n anaphase bridge.

A fragment of considerable stze accompanies the dicentric

bridge ( Pig. 32 ). Manna ( 1954 ) reported the occurI'ence

of dicentric gridge without any accompanying fragment in

T. arululata. The dicentric bridge involves an intricate

terminal association of only one of the daughter chrometids

of a homologue with the corresponding one of the other.

~ - ... \~ ,,"- • ~r .... ,\.'" .. • , ex ,f '" .' 0' " \ . • •

. f .. "', , .

" . .. ,. -0 -, ..

, ." .-• , F1g.2 '7

-i"'" -~,

\' eX , f ',"

i.

•

F1g.28

, ~ ';'1 t-... .,.. -¥ , ... r r •

'~ .~~\ I • "~I ~ . ' -' \ ~ .. -

• - .... ~ y~. .~

? ~:/~- ~ c..

~ . -"" ... 1r., , '" .I..,,'"

~ ~'';'

, "

"- " '~ ~ .§.'

"'J .~:-

Fig. 29

-r

,,J( x..,,' ,'-

I

- • - , •

~ " W

~ t. p ,c.

\

~~ I ~ 'f , f)

• ..... ~ .~

.... .,

~

, .

...

~ ,.

-i

/ tp ,c

t..' . , .. ' ~, . . <.

~ ~ "..0}

• .. ~~;

~ ~ 1,':

,'.- ~:-

F1g.30

-I

~ ... • + I -Fig. 31

-42-

The accompanying fragment is a small body lying at the

equator of the spindle. The bridge is stretched between

the two poles of the spindle, the other daughter half of

each homologue lying free, giving it a characteristic

appearance. The two limbs which form the bridge are asso­

ciatedat their terminal ends. The fragment seems apparen­

tly acentric in nature.

(iv) Chromosome breakage: In a single individual, one

cell at early anaphase It shows a breakage in the sex

chromosome (Fig. 33 ). The breakage is at the chromatid

level and is confined to the distal region. The fragment

lies close to the arm and there is no visible connection

between the two.

Table 3 : Frequency distribution of abnormalities with

their percentages.

Types of' Stage of Total Cells Total of Abnormality Meiosis Studied Abnormal Cells

Extra Metaphase

I Fragment I 35 3

I

Percen-tage of Aberration

a.51 %

I1 Diakinesis I Polyploid

I 64·.00 % Cells 25 I

16 ~'-'

Anaphase Anaphase Bridge I 99 1 l.vl %

Chromosome Anaphase Breakage I 71 1 1.40 %

-I I

~~~ fr. ch

r~ / r . frj aS5o .ch- e-

\ b \J

l: '(i: • (

- -I Fig. 32

.. " A :>, •

~ ~ .,,, ' br I! , .. ,. . .

. ~ ~;... ,:

Fig. 33

-43-

o. Mitosis: At prophase, no ohromatin 'threads' are visible.

The whole nuolear volume is filled up with heterogenous

material. In the male tissue, the single X-chromosome remains

isopycnotic during interphase and hence no Barr body oan

be seen ( Fig. 34 and 35 ). In the female tissue, however,

one of the two X-chromosomes becomes heteropycnotio. Thus

the behaviour of the X-ohromosomes in somatic cells is exa­

otly opposite to that Observed in germ oells ( Mukherjee,

1965 ). At metaphase, the chromosomes beoome visible as long

bodies with a distinot split between their chromatids. The

degree of oondensation is less as oompared to the germinal

chromosomes. A comparison of the lengths of germinal

( spermatogonial metaphase ) ohromosomes and that of the

somatio ohromosomes ( Fig. 36 and 37 ) makes it olear that

the latter are approximately one micron longer than the

former. There is a slight indioation of the presence of

small arms in some of the longer chromosomes ( Fig. 34 and 35,

arrow marks ). No anaphase and telophase oould be found.

2. Chrotogonus traohypterus Blanohard.

The diploid number of chromosomes in the ma.le germ

oells of Chroto8onus traohypteru8 is nineteen (Sharma,

Parshad and Bedi, 1962 ). Measurements of ohromosomes

were made froml twettty olear well-plaoed spermatogonial

metaphase plates ( T8ble 4 ). The chromosomes oould be

classified into three long, six medium and one short

chromosomes ( Fig. 38 ). The sex chromosome i~ the longest.

a.. Meiosis: The meiosis hCis already been worked out

Fig. 34

Fig. 35

7

'" 6 '-u

L 5 .<: 4

~ 3 t::n c Cl>

.....J

'"

8

7

2

~ 6 L 5 c .- 4

..r:::: ~

~3 Cl>

...J 2

2'1

2 3 4 5 6 7 8 9 10 11 X C hro mo so m e Number

Flg.36

L.... L- I- L- LL I-L- ...... L-I-I- I-

1 2 3 4 5 6 7 8 9 10 11 ·x' Chromosome Number

Fig. 37

-44-

Table 4 I Mean length in micra of the spermatogonial metapha.se chromosomes.

Chromosome No. Length + s. D.

1 4.90 + 0.65 -2 4.53 + 0.20

I

-3 3.90 + 0.13 -

I I

I I

4 3.60 + 0.08

5 3.43 + 0.08 -6 3.19 .! 0.19

7 2.95 + 0.11 -8 2.59 + 0.13 -9 1.75 + 0.08 -

I ' X' 5.85 + 0.14 I -

hence it is omitted by the author here. A few points of

interest are, however, being mentioned. The 'megameric'

bivalent, which is a diagnostio feature of the Ottu (Punjab)

population (Sharma, Parshad and Gupta, 1965) and is absent in

the Delhi population (Sharma, Parshad and Bedi, 1962), is

absent in the local popu12tion of Bhopal too. In the early

prophases, two heteropycnotic bodies oan be observed - one

large and one small (Fig. 39). The former is obviously the

2 3 4

6 7 8 9

AA n .. A" ....

\ top,

Fig. 38

_____ - C.C

F1g.39

~ O-p.c \/,..0 • \ ,;J

... ~'X'

Fig. 40

5

a

-45-

Table 5 Chiasma frequency at different stages of meiosis in the Bhopa1 population of Chrotogo!lUS trachYl;lterus.

Number of stages of Total Mean chiasma frequency Nuclei Meiosis Chiasmata per nucleus

10 Diplotene 155 15.5 .:!: 1.21

13 Diakinesis 146 11.23 + 1.04 -15 Metaphase I 160 10.66 + 0.25 -

Table 6 s Mean chiasma frequency per nucleus in the three popu1ations of Chrotogonus trachypterus.

Mean chiasma frequency per nucleus Population

Diplotene Dakinesis Metaphase I

Delhi 15.25 + 0.12 12.15 + 0.31 10.95 + 0.94

Ottu(Punjab) 15.86 + - 11.93 + - 11.20 + --Bhopa1 15.5 + 1.27 11.23 + 1.04 10.66 + 0.25 -

-

sex chromatin 2nd the latter probably belongs to the supernu­

merary elements. At anaphase I, the formation of 'pseudo­

chiasmata' is found to be a regular feature (Fig. 40, 41, 42

and 43). In the other popu1ations, reported earlier, this

characteristic is not so prominent.

In the present study the loss of chiasmata is

str.onger between diplotene and diakinesis (Table 5) and mean

chiasma frequency per nucleus differs from that of the Delhi

• .. ( ,

"::>- X

,. -

~ 0 • •

• p.c-"

• I

Fig. 41

Fig.42

Fig.43

( , X I , .. f.-

•. ~ , -•

# •

-46-

popula.tion but shows a distinct similarity to tha.t of the

Ottu population ( Table 6 ). ,

b. Abnormalities in the natural E~Eulation :-

A total of eighty-six individuals were investigated

to locate the aberrations. A list of various typys of

aberrations in the Delhi and Ottu population and their

presence or absence and frequency in the Bhopal population

is given in Table 7.

(i) Supernumerary Chromosomes: Only one individua.l

revealed extra elements in its cells, which are smaller than

the smallest autosome and are positively heteropycnotic

during diplotene and early diakinesis. Extra elements with

such oharaoteristics have been designated as supernumerary

chromosomes by a number of scholars ( Carothers, 1917;

Carrol1, 1920; White, 1949; Rothfels, 1950; Rees and Jamieson,

1954; Srivastava, 1954; Evans, 1954; Ray-Choudhuri and Guha,

1955 a.nd Nur, 1963 ). Extra chromosomes with similar bahaviour

have been described as 'B' chromosomes by Lewis and John(1959).

The number of supernumerary chromosomes in the

meiotio cells of indiVidual No. lA varied from one to six.

which shows that they are mitotically unstable. resulting

in their accumulation during the pre-meiotic division. In

the indi.vidua1 under consideration. 68 cells out of a te.tal

of 91, carry two supernumerary chromo30mes ( Tables 8 & 9 ).

Sharma et sI ( 1965 ) reported the occurrence of 1 - 7

supernumerHrY elements in the Ottu population of Chroto~onus

trachypterus.

-41-

Table 1 I Various aberrations and their distribution in the three populations of Chrotogonus traohlEterus.

I Nature of aberration Presenoe (+)/Absence (-) Number of in the population of Individuals

Delhi I Ottu Bhopal (Bhopa.l)

1. Supernumerary Chromosomes (-) ( +) ( +) 1

2. Aneuploidy ( +) (+) ( +) -33. Polyploid Cells (+) ( +) ( +) 3

4. Suppression of paohytene pairing ( +) (-) (-) -

5. Anaphase laggards ( +) ( +) (-) -6. Anaphase bridges (+) ( +) (-) -

--7. Non-disjunotion (-) I

(+ ) (+ ) 1

8. Understained Regions (-) ( +) ( +) 3

9. Chromatid bree,kage (-) (+ ) (+ ) 1

10. Extra fragment (-) ( +) ( +) 1

11. Unequal bivalent ( +) (-) (-) -

n

-48-

Table 8 : Frequenoy distribution of the two supernumerary ohromosomes at various stages of meiosis.

stage of meiosis Number of Cells with Peroentage Cells S + 2s

l1li1 --Diptotene 20 13 65 %

Diakinesis 52 4,3 82.69 %

Anaphase I 4 3 75.00 ~

Meta.phase I 15 9 60.00 ~

Column Total J 91 68 74.70 ~

Table 9 : Frequenoy distribution of the supernumerary ohromosomes in the meiotio oells ( pooled data ).

Chromosome

I

Complement S + 1s S + 28 I s + 3s s + 4s s + 58 S + 68

I

Number of Cells 12 68 3 5 ;fl 2

Percentage ~.18; - -

74.70% 3.29% 5.49% 1.09'?, 2.19%

-49-

The supernumerary element. when single t

( unvalent ), orientates normally at the equator. At anaphase 1\

I and 11 it moves undivided to either of the poles along

with the autosomes showing a double reduction. In a long

cell st teloph~se II~ the univalent is found to l~g

behind ( Fig. 44 ).

~ben represented more than once, the supernume­

rary chromosomes usually form bivalents, each with a

single terminal chi~sma. The single bivalent in the n + 18a

condition 1£ found to divide reductionally at anaphase 11

( Fig. 45 ). In the triplioate oondition, one bivalent i~

accompanied by a univalent. In a sine~e cell out of three,

the bivalents exhibit non-disjunction at anaphase 11 and

pass to the same pole, whila the single univalent passes

to the other pole ( Fig. 45 ).

In the n + 4s condition, all the five cells

have the supernumeraries in the form of two bivalents. The

latter show double reduction and pass to the opposite poles

in an undivided state ( Fig. 46 ). This behaviour is not

noticed 1n the Ottu population. In the pentapl1cate condi­

tion one bivalent is found at the other pole at anaphase

11 ( Fig. 46 ). The behaviour of the pentaplicate is

undetermined in the Ottu population. When. present in

hexaplioate, the supernumeraries form two bivalents and two

univalents ( Fig. 45 ). Their behaviour at anaphase could

not be determined as both the oells possessing them were at

early anaphase II ( polar view). The hexaplioate condition

~ 1\ • s_ •

it ~

,'t-~

"-,,,,,-~ ~ ., ~

-~

..1-4["

-:...\ 11 - ,

\ ... ~-\' \" I

.... ;'-- -ss I s

. --.

.. • •

t ...

-......

.4 Fig. 44

Fi g .45

Fig.46

~ .,~

~

.1Jj-

'1-

• . 1

" . .

-50-

ie not reported in the Ottu population, while the heptapli­

oate oondi tj.on is not found in the Bhopal population.

The behaviour of supernumeraries during anaphase

I was similRr to that of the Ottu ( Punjab ) population.

(ii) Polyplo~.dy:

Soattered polyploid oells were obssrved in three

individuals, oa,rrying otherwise the standard oomplement in

the meiotio I ( 9 AA + X ) and meiotio II ( 9A + X I and (9A)

cells. The frequenoy distribution of the polyploid cells is

given in Table 10.

The two tetraploid oells at diakinesis are ohara­

oterized by the absenoe of any multivalent ( Fig. 47 ).

The two sex chromosomes lie away from eaoh other. The lone

tetraploid oell at metaphase II oontains 36dA + 2dX

oomplement ( Fig. 48 ). The daughter sex chromosomes show

a different degree of condensation and the autosomal

univalents reveal a olear split between the daughter

autosomes. In the third case of polyploidy, the first

spermatooyte nuclei are contained within a OOIIl1!lOn oell wall

( binuoleate condition) and at anaphase 11 divide into

9dA + dX - 9da + dX and 9dA - 9dA ( Fig. 49 ). The latter

again remain within the oommon oell wall, resulting in a

tetraploid condition.

In the Delhi and Ottu populutions, a low frequenoy

of polyploidy was noted in the spermatogonial metaphase and

first meiotio prophase and metaphase.

(iii) BPn-disjunotion: In a single individual 3 oells,

.. ~

" ,., . ''\; --:::

• . ~.

c.c..w--:-

, .-:

• . --'/ . /, . r (x •

Fi g. 47

Fig. 48

, , .. , ' . -- I~ ~

'" , --I "

~ '" - --\ , , - .. • Fig. 49

-

-51-

Table 10 : Frequency distribution of polyploid cells during various stages of meiosis.

stage of Iotal cells Cells 'l-lith Perc:entage Meiosis counted polyploidy aberration

-- .. _- -. .-Diakinesis 29 2 6.89 %

-_._"---._-------- .-Metaphase II 11 1 9.09 %

Anaphase II 32 5 15.62 % - ._---_ .. _-- ---

out of a total of 19 ( 15.78 % ), eDlibit non-disjunction

during anaphase II. The chromosomal configuration of two

cells is 8dA and 10da, where two daughter autosomes of a

univalent pass to one and the same pole ( Fig. 50 and 51 ).

In the tbird case, non-disjunction mi&~t have occurred at

anaphase I with "lA + 2AA + X at one pole and 7A at the other.

The latter again underwent non-disjunction at anaphase II,

when the two daughter 8utosome8 of a univalent passed to

one and the same pole. This resulted in cA and 8A oon£igu­

rations ( Fig. 52 ).

(i v) Unde!:~!§:!.l!:~.9:)i~;$!.~,!:

Three ind:l. viduals possessed understained regions

in certain autosomes during anaphase I and 11. These are

invariably situated sub-terminally ( Fig. 53, 54 and 55 )

and affect only one member of a pair of homologues except

, \,

• \ non, di5 " .-/

I't~. 1'~1

Fig. 50

loo , "

, :.c ~ 'if

~ ,

* :/<' " .,

Fig. 51

, Y\OY'l. di5 __ .. , :

,) ~" ,~

Fig.52

,

Fig.53

•

Fig.54

/'f I \) - /" dX

~ I ~~)~ u . s . r

Fig. 55

-52-

in one oase ( Fig. 53 ). A small chromatic connection can

be seen between two yarts of the chromosome arm. The

frequenoy of oells carrying understained regions in the three

individuals is given in Table 11.

Table 11 : Frequency distribution of understained regions during different stages of meiosis.

Individual stage of Total Cells Cells with Peroen Number Meiosis oounted I understai- tage

i nad region I i

1. Anaphase I 15 4 26.66%

---2. Anaphase 11 22 8 )6.36%

...

3. Anaphase 11 19 6 31.51%

(v) Chromatid Breakase :

In a single individual. out of 21 cells at

anaphase 11, one oe11 carried a breakage. The latter is at

the proximal end of a daughter chromosome ( Fig. 56 ). The

fragment is not associated with the other part and lies a

little away from it, at a different angle.

(vi) Extra Fragment :

Out of 10 oells, studied during metaphase 11, two

cells h~ve been found with an extra fragment ( Fig. 51 ).

The fragment lies free at the periphery of the spindle, out

•

• I I,

(I J\"

"." /" "

f 1-' . br

Fig . 56

.-

• r.:., /

~~ ~ • • ~

".!.. ... ! , - \ , .. ' -

"*'* ,~ I

- .('

Fig. 51

int .nu

Fig.58

-53-

side the circle of metaphase 11 chromosomes. It is round

in shape and smaller than the supernumerary chromosomes.

c. Mitosis:

The prophases do not show any heteropycnotic body

in the male somatic tissue' Fig. 5d ). At metaphase, a

clear split is visible in all the chromosomes. The sex

chromosome can not be differenciated from the autosomes on

the basis of degree of condensation which is uniform in

both, but it can be differentiated on the basis of length.

No telophases and anaphases could be found.

A comparison of the lengths of spermatogonial and

somatic ( gastric caeca ) metaphase chromosomes, reveals

that the former are much more condensed than the latter.

The histogram of spermatogonial metaphase chromosomes

( Fig. 59 ), based on the data of Table 4, shows that the

longest chromosome measures about 6 micra. while the same

chromosome measures about 15 micra in the somatic tissue

( Fig. 6J ).

In the rod-shaped chromosomes, though reported

to be 'acrocentric' , no second arm could be seen even in

the somatic chromosomes which are loosely coiled.

3. Leva indica Boliver.

The diploid number of chromosomes in the male germ

cells of Leva indica is twenty-three ( Fig. 61 ). Measurements

of chromosomes were mDde from twenty well-placed sperma­

togonial metaphase plates ( Table 12 ). The chromosomes can

be classified into three long, six medium and three short

5 ~ L-L)

4

L 3 c:

2 -C. ...... = c: 4J

.....J

2 3 4 5 6 7 8 9 'X'

Chromosome Number

Fig.59

15

12

'" L-U

9 :i c: ._. ~ E, ... C7' c: QJ

...J 3

2 3 4 5 6 7 8 9 ·x' Chromosome Number

Fig. 60

-54-

Table 12 : Mean length in micra of the spermatogonial metaphase chromosomes.

Chromosome No. Length::!: S.D.

1 4.86 + 0.40 -2 4.48 + 0.48 -3 3.55 ! 0.54

4 3.09 + 0.44 -5 2.69 + 0.33 -

I ! 6 2.36 + 0.20 -7 2.14 + 0.30 -.--8

I 1.75 + 0.20 -

I

9 1.34 + - 0.09

10 1.18 + 0.30 -11 1.10 ! 0.31

• X' 5.40 + 0.47 -

chromosomes. ( Fig. 62 ). The sex chromosome is the

longest.

Fig. 61

2 3 5 6

7 8 9 10 11 'X'

n6 •• .... .... .. .. 0 10fL

I

Fl g .62

-55-

a. Meiosis:

The general course of meiosis is of the classical

type. During interphase stage, the sex chromosome appears

as an intensely positive heteropycnotic body. This behavi­

our is maintained till diplotene. At late spermatogonial

metaphase, the sex chromosome oan be distinguished from the

autosomes ( Fig. 61 ) due to its length. The arrangement of

the first spermatocyte metaphase chromosomes is normal and

regular. Bivalents of various eh&pe~ and sizes are arranged

equ1distally from the two poles. The sex ohromosome lies

either 'above' or 'below' the equatorial plate. The

'pseudo-ohiasmata' have not been observed at anaphase I.

The long univalents are arranged at random on the radial

metaphase 11 oonfiguration. No asynchronous behaviour has

been noticed at anaphase 11.

A study of ohiasma frequency reveals that the loss

of ohiasmata is stronger between diakin.esis and metaphase

I ( Table 13 ). The long bivalents possess 2-3 chiasmata,

while the medium ones never have more than two chiasmata.

The short bivalents possess one chiesme each.

b. Abnormalities in the natural populatio~ ,

A total of fifty-two individuals were studiea.

The freouenoy of aberrations is very low ( Table 14 ). The

aberrations can be analyzed under the following headings :

(i) Extra fragment: In a single individual, 4 cells out

of 54 at diakinesis, have been found with an extra fragment.

The latter is round in shape and smaller than the smallest

-56-

autosome ( Fig. 63 ).

(ii) Polyploid cells : In a sin~;le follicle of an

individual 11 cells out of 48 at anaphase 11 are tetraploid

in nature. Forty-four daughter chromosomes were contained

within a common cell wall ( Fig. 64 ). Similar type of

tetraploidy has been found in C. trachypterus by the

author. But in the present case, the origin of polyploidy

seems to be different. Here the complement includes 44dA

instead ot the expected 44d.A + 2dx.

Table 13 : Chiasma frequency at different stages of meiosis.

Number of stage of Total Mean ohiasma frequency nuclei meiosis Chiasmata per nucleus

-10 Diplotene 136 13.6 .! 1.49

12 Diakinesis 144 12.;)0 + 1.12

1 Metaphase I 68 9.71 + 1.11 -

(i1i) Abnormal Spindle: At anaphase 11, 30 cells out of

a total of 90, possess an abnormal spindle structure. One

pole is normal with 11/12 daughter chromosomes, while the

other pole is 0ifid ( Fig. 65 ). At this pole the daughter

chromosomes are arranged in two groups. A wide gap is

present between the two groups of chromosomes.

... I r

[.~ ~ l eJ

" I ~ .. , ...... (J •

~ 1 J -- -

Fig.63

-c.W

Fig.64

-51-

Table 14 : Frequenoy distribution of abnormalities in the natural population of Leva indioa.

--.

Types of I Total oells I Stage of Cells with Peroentage a.bnormali ty I meiosis studied tberratiOn

of aberration

.- -Extra Diakinesis 54 4 1.40 % fra.gment

I

AnBPhBse-t +17 •

"' . Polyploid 48 35.41 %

i Cells

I I I

III i Abnormal I Anaphase 90 30 33.33 % ! Spindle

Breakage Spermatogo- 11 5 29.41 % nial metaphase

--

(iv) Bree,kage: At late spermatogonial metaphase, 5 cells

out of a total of 17. possess breaks in the long ohromosomes.

Usually the two pairs of long autosomes are affected and the

sex ohromosome remains unaffected. In Ol1e oase, only one

ohromosome of a pair is affeoted ( Fig. bb ) t but usu~1l1y

the breakage is found in more than one chromosome ( Fig. 6'1 ).

In &11 the ceses, both chromatids of the affected chromosome,

possess the breakage at the same locus.

c. Mitosis:

The prophases do not show any hetropyonotic mass

c..W

~

Fig.65

Fig. 66

Fig.67

.. , ) . , . ,

." . ... I · . ~. 'x'

o ''' :~ ~ . ~ , . " , • h.b

Fig. 68

-58-

in the male somatio tissue. But the cells of the femele

tissue do possess a single indistinct hetropyonotio mass

( Fig. 68 ). At early metaphase the chromosomes attain a 1

leaser degree of condensation as compared to the germinal

tissue, and are long and slender. A distinct split is

present between the chromatids. The homologous chromosomes

lie apart. The sex chromosomes can not be distinguished

from the autosomes on the basis of condensation. Being the

longest in the complement, their identification is possible

by comparative measurements ( Fig. 68 ). At ll:ite metaphase

a greater degree of condensation is achieved and the split

between the chromatids does not look very distinct.

No asynchronous movement has been noticed at

anaphase. The short chromosomes reach the poles first. At

telophase, the daughter chromosomes at the poles can not

be oounted easily beoause of a olose grouping and the onset

of deoondensation. The two daughter cells get separated by

a constriction at the equatorial region. A smooth outline

/' is squired by the daughter nuclei at interphase.

A comparison of the lengths of germinal (spermE,to­

gonial ) and somatic ( gastric caeca ) chromosomes reveals

that the difference is not much. The sex chromosome in the

former measures about 5.50 micra, while in the latter about

6.75 micra. ( Fig. 69 and 10 ).

4. Attraotomoryha crenulata Fabricius.

The diploid number of chromosomes in the m81e

germ cells of Attractomorpha crenulata is nineteen ( Fig. 11).

~ 5 <J

== 3

6

'" 5 '-.9 2:

4 c:

..c 3 .., eT> c: 2 '" -l

2 3 4 5 6 7 8 9 10 11 X

Chromosome Number

Fig.69

2 3 4 5 6 7 8 9 10 11 'X'

Chromosme Number

Fig. 70

-59-

Meaauremellts of chromosomes were made from twenty well-spread

spermG.togonial metaphase plates ( Table 15 ). The chromosomes

C&ll be classified into three long. six mediUlll and one short

chromosomes ( Fig. 72 ). The sex chro~osome is the largest

and can be distingu,ished easily i'rom the autosomes ( Fig. 71)

because of its length.

Table 15 : Y..ean length in micra of the sperme.togonial meta.phase chro:n~so~E's.

ChromOSOm~~No. I

I Length .:!: S. D.

i

1 7.78 + 0.96 -2 7.16 + 0.48 -3 6.14 + 0.93 -4

I 5.30 0.42 + -

5 4.98 + 0.41 ,

--_. 6 4.84 + 0.47 -7 4.37 + 0.50 -8 3.67 + 0.41 -9 2.55 + 0.17 -

'X' 8.64 + 1.05 -

•

•

1

6

~A

, ,

2

7

Fig.71

3

8

I'A •• 10)1-, ,

Fig . 72 . '

(

4 5

9 ·X'

•• 0

-6·)-

Meiosis ia not being dealt with by the author except for

a few points of interest.

a. t\b~~!:llalities in the natural population :-

In the natural population of Attraotomorpha orenu­

~, polyploidy is a oommon feature. The behaviour of

diploid Rnd tetraploid spermatocytes was reported by

Ray-Choudhuri and Bose , 1943 ). In the present aocount,

therefore, varir:Jtions other than polyploidy 8I'e being

reported.

Only four individuals out of a t~tal of thirty-

five, possessed abnormal cells. The frequency of aberrations

is quite low ( Table 16 ).

Table 16 : Frequency distribution of abnormalities in the na.tural population of Attract?Dlorpha orenulata

I Types of stage of Total oellS~lle with peroentagel of

I

aberration meiosis studied aberra.tion aberration

-AnaphaS~

~ . .. , . . Chromatid I 3 3.33 % Breakcge

-Chroma.tid Anaphase I 90 35 38.88 % Separation

--,---

(1) Chromatid breakase I At anaphase I. the largest

ohromosome, that is the sex ohromosome, possesses breakage

" " t!., (

br f'

. rO~ ,,~~~X' . \) "Vt..'

t' ",. -

Fi g . 73

Fig. 74

... I

1'\

-61-

in one ohromatid ( Fig. 73 )." The break ooours in the distal

region. The fragment lies a little filway from the heteromor­

phio univalent and no oonneotion is found between the two.

The broken pieoe does not lag in any of the cells.

(ii) Chromatid Separation : The separation of the two

daughter halves is found in 38.8 % oells at anaphase I

( Fig. 74 ). Thus there is a olear indioation of preoocious /\

behaviour. The daughter halves do not drift away from each

other. The separated ohromatids invariably pass to the same

pole after anaphase I in all the instances noted by the

author.

b. Mitosis I

The prophases do not possess any heteropycnotio

maSf:3 in the me.le tissue. At pro-metaphase the ohromatin gets

resolved into olosely paoked blooks inside the nuolear wall.

At metaphase, the ohromosomes look thioker and longer

( Fig. 75 and 76 ) than the spermatogonial metaphase

ohromosomes. The size differenoiation between the two is

appreoiable. The sex ohromosome measures 8.64 miora in the

germinal tissue and 10.69 miora in the som2tic tissue.

Similarly the smallest autosome me8.Sv.res 2.55 micra in the

germinal and 3.72 miora in the somatic tissue. The centromere

is grea.tly stretohed in some of the ohromosomes, revealing

a lightly stained gap between the two chromatids •• Anaphase

and telophase stages were not available.

5. Poekilooerus piotus Fabrioius

Spermatogonial metaphase plates show the presenoe

Fig. 75

F1g.76

~

8

7

6

t; 5

2: 4 c:

'': 3 ..... ~2 Cl.>

-11

<0 c.... <.>

~

c::

~ ..... t:7\ c:: cv -1

10

9 8 7 6 5 4

3 2.

23456 7 8 9 X Chromosome Number

Fig. 77

2 3 4 5 6 7 8 9 'X'

Chromosome Number

Fig. 78

-62-

of nineteen rod - shaped chromosomes ( 18A + x ) whioh can

be divided into three long, six medium 8nd one short

ohromosomes.

The meiosis of this speoies has been investigat"ed

by a number of soholars ( Asana and Makino, 1934; Rao, 1937;

Dutt, 1955; Srivastava, 1960, and others ). White ( 1940 )

reported that the negative heteropyonosis of the X-chromo­

some was either less pronounced or even absent in the last

spermatogonial. division. The small arms, though reported to

be present in all the chromosomes by Dutt ( 1955 ). could

be observed in rare instances. Their presenoe, however, is

said to be well-marked in the smallest autosome by Darlington

( 1936 ) and Coleman ( 1943 ). The smallest autosome shows

positive heteropyonosis at diakinesis, along with the

X- ohromosome ( Mangalangi and Subramanium, 1963 ) though

the latter stains relatively lightly.

The present aocount includes only mitotic oaryo­

logioal. study and the banding pattern.

a. Mitosis: The pattern of mitosis is similar to that

reported by the author in other grasshoppers. At ee.rly

metaphase, however, distinct second arms are visible in one

of the smallest autosomes and in one modium f:'}utosome

( Fig. 79 ). Darlington ( 1936 ) and ColE)IDhn ( 1943 )

reported the oocurrence of second arms in the smt.311est

autosome only.

The length of the minute arm, in the present

investigation, is about 0.9 to 1.00 micron. A comparison

]'1g.19

-

• 14

12. I'\l L.. U

10 ~

c: 8 .c .... 6 C'l c: ( . ' .. QJ

.-J 4

2. ') .' I ..

2. 3 4 5 6 7 8 9 ·x' Chromosome Number

Fi g .BO

20

."

~15 ~

c ,-10 .c

...... t:n C C1.I

....J 5

, 1

2 3 4 5 6 7 8 9 ·x' Chromosome Number

Fig .al

-63-

of the lengths of somatio metaphase ohromosomes with that

of the spermatogonial metaphase ohromosomes, makes it olear

that the former is almost double that of the latter ( Fig.

80 and 81 ).

The degree of condensation is uniform in the

autosomes and the sex ohromosome, henoe the latter oannot

be distinguished from the long autosomes easily. The well­

separated, thread-like ohromatids ooil around eaoh other at

early ~etaphase and resemble the oOnIigurations aoquired

by the bivalents at diplotene in meiosis ( Fig. 79. arrow

marks ). \

A greater degree of oondensationis achieved at

late metaphase. The maximum reduotion in length is observed

at anaphase. No asynohronous behaviour has been notioed

during anaphase and telophase.

b. Banding pattern c The nature of oentromere has been

observed in all the stages of sperma toger~esis with the help

of Giemsa-staining ( Sumner. 1972 ). The observations not

only differ in many respeots from the routine analysis of

oaryotype. but also oOIJ.tradiot a few points. The various

stages are being analysed systematioally :-

spermatosonial metaphase: As stated earlier, the ohromoso­

mes of P. :piotua oan be divided into three oa.tegories, viz.,

long, medium and short. For the sake of oonvenience, there­

fore t eaoh ohromosome is given a nomeno18.ture aocording to

ita oategory. Fig. 82 shows all the ohromosomes of the ~ diploid oonpliment with their respt:lotive numbero 8.l1d .A

oategories. There are two pairs of long chromosomes (L1 and L2 )

.1

Fig.82

Fig.83

. •

-64-

six pairs of medium chromosomes (Ml to 1V16 ), and one pair of

short ohromosomes (SI). Besides these eighteen autosomes,

the single X-chromosome, long(:st in the oomplement, is e8sily

distinguishable. Giemsa stain has demaroated a de.rk band of

repetitive DNA sequences in the centromerio region of all the

ohromosomes. This band of heterochromatin, after a closer

observation, is seen to be oomposed of four subunits ( Fig. 82,

X and M4 ). Besides the centromerio heterochromatin, a less

porminent band is present at the tolomerio end of the X­

ohromosome. The smallest autosome. (Fig. 82, SI) is the most

distinguished of all because of the presence of dark blobs

of heteroohromatin at the telomeres.

Leptotene : The darkly stained m~~ss at the left hand end of

the leptotene-nucleus ( Fig. 83 ) can be e8sily mistaken

for the positively heteropyonotic sex-chromatin. But the

latter is actually at the opposite end ( Fig. 83, h.b. ).

The dark mass is the outoome of the 'bouquet' arrangement,

so characteristio of leptotene staee, being formed by the

polarization of centromeres and telomeres. The ooourrenoe

and orientation of the loops ( Fig. 83, Ips. ) indicates

that the ohromosomes have doubled on themselves and both

of their ends are polarized towards, and oompose, the

'dark pole' ( Fig. 83, dk. pI. ) simultaneously. The four

sub-units of eentromerio heterochromatin are well-marked

in one of the ohromosomes ( Fig. 83, arrow-mHrk ).

Pachytene: (Fig. 84 and 85 ) Late paohytene nuolei reveal

more details about the oonstitutive heteroohromatin. The

Fig.84

Fi g . 85

-65-

latter is separated from the main chromosome by :3 constric­

tion ( Fig. 84, a.rrow mark ). The darkly staining centromeric

chromomeres a..ppear like the t horns of 8n8il' and may easily

be mistaken for the ·second arms'. The smallest autosomal

bivalent can be distinguished without any difficulty because

of both its size and the prominent telomeric band of hetero­

chromatin ( Fig. 84 ). The sex chromatin hp..s acquired et

peculiar, geometrioal configura.tion in the interphase

nuoleus ( Fig. 85, arrow mark ). At late pachytene, the sex

chromatin appears dumb-bell shaped and eta,ins lighter than

the constitutive heteroohromatin.

Two more oells at pachytene ( Fig. 86 and81 )

show that the cODst1 ttltive heterochromatin also includes the

proximal part of chromosome arms besides the centromeric

heterochromatin, in almost all the bivalents. The SI

autosomal bivalent appears uniformly dnrk because of the

approximation of centromeric tUld telomeric bands of

heterochromatin ( Fig. 81, SI ).

The band of oonstitutive heterochromatin at the

proximal region of chromosomes is more clearly revealed in

Fig. 83 ( arrow marks ). Most remarkable is the 51 bivalent,

which possesses two distinct, close bands in the distal

region ( Fig. 88, dis. bl and die. b2 ). The M6 bivalent

also possesses bands in its dietal region, each ~~ivalent

having a single band situated sub-terminally. The stain of

these bands is lighter than the other bands.

The bivalents at diplotene Gnd diakinesis normally

"'- pl"O . b. het "'-t .e

"- ,/

F1g . 86

-

F1g . 81

-66-

show a greater affinity for dyes. With the use of Giemsa,

however, only those regions take the stain which possess

highly repetitive DNA sequences. Rest of the portions take

a lighter stain ( Fig. 09 ). The SI hi~alent, the single

intersti tial ohic-:lsma being ter:ainalized. is darker than the

other bivalents, espeoially a't the proximel and distal

regi0ns ( Fig. 89, SI ). The u.n1va1en~ 'X' lies coiled upon

itself in the middle of the plate ( Fig. 89, X ), and looks

darker than expected because of the overlapping.

At diakinesis and metaphase It the bands beoome

almost indistinguishable because of the contraotion of the

ohromosomes. still, the heterochromatin can be loce,ted at

the oentromeric end ( Fig. 90, cen. het. ). The arrangement

of bivalents at metaphase I is very clear in Fig. 90, where

the sex ohromosome and the SI autosomal bivalent lie in the

centre of the spindle, while Ll , L2 and Ml to M6 bivalents

occuVy the peripheral region with their darkly stained

centromeric ends directed towards the oentre of the spindle.

The 'X' possessei:3 a distinct centromeric band ( Fig. 90,bd ).

Another view of metaphase I in profile is

illustrated in Fig. 91. Here the bands oarmot be demarkated

in the bivalerlts. The univalents of SI p..nd M6 pairs have

already started moving towards the opposite poles ( Fig. 91,

SI and Si ; M6 and M· 6 ). The sex chromosome shows a

split between its ohromatids a.nd dark regions at both the

liistal and proximal ends.

In the next stage, i.e., anaphase It the slightly

Fig.88

Fig.89

cent . het

Fig . 90

Fig . 91

Lt

~Cent . hel:

~ ...... ---------------61-

heteropyonotic 'X' can be seen at the t1ower' pole ( Fig.

92 ). Its oentromeric band looks darker than the telomeric

ones of the separated chromatids. While all the M-univalents

and L2-univa1ents have reached their respective poles,

Ll-univalents have just departed from each other at the

equator ( Fig. 92, L1 ). The oentromeric heterochromatin

in the L- and M- un1va1ents appears like two dots, lying

side by side. The Sl -univalents possess Cl. comparatively

light region separating the two d::)rk bands a.t the two ends

( Fig. 92, bdl , bd2 ). The heteropycnotic behavioQr of the

'X' persists only upto anaphase 11. Afterwards. the degree

of condensation is found to be uniform in all the chromo-

somes.

At ane.phase 11, the division of the centromere

is superbly revealed by the separate 'daughter' bands at

the proximal end of the divided chromatids ( daughter

chromosomes ) in Fig. 93. The dc;ughter chromosomes of M4,

M5 and M6 have shifted apart while those of Sl lie overla-

pped ( Fig. 93 ). The sex chromosome shows a gree of

condensation equivalent to that of the autosomes. The

orientation of Llt L2, Ml , M2 and M3 suggests that the

centromeres are directed towards the centre of the spindle.

B. Effeot of Drugs

1. Quanti tative stu~l : Caff'eine

Caffeine is extensively used as a therapeutic

agent and because of the widespread consumption of

Fig.92

M2 '--1- ( Mc;.

. . Ml

c.ent .het . x/ tel . he.\:

" Mo\:

Fi g .93

-63-

oaffeinated beverages a major part of the human raoe is

ohronioally exposed to this compound.

In the field of oaffeine mutagenesis there is

some oonfusion due to several contradictory reports. A

survey of available reports reveals interesting findings

on the nature of this methyl xanthine. Kihlman and Levan

( 194d ) observed two main features in the action of

caffeine - (1) the formation of cell wall after mitosis

was impeded although the spindle functioned normally

(2) chromosome bridge formation at anaphase started

appearing after a few hours of treatment. Most of the

work that followed contained genetical and biochemical

results.

Axelrod and Reichanthal ( 1952 ) studied the

fate of caffeine in human subjects. Some of the important

findings were - (1) caffeine was rapidly and essentio.lly

completely absorbed from the gastro-intestinal tract; (2)

caffeine was almost entirely transformed in ms.n, only

about 1% being excreted in urine; (3) the drug pasiSed

rapidly into the central nervous system ; and (4) there

was no day-to-day acculllulation of the drug.

Goldstein and ~arren ( 1961 ) cQnsidered

mutagenecity of caffeine in mammals on indirect ground.

It was stated in their work that since caffeine was known

to be mutagenic to bacteria, fungi and Drosophila, it

could be mutagenic in man because of the bpsic similarity

of genetio material in all forms of life. Their experiments

J

-69-

on pregnant hum2D subjects clea1'ly demonstrated tfH:;t

caffeine equilibrates fret.ly between maternal plasma t:1.L:.d

1-8 week foetus and the exposure misbt be hC;izardous at this

stage - when the segregatiJn and prolifE:iration of germ cells

takes place.

Cattanach ( 1962 ) found caffeine weakly mutage­

nic in mice, for only two possible translocatioll8 were

detected. Ostertag et al ( 1965 ) found the drug mu.tagenic

in man as it could induce breakage in the human ( HeLa )

cells in tissue culture.

Clerk and Clark ( 1968 ) reported that caffeine

could induce ohromosome loss in both male and female

Drosophil~ B.nd suggested that this drue might be a oontri­

butory faotor in the induction of Turner's syndrome ( XO )

in man. Kuhlman et al ( 1968 ) in their experimEmt u.pon

human oells in cu.l ture, ±'ound that the action of oaffein

is analogous to that of radiation.

A reoent report by Slizynski ( 1911 ) is based

on direot cytological and chromosolUtil observation on the

rat testis. The tissue was, however. maints.ined outside the

body in a short-term culture. The results obtained were

both positive ( clUt'1lping, stickilless and ohromatin extru­

sion from the {luolei ) a.nd r.:eg~i.,.tve ( gap formation,

fra.gmentation and v~ouolar extrusion of material from the

nuclei ).

Most of the reports are not purely oytolo;gical

and give an idea of the nature of the drug itself. Among

-70-

the direct cytological studies, that of Kihlman nnd Leva,n

( 1943 ) on plcmt chromosomes is worth mentioning. In the

case of animals the tissue was invariably treated outside

the body in a culture. ( Ostertag et 81, 1965; Kuhl.ITV n

et aI, 1968 and Slizynski, 1971 ).

In the present work, the tioi.,ue wes trel:1.ted in

a natural state, still at·taohed to tlie body and the division

oycle was also taken into considerati0n for treatment.

In order to make a. comparative study of the

effeot of known concentrationq of caffeine upon the testes

cells of o. trachy;eterus, the frequenc.y of three types of

abnormalities, e.g. fragmentation, clumping end polyploidy,

has been determined. In this experiment, mortality occurred

in the higher dosage range ( 0.5 ,%, ·).6 ;1&,·).'7 %, 0.:3 ~t

0.9 10 and 1 % ). Details of the experin,e:nt are given in

Table 11.

The frequency is scored et d1plotene, diakinesis

a...'1d metaphnse I .i;or each type of e,bnor:n:.-.li ty. The dl:it:::~ r-lre

po~led and averaged according to the number of s.rv1ved

specimen for each concE':ntration of caffeine. f,ll the

ceoncentrations were introduoed at prophase and the effect

waa studied after six dpys, when, according to the division

oycle ( Fig. 3 ) the cells are at matepna.se r. An. analysis of the ~bIJorm81i ty data reve~ds some

interesting facts about the relation of tbe varying concen­

tration to the fixed time of tr~at~e~t. The frequency

distribution of the three abnormalities for 0.1 %

-71-

Table 11 : Details of experiments of the control and treated specimens of c. trachypterus.

Concentrations Number of specimen Number of specimen of Caffeine used in Experiment survived after

treatrr.ent

"--, Jcontrol Tref1ted Control Treated

0.1 .~ I 5 5 5 5

0.2 ~ I

5 5 5 5 i i

- i

0.3 % I 5 I 5 5

I 5 i

I !

0.4 % i 5 5 5 5 -.- I

0.5 % ! I 5 5 5 4 I

0.6 to I 5 5 5 I 4

I

0.7 '% I 5 I 5 5 2 , ,

0.8 % I 5 5 5 1 ~- -

0.9 to 5 5 5 0 !

~ : ---_.-_.

>: r-1.0 'fo 5 5 0

- ..

caffeine is given in Table 13. Vfuile the occurrence of

£ragmentation is nomin~l ( 4.44 ~ ), the other two

Total apeci-men sur-vived

10

10

10

10

9

9

7

6

5

5

-72-

abnormalities are quite significant, clumping being 16.44 %

and polyploidy 20 %. The fragmentation involves the chroma­

tids only and all the stages are easily recognizable. The

olumps are formed by the stioking together of two or three

chromosomes. Polyploidy is at the level of tetraploidy. The

latter state is devoid of multivalent configurations and

non-assooiation of the sex chromosomes. A comparison of the

treated series with the control series reveals that there

is a slight depression in the rate of progression of

prophase into metaphase. The latter stage is numerioally

higher in the oontrol series. The cell-wall is intact in

all the stages.

Table 18 I Frequenoy distributions with their percentages of some types of abnormalities produced by 0.1 % caffeine ( data. pooled and averaged for the five survived speoimens ).

Types of Total cells Total Peroentage abnormality studied abnormal cells frequenoy

Fragmentation 225 10 4.44 ~

Clumping 225 37 16.44 " Polyploidy 225 45 20.00 %

The concentrations, 0.2 %. 0.3 % and 0.4 % show

a gradual rise in the percentage frequencies of clumping

-13-

Table 19 : Frequenoy distributions with their peroentages of some types of abnormalities produoed by

0.2 % oaffeine ( data pooled and averaged for the five survived speoimens ).

Types of Total oells Total percentage abnormality studied abnormal oells frequenoy

Fragmentation 206 12 5.82 %

Clumping 206 45 2l.84 to

Polyploidy 206 68 33.00 ~

and polyploidy ( Tables 19, 20 and 21, respeotively ).

But the frequency of the former is always lower than the

latter. The inorease in the frequency of fragmentation is

gradual from 0.1 ~ to 0.2 % , but at 0.3 % it zooms up,

being almost double the frequenoy at 0.2 %. Its inorease

is steady and signifioant at 0.4 % also. upto the latter

oonoentration, no mortality ooours and oell-wall formation

has impeded notioeably at 0.4 % oonoentration, where the

degree of ploidy has inoreased to 52.43 %, that of olumping

being 49.26 %. Mortality ooours in the higher dosage range. In

0.5 % oaffeine - treated series, a single individual died

after five days of treatment. Other individuals reveal that

the fragmentation involves more oells though at a slower

-14-

Table 20 : Frequency distributions with their peroentages of some types of abnormalities produoed by 0.3 % caffeine ( data pooled and averaged for the five survived specimens ) •

. --Types of Total cells Total Peroentage abnormality studied abnormal oells frequenoy

Fragmentation 300 35 11.66 %

Clumping 300 121 40.33 %

Polyploidy 300 125 41.66 %

Table 21 I Frequency distributions with their percentages of some types of abnormalities produced by 0.4 % caffeine ( data pooled and averaged for the five survived specimens ).

Types of Total cells Total Percentage abnormality studied abnormal cells frequency

Fragmentation 410 103 25.12 'to

Clumping 410 198 49.26 %

Polyploidy 410 215 52.43 %

-75-

Table 22 I Frequency distributions with their percentages of some types of abnormalities produced by 0.5 % caffeine ( data pooled and averaged for the four survived specimens ).

Types of Total cells Total Percentage abnormality studied abnormal cells frequency

Fragmentation 360 101 28.05 %

Clumping 360 190 52.17 %

Polyploidy 360 212 58.88 %

Table 23 I Frequency distributions with their percentages of some types of abnormalities produced by

0.6 % caffeine ( da.ta pooled and averaged for the four survived specimens ).

Types of Total cells Total Percentage abnormality studied abnormal cells frequency

Fragmentation 350 98 28.00 %

Clumping 350 195 55.11 %

Polyploidy 350 210 60.0J %

-16-

rate ( Table 22 ). The other two abnormalities have, how­

ever, inoreased significantly, olwmpimg being lesser than

polyploidy. In this series, the de.:?;ree of ploidy could not

be distinguisLod as the groups of chromO[lomes were closely

approximated. Clumping usually involves all the chromosol{,es

of a set, so that the individuul indentity of the chromo­

somes becomes impossible. Moreover. the heteropycnotic

behaviour of the sex chromosomes is not recognizable due

to general over-condensation.

In 0.6 % caffeine treated series the mortality

is similar to the 0.5 % caffeine-treated series but the

aberration has obviously increased at least in the cases

of clumping and polyploidy. The percentage-frequency of

fragmentation is, however, equal to that found in :).5 %

series ( Table 23 ). In 0.1 % series, all the three abnor­

maliti~s show an increase in percentage-frequency

( Table 24 ). Only two ~peoimens survived after treatment,

in this series. The 0.8 % solution of caffeine see::ms to

be significantly toxic, as here 80 % mortality oocurred.

The only surviving individual possessed the freouency­

distribution given in Table 25. The percentage-frequency

of fragmentation has gone down in this case, while that of

clumping a.nd polyploidy han risen to 8 striking level. The

cell-wall fOrnl:.:.,tion hbs been found to be completely

impeded in this series. The recognition of cell-stages was

very diffioult due to high degree of ploidy and stiokiness

involving a number of nuclei.

-'77-

Table 24 : Frequency distributions with their percentages of some types of abnormalities produced by 0.7 % ca~feine ( data pooled and averaged for the two survived specimens ).

<- -Types of Total cells Total Percentage abnormality • studied abnormal cells frequency

Fragmentation 200 65 32.50 % -

Clumping 200 138 69.00 %

Polyploidy 200 149 74.50 %

Table 25: Frequency distributions with their percentages of some types of abnormalities produced by 0.8 % caffei.oe ( data. pooled for the only survived specimen ).

ITot~ cells -- --

Types of Total Percentage abnormality studied abnormal cells frequency

Fragmentation 90 22 24.44 j,

Clumping 90 65 72.22 % -

Polyploidy 9J 70 77.77 % --

-78-

A systemCltic quantitative am::lysis 08!l1l0t be

oarried out with the series of the lEst two concentrations,

where absolute mortality oocurred • In both the oases, the

speoimens died within one Or 'two d;;:ys of treatment. However,

an examination of the tissue of tbe dying specimen throws

some light on the state of the tissue. It is found that

the oells either do not progress beyond early prophases,

or if they do, their reoo~~ni tion is not posoible. The

ohromatin extrudes out from the nuclei, giving them a

'notohed' uppearanoe. Similar unusual appearance has been

reported by Slizynski ( 1970 ) in mouse ohromosomes after

caffeine-treatment. Clumping is a generF,l feature and

sometimes club-shaped bodies protrude out from the clumped

masses. Pachytene ohromosomes, whenever reoognizable,

possess several ga.ps. It is found that the effect of

caffeine, though highly toxic in the 0.9 fo and 1.0 %

oonoentrations, does not interfere with pairing of

chromosomes at pe_ohy-tene. Frequently the p[;chytene

ohromosomes disintegrate into numerous stron:rly staining

fragments of different sizes.

2. Qualitative studl : Caffein~

The seoond lot of' the 35 - days old males of

C. traohyoterus was used for this analysis. It is observed

in the quantitative analysis that the 0.4 % conoentration of

oaffeine is cuite potent. Moreover, it does not oause

mortality. But with 0.5 % ooncentration onwards, the percen­

tage of mortality increases. Beopuse of these two reasons,

-79-

0.4 10 caffeine has been seleoted to study the effect w.nen

the treatment time is being altered. Testes from five males

of the oontrol- and treated-groups were removed at the follo­

wing intervals: 4, Id t 43, and 72 hours Emd 6, '7, 8, 14,

and 21 dDYs post-injection. As is clear froIn Fig. 9,

these intervels involve 8,11 thE) stages of spermHtogenesis.

The abnormalities can conveniently be put into four

categories -

(i) mitotic changes;

(ii) changes in the meiotio prophase stages;

(iii) changes at metaphase I and 11;

(iv) changes at first and second post-metaphase stages.

u. Control Series :

In this series, except 'pseudo-chiasmata', no

perceptible ohanges from the normal meiotic behaviour of

the chromosomes h~.s been noticed. There is some general

effect, ho~ever, in comparison to normal individuals. The

disposition of chromosomes in the control series is

relatively clear with some shortening effect.

b. Treated Series :

(i) Mitotic ohanges : not found.

(ii) Changes in the meiotic prophese stages :-

1) High condensation: This abnorm::;l! ty

generelly afieots all the bivalents at

diakinesis. The chromosomes :c;re so thick

that no hollow, oentr~d sp;;l.ce CBn be

-dO-

observed in tho.3e bivc.l(;nts where the two

interstitial chi&sm~ta huvs termin~lized.

The ·l·~.mp-brush' 8flpearance is COIDI)letely

miss.ing and the po;::;i ti vc heteropycnosis of

the sex-chromosome is not distinguishable

( Fig. 94 ).

2) Lesions J Erosion of chromatin occurs at

diakinesis ( Fig. 95 ). About three biva­

lents of each plate possess this abnormality.

Lesions a.ppear as stain-less notches or

bands along the arms of the chromosomes.

Sometimes the outline of the chromosomes

becomes wavy or irregular due to the

dechromatization.

(iii) Changes at metaphase I and II :-

These stages are most vulnerable to the effect

of caffeine. The chromosomes ore relatively

contracted. The abnormalities app::.rently

depend u.pon the duration of treatment. with

the increase in treatment-time, more and more

cells ere victimized.

1) P?lXEloidl : Most of the cells at metaphase

I1 possess tetreploid complement. The latter

has the chromosomal configu.ration

36 A + 2 X. It is not possible, however, to

distinguish the sex chromosomes ( }'ig. 96 )

because all the chromosomes 8re over-

L~~ __ _

e; ~ ~ , ,

~

I ,. •

• ::. -/ l

,

F1g.94

'. -" " .. \ ..

•

.. ) ( ___ e

~*- "I /. . )

Q ' "- ,~

..... Q '1

( .., . e.. . . . ;.

Fig.95

Fig.96

v

,~~ , •

•

o " -

I

-81-

condensed end stain darkly. It is clear

from the disposition of the ohromosomes

that the spindle is um~ffeoted, though it

might h~1ve failed at anaphase 1. The

chromatids ( daughter autosomes ) of the

univalents divide and lie parallel to each

other. Sometimes the p.artners m<~iy drift

apart. The cell wall is fOund to be intact

in most of the oaoes.

2) Q-1~.E~nt$: This is the most frecuently

occurring abnormality, involving more and

more cells with the increa.se in treatment­

tirr:.e. Ini-tially, only three or four chromo­

some. of the metc,phflse 11 plates 8re involved

( t'1g. 97 ). Later on, the clwnping is seen

to involve all the chromosomes ( Fig. 98 ).

The i:ient1ficctioll of the stage ar:rd chromo­

somes is, however, possible und a few

norm~?l cells C[,;ln ca observed in the vicinity

of the affected cells ( Fig. 90 ). After a

further increase in the troatment-time, the

clumps become more intense. The identifica­

tion of the stage is possible but the

indi vidu~Jl iden"ti ty of the chromosomes is

extremely diffioult. Moreover, the cell walls

cannot be locGted ( i'ig. 99 ).

3) Spindle abnormfci.li t.z: Irwo 3ub-typ€;s of this

, ~ .. ,~f "t. k ..r ':, ... { .. ...... • ~.

• r

.," .. ~ ... J:Oll-~ ""

~, ... ~

~r-

~ ,~

6 "0-

~ t"*

c • ~ /1/ \\\1 • ~~ t ~.

C • ~-

If ...

tr ":" \ • ,. •

#

~ '" • • F1g.91

- I 1-

~ • 'I:'

IIj * ~'t

,~ t;r ~~ -e

-I Iv -Fig.98

-

I -I , Fig.99

-82-

abnorm:.31i t,y ere met VIii th in the prosent

study. In one sub-type, t~e spindle is still

intact but the irr·egul~.lri ty in the arrange­

ment of metaphase 11 - chromosomes reveals

that the fibres of spindle are not in align­

ment ( Fig. 100 ). In the other type, the

disruption of the whole spinde apparatus has

occurred ( Fig. 101 ). The univalents with

their tii vided chromatids lie haph"lzardly t

usually beyond the norma.l dimensions.

(iv) Changes at first and seoond post-metaphase stages :-

1) Breakage: This abnorIDslity is not very

frequent and usue:lly the smallest, and

sometimes one medium, Butosomes are involved.

The broken piece, though well-separated from

the heteromorphic univalent, passes to the

same pole ( Fig. 102 ).

3. Qualitative studl : Enteromyoetin

(1) Mitotio changes

1) Lesions I This abnormality affects the longer

chromosomes of the complement, especially the

sex-chro~some ( Fig. 133 ). In the latter

case, the lesion occurs in both arms at the

same locus, murked off cler:~rly by stain-less

bands. The fact tl1at the same region is

most susceptible to the effect of enteromyoetil'

.. •

•

~ . •

,. ,..

Fig.lOO

\

Fig.lOl

•

,

•

Fig.102

-33-

is ms·de clef:r in ~ig. 104. This figure also

ShORS the lfurgest autosome with lesions in

both chromatids at the same locus.

(ii) Changes in the meiotic prophase stages :-

1) :area.ka~e : The antibiotic produoed breakage

at the ohromosome-level at diplotene. The

broken piece lies well np~rt from the

heteromorphic bivalent ( Fig. 105 ). ~o

chromatic connection can be seen between

the two. This abnormality usually involves

2)

the longer bi valen ts 8.nd the number of

breaks per cell never exceeds one.

EollEloidZ : A good number of cells tit

diakinesis are tetraploid in nature (Fig.106).

Such cells aoquire greater dimen~1ons and

the bivalents lie haphazardly within. The

chromosomal configurations are.

4n = 38 ( 36 A + 2 X ). No multivalent confi­

gurations [;ire met wi th. The sex chromosomes

lie ~part, never showing any tendency to

synepse. The degree of heteropycnosis is

relatively less marked in the sex chromoso­

mes, which stain almost (:l.S intensely as the

autosomal bivalents.

(iii) Changes at metaphase I and 11 :-

1) FraiZIDentation : Metaph£cse 11 is found to be

most vulnerable to the effects of this

•

·'1. .. , ~. '"

Fig.lD3

-Fig.1Q4

') " c

,

Fig.105

r~ ...

;

-84-

antibiotic. A study of the victimized cells

shoV'.s that this effect shows no response to

the increase in treatment time. The gradual

steps of fragmentation can be seen ill

Fig. 101. Here, the first step comprises

'notches',. The latter provide a pseudo­

appe;::,rance o:f • ea.telli te' to the daughter

chromosomes. In the next step, the fra~nent

shows the signs of detachment because an

achromatic gap appears ( Fig. 107, arrow

mark ). In the lest step, the fragment lies

freely after detachment. Four or more

dt:ughter chromosomes show this type of

fragmentation. Another pl[lte at the rig.t.t haud

side ( Fig. 101 ) shows a number of frag­

ments.This type of abnormality comes under

the delayed effect of the antibiotic.

(iv) Changes at first and second post-metaphase stages :-

1) Stickiness: This abnormality he.s been found

in combination with polyploidy at (::naphase

I. The latter stage possesses the tetraplOid

complement. It is seen tbat at the beginning

of anaphase It the bivalents get arranged

on the eoubtor, but are unable to separate

( Fig. lC)8 ). All the chroi'tosomes appear

highly condensed so that no separetion CCln

~ .

1'\

" " 1 -t.~.c..

<.~ -: +:

• ,.' c. ••

,. ~ c: .. • c.. .,..~

~ 1'\ ~\ , - .. '" -J.j

, e -', t\

~

~ {

F1g.106

r

•

Fig.107

/"'~,i~' sep . univ

i" Fig. 108

, '

-d5-

be observed in between the chromatids. The

morphology can be mistaken for the metapha.se

11 except for the orientation of the bivalents,

The latter show the polarity characteristic

of anaphase I stages. Similar tipe of

stickine::;s h,3s been reported by Manna and

Bhunya ( 1971 ) in their study on the effects

of tetracycline on the chromosomes of

Spathosternum praain1ferum.

2) Spindle abnormalit~: This a.bnormality is

most common in anaphase I cells. When the

complement divides normally, the two poles

of the spindle receive 9A + X and 9A

complements. In the affected cells, the

9A + X- pole is always found to be normal

( Fig. 109 ) but the other pole is bifid.

Such cells acquire a tripolar appefirance. e.

The bifid pole usually shows some spcificity 1\

about the distribution of chromosomes. The

smaller group contains three, and the bigger

six, Butosomes ( Fig. 109 ). Moreover, the

chromosomes of the bifid pole enter telopha-

se earlier than the chromosomes of the normal

pole ( Fig. 109, right cell ). This type of

spindle abnorml:\li ty ha.s not been reported

so far.

.. <,

·~ •

, bN-. , bi f. pl ---

bif .pl ___

Fig.109

'~'---- ·chd . br

Fig.110

Fi g. III

-36-

4. Qualitative study: Formaline

(i) Mitotic ch8nges : not found.

(ii) Chani:~es in the meiotio prophase stages :-

1) Dechromatization and breakase : This non­

delayed effect projects a number of achro­

matic regions in the diplotene-chrolliosomes.

The lat-ter weaken the Etrchi tecture of the

chromosomes ruld usually caU8e breakage at

the level of chromosom.es ( Fig. 110, arrow

mark ). The bi vEtlents usually lie far apart

indicating that the cell-wall has been

destroyed by the chemical. The oharacteristic

'lamp-brush' appearance is also reduced or

missing from the diplotene stages.

2) Interbivalent connection : This is also a

non-delayed effect, in which two or three

biva.lents get conneoted ( Fie. III ). The

connection is revealed by a clrromatin-

thread which stains lighter than the bivalents,

Manna and Parida ( 1965 ) have reported

similar type of connections in their study

on the differentially administered colchi­

eine effects upon the testis cells of

Spathosternum prasiniferum.

3) Breaka~e : This effect, though non-delayed,

is of lesD frequent occurrencey The break is

at the chromutid level ( Fig. 112 ) and is

Fig.112

Fig.113

lI" o/COnbV~

~ . ·fA

Fig.114

-'37-

found in one of the medium bivalents. The

sex-chrowosome remtins unaffeoted. Forma­

line does not influence the frequency of

chiasma at diplotene sta.ge.

4) ClUlllpintS : The abnormality is found mostly

in the cells at diakinesis. The bivalents

thicken and stain darkly. Besides, the sex­

chromosome ls identifiable on tee basis of

morphology alone and not because of its

heteropycnosis. It stains 8S da.rk bS the

autosomal bivalents, The latter form small

'clumps'. The intensity of clumping increa­

ses with the time of treatment. In the

e;-;!rlier stages two or three bi valer.. ts stick

together ( Fig. 113 ). Their morphology is,

however, quite distinct.

(iii) Changes at metaphHse I and 11 :-

1) Inter-bivalent connections : This effect is

similrir to the one desoribed earlier for

meiotic proph::;!se stages. But here five. and

sometimes six, bivalents are connected in a

linear fashion ( Fig. 114 and 115 ). Besides

the signs of erosion of chromatin-ma.terial

are also there. The 'lamp-brush fibres' are

obviously les8 prominent. This trpe of

appecJI'ance in the prometaphafJe stage has been

reported by Manna and Parida ( 1965 ) in the

LO .-,.

-. F1g.115

F1g.116

F1g.117

-88-

spermatocytes of Spathosternum~r~~1n1fer~ /",

Fig. 115 shows the occurrence of breaks

besides the ohain of three bivalents.

2) Destruction of morphologz: The chromosomes

of metaphase 11 stage are most affected by

formalin and possess a number of abnorma-

lities. It is observed that the chromosomes

appear thicker than usual and are a.bnormal

in their orientation ( Fig. 116 ). Their

outline is fuzzy and irregular. Usually two­

to-three chromosomes stick together to form

small groups. In one cell, four such groups

have been found at ec~ual distances in a

ouadrangle ( Fig. 116, upper cell ). It

gives a 'pseudo-tetranucleate' appearance

to the oell. The stage can be identified,

however, because one or two of the chromo­

somes maintain their identity. A pachyten,e

nucleus in Fig. 116, exhibits the presence

of 'snail-horn'-like centromerio-chromomeres

in the synapsed homologous chromosomes.

3) Lesions: This abnormality is a common

feature in the metaphase II chromosomes. It

shows a speoifio affinity towards the sex­

chromosome. The latter possesses either one

( Fig. 117 ) or two ( Fig. 118 ) lesions.

Manna (1969) has reported that chemicals

•

•

•

•

F1g.118

". "'-u.s .r , • ,I * .,. J ~ slr."'n

,.

• . - .

I

-89-

like alcohol, 108d nitrate, terramycin, maleic

hydrazide, BUDR. corline and formaline, etc.

affect the X chromosome more than the auto-

somes. The present observation confirms this

specific nature of formaline. The autosomes,

though devoid of lesions. show tUl aberrant

morphology ( Fig. 111 and 118 ). Their

orientation is, however, normal.

4) stretched centromere and dechromatization :

These two abnormalities are also confined to

metaphase 11 chromosomes. The erosion of

chromatin depends upon the time of treatment.

In the beginning it is less pronounced,

oocurring in onl~ some of the chromosomes

( Fig. 119 ). Large achromatic gnps can be

seen in a few of the chromosomes. The

stretching of oentromere is confined to the

longer ohromosomes ( Fig. 119 ). With the

inorease in treatment-time, all the chromo­

somes look wiry and lightly-stained (Fig.120).

Moat noticeable is the X-chromosome with

m[J.ximum dechromatization. The oocurrence of

darkly staixling blobs at the telomerio ends

indioates that perhaps the process of

dechromatization proceeds from centromere

tow6rds telomeres ( Fig. 120 ).

5) star-shaped .confi~rati~ : The metaphase 11

-90-

chromosomes 2ssume 'ball' or 'star' shaped

configurations ( Fig. 121 ). In this

abnormali ty the proxirn5.1 ( centromeric )

end~3 of the chromosomes are direoted towards

a sinele pOint in the cell. This polarity

along with the olose approximation of the

ohromosomes results in the darkly staining

• start oonfiguration. The stage oan be

identified because of some of the chromo­

somes which have not contributed to the

star-oonfiguration ( l'ig. 121, arrow mGrks).

Such type of abnormality has been reported

to be caused by oolohicine ( Manna and

Parida, 1965 ). Fig. 121 8180 shows another

metaphase 11 cell ( left-hand side) with

aberrant morphology of the chromosomes.

(iv) Changes at first and second post-metaphase

stages: not found.

5. Qualitative Study: Nicotine

This is found to be the most potent drug and

resulted in mortality if the treatment-time exceeded one

week.

(i) Mitotic changes :-

1) Fragmentat.!2!! : The drug inflicts heo.vy

fragmentation in the gonial metaphases.

The breaks are entirely of the chromatid­

type ( Fig. 122 ), and do not show any

•

•

•

st . confg~

.... ,. F1g.121

F1g.122

br . p I • -f

"I

I br . p- br

Fig.123

•

-91-

reunions. The latter observation is based on

a large number of cells having numerous

fra,gments. No ring-like structures and no

exchange type configurE'.tions have been found.

The broken ends thus appear not to have the

oapao! ty to reunite, f.l.or probably to resti­

tute. Similar type of effect has been repor­

ted to be caused by Actinomycin-D on the

chromosomes of Sohistocerca gresaria ( Jain

and Singh, 1961 ). The sex-chromosome, in

the present investigation, remains unaffeo­

ted and shows its oharfloteristic negative

heteropycnosis ( Fig. 122 ).

(ii) Ch2n,zes in the meiotic proI?hase st~5es :-

1) Brea.kage : This abnormality is observed in

a large number of diplotene stages. The

breaks oocur in two or three of the bivalents.

The broken pieces lie far away from the main

part ( Fig. 123 ) and are apparently

acentric in nature.

2) ClumI!in~ : It ha.s been observed in diplotene

and diakinesis stages. The bivalents appear

muoh thickened and close together forming

f bf.llls t ( Fig. 124 ). The stages can be

identified, however, because of single

strr,y bivalents lying outside the clumps.

I

Fig.124

i ,

x--

" 1

Fig.125

,~"

I )

••• ,~ : .. , , ~ -•

le-{~ • • t".

) (,

I -11

Fig.126

-92-

(ii1) Cha.nges at metaphase I and 11 :-

1) Clumping: This abnormality is very common

in meta.phase I ste.ges. The intensity of

clumping is not much, however. Usually two

or three bivalents stick together forming

one or two ,groups ( Fig. 125 ). The

orientation of the bivalents remairls

unaffected. The sex chromosome, in most of

the oases, does not include itself in the

olumps.

2) Lesions : This effect is most frl:quent in

metaphase I1 oells. All the ohromosomes,

except the smaller ones, possess lesions.

Fig. 126 shows a diploid cell with lesions

in the chromosomes. The latter have either

wavy or notched outline. Some of the chromo­

somes h8ve divided cmd the daughter

chromosomes have drifted ap2rt. The sex

chromosolLe carl not be identified. The

disposition and orientation of ohromosomos

remains normal.

(iv) Changes a.t first and second post-metaphase

stages: not found.

::.::*=*:::*=*:::*=*=*= =*=*=*=

=*=

VI

RESULTS AND DISCUSSION

-93-

RESULTS AND DISCUSSION

A. Meiosis and Mitosis

The speoies of grasshoppers included in the present

investigation can be split into two major groups - 23 -

chromosomes types and 19- chromosomes types. The former

group includes TriloEhidia annulata and Leva indic~. The

latter group includes Chroto5?nus trachypterus,

Attractomorpha orenulata end Poekilocerus pictu~. The analysis

of caryotypes in ::ill the species includes a straight compa­

rison of the dimensions and morphology of meiotic and

mitotio complements. Any establishment of relationship of

caryotypes in the speciel2>, is not the Elim of this work. The

main interest of the auther lies in a critical review of the

orthodox beliefs about the caryotypic stability and nature

of centromere in the Acrididae.

The oryptosaccine grasshoppers, T. annulata and

L. indica, possess 22 autosomes aIld single X chromosome ill /',

their male diploid complements. The chromosome behaviour

during the spermEtocyte-divisions is orthodox and is in no

way differen~ from the other species of grasshoppers with

23 chromosomes. The two apeoiBs differ in t.he length of the

X chromosome. The latter, in ~~ annulata, stands seoond in

, order of length, while in L. indica, it 1sI"longeat in the t complement. The lengthwise grouping of the chromosomes is

also different in theSE} species. T. annul~;lta possesses 3

long , 7 medium and 2 short chromosomes ( sub-family

-94-

Oedipodin[:e ) and ~. indics possesses 3 long, 6 medium. and

3 short ohromosomes ( sub-family Gomphocerinae ). A oompari­

son of the lengths of' both the serminal and somatio ohromo­

somes reveals that the ohromosomes of L. indioa are smaller

than those of T. annulata. The dimensions of the longest and

smallest chromosomes in the germinal tissue of' T. annulata

and L. indics are 7.38 miora/ 5.40 micra and 1.37 micra/

1.10 miora, respeotively. Likewise, in the somatic tissue,

the dimensions are 8.39 miora! 6.<57 miora and 2.47 micra/

2.41 micra.

It is obvious f'rom the oomparison of' lengths that

in both the species, the somatio chromosomes condense to a

lesser degree at metaphase than the gonial metaphase chromo­

somes. The behaviour of the sex chromatin differs in the

germinal and somatic tissue. In the former, it squires a /',.

distinot 'horse-shoe' shape during meiotio prophase I. No

heteropyonotio tissue can be found in the somatic tissue of'

male grasshopper. In the females, however. an indistinct

heteropycnotic mass is detectable. A related disoussion

follows in the foregoing account.

A study of chiasma frequency in T. annulate and

L.indica reveals that the loss of chiasmata is more between

diplotene and diakinesis in the former, while in the latter,

it is stronger between diakinesis and metaphec,se I. The number

of chiasmata in both the~ecies never exceeds three in the

long bivalents Hnd two in the medium bivalents. The chhsma

frequency of T. s.nnu18.ta is a little higher than that

-95-

of L.indica.

All attempts to locate the second arms, especialll

1n the less condensed metaphases of the somatic tissue,

proved fruitless. It 1s most tempting to regard the tsnail

horn~- like appearance at paahytene stages of T.annulata

( Fig. 34, 35 ) as second arms. Such structures are,

however, possessed by the centric chromomeres ( Marks,1957),

which become very prominent when sister centromeres are

in a state of repulsion.

The chasmosaccine grasshoppers, c. trachypterus,

A. crenulata and P. pictus, possess 18 autosomes and single

X chromosome in their male diploid complements. The behaviour

of the chromosomes during the spermatocytic divisions is

orthodox and is in no way different from other species of

grasshoppers with nineteen chromosomes. The caryotypes of

c. trachypterus and A. crenulata differ in the length of the

X chromosome. The latter, in c. trachypterus, 1s the longest

in the complement, while in A. crenulata it is second in

order of length. The lengthwise categorisation is,however.

similar in all the three species( sub-family Pyrgomorphinae),

consisting of 3 long, 6 medium and 1 small chromosomes. A

comparison of lengths of chromosomes in the three species

shows that the chromosomes of c. trachypterus are smallest

in size. The longest chromosome in the germinal tissue of

c. trachypterus, A. crenulata and P. pictus measures

5.85/8.64/9.11 micra, respectively. The smallest chromosome

in the germinal tissue measures 1.15/2.55/3.14 miora in the

-96-

same order. The data from the somatic tissue are 15.64/10.69

/14.16 micra for the longest chromosome and 3.88/3.12/5.64

micra for the smallest chromosome.

The above oomparison makes it clear that in all

the species, the somatic chromosomes condense to a less

degree at metaphase than the gOnial metaphase chromosomes.

c. traohlPteru8 and A. crenulata show some inter-individual

differences. The lengths of germinul chromosomes of the

latter species are higher than those of the former speoies.

But the order is reversed for the somatic tissue, where

the chromosomes of c. traohypterus are longer than those of

A. crenulata. An apparent reason for this behaviour seems

to be tha,t dehydration at late prophase, which results in

the gradual condensation of ohromosomes, is less in the

somatic tissue of A. crenula,ta than in c. trach,ypterus.

The somatio-metaphase ohromosomes of P. pictus are remark­

able in a.oquiring a length almost double that of their

germinal oounter-parts.

A study of chiasma frequency is worked out for the

local ( Bhopa.1 ) population of c. trachlEterus and is

oompared with the populations of the same species belonging

to Delhi and Ottu ( Punjab ). In the Bhopal - population

the loss of ohiasmata between diplotene and diakinesis is

stronger, as in the other two populations, but the mean

chiasma frequency per nucleus differs slightly.It is less

than the Delhi-population, and almost similar to that 0' the Ottu ( Punjab ) - population.

-97-

In all the three species, the sex - chromosome

does not show posi~1ve-heteropycnosis in the prophases of

male somatio tissue. At somatic metaphase, the degree of

oondensation is uniform in the autosomes and the sex

ohromosome. The identification of the latter, therefore,

depends solely on the morphometric data of the somatic

ohromosomes after a oomparison with the germinal chromo­

somes.

All attempts to locate the seoond arms are made

futile in C. trachypterus and A. orenulata. The former

speoies possesses the 'snail-horn'-like structures in its

pachytene nuolei ( Fig. 116 ) oomparable to similar struo­

tures of !. annulate. In P. pictus, however, the smallest

autosome, along with one of the medium autosomes, displays

distinct second arms and makes the term 'acrooentrio'

partly justifiable. But there is some oontroversy about the

oocurrenoe of seoond arms in this speoies and a variety

of opinions is available. While Darlington ( 1936 ) and

Coleman ( 1943 ) agree about the • true' acrocentric nature

of the smallest autosome, Dutt ( 1955 ) olaims that in rare

instances, the seoond arms are visible in all the chromosomes.

In the present study, however, seoond arms OanllOt be located

in all the chromosomes.

The heteropycnotio behaviour of the sex ohromoso­

me, in the prophases of somatio cells, is a oontroversial

matter and has been reviewed by a number of scholars from

time to time. The mysteries involving the funotional and

-98-

morphologio differentiation of the X-chromosome have begun

to unravel only in the earlier decades of this century.

The oytologio evidenoe for its peculiarities of behaviour

was observed even before its role in sex - determination

could be reoognized. Henking in 1891 ( Wilson, 1925 )

first observed a "peouliar ohromatio - element" in the

gonadal oells of Plrrhocoris apterus which during sperma­

tocyte-diVision became greatly condensed, divided equationa­

lly in the first division but moved undivided to one pole

during the seoond anaphase. Aooording to McClung ( 1902 ),

"Montogomery ( '98 ) deserves the credit for observing that

this body is merely a chromosome of the spermatogonia that

pursues a somewhat different course from the others".

The unusual heteropyonotio behaviour of the

X-ohromosome which Henking first observed in the spermato­

oytes of Pyrrhoooris is now known to be a widespread

charaoteristio of the X-chromosome of many speoies of inseots

and other animals ( Wilson, 1925 ). sttldies on female

germinal cells reoeived co~paratively little attention but

oooytes from various Hemiptera failed to show any intensely

staining ohromosomes that were so characteristic of the

spermatooytes ( Wilson, 1906 ).

Conspiouous heteropyonosis of the X-chromosome

was observed not only in gonadal cells but also in somatio

ntlolei. Geitler in 1937 ( Mittwooh, 1964 ) found that in

somatio interphase ntlclei from both heterogametic ( male )

and homogametic ( female sexes of insects, the X chromosome

-99-

remains heteropyonotic. The oells from the female show two

heteropyonotic bodies and from the males only one. Later

numerous investigations on the chromosome constitution and

sex ohromatin of mammals revealed that the number of sex

ohromatin bodies in diploid oells is always one less than

the number of X-ohromosomes ( Mukherjee t 1965 ). It

gradually became very olear that the behaviour o£ the

x- chromosomes in somatio oells is exaotly opposite to that

observed in germ cells. The single X- chromosome in

heterogametic males becomes heteropycnotic in spermatocytes

but remains isopycnotic in the somatic cells, whereas both

X-chromosomes in homogametic females remain isopycnotic in

oocytes, but one of them becomes heteropyonotio in the

somatio cells. The latter faot applies to the present

investigation also.

An attempt was made to explain this behaviour of

X-chromosome in the female somatic cells through the

"single active X hypothesis" ( Lyon, 1961; Russell, 1961 ).

The hypothesis states that the heteropycnotic X-chromosome

of the female somatic oells is genetioally inaotive. Suoh

an inactive X oan be either paternally or maternally

derived in different oells of the same animal. This

differentiation of the X-chromosome takes plaoe early in

embryonic development a.nd remains oonstant f'or all

desoendants of the differentiated oell. Further observations

showed that the sex chromatin mass in the interphase nuolei

of females is late replioating ( Atkins et al, 1962; --

-100-

Mukherjee and Sinha, 1963 ). The present status of the

hypothesis also inoludes two more statements that " more

probably, heteropyonosis alternates between the two XSs

in a female somatio nuoleus regardless of their parental

derivation tI ( Ohno and Hauschka, 1960 ) and that the

differenciation of the inactive X takes place some time

during the early embryonio life ( Lyon, 1961, 1962 ).

The nature of the centromere is the most contro­

versial aspeot of the Acrididae. Two oonflioting views

relating to the rod-shaped ohromosomes exist. Aooording to

McClung ( 1914 ) and Makino and Mamma ( 1950 ) some, at

/' least, of these ohromosomes are genuinhely telocentric.

White ( 1914 ), Darlington ( 1936 ) and Coleman ( 1943 ),

on the other hand, have all argued that these rods are

acrocentrio but that faulty fixation and unfavourable

staining frequently obscure their real struoture. The

latter opinion was based on two lines of evidenoe :-

(i) Small heads are frequently visible at

metaphase both in spermatogonial and at

seoond meiotio division.

(ii) So-oalled ditaotio bivalents are sometimes

produoed, in whioh assooiation is maintained

exolusively by what is interpreted to be

the short arms.

It id worthwhile, at this pOint, to review the

oriteria chosen for distinguishing objeotively between

telocentrio and aorooentrio ohromosomes :-

-101-

(i) The oocurrenoe of small granules at the

oentrio end of rod ohromosomes is not an

adequate indication of short arms sinoe

olearly these oould represent oentromerio

ohromomeres, as was believed by Makino and

Momma ( 1950 ) also. Indeed in terms of the

known struoture of metaoentrics ( Lim~-de­

Feria, 1956 ), a telooentric ohromosome is

expected to contain centric chromomeres and

there oan be little doubt that these have

frequently been taken to represent a seoond

arm. It is on the basis of such granules that

White has repeatedly argued for the occurrenoe

of short arms in orthopteran rods.

(ii) As Ostergren, Mole-Bajer and Ba.jer ( 1960 )

po:l,nt out it is possible in fixed and stained

preparations "to see a specieol fibrillar

material that is attaohed to 6ach oentromere

and extends from it in the direotion of the

spindle pole toward whioh the oentromere is

oriented".

(iii) Sinoe oentromeres are reletively unooiled at

metaphase of mitosis they appear both thinner

~nd less deeply stained than the main

chromosome arm. Moreover, as Jones and Colden

( 1968 ) point out, a terminal centromere oan

be expeoted to show enhanced cle.ri ty of

-102-

structure compared with that of metacentrios,

beoause unlike the metacentric, its centromere

region is not subjected to the effect of arm

coiling on both sides.

(iv) At anaphase short arms sbould flex as do long

ones. Telocentrios on the other hanl, are

expeoted to move with their centric ends

pointing directly to the poles ( Levan, Fredga

and Sandberg, 1964 ).

That these oriteria are justifiable is confirmed

by Marks' ( 1957 ) observation on Oxalis dispar. Here two

small stained bodies are visible at the centric end of the

telocentric members. When sister chromatids are in a state

of repulsion they produce a structure "reminiscent of the

horns of a snail". When repulsion is not evident the terminal

oentromeres appear as small, faintly stained. tapering ends.

The present study oonfirms these points.

White ( 1957 ) has argued that "failure to observe

the minute seoond arms should not neocessarily be regarded

as proof that they do not exist, since even in those forms

in whioh they undoubtedly oocur they oan only be seen in

exceptionally well-fixed oells." On the basis of the oriteria

listed above one may feel disinclined to believe in invisible

short arms. The demonstration of absbnce of short arms is

unlikely to convinoe those prep8red to believe in the

invisible. Beoause of the same reason, one cannot accept

White's seoond rule for it is abundantly clear that in many

./

-103-

species the centromere does not lie in a heterochromatio

region.

In many telocentrics the struoture of the centro­

mere appears to be equivalent to a half, or at least less

than the whole, of a metaoentrio ( John and Hewitt, 1966b ).

This suggests either an origin by misdivision of a metaoen­

tric type or else the production of the metacentrio by the

fusion of two telooentrio centromeres. In other oases the

telo-oentromere seems to be essentially identical in

structure with that of the metaoentrics found in other

species ( Tjio and Levan, 1954; Johes and Colden, 1968 ).

This oould be taken to imply either that the telooentrics

have arisen not by misdivision but rather by whole arm

erosion following breakage proximal to the centromere. Such

an event might be expected to oocur more readily in acro­

rather than meta-centric since the extent of material lost

would be small.

Actually the ideas concerning relation-..:,ship

between the rod-shaped and V-shaped chromosomes is clearly

dominated by the belief that strictly telocentric chromosomes

either do not exist ( Nawaschin, 1916; Lewitsky, 1931;

Muller, 1940 ), or if they exist, are unstable ( Rhoades,

1940.)' or if stable they are rare ( white, 1957, 1959 ).

There is, therefore, a long and unfortunate histary to the

whole issue and one which still prejudices contemporary

thinking on the subject.

As is so often the case in science the truth may

-100-

well lie between the two viewpoints discussed earlier. The

author feels more inclined to regard the 'snail-horn' like

structures as oentrio ohromomeres and not the seoond arms.

This clearly demaroates the chromosomes of T. annulata , . c. tra~ypterus, L. indioa and A. orenulata as telooentrio

in nature. This assumption includes the positive evidence

provided by the chromosomes of the somatio tissue ( gastrio

oaeca ). The ohromosomes of P. pictus, however, apparently

form an asymmetrical oaryotype, because the seoond arms

are visible in two ohromosomes only. The details of the

nature of oentromere in this speoies are discussed in the

section of 'banding pattern-.

B. Banding Pattern of P. piotus

In the present investigation, the germinal

chromosomes were subjeoted to Giemsa staining (sumner,

1972 ). The regions with oonstitutive heteroohromatin

stain heavily with Giemsa and a general pioture of the

banding pattern oan be drawn.

From the observation of the behaviour of the

X-ohromo some , it beoomes very clear that the condensed

stat~ is not responsible for the staining reaotions.

Normal staining reactions distinguish X as negatively

heteropycnotio at gonial metaphase, meiotic metaphase I

and anaphase I; and positively heteropyonotio at meiotic

prophase I in P. pictus. But after the treatment for

-105-

C-banding, the X ohromosome stains poorly except for the

centromeric and telomerio bands of heterochromatin. Its

general state of condensation is, however, distinguishable

from the autosomes after applying this technique. Besides,

the teohnique is specifioally reliable for the location

of constitutive heterochromatin.

The characteristic 'bouquet stage' oan be demonstra­

ted with this staining method because the orientation of

the prOximal and distal ends with the dark bands becomes

very olear ( Fig. 83 ). Another interesting point noted

by the author is that the sex chromatin, which stains

lighter than the euohromatin, occupies a position opposite

to the • dark pole' B,t leptotene. The • dark pole t is a

oonsequence of bouquet arrangement, formed by the approxi­

mation of centromeric and telomeric ends. Since all the

centromerio ends bear darkly staining bands, their

approximation results in a prominent body, referred by the

author as 'dark pole'.

The differentiated oonstitutive heterochromatin

comprises the oentrio ohromomeres and the heterochromatio

region of the proximal part of the chromosomes ( Figs. 84,

85, 86, 87, 88 and 89 ). This oan explain the four SUb-units

of the C-band visible in the X-chromosome ( ~ig. 82 ), out

of which two proximal ones belong to the centrio chromomeres

and the two distal one:::: to the proximal-heterochromatio­

bands of the two chromatids. The sub-units come to lie

together when the ohromosomes are comparatively oondensed,

,/

-106-

as during gonial metaphase, diakinesis, metaphase I and

anaphase I, and metaphase 11 and anaphase 11. On the other ,<

hand, the sub-units beoome separated when the ohromoaomes

are less oondensed as during leptotene ( Fig. 83 ),

zygotene, paohytene ( Fige. 04-88 ) and diplotene ( Fig. 89).

That the bands oomprise four sub-units, is further confir­

med at metaphase 11 where eaoh ohromatid ( daughter

ohromosome ) contains only two sub-units ( Fig. 93 ). The

latter look like one due to condensation of the chromosomes,

exoept in M4• The oocurrence of four sub-units of

C-bands was reported in the metaoel1trio ohromosomes of

Mz!meleotettix maoulatu:! by Gallaghar, Hewitt and Gibson

( 1973 ). They made no mention of the sub-units in the

telooentrio ohromosomes of the same species. It is felt by

the author that the 'four' sub-units of the metacentric

ohromosomes of M. maoulatus ( where no separation of the

ohromatids was visible ), would have resolved into 'six'

sub-units if the ohromatids were separated. < Thts demons­

tration is possible in the lesser condensed stages. On the

other hand, if it is preferred to count the individucd

sub-units 9f the two ohromatids as one, then the four

sub-units found in the present study, may be counted as

three. Out of these three sub-units of C-band, -two belong

to the oentrio ohromomeres and one to the proximal

heteroohromatin of the chromosome.

It is possible to locate the heterochromatin at

all stages of meiosis. At diplotene, the chiasma is found

;' !

-107-

to oocur dietsl to the centric ohromomeres .and proximal to

the proximal heterochromatin of the chromosome. Similar

("'V '" behaviour was noted in Brlodema tuberculcta Klaaterska,

\\n·,

Natarajan and Ramel, n in son::e of the chromosomes.

The size oft the densely staining C-band.::! aeE::.JlS

to be uniform in all the chromosomes. The Giemsa-staining

of the rest of the genome, apart from the constitutive

heterochromatin, is generally quite diffuse. Faint telomeric

blobs of stain t:1.re visible particularly on the X chromo-, some. These have no obvious significance other than the

demonstration of repetitive DNA-sequences. Similer blobs

have been reported in Drosophila ( Hennig, Hennig and

Stein, 1970; Gall, Cohen and Polan, 19'71 ).

The Sl chromosome shows a pattern quite different

from the other chromosomes at all stages of division. Besides

the dark C-band, rest of portion also acquires an equally

dark stain, except for a narrow stain-less region distsl

to the C-band ( Figs. 84, aa and 89 ). The heterochromatin

of the chromosome arm of SI forms two bands ( Fig. a8 ).

Thus Sl is the only autosome in the genome which oontains

oomparatively maximum heterochromatin.

As regards the 'second arms', the present inves-

,-' tigation denies their ocourrenoe. 0rourse. if the centric

ohromomeres are to be regarded as second arms, as White and

followers do, then all the chromosomes are acrocentric in

3ensu stric'tu. The author, however, feels disinclined to

believe in the view. A more intensive investigation is

-108-

needed for a. definite statement about the true nature of the

chromosome.

The abnormalities reported in the present study

inolude :- extra fragment, polyploidy, breakage, anaphase

bridge, abnormal spindle, supernumerary chromosome, non­

disjunotion and chromatid-separation.

The extra fragment is found in ~. annulatu,

L. indica and c. t~a~hypterus. In 1_ annul~ta only one

individual out of hundred and six possesses it in 8.57 %

cells at metaphase I. In L. indioa one individual out of

fifty-two possessed the extra fragment in 7.4% cells at

diakinesis. In c. trachlEteru~ one individual out of

eighty-six possesses it in 2.85 % oells at metaphHse 11.

As is apparent from the data, the percentage of aberration

is not very si~lificant in all the three species. The

fragment, in all cases, is the smallest element, in the

oomplement. It is never found to leg behind during anaphase

a!ld thus a.pparently does not interfere wi lih spindle elonga­

tion and consequently with cleavage.

The frequency of polyploid cells is also quite

low. In T. annulata 1/106 individual possesses thirteen -tetraploid cells at diakinesis in a single follicle. In

L. indica 1/50 individual possesses 35.41 % tetraploid cells

clt metaphase 11. In C. trachypterus, the polyploid cells are

-109-

observed at diakinesis, metaphase 11 and anaphase 11. The

pooled data show the frequenoy to be 11.10 ~. It is stated

by Rothfels ( 1950 ) and White ( 1951 ) that the

"polyploid oells, in partioular, are not unoommon in

aoridoids". Moreover, ohromosome variations up to 5 ~ are

expeoted in all the individuals from whioh large oell

samples are taken.

The ooourrenoe of polyploidy oan be traoed along

two lines. If the spindle fails to elongate during the

mitotio and meiotio divisions, the tetraploid oells

originate. The latter are quite frequent in the populations

of grasshoppers. Seoondly, if there is a failure of oleavage

during mitosis, the binuoleate oells are formed. The

latter enter prophases separately during meiosis. On

approaohing metaphase, as their nuolear membranes dis­

appear, the ohromosomes of both the nuclei come to lie on

a single segment, so that the resulting metaphase is

tetraploid. It is noteworthy that if polyploidy ooours

due to the first reason, the tetraploid oells entering

meiosis oarry a varying number of multivalents when the

spindle fails in one of the mitotio divisions. In

c. traohypterus the tetraploid metaphe.se 11 oell ( Fig. 48)

may have arisen aocording to the given sohedule :-

Step (i)

step {ii}

9 AA + x

9 A + x ; 9 A at anaphase I, due to

failure of spindle elonga.tion.

step (111)

-110-

9 dA + dx ; 9 dA

9 dA + dx ; 9 dA at metaphase 11.

Thus step (iil) gives rise to 36 dA + 2 dx oonfiguration

at metaphase 11 ( Fig. 48 ).

The tetraploid oell at anaphase 11 ( Fig. 49 )

seems to have arisen in a somewhat different manner :-

step (i)

step (li)

Step (iii)

9 AA + x

9 A + x , 9 A • The daughter nuolei

separate after 8,naphase I, but remain wl thin the

same oell-wall due to failure of oytokinesis.

9 ciA + dx 9 dA

========- -- The daughter nuolei 9dA+dx 9dA

of step (1i) divide on

separate spindlesat anaphase 11. This aooounts

for the different orientations of the splndles.

In L. indioa the polyploid oell at anaphase 11 .... , . oontains 44 daughter ohromosomes. The origin of this oonfi­

guration can be traoed in the following way :-

Step (1) 11 AA + x ; 11 AA + x

Step (ii) 11 A + x + 11 A .' 11 A + 11 A + x ,

at anaphase I.

Step (iil)

step (iv)

11.A ,

11 dA

11 dA

-111-

11A due to fusion of cells.

11 d.A

11 dA = 44 dA at anaphase 11.

In none of the cases, the polyploid condition

originates due to failure of spindle elongation at mitotio

divisions. Such cases carry multivalents which are not

found in the present study.

The breakage is found in T. annulat~ in 1/106

individual in 1.4 % oells at anaphase I. It Is at the

chroma.tid-level and the fragment lies close to the arm. In

L. indica this abnormality is found in 29.41 % oells at

spermatogonial metaphase and invariably affects the longer

chromosomes only. Both the chromatids possess the breakage

at the same locus. Here also the fragments do not lin6er

away from the arms. In C. trachypterus 4.76 ~ oells at

anaphase 11, and ill A. crenulata 3.33 % cells at anaphase I,

possess breakage at the chromatid - level. In these two

instanoes, the fragments not only form an angle with the

main arm but also lie away from it.

The question whether the breakage is • complete'

Is a difficult one to answer. In T. annulate and L. indica

the broken ends are still quite close together and one

might conclude that no real fragmentation has oocurred.

-112-

White ( 1951 0 ) has desoribed "elastio oonstriotions" in

some speoies of morabine grasshoppers where the two portions

of a ohromosome may sometimes be pulled out to enormous

lengths at first metaphase and anaphase. But in suoh oases

it was oono1uded that the two portions of the ohromosomes

were still connected by a fine thread. In the present case

no thread could be seen in any case, but an invisible

oonneotion may still exist. This seems to be true for

C. traohlEteruB and ~orenulata, beoause the fragment is

never found to lag behind on the equator, neither does it

move to the opposite pole.

The dicentric bridge is found in a single indivi­

dual of ~. annula,tl!- , in one cell out of a total of ninety­

nine. Similar bridge has been reported in Chr~to~onus

trachlpteru!!, ( Sharma, Parshad and :Eedi, 1962 ). A number

of explanations have been put forth to account for the origin

of dicentric bridge. The latter with or without 1:1 fragment

may occur spontaneously ( Darlington and Upcott, 1941 ;

Rees, 1953; and Manna, 1954 ), or may be induced either by

irradiation ( LaCour and 'Rutisha.user, 1954 ; and Ray­

Ch~udhuri, 1961 ) or by chemicals ( De.rlington and Koller,

1941 ; Dar1ington and McLeish, 1951 ; and Steffensen, 1955 ).

The bridges without any fragment, found during

the first and seoond meiotic divisions of Aio1opus ap. and

Tri10phidia annu1r;ta ( Manna, 1954 ), are formed just out of

stickiness in the separating elements. In the present

study t the dioentric bridge of T. annu1~:1ta is accompanied

r

-113-

by a. fragment of oonsiderable size. The bridge does not

appear to be the outoome of B mere stiokiness. It has been

found that the bridge oocurring 8t the first meiotic

division, may arise because of the following reasons :-

(i) Inter/intra chromosome crossing over in re13tively

inverted segments.

(ii) Inverted crossing over.

(iii) Chromosome breakage with sister ohromatid reunion.

(iv) P~.rtial reciprocal or inverted crossing over

between homologues.

(v) Localized a.dhesion between sister or non-sister

chromatids with or without breakage.

(vi) Interlocking of plectonemically ooi1ed stra.nds.

In the first three causes, the bridge is

aooompanied by a fragment ( John and co-workers, 1960 ).

The ooourrence of"" dicentric bridge in the present investi­

gation, can only be attributed to second and third causes

because the bridge is not formed by an asymmetrical bivalent

and it has no lobe in the middle. The first cause is not

applicable because the inversion in heterozygous condition

must be represented uniformly in the entire testis or at

1e~st in a port of the testis. In the case under consi-

deration, however, the bridge is found in a single cell of

a follicle. The cause of bridge-formation can, thus, "be

attributed to either the inverted crossing over or ohromo­

some breakuse with sister chromatid reunion.

The abnormal spindle is found in L. indica in

-114-

33.33 ~ cells at anaphase 11 in one individu8.1 out of C:i

total of fifty-two. The only probable cause seems to be the

use of colchicine for chromatid separation. But the author

has not came across a similar insta.nce of bifurcation of

one pole of spindle attributed to the use of colchicine.

The occurrence of supernumerary chromosomes is

an interesting feature in the caryology of c. trachlpterus.

The Delhi-population of this species contains 1-7 super­

numerary elem8nts ( SharmB, Parshad and Bedi, 1962 ) and

the Ottu ( Punjab ) - population contains the same number

of' supernumeraries along with a megamaric bivalent. In the

present investigation, the Bhopal-population of this

speoies is found to contain 1-6 supernumeraries and no

\ megamerio bivalent. Only one out of a total of eighty-six

\individuals investigated. In the individual under conside­

ration the S + 2s condition is most frequent ( 74.70 ~ ).

Rest of the configurations are of lesser frequency. The

S + 6s oonfiguration ienot found in the Ottu ( Punjab)

population, while the S + 1s configuration of the latter

popUlation is not found in the Bhopal-population.

The supernumerary elements show positive hetero­

pycnosis at early prophase I ( Fig. 39 ). In the later

stages they become indistinguishable in their staining

intensity with other autosomes and can be differentiated only

due to their size and behaviour. They do not pair with the

X-chromosome or any other autosome of the complement, but

pair with each other. The supernumerary bivalents possess

-115-

single terminal chiasmata.

White ( 1954 ) has classified the supernumerary

chromosomes into mitotically stable and unstable types. In

the former category, all the cells of the germ line contein

the same number of supernumeraries, while in the latter,

there are intra-individual variations between different

follicles, but not within the follicles themselves. The

present material belongs to the latter category.

As regards the oriein of supernumerary elements,

there are two schools of thought. One believes in the

origin of the supernumerary from the X-chromosome

( Robertson, 1917 ; Rothfels, 1950, and others ) on the

bs,sis of tendency of the former to syna.pse with the latter.

The other school believes in the origin of the supernumery

from an autosome ( Carroll, 1920; h~ans, 1954; Ray-

" Ch2~dhuri and Guha, 1955 ). The former sohoo1 of thought

is strongly opposed by Ray-Choudhurl and Guha ( 1955 ) on

the ground that the supernumerary does not exhibit

negative heteropycnosis at early spermatogonial metaphase

( as oonfirmed in the present investigation also ) which

is a oharacteristio feature of X-chromosome in Acrididae.

In the Ottu ( Punjab ) - population of

~ trachY.:Eterus , the origin of super::umeraries is attributed

to the megamerio chromosomes. Since the Bhopa1-popu1ation

lacks the megamerio ohromosomes, the only possible donor

seems to be extra autosomes, included in the standard

complement tl~ough non-disjunction. Suoh autosomes form

-116-

heteromorphio bivalents due to deletion or defioiency and

give rise to supernumeraries. Similar instances have been

reported earlier in Orthoptera - ( Robertson, 1915 ;

Darlington, 1936, and Sharma Parshad and Bedi, 1962 ).

The supernumAraries do not induoe any exophenoty­

pie variations with whioh one oan easily study their adaptive

significance. The speeding up of ecrmination hL~f:'l bSE.:n

attributed to their OC(1)xrencs. In the absence of benefioial

effects, the supernumeraries can perstst in the popu1<::ltion

only by oertain acoumulation meohB.uisms ( Nur, 1963, 1969 ).

At th.e same time there are eliminb.tion mechanisms also

t:.D..rough which selection pressu.re is brought on the supor­

nu..merariE;s. By the irrterHction of these two opposing foross

the supernumeraries are maintained b.t alj optir.a.wll level in

tae population.

The phenomenon of 11on-disjunctiol:! opel'ates in

the standard complement gl~o besides occurring in the

supernumeraries. Thus in c. traohl12teru§. 15.78 ;J; cells at

anaphase 11 possess variable or-.xomoBomal configurations.

In one instance the two pol(;;;s at anaphase 11 contain 8 dA

and 10 dA instead of 9 dA each ( Fig. 5), 51 ). Another

instance shows 6 dA and 8 dA configurations ( Fig. 52 ).

The latter condition has (Jome into being through the

following steps:-

step (i) ( 9 AA+ x ) at Illt';tHphHse I.

Step (ii) ( 2 AA + 1 A + x ) ; ( 1 A ) E..t anaphase I

instead of ( 9 A + x ) and ( 9 A ) , due to

-117-

non-disjunction of two bivalents.

step (iil) Splitting of ( 7A ) configuration of step (ii)

into :- ( 6 dA ) and (8 dA ) instead of

( 7 dA ) and ( 7 dA ) due to the non-disjunction

of one univalent.

The separation of chromatids has been observed

in A. crenulata in 38.8 % oells at anaphase I ( Fig. 74 ).

The separated chromatids s~ow a precocious behaviour but

do not pass to the opposite poles, till anaphase 11.

A review of abnormalities in the natural popula­

tions df the selected grasshoppers makes it olear th8t

maximum abnormalities occur in c. trachypterus. The reason

may be attributed to the inability of these hoppers to

cover long distances. The females virtually do not take fli­

ghts beoause of their heavy bodies and rem:7in within li.mi ted

artSs. The meles, though comparatively more active. are

not fast fliers and usually hop, rather than fly, in their

surroundings. Thus, these grasshoppers live as small

olosely breeding populb.tions more or less in se;rregation.

The aberrations, once originated, oontinue to exist in the

germ-line.

D. Effect of Drugs

The O,uanti tative study of efl'ects of caffeine

includes those freauently occurring aberrations which are

obtained when the treatment-time is kept constant and the

-118-

ooncentration of the drug is changed. The aberrations

selected for quantitative analysis, include fragmentation,

clumping and polyploidy. The data have been scored at

diplotene, diakinesis and metaphase I, and pooled and

averaged for the total number of surviving speoimen.

Out of the ten concentrations of caffeine,first

four ( ~.l % to 0.4 % ) do not cause any casualty. From

0.5 % onwards, the percentage of casualties increases.

Twenty percent specimens die after treatment with 0.5 % and 0.6 % caffeine. The peroentage of mortality with 0.7%

and 0.8 % is 60 % and 80 %, respectively. Absolute

mortality inour. with 0.9 % and 1.0 % caffeine. In the

last two ooncentrations, the mortality does not ocour

immediately but after 1-3 days of treatment. The speoimens,

therefore, do not reach the desired stage of division and

~ have to be studied before the scheduled time. Thus a r ~

systematic quantitative analysis is not possible at the

0.9 % and 1.0 % ooncentrations.

A survey of the data of fragmentation at the

different concentrations reveals that it is not easy to

establish a relationship between the percentage frequency

of aberration and the increasing concentration. There is a

grad.ual inorease in the beginning - 4.44 % with 0.1 %

caffeine and 5.82 % with 0.2 % oaffeine. This is followed

by an irregular increase - 11.66 % with 0.3 % oaffeine and

25.12 % with 0.4 % caffeine. With 0.5 % and 0.6 %, the

'peroentage rema.ins the same - 28.05 % and 28.0 %.respectively.

-119-

A further increase is noticed with 0.7 % , i.e. , 32.50 %. With 0.8 ~ oaffeine, however, only 24.44 % aberration is

obtained. In this case the mortality was 80 ;, and the data

were soored from the only surviving specimen. It can be

assumed that the peroentage frequency could have been

higher, if the dAta could be scored from all the treated

specimens. A oonfirmative evidence to this effect is

provided by the tissue of the :).9 % and 1.0 % caffeine

treated specimens, which were fixed before the scheduled

time. Here the divisional stages either do not progress

beyond early prophases, or if they progress their recogni­

tion is not possible. The pachytene stages reveal heavy

fragmentation in almost all the chromosomes. The fragments

are of different sizes and stain dGrkly. Where not fragmented

the chromosomes bear 'gape' at short intervals, which are

considered the forerunners of fragmentation. In such nuclei,

the pairing is found to be normal. Thus, caffeine primarily

does not affect the behaviour of the chromosomes but shows

a negative effect on the chromatin material by inducing

gaps and fragmentation. Slizynski ( 1970 ) had obtained

similar type of fragmenta.tion at pachytene stage in the

mouse - testes cells, after usirtg saturated caffeine

solution.

The other two abnormalities - clumping and

polypl.idy, show a more defined relationship with the

increasing concentrations of caffeine. More and more cells

are viotimized as the concentration is increased. starting

-120-

from 16.44 % with 0.1 % oaffeine, the peroentage frequency

of cl~~ping inoreases gradually to 21.84 % ~ith 0.2 % ,

40.33 % with J.3 " , 49.26 % with J.4 fo , 52.1'1 % with

J.5 % , 55.11 % with 0.6 % , 69.00 % with 0.7 % , and

72.22 % with 0.8 % oaffeine. Simile.rly, the d8ta for

polyploidy are 20.0 % ( 0.1 % ), 33.0 % ( 0.2 % ), 41.66 %

( 0.3 ~ ), 52.45 % ( 0.4 % ), 58.88 % ( 0.5 % ), 60.0 % ( o. 6 % ), 14.50 % ( 0.1 % ), and 77.17 % ( 0.8 % ).

Thus, at eaoh oonoentration, the percentage frequency of

polyploidy is higher than that of clumping.

Clumping results primarily due to the aooumulation

of exoessive ohromatin on the chromosomes. The faot that

olumping may second1:1rily be induced by the olose approxima­

tion of chromosomes in the highly polyploid cells , is

evident from the data of percentage frequencies of

clumping and polyploidy. Clumping inoreases in accOrdatloe

with the inorease in polyploidy and is always numerioally

lower than the latter. This is expected beoause a close

approximation of ohromosomes along with an acoumulation

of exoessive ohromatin is destined to result in heavy

clumping.

The qualitative analysis is carried out with

0.4 % caffeine, a conoentration, which besides being

effective, does not result in mortality. keeping the

concentration constant, the time is varied at the given

intervals - 4, 18, 48 and '12 hours and 6, 1, 8, 14 and 21

days post - injeotion. These timings involve all the

-121-

divisional stages of spermatogenesis. The gradual steps of

mode of action and intensity of effect of caffeine can be

studied by the present method. The earliest signs of

olumping appear at late prophase ( Fie. 94 ) when tha

chromosomes look thicker than usual.Clwnping inoreases after

metaphase 11 and gradually victimizes all the chromosomes

of a complement ( Figs. 9'7 t 98 and 99 ), so that the

individual identity of the chromosomes is lost ultimately.

The negative effect of caffeine first appea.rs

at late prophase in the form of lesions ( Fig. 95 ) and

culminates in definite breaks in the l8ter staees

( Fig. 102 ). It appears that the breakage caused by

caffeine is not reparable. Cattanach ( 1962 ) has disoussed

this possibility in the mouse chromosomes. while Kuhlman

et sI ( 1968 ) report the fftot for mammalian cells that

ohromosome - breaks cHused by caffeine hr::3.ve lost the

oapaoity to rejoin. Oocurrence of spindle - abnormality

and C- mitotic effect indicate that caffeine is similar in

its action to colchicine, in this respect.

The positive effect of caffeine depends on the

existence of extra chromosomal DNA. The latter may come

from the nuclear sap, where these is DNA in the amount

comparable to that of the chromosomes ( Izawa et aI, 1968 )

or it ma.y be a by-product or a surplus from an unscheduled

synthesis. The present investigation indicates that the

positive and negative actions of caffeine can proceEd

simul taneously in tIle same nucleus. The negative changes t

-122-

in which the chromatin extrudes from the chromosomes, may

lead to chromosome breaks by influencing certain nucleo­

tides. The ohoice of the effect may depend on the state of

the nucleus and on the region81 characteristics of the

chromosomes.

The effects of the antibiotic Enteromycetin

( Chloramphenicol - Dey's ) are manifested in the chromo­

somes by lesion, breakage, fre,gmentation and stickiness,

and in the spindle apparatus by polyploidy and spindle

destruotion. An analysis of the effects reveals that in its

negative action, enteromycetin involves particular sites

of the larger chromosomes, espGcially that of the sex

chromosome ( Figs. 103 and 104 ). This effect is more

specific in the first part of meiosis. During the second

part, however, the autosomes also get victimized. Here the

break is not preceded by lesion. Instead, notches appear

at the terminal end and become deeper gradually, thus,

constricting off sme!ll fragments ( Fig. 101 ). This effect

is obtained when the treatment exceeds 8 days,

The chromosomes acquire a sticky quality, most

probobly due to some physiological changes. At anaphase I,

the chromosomes orientate normally but are unable to move

towards the two poles, because their distal ends stick to

each other ( Fig. 108 ). In the longer bivalents, the

stickiness involves the sub-terminal parts as well, so tht=Jt

a peculiar tri-rediale appear~.::Jl1ce is acquired by the

chromosomes. Tetracycline has been reported to cause

-123-

simil:':ir effecte on the chromosomes of Spathosternum

pre.siniferum ( Manna and Bhunya, 1971 ) • The e.ction of

enteromycetin differs from tetracycline in showinS I:m affi­

nity for the X-chromosome.

In its mild polyploidj.zing e.ction, ecteromycetin

is similar to colchicine, but the type of spindle destruc­

tion obtained seems to be unique. The bifid pole possesses

the haploid set minus X, in two groups of 6 and 3 univalents

No probable reason for this behrviour could be found Oltt.

The tOxicity of this antibiotic becomes more

obvious if the treatment is continued for a long time.

when it starts fragmenting the chromosomes.

The chemical- formaline incurrs a variety of

aberrations, mostly mar.lifested in the chromosomes. The

erosion of chromatin from the nuclei is one of the IDfJ.jor

effects. At late prophase, the bivalents show a number of

regions which do not take the stain ( Fig. 110 ). Such

regions result in breakage in the same stage. There is an

indication of recov{~ry from this effect, 8S at metaphase 11

only lesions ( Figs 117 and 118 ) and no breakage are

obtained. The forerunner of lesions, the dechromatization,

brings about a chB.nge in the morphology of the chromosomes

( Fig. 116 ).

Another interesting abnormality obtained with

formaline. is the inter-bivalent connection. The latter

starts appe~::ring at diplotene ( Fig. III ), where usually

two or three bivalents get connected. Later on, more

-124-

bivalents are involved ( Figs. 114 and 115 ) forming a

long ohain. Thin, lightly staining conneotions oan be seen

at the linking points. Similar behaviour has been obtained

with oolohicine in the pro-metaphases of SEsthosternum

prasiniferum ( Manna and Parida, 1965 ). Suoh oonneotions

seem to be of temparary nature beoause later stages are

devoid of them.

Though olumping is obtained with formaline at

late prophase, it is not very signifioant. The bivalents

appear thiokened due to the acoumulation of excessive

DNA, and the sex ohromosome loses its heteropyonotio

behaviour and stains as intensely as the autosomes ( Fig.

113 ). The oel1a reoover from this positive effect of

formaline in the later stages which do not show olumping.

Formaline sholf,s a definite effini ty for metaphase

11. Moreover, this investigation oonfirms the earlier

report that this ohemical affeots the sex ohromosome speoi­

fioal1y ( Manna, 1969 ). The oentromere of the sex

ohromosome is greatly stretched at metaphase 11 ( Figs.

119 and 120 ). The ohromosomes appear very thin and

stain lightly due to the erosion of chromatin. Aohromatio

gaps ooour in all the ohromosomes. The telomerio ends are ,

however, the darkest part of ohromosomes, indicating that

dechromatization commences from the proximal end and

proceeds towards the distal end. Thus, nost of the effeots

of forma1ine are of the negative type, originating due to

the produotion of antimetabolites.

-125-

In one instaLce, fo~aline is obsGrved to effeot

the spindle also resulting in 'star'- oOLfigurations

( Fig. 121 ). Suoh aberrations are oharacteristic of the

alkaloid oolchicine ( Manna and Parida, 1965 ).

A preliminary observation on the effect of

nicotine reveals that it is the most potent of the chemicals

used in this investigation. The effects Bre mostly non­

delayed. Niootine enflicts heavy frac;mentation at gonial

metaphsses ( Fig. 122 ). In the absence of exchange - type

configurations and ring-like structures, it can be stated

that the broken ends I::!.re uns.ble to reunite. The sex

chromosome remains unaffeoted. Some other abnormali~ties of

lesser intensity occur at diplotene ( Fig. 123 ), metaphase

I ( Fig. 125 ) and metaphase I1 ( Fig. 126 ). TheSe include

the positive effect ( clumping) a."ld the negative effect

( lesions, breaksge ).

The effects of the four chemicals used in this

investigation provide sufficient evidence to conclude that

ttey affect synthetic activities at one stage or the other.

Th.e binding of the chemioals with DNA in the ohromosollles is

expeoted to bring about structural alterations, whioh may

result in the disruption of chromatin material. In all the

instances of break;3ge. reunion is absent. This interpretation

suggests that the break£::ge. and the subsequent failure of

repair mechanism, both result from a block in some essential

synthetic activity, probably both of protein and DNA.

I VII I ~~

-126-

StThfEARY

A. Caryo1ogica1 study :

This study includes the mitosis and meiosis of

five species of grosshoppers belonging to the family

Aorididae ( Order : Orthoptera ) t namely, Tri10phidia

annulata ( sub-fsmily : Oedipodinae ), Ch~otogonus

trachypterus ( sub-family: Pyrgomorphinae ), Leva indica

( sub-family : Gomphooerinae ) t ~ttractomor:eh~ crenulata

( sub-family • Pyrgomorphina.e ) and Poekilocerus pictus • -( sub-family • Pyrgomorphinse ) . •

The diploid number of chrom.osomes in the male

germ cells of T. annu1ata is twenty-three. The chromosomes

can be classified into three long, seven medium and two

short chromosomes. The sex chromosome is the third in order

of length. Meiosis follows the classical pattern. Paohytene

ohromosomes possess 'snai1-horn'-like centric chroiIlomeres,

which give the impression of second ar:ns. The later stages

of meiosis do not show any indication of presence of

second ar'-IlS. ~I!ean chiasma fre::uency per nucleus at

diplotene, diakinesis &.nd metaphase I is 16.00 ! 0.02,

11.46 ! 1.24 and 11.35 ! 1.26, respectively. The

• pseudo-chiasmfita' occur at eHrly anaphase I. During

mi tosis the single 'X' chromosome reml.::.ins isopycnotic at

prophase. The longer Butosomes indicate the presence of

small arms in the somatio tissue ( gastric caeca ). A

comparison of lengths of germina1- and somatic-

-12'1-

ohromosomes makes it olea.r tl'.lEit the lu.tter are about one

micron longer tllan the former at metaphase.

The diploid number of chron:,08omes in the male

germ cells of Chroto$onus trachlP~s is nineteen. The

chromosomes can be classified into three long, six medium

and one short chromosomes. The meiosis of this species

has been worked out in the populations of Delhi state

and Ottu ( Haryana state ) of India. The present accowlt

includes the morphometric data a!jd a few points of interest

in the meiosis of the local ( Bhopal ) populgtion of M.P.

state. The results are compared and contrasted with

those of the other two reported populations. Mean chiasma

frequency per nucleus in the Delhi-, Ottu- and Bhopal

- populations, respectively, is , 15.25 ! 0.'12 / 15.86 /

15.50 + 1.27 at diplotene, 12.'15 ! a.dl / 11.93 / -11.23 + 1.04 at diakinesis, and 10.95 ! 0.94 / 11.20 / -10.66 + 0.25 at metaphase I. No second a.rms are visible -e.t a.ny stage. The pachytene nuclei possess t snail-horn'­

like centric chromomeres. During mitosis. the prophases

do not show any heteropycnotic body in tbe m:::lle somatio

tissue. The sex chromosome can be distinguished only on

the basis of its length. Second arms ~,re not visible in

any chromosome • A comparison of the lengths of germinal­

and somatic- metaphase chromosomes reveals that the latter

are two times longer than the former.

The diploid number of chromosomes in the male

germ cells of L. indica is twenty-three. The chromosomes

-128-

oan be olassified into three long, six medium and three

short ohromosomes. The sex ohromosome is the longest.

The general oourse of meiosis is of the classical type.

Mean ohiasma frequency per nucleus at diplotene, diakinesis

and metaphase I is 13.6 ! 1.49, 12.JO ! 1.12 and 9.71 ! 1.11

No seoond arms are visible at any stage. During mitosis,

the prophases of female somatio tissue exhibit an indis­

tinot heteropyonotio mass. The second arms are not visible.

A oomparison of the lengths of germinal- and somatio­

metaphase ohromosomes indioates that the ooiling is much

more loose in the latter.

The diploid number of ohromosomes in the m:~le

germ oells of A. crenul:;;ta is nineteen. The chromosomes

oan be olassified into three long, six medium and one

short ohromosomes. The sox ohromosome is the longest.

Meiosis has been reported elsewhere. During mitosis, the

sex ohromosome remains isopycnotic in the m81e somatio

tis3ue. The length of the somatic chromosomes at metapha.se

is almost double that of the spermatogon:i.al metaphase

ohromosomes.

The diploid number of chromosomes in the mole

germ gells of P. pictus is nineteen. The chromosomes can

be classified into three long, six medium ruid one short

chromosomes. Meiosis hss bean reported elsewhere. Mitosis

reveals the occurrence of second arms in the smallest

and one of the medium autoSOrlLes. The length of the second

arm varies from 0.9 to 1 micron. Different views are

-129-

available re)~aI'ding the po si tion of the centromere. There

is appreciable difference in the lengths of metaphase

chromosomes from the germinc:;l ;:;!1d somCltic tissues.

B. Abnormalities in the Natural ~~E~ations :

The abnormalities are scored inthe natural

populations of T. annUlate. t L. indlca, C. trachypterus

and A. crenulata a,mong one hundred - aud - six, fifty-two

eighty - six I:-illd thirty - five individuals, respectively.

T. armulate. cor.taiua extra fragment polyploid

cells, anaphase bridge [md breakage in its population.

~. indica contai!ls extra fragment, polyploid cells,

abnormal spindle and breakege. c. trachypterus contains

supernumerary chromosomes, polyploid oells, non-dif3junction,

breakage and extra fragment. Lastly, A. eranulata contains

breake:-lge and chromatid separation.

The percentage frequency of extra fragment is

nQ,t very significant in the species. It h&s not been found

to lag behind and interfere in spindle elongation.

Polyploidy is at the level of tetraploidy and originates

most probably due to failure of cleavage during mitosis.

No multivalent confi.gurations bre found and the sex

chromosomes do not show any tendency to synapse in the

tetraploid cells. The occurrence of brakage is significant

/'~ in L. indica only ( 29.41 'to ) • Here the breaks are Observed {

in the gonial metaphases affectinG both the chromatids of

-130-

the sex chromosome and the largest 8utosO,IJe. The

I oompleteness' of the break,:ige is doubtful in all the

instances. The dioentrio bridge has been observed in a

single oell in T. annulata.. A fragment of considers.hle

size aocompanies the bridge. Abnormal spindles with one

bifurcated pole hs.ve been found in L. indioa, the

peroentage frequenoy being 33.33 % • The origin of this

abnormatity is uncertain. The supernwnerary ohromosomes

occur only in C. trachlEter~s. Their number varies from

one to six in the oomplement. The behaviour is compared

and oontrasted with that of the supernumeraries of the

Ottu ( Haryana state ) - popul::'tion. Non-disjunotion

introduoes variable oonfigurations in the meiotio oells

of c. trachyPterus. The chromatids of the smallest

autosomal univalent divide preoociously in A. orenulata.

c. Banding Pattern of P. pi~ :

The nature and behc"Jviour of the ce tromere bi.:lS

been traoed in all the stages of spermatogenes~s with the

he~p of Giemsa staining. The o'bservations not only differ

in many respeots from the routine analysis of caryot;lpe,

but also oontradiot a few pOints. All the ohromosomes

possess well - marked C-bands, comprising the centric

cnromomeres and proximal band of heterochromatin of the

chromosoale arms" The sex chromosome possesses telomeric

bands 8S well. The smallest autosome has two bcll1ds in tbe

-131-

distal part. The C- band consists of four sub-units. The

centrio ohromomeres appear like 'snail - horns' at

paohytene. The sex chromosome, though showing hetaropyono­

sia after general staining, stains as intensely as the

auto8omes with Giemsa. The prooedure is, therefore,

reliable for the looation of constitutive heterochromatin.

D. Effects of Dru~s :

The investigation includes the q,uantitative

analysis of the effects of caffeine and qualitative analyses

of the effeots of oaffeine, enteromycetin, formaline and

niootine on the germ oells of C. trachlE!~. The division

oyole has been consulted for caloulating and adjusting the

treatment - time for the analyses.

Ten conoentrations of oaffeine have been tested

quantitatively, keeping the treatment - time fixed.

Mortality ocours in the higher dosage range ( 0.5 % to 1.0 %)

The frequenoy of three types of abnormality has been soored

- fragmentation, clumping and polyploidy. Data are oollected

from diplotene, diakinesis and metaphase I stages, and

pooled and averaged acoording to the number of survived

speoimens, for each concentration of caffeine. The

frequency of olumping and polyploidy show a definite rela­

tionship with eaoh other end with the inorease in oonoen­

tration. No simple relationship oan be drawn, however, from

the frequency - study of fragmentation.

-132-

For qualitative analyses of the four drugs,

following ooncentrations have been selected - J.4 % oaffeine, 0.25 ~ enteromyoetin, 3.0 % formaline and 0.5 % niootine. The testes were fixed at the intervals of 4, 18,

48 and 72 hours and 6, 7, 8, 14 and 21 days post - injeotion.

The abnormalities have been oategorised into (i) mitotio

ohanges , (ii) changes in the meiotio prophase stages,

(iii) ohanges at metaphase I and II, and (iv) ohanges at

first and seoond post - metaphase stages.

Niootine has been found to be the most potent

drug of all. Probable modes of aotion of the various drugs

have been disoussed.

=*=* =* =: *= iI::: *= -lI::*= *=*=

VIII

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-133-

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/'

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-141-

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-148-

and neo - Y type male • It THE NUCLEUS.

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.,/ 115. Manna, G.K. and S.C. M.azumder. 1964. It Ethyl alcohol

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~,.-, <

\ /'" NATURWISSLN. 51 (24) : 646.

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\....-/ 111. Manna, G.K. and S. P. Bhunya. 1911. It Tetracycline induced

/ /

/

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/ J. SOC. CYTOL. t GENET.

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-15J-

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/'

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\

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=*=*=*I:=f= ~ *="*=:: :11: V =*=

IX

EXPLANATION OF FIGURES

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Fig. 1

Fig. 8

Fig. 9

Fig. 10

Fig. 11

Fig. 12

-li4-

EXPLANATION OF FIGURES

Photograph of male speoimen of T. annulata.

Photograph of male specimen of

c. trachypterus.

Photograph of male specimen of L. indica.

Photograph of male specimen of A. crenulata.

Photograph of female specimen of P. pictus.

Photograph of cage used for rearing smaller

grasshoppers ( sliding glasS-door removed

from the front ).

Photograph of oage used for rearing P. piotus.

Photograph of diagrammatic representation of

approximate schedule of spermatogenesis in

c. trachlpterus.

Photograph of diagrammatio representation of

the method of counting metaphases ( or any

desired divisiona.l stage ) in the squash of a

testis - follicle.

X 800. Photomiorograph of spermatogonial

metaphase of T. ann~lata.

T. annulata. Photograph of spermatogonial

metaphase chromosomes arranged in order of

length. Sex chromosome is at the right hand

end.

X 800. T. annulata. Photomicrograph of

unsplit. heteropyonotio 'X' ohDomosome at

Fig. 13

.Fig. 14

Fig. 15

Fig. 16

Fig. 11

Fig. 18

Fig. 19

-165-

spermatogonial metaphase. A pachytene nucleus

at upper right - hand corner.

X 320. T. annulata. Photomicrograph of

pachytene nuclei with distinct positively

heteropyonotic sex - chromatin.

X 800 T. annulata. A pachytene nucleus with

clear split between the synapsed homologous

chromosomes.

X 800. T. ar~ulata. Pachytene nuclei with

centromerio ohromomeres which appear like

seoond arms t and interstitial blocks of

heterochromatin in the bivalents.

X 320. T. annulata. Photomicrograph of polar

view of metaphase I.

X 800. T. annuls!!. Photomicrograph of

bivalents at diplotene showing various confi­

gurations aoquired due to varying number of

ohiasmata, the maximum number being three

( marked with arrow).

X 320. T. annulate. Photomicrograph of profiles

of metaphase I. The sex ohromosome lies

either 'above' or 'below' the equatorial

plate.

X 800. T. annulata. PhotomiorQgraph of

formation of • pseudo-chiasmata' by the distal '

ends of the separating chromosomes. at

anaphase I.

Fig. 20

Fig. 21

Fig. 22

Fig. 23

Fig. 24

Fig. 25

Fig. 26

Fig. 27

-166-

x 800. T. annu1ata. Photomicrograph of

beginning of movement of chromosomes towards

the opposite poles. A slightly heteropyonotic,

split 'X' chromosome can seen at the 'upper'

:.Dole.

X 800. T. annulata. Photomiorograph of late

anaphase I with 'X' - chromosome in the

split oondition at the right - hand side pole.

X 800. T. annulata. Photomicrograph of early

anaphase I with 'X'- ohromosome in unsplit

condition.

X 320. T. annulata. Photomiorograph of polar

view of metaphase 11 ( left oe11 ) and side

view of anaphase 11 ( right cell ) within the

marked area.

X 800. T. annulata. Photomiorograph of

magnified view of the area marked in Fig. 23,

showing metaphase 11 ( Right ) and anaphase

11 ( left ).

X 320. T. annulata. Photomiorograph of

telophase I with darkly stained spindle -

fibres stretched between the two poles.

X 320. T. annulata. Photomiorograph of late

telophase 11 ( three oells in the middle ).

X 320. T. annulata. Photomicrograph of

metaphase I ( left cell) with an extra

fragment at the equator.

Fig. 28

Fig. 29

Fig. 30

Pig. 31

Fig. 32

Fig. 33

Fig. 34

Fig. 35

Fig. 36

-161-

X 320. T. anntt1ata. Photomiorograph of two

metaphase I oel1s ( left and middle ) and one

anaphase I.,oe11 ( right ), eaoh wi th s.n extra

fragment.

X 320. T. annulat~. Photomiorograph of a

tetraploid cell ( middle ) at diakinesis.

X 320. T. annulata. Photomiorograph of two

tetraploid cells at diakinesis.

X 320. T. annulate. PhotomicrogTeph of one

tetraploid ( centre left ) and one normal

cell ( oentre fight ) at diakinesis.

T. annulate. Photograph of anaphase I with a

dioentric bridge aocompanied by a fragment.

X 800. T. annulata. Photomicrograph of polar

view of early anaphase I showing ohromatid -

breakage in the negatively heteropycnotic

sex chromosome.

x 800. T. annttlata. Photomicrograph of thirteen

chromosomes of a metaphase plate from the

male somatic tisstte ( gastric caeca ). Arrow

displays the indication of occttrrenoe of

second arms.

x 800. T. annula.ta. Photomicrograph of the

remaining ten ohromosomes of the same

metaphase plate shown in Fig. 34.

T. annulate. Photograph of diagrammatic

representation of the lengths of spermatogonial

Fig. 37

Fig. 38

Fig. 39

Fig. 40

Fig. 41

Fig. 42

-168-

metaphase chromosomes, based on the data

given in Table 1.

T. annulata. Photograph of diagrammatic

representa.tion of the lengths of' aomatio

metaphase chromosomes of male.

o. traohYRterus. Photograph of spermatogonial

metaphase chromosomes arranged in order of

size. Sex ohromosome at the right - hand end.

X 800. c. trachypterus. Photomicrograph of

paohytene nuoleus showing positively

heteropycnotic sex chromosome along with

a.nother similar, smaller mass of chromatin.

Destinct centromeric chromomeres can be seen.

X 800. o. trachyPterus. Photomicrograph of

side-view of early anaphase I, showing

'diamond' - shapes ( psuedochiasmata ) formed

by the bivalents. The cell at right - hand

end shows metaphase I.

X 800. c. trachypterus. Photomicrograph of

polar view of two cells at early anaphase I.

Pseudo chiasmata oan be seen in all the

bivalents. Sex chromosome shows a clear split

in the midcUe.

X 800. c. trachypterus. Photomicrograph of

eelrly anaphase one in the marked area, showing

the configuRations of the bivalents • Another

oell at metaphase can be seen at rl8ht-ha_d end.

Fig. 43

Fig. 44

Fig. 45

Fig. 46

Fig. 47

-169-

Photomicrograph of early anaphase I, shown in

Fig. 42, enlarged photographioally to reveal

the ns,ture of asoocia.tion of separating

chromosomes. Sex ohromosome shows a split

between its chromatids.

X 320. c. traohypterus. Photomiorograph of

cells at telophase 11. The single supernume­

rary element can be seen lagging at equator

in top central oell.

X 800. c. tra.ohypterus. Photomiorograph showing

reduotion division of supernumerary bivalent

( right oell ) with 9dA + s at each pole;

and ( left oell ) with 9dA + aa at one pole

and 9dA + s at the other. Both the cells are

at anaphase I!.

X 320. o. trachlpterua. Photomiorograph

showing different behaviours of the super­

numerary elements. In the n + 4s oondition

( Central cell ) 9dA + 1 es pass to eaoh pole.

In the n + 5s oondition ( upper right oell )

9dA + 1 ss pass to one pole and 9dA + 1 ss + 1 a

pass to the other. In the n+6s oondition,

ladA + 2ss + 2s oan be seen in the polar

view of anaphase 11 ( lower right oell ).

X 320. c. traohypterus. Photomiorograph of

two- tetraploid cells at diakinesis. The

chromosomal configJJ.ration in both oases is

Fig. 48

Fig. 49

Fig. 50

Fig. 51

Fig. 52

-110-

l8AA + 2x. The two sex ohromosomes lie away

from each other and show negative heteropycno­

sis. Two normal oells at diakinesis can be

,seen ( extreme left and upper centrol

regions ).

X 800. c. traohypterus. Photomicrograph of a

tetraploid cell at metaphase 11. The

chromosomal configuration is 36dA + 2dx.

X 800. c. trachypterus. Photomiorograph of a

tetraploid oell at anaphase 11. Derived from

the binucleate oel1, the chromosomal

configurations of the two sete are

9dA + dx - 9dA + dx ( upper, right side ) and

9dA - 9dA ( lower side ), respectively.

X 320. c. traohYEterus. Photomiorograph of an

anaphase II oell with non-disjunction. The

ohromosomal oonfiguration is 8dA ( upper

pole) and lOdA ( lower pole ).

X 320. c. traohypterus. Photomiorograph of

ooourrenoe of non-cisjunotion at anaphase 11.

The ohromosomal oonfiguration is 8dA

( left pole ) and 10 dA ( right pole ). Other

oells at anaphase 11 show normal disjunotion

of the doughter ohromosomes.

X 800. c. traohlPterus. Photomicrograph of

oocurrenoe of non-disjunction at anaphase 11,

a oase where non- disjunotion had already

Fig. 53

Fig. 54

Fig. 55

Fig. 56

Fig. 57

-1'71-

occurred at anaphase I also. The latter oase

might have had 7A + 2,AA + x at one pole and

7A at the other. The 7A configuration

intead of. splitting into TdA - 7dA, splits

into 8dA - 6dA due to IJ.on-disjunction. A

normal cell at a:naphasf; 11 ( 9Wi + dx - 9dA

+ dx ) is seen on the left-hand side.

X 800. c. traohlEterus. Photom.iorograph of

anaphase I showing u..r..der - stained regions

in the ohromatids of' autosoma~ univa.lenta.

The latter are homologous and contain the

under - stained region in one chromatid only,

but at the same locus.

X 800. o. trachll?terus. Photomicrograph of'

anaphase 11 showing under - stained region

in one daughter univalent.

X 800. c. trachlpterus. Photomicrograph of

anaphase 11 showing under - stained region in

the daughteI' sex chromosome ( lower pole ).

X aOO. C. tr·aoh;YEtel'us. Photomicrograph of a

cell at anaphase 11 with a breakage in the

proximal region of a daughter autosome.

X 320. o. traohypterus. Photomicrograph

showing the presence of extra. fra§]Iler!t in two

oells at metaphase 11. Surrollnding oells

show different s-tages of' spermatogenesis.

Fig. 58

Fig. 59

Fig. 60

Fig. 61

Fig. 62

Fig. 63

Fig. 64

Fig. 65

-172-

x 800. c. traohlEterus. Photomiorograph of

metaphase from the gastrio oaeca of male

grasshopper. The sex ohromosome , being the

longest in the oomplement, oan be identified

only beoause of its length. Interphase oells

possess no positively heteropyonotio body.

c. traohlpterus. Photograph of diagramm&tio

representation of lengths of spermatogonial

metaphase chromosomes, based o~ the data

given in Table 4.

o. traohy?teras. Photograph of diagrammatio

representation of lengths of ohromosomes of

the somatio metaphase ( gas trio oaeoa ) of

male grasshopper.

X 800. Photomiorograph of spermatogonial

metaphase of Leva indioa.

L. indioa. Photograph of spermatogonial

metaphase ohromosomes arranged in order of

size. Sex ohromosome at the right - hand end.

L. lndioa. Photograph of diakinesis stage

showing an extra fragment.

~. indioa Photograph of polyploidy at anaphase

II. The total ohromosomal oonfiguration is

44dA, that is, lldA at Gaoh of the four poles.

L. indioa. Photograph of a oell at auaphase I,

posaessing an abnormal spindle. Ohromosomal

oonfiguration of the normal and bifid pole

Fig. 66

Fig. 67

Fig. 68

Fig. 69

Fig. 70

Fig. 71

Fig. 72

-173-

are lldA and 8dA - 3d., respectively.

L. indica. Photograph of spermatogonial

metaphase with breaka.ge in both the chromatids

of the long autosome.

L. ind10a Photograph of spermatogonial

metaphase with breakage in three long auto­

aomes.

X 3~O. L. indica. Photomicrograph of

metephe.se from the gastric caeca of female

grasshopper. The two sex chromosomes oan be

identified only beceuse of their length,

being the lonBest in the oomplement. An

interphase cell at the bottom, shows an

indistinct heteropycnotic mass.

L. 1nd1ca. Photograph of diagrammatio

representation of lengths of spermatogonial

metaph~:!.se chrOG.lC'sC'IDE'!S, be.sed on the data.

given in Table 12.

L. 1ndica. Photograph of diGgrammatio

represente,t1on of lengths of chromosomes of

the SOI!l&tio metaphase ( gastrjc oaeca ) of

male grasshopper.

X 000. Photomicrograph of early spermatogonial

metaphase of Attr~tomorEha.~re~ulata.

A. cr~pu.lat~. Photograph of spermatogonial

metaphase chromosomes arranged in order of

length. Sex chromosome at the right - htomd end.

Fig. 13

Fig. 14

Fig. 15

Fig. 17

Fig. 18

Fig. 19

Fig. 80

Fig. 81

-174-

A. crenulata. Photograph of a cell at

anaphase I with a breaka.ge in one chromatid

of the sex ohromosome.

A. crenulata. Photograph of a oell at

anaphase I in the polar view. The chromatids

(dA) of the smallest autoBomes have

disju.ncted preoociously.

Photograph of early metaphase stage from the

gastric oaeoa of male nymph of A. orenulata.

Photograph of late metaphase stage from the

gastrio caeca of female nymph of A. crenu.lata.

A. orenulata. Photograph of diagrammatio

representation of lengths of spermatogonial

metaphase ohromosomes, based on the data

given in Table 15.

A. crenulata. Photograph of diagrammatic

representation of lengths of ohromosomes of

the somatic metaphase ohromosomes ( gastric

caeca ) of male grasshopper.

Photograph of somatic metaphase from the

gastrio caeoa of female nymph of P. pictus.

P. Eictus. Photograph of diagrammatic

r~presentation of the lengths of early

( black bars ) and lete ( dotted bars )

sperm:::1to::sonial metaphase ohromosomes.

P. pictus. Photograph of diagrammatically

representation of the lengths of the somatio

Fig. 82

Fig. 33

Fig. 84

Fig. 36

-175-

metaph~se chromosomes ( gastric caeca ) of

male grasshopper.

X 800. P. pictus. Sperma.togonial metaphase

with distinct bands of centromeric

heterochsomatin revealed by Giemsa staining.

The four subunits of the band can be clearly

seen in X - chromosome and M4 autosome.

X 800. P. pictus. Leptotene stage with the

oh~r~cteristic 'bouquet' arrangement,

Centromeres and telomeres centralize to form

the 'dark pole'. The positively heteropycno­

tic sex ohr'omCltin lies at the oppoai te pole.

X 800. P. pictus. Pachytene stage showing

nine autosomal bivalents with distinct

oer:tromeric chromomerea and a jumb-bell

shaped sex - ohromatin body. Smallest

autosoID8J.. bivalenl; (SI) with clear telomeric

band.

x 800. P. pictus. Pachytene stage showing

distinot oentromeric chromomeres, oonstricted

off i'rom the main body of the chromosomes.

Another cell .at interphase ( 3xtrame left )

possesses a peculd:a.r, geometrio£;,lly sha.ped

heteroPycLotic body.

X jQ,). P. pictus. .Pa.cflytene nucleus showing

the presence of' a narrow, dark band at the

1;lroximal region t)f the chromosome (&.rrow marks).

Fig. 87

Fig. 88

Fig. 89

Fig. 90

Fig. 91

Fig. 92

-176-

X 80). P. pictus. Pachytene nucleus with the

centromeric chromomeres and proximal band of

heteroohromatin. Smallest bivalent ( SI )

looks uniformly dark because of the approxi­

mation of diatal ~ proximal bands.

X 800. P. Eictus. Pachytene nucleus with very

distinct blobs of proxime.l heteroohromatin in

all the bivalents. Two dark bands are visible

in the diatel region of Sl* and 1 light band

in the sub-termim:tl distal pert of M6-

X 800. P. pictua. Dialiinesis stage showing

ole~rly the centromeric bands only. SI shows

almost uniform dark staining_ ·x· lies coiled

upon itself.

X 800. P. pictus. I'olar view of metaphase I.

The negctively heteropycnotic 'x· shows a dis­

tinct oentromerio band. The bivalents also

show the oentromerio bands, which are not

very distinct are direoted towc~rds the oentre of

the spindle.

X 800. P. pictus. Side view of metaphase I.

Bands oannot be located in the much condensed

and over-stained biv':llents but the t X' does

possess dark regions at both the terminals.

X 800. P. pictus. Anaphase I with the sex

chromosome at the 'lower' pole. The bands of

·x· comprise one lighter band at the telomeric

end and one darker band at the proxima,l end.

The latter type of band is composed of 4.

Fig. 93

Fig. 94

Fig. 95

Fig. 96

Fig. 97

Fig. 98 I

-111-

subunits. Long and medium univalents have

olear oentromerio bands, while Sl-univalents

have both oentromerio and telomerio bands.

X 800. P. p1otus. Polar view of anaphase 11.

The ohromatids ( daughter ohromosomes ) show

oontraoted oentromerio bands at the proximal

ends, direoted towards the oentre of the

spindle. The two subunits of o-bands oan be

olearly seen in M2 and M5•

X 800. Effeot of oaffeine : High oondensation

of bivalents at diakinesis with oomplete

loss of 'lamp-brush' appearanoe. X -

ohromosome not distinguishable.

X 320. Effeot of oaffeine : Lesions in the

bivalents at diakinesis. Deohromatization

has resulted in the wavy outline of some of

the chromosomes.

x 800. Effeot of oaffeine : Polyploidy at

metaphase 11. The ohromosomal oonfiguration

of the tetraploid oell is 36A + 2X. The sex

ohromosomes oannot be distinguished on the f

basis of heteropyonosis as their staining

intensity is equal to that of the autosomes.

X 320. Effeot of oaffeine : Early stage of

olumping involving three or four ohromosomes

of eaoh oomplement at metaphase 11.

X 320. Effeot of oaffeine : Later stage of

Fig. 99

Fig. 100

Fig. 101

Fig. 102

Fig. 103

Fig. 104

-178-

clumping, involving almost all the chromo­

somes of metaphase 11 cells. The identifica­

tion of the divisional - stage is, however,

possible. Cell walls are not visible.

X 320. Effect of caffeine : Intense clumping

at metaphase 11 obtained after increasing the

treatment - time. The individual identity of

chromosomes is lost completely and the stage

can be identified with muoh difficulty. The

cell walls are not visible.

X 800. Effect of caffeine : Irregularity in

the arrangement of metaphase 11 chromosomes

because of the non-alignment of spindle -

fibres.

X 800. Effect of caffeine : Spindle dissolu­

tion at metaphase 11, causing a scattering

of daughter chromosomes beyond the normal

dimensions.

X 800. Effect of caffeine : Breakage in the

smallest autosome~ Both the fragment and the

heteromorphic univalent pass to the same pole.

X 800. Effect of enteromycetin : Lesion in

the sex chromosome at spermatogonial

metaphase.

X 800. Effect of enteromycetin : Lesions in

both chromatids in the sex chromosome and one

of the longest autosomes, at spermatogonial

Fig. 105

Fig. 106

Fig. 107

Fig. 108

Fig. 109

Fig. 110

-179-

metaphase.

X 800. Effeot of enteromyoetin : Chromosome

-breakage at diplotene.

X 800. Effeot of enteromyoetin : One tetra­

ploid oell at diakinesis ( right - hand side )

with the ohromosomal oonfiguration IBAA+2X.

The sex ohromosomes do not show any tendenoy

to synapse.

X 800. Effeot of enteromyoetin : Fragmentation

at metaphase 11. The "pseudo - satellites"

are due to the notohes whioh are the first

signs of the beginning of fragmentation.

X 800. Effeot of enteromyoetin : A tetraploid

oell at anaphase I showing stiokiness in the

telomerio ends of ohromosomes. All the

ohromosomes are highly condensed and no

separation between the ohromatids is visible.

X 800. Effeot of enteromyoetin : Two oells

at anaphase I with abnormal spindles. The

latter have one normal pole with the

ohromosomal configuration 9A+X. The other

pole is bifid and oontains 3A in the smaller

group and 6A in the larger group. The

univalents at the bifid pole enter telophase

earlier than the univalents at the normal

pole ( right cell ).

X 320. Effect of formalirle : Soattered

Fig. III

Fig. 112

Fig. 113

Fig. 114

Fig. 115

Fig. 116

-180-

bivalents at diplotene show dechromatization

and breakGge at chromosome level. There is an

indication of the destruction of the cell

wall as the bivalents lie scattered.

X 800. Effect of formaline : Diakinesis stage

showing the occurrence of interbivalent

connection. The latter comprises a thin

thread of chromatin connecting two of the

bivalents.

X 800. Effect of formaline : Late diplotene

stage with break2ge in one chromatid of a

medium bivalent.

X 800. Effect of forma1ine : A cell at

diakinesis showing clumpillg. The clumps are

formed by the grouping of 3 - 4 bivalents,

the identity of which is not completely lost.

The sex chromosome stains as intensely as

the Butosomes.

X 800. Effect of formaline : A cell at

prometaphase showing the linking of five

bivalents and the sex chromosome. Erosion of

chromatin material is also visible.

X 300. Effect of formaline : Metaphase I with

a chain of three bivalents and breakclge in

the clump ( left - hand side )

X 800. Effect of formaline : Destruction of

morphology of metaphase II chromosomes. A

Fig. 117

Fig. 118

Fig. 119

Fig. 120

Fig. 121

-181-

'pseudo-tetranucleate' appearance is found in

the cell ( upper right ) due to the grouping

of chromosomes. A pachytene nucleus in the s&me

photograph shows the 'snail-horn'-like

appearance.

X 800. Effect of formaline : Occurrence of

lesion in the sex chromosome at metaph&se 11.

The Butosomes hGve slightly affected morpho­

logy.

X 800. Effect of formaline : Occurrenoe of

lesion in the sex ohromosome at metaphase 11

( right oell ). Another cell at metaph&se 11

( left-hand side ) shows a high degree of

dechromatization.

X 800. Effeot of formaline : Metaphase 11

showing a number of abnormalities. A promi­

nent under-stained gap is present ill an

autosome. The sex chromosome shows a

stretohing of its oentromere. All the

chromosomes possess under-stained spots due

to dechromatiz~tion.

X 320. Effect of formaline : cells at meta­

phase 11. The centromere of the sex ohromosome

is stretched to a great extent. Almost all

the chromosomes appear under-stained due to

extreme deohromatization.

X 300. Effect of formaline : 'star' configura-

Fig. 122

Fig. 123

Fig. 1~4

Fig. 125

Fig. 126

-182-

tion at metaphase 11 ( upper right corner ).

The stage is identifiable because some of the

univa1ents maintain their morphology by not

joining the tstar'. Another metaph&se 11

( lower left cor-ner) shows aberrant morphology

of the chromosomes.

X 800. Effects of nicotine : Fragmentation at

gonial metaphase. The sex chromosome is untou­

ched and m~intains its negative heteropycnosis.

Almost all the autosomes have fragmented at

the chromatid-level.

X 800. Effect of nicotine : Diplotene stage

showing breaks in two bivalents. The fragments

lie away from the heteromorphic bivalents.

X 8JJ. Effect of nicotine : clumping at

diakinesis. The bivalents become thickened and

ag;~rega_te to form 'ball'-like structures.

X 800. Effect of nicotine : Clumping at

metaph<:.se I involving 2-3 bivalents.

X 800. Effect of nicotine : A diploid cell at

metaphase 11 with lesions in all the

chromosomes except the smaller ones. Some

chromosomes possess wavy outline.

X

ABBREVIATIONS

A

AA

asso. ch

bd

bif. pI

br

br. p.

c.c.

c.c.w.

oent. het.

chd. br

chr. br

cl

oon. bv

dA

dis. bI

dis. b2 dk. pI

dX

e.f

fr. ch

frg

g

h.b

into bv. con

-183-

ABBREVIATIONS

=

= = =

= =

= =

=

= = = =

= = = = = = = = = = = =

autosomal univalent.

autosomal bivalent.

assooiated ohromatids.

band.

bifid pole.

breakage.

broken piece.

oentrio ohromomeres.

oommon oell wall.

oentromerio heteroohromDtin.

ohromatid breakage.

chromosome breakage.

clump.

oonneoted biva.lents.

daughter autosome.

first distal band.

seoond distal band.

dark pole.

de,ughter sex ohromosome.

extra fragment.

free ohromatid.

fragment.

grass.

heteropyonotic body.

inter-bivalent connection.

into oh

into nu

Ll

L2

l.b.p.

le

lps

MI

m.l

m.s.

n

non. dis

p.c.

pro. b. het

prox. het

=

= =

::

= = ::

::

::

=

=

=

=

::

::

=

=

=

::

=

-184-

inters,ti tial ohiasma.

interpha.se nucleus.

first long autosome of P. piotus.

second long autosome of

P. piotus.

lagging broken pieoe.

lesion.

loops.

first medium autosome of

P. pictus.

seoond medium autosome of

P. pictus.

third medium autosome of

P. piotus.

fourth medium autosome of

P. :eictus.

fifth medium autosome of

P. Eiotus.

sixth medium autosome of

P. pictus.

movable lid.

moist sand.

haploid oomplement.

non-disjunction,

pseudo-chiasma.

proximal band of heteroohromatin.

proximal heterochromatin.

r. s.

S

SI

s

S. D.

sep. chd

sep. univ

sI. g. d.

sp. f

ss

st. confg

str. cen

tel. het

ter. ch

t.n.c.

t.p.c.

u.s.r.

w

X

-185-

=

= =

=

= = = = = =

=

=

=

=

= =

= =

reductional split.

standard diploid complement.

short autosome of P. piotus.

supernumerary univalent.

standard deviation.

separated ohromatids.

separating univalents.

sliding glass door.

spindle fibres.

supernumerary bivalent.

star oonfiguration.

stretohed centromere.

telomerio heterochromatin.

terminal chiasma.

texra-nucleate oell.

tetraploid oell.

understained region.

water.

sex chromosome.