Biochemistry and genetics of insect resistance to Bacillus thuringiensis insecticidal crystal...

1 Nov 2001 10:16 AR AR147-17.tex AR147-17.SGM ARv2(2001/05/10)P1: FJS

Annu. Rev. Entomol. 2002. 47:501–33Copyright c© 2002 by Annual Reviews. All rights reserved

BIOCHEMISTRY AND GENETICS OF INSECT

RESISTANCE TO BACILLUS THURINGIENSIS

Juan FerreDepartment of Genetics, University of Valencia, 46110-Burjassot (Valencia), Spain;e-mail: [email protected]

Jeroen Van RieAventis CropScience, 9000-Gent, Belgium; e-mail: [email protected]

Key Words microbial control, resistance mechanisms, resistance management,transgenic crops, insecticidal proteins

■ Abstract Bacillus thuringiensis(Bt) is a valuable source of insecticidal proteinsfor use in conventional sprayable formulations and in transgenic crops, and it is themost promising alternative to synthetic insecticides. However, evolution of resistancein insect populations is a serious threat to this technology. So far, only one insectspecies has evolved significant levels of resistance in the field, but laboratory selec-tion experiments have shown the high potential of other species to evolve resistanceagainstBt. We have reviewed the current knowledge on the biochemical mechanismsand genetics of resistance toBt products and insecticidal crystal proteins. The un-derstanding of the biochemical and genetic basis of resistance toBt can help designappropriate management tactics to delay or reduce the evolution of resistance in insectpopulations.

CONTENTS

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 502SELECTION FOR RESISTANCE TOBt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 503

Lepidoptera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 503Diptera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 506Coleoptera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 506

BIOCHEMICAL BASIS OF RESISTANCE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 507Altered Proteolytic Processing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 507Binding Site Modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510Other Mechanisms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513

GENETICS OF RESISTANCE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513Intraspecific Variation in Baseline Susceptibility. . . . . . . . . . . . . . . . . . . . . . . . . . . 513Variability for Resistance Genes as Detected by Laboratory Selection. . . . . . . . . . 514Estimation of Resistance Gene Frequency. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514Mode of Inheritance of Resistance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 515

0066-4170/02/0101-0501$14.00 501


502 FERRE ¥ VAN RIE

Stability of Resistance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 520Simultaneous Occurrence of Different Resistance Genes. . . . . . . . . . . . . . . . . . . . . 521Identification of Resistance Genes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 522

CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 523

INTRODUCTION

Biological insecticides, such as sprayable formulations based onBacillus thurin-giensis(Bt), can provide a valuable alternative to synthetic insecticides that suffersome disadvantages related to environmental damage and health hazards. However,owing to their limited field stability, lack of capacity to reach cryptic insects, andnarrow spectrum of activity,Bt sprays still represent only a minor fraction of theinsecticide market. Transgenic plants expressingBt insecticidal crystal protein(ICP orcry) genes overcome the first two drawbacks. Recently, several varieties of“Btcrops” (cotton, corn, and potato) expressingcrygenes have been commerciallyreleased in the United States and other countries (49). The use ofBt crops isexpected—and has been reported (14, 28, 90)—to lead to a reduction in the useof synthetic insecticides. Perhaps the most serious threat to the durability of thisnovel insect control technology is the potential of insect populations to developresistance toBt Cry proteins.

Indeed, insects have demonstrated their enormous genetic plasticity with over500 insect species resistant to one or multiple insecticides (26). In 1985, the firstreport on insect resistance toBt was published (71). Since then, many more suchcases have been reported (9, 20, 24, 94, 101, 125). Most of these colonies wereselected for resistance under laboratory conditions. Laboratory colonies usuallyhave limited genetic variation and may not contain all resistance genes presentin field populations. There are in fact several examples of selection experimentson laboratory colonies of the diamondback moth (Plutella xylostella) that failedto selectBt-resistant insects (14a, 53a), although this very insect species was thefirst, and still is the only, species to develop resistance toBt applied as a biopes-ticide. Laboratory selection experiments do not predict if resistance will developin the field or which resistance mechanisms will be selected, but they can in-dicate the repertoire of resistance mechanisms available in a certain population(25). These experiments are essential to study inheritance of resistance genes. Fur-thermore, resistant insect colonies can be extremely valuable in the experimentalevaluation of the validity of proposed resistance-management tactics (56, 87, 95,118).

The toxic pathway of Cry proteins involves several steps: On ingestion by sus-ceptible insects, crystals are solubilized and protoxins are released. These protoxinsare then processed by midgut proteases into a protease-resistant core fragment, thetoxin, which passes through the peritrophic membrane and binds to a specific re-ceptor located on the brush border membrane of midgut cells. Binding, followedby (partial) insertion of the toxin into the membrane, leads to pore formation, celllysis, and eventually insect death (94).


INSECT RESISTANCE TOB. THURINGIENSIS 503

Detailed insight into the biochemical and genetic basis of resistance, combinedwith the ever-increasing knowledge base on the mode of action of Cry proteins, canprovide valuable information for designing and fine-tuning resistance-managementtactics for the deployment ofBt sprays and crops aimed at safeguarding this insectcontrol technology (30).

SELECTION FOR RESISTANCE TO Bt

Selection experiments on laboratory or field-collected insects have been performedusing a variety ofBt products such as formulated spore-crystal mixtures, encap-sulatedPseudomonas fluorescenscells expressing Cry protein, Cry protoxins,Cry toxins, and material derived from Cry protein–expressing crops. A fairlycomplete overview of the results from these experiments is shown in Table 1(see the Supplemental Material link in the online version of this chapter or athttp://www.annualreviews.org/). In Table 2 of this paper, we limit the data to thoseinsect colonies for which relevant complementary data on the biochemical mech-anism of resistance is available.

Lepidoptera

The first thoroughly studied case of resistance toBt was the 100-fold decrease insusceptibility (ratio between the final and initial LC50 values) to Dipel [a commer-cial Bt product based onBt var. kurstaki (Btk)] in an Indianmeal moth (Plodiainterpunctella) population from grain bins following 15 generations of laboratoryselection with Dipel (71). Resistance reached 250-fold (ratio between the LC50 ofthe selected colony and the LC50 of a control colony) after 36 generations of selec-tion with Dipel (72). It is interesting that the Dipel-resistant colony (colony 343-R)was still susceptible to certain otherBt strains, at least some of which contain Cryproteins different from Cry1A (73). Relative to the unselected control colony, 343-R was more than 800-fold resistant to Cry1Ab, but it was nearly fourfold moresusceptible to Cry1Ca (127). Selection of another colony (Dplr) with Dipel in-creased resistance to Cry1Ab over 200-fold without increasing susceptibility toCry1Ca (45, 74, 75).

P. interpunctellacolonies have also been selected for resistance to variousotherBt strains, includingBt var.entomocidus(Bte) HD-198 andBt var.aizawai(Bta) HD-133 (colonies 198r and 133r, respectively) (74, 75). Although bothBtstrains contain Cry1C and although Cry1Ca is highly toxic toP. interpunctella,resistance levels against Cry1Ca were low in colonies 198r and 133r. PerhapsCry1C-resistance alleles are less frequent in this species than are Cry1A-resistancealleles. Both resistant colonies showed relatively higher resistance levels to Cry1Acthan to Cry1Ab, although Cry1Ab, but not Cry1Ac, is present in bothBt strainsused for selection.

The diamondback moth (P. xylostella) is the only insect species that has evolvedhigh levels of resistance toBt in the field. The first case of field resistance was



reported from Hawaii. Populations from areas heavily treated with Dipel provedless susceptible than populations that had been treated at lower levels, with thehighest level of resistance at 30-fold (102). Laboratory selection using Dipel in-creased resistance rapidly to over 1000-fold (104, 108). The resulting colony (NO-QA) displayed high levels of resistance to Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa,and Cry1Ja, whereas resistance to Cry1Ba, Cry1Bb, Cry1Ca, Cry1Da, Cry1Ia,and Cry2Aa was not considered significant (106, 108, 115). Leaves of Cry1Ac-expressing canola killed all susceptible larvae exposed to them, whereas 90% ofthe larvae survived on leaves of normal canola. In contrast, NO-QA larvae devel-oped equally well on both types of leaves (89).

A diamondback moth colony (BL) derived from a field population in thePhilippines regularly exposed to Dipel showed more than 200-fold resistance toCry1Ab (21). The same colony was still fully susceptible to Cry1Ba and Cry1Ca. Inanother colony sampled later from the same location in the Philippines, resistancewas limited to Cry1Ab and did not extend to Cry1Aa, Cry1Ac, or Cry1Ba (7). Fol-lowing additional selection with Cry1Ab and later with a hybrid Cry1A protoxin,this colony (PHI) was observed to be partially resistant to Cry1Aa, Cry1Ab, andCry1Ac, and still susceptible to Cry1Ca, Cry1Fa, and Cry1Ja (113).

A P. xylostellacolony (Loxa A) from Florida was found to be more than 1500-fold resistant to Javelin (a commercial formulation ofBtk NRD12) in the secondgeneration after the colony was collected from the field (121). Resistance rapidlyfell to about 300-fold in the absence of selection but remained stable at this level insubsequent generations. Insects with this level of stabilized resistance were morethan 200-fold resistant to Cry1Aa, Cry1Ab, and Cry1Ac, but they were still fullysusceptible toward Cry1Ba, Cry1Ca, Cry1Da (121), and Cry9Ca (54). Whereassurvival of susceptible larvae at 72 h on leaf disks from Cry1Ac-expressing broccoliwas much reduced compared with survival on leaf disks from normal broccoli,survival of resistant larvae did not differ between the two types of broccoli (119).In fact, resistant larvae could complete their development from egg to adult andcould cycle for multiple generations equally well on both plant types (76, 118,119).

Insect resistance inP. xylostellapopulations toBtk products has resulted inextensive use ofBta-based insecticides in certain locations.Bta strains typicallycontain, in addition to Cry1A toxins, Cry1Ca. Insects in two colonies (NO-93,NO-95) from one such location in Hawaii displayed up to 20-fold resistance toCry1Ca (62). These Cry1Ca-resistant colonies were only two- to fourfold lesssusceptible toBta, somewhat less susceptible to Cry1Ab, and 50- to 130-foldless susceptible toBtk formulations when compared with a susceptible colony.Following selection of NO-95 larvae with Cry1Ca (57, 60), this colony (Cry1C-Sel) was 19-fold resistant to the Cry1Ca toxin and 48-fold resistant to the Cry1Caprotoxin. Another Cry1Ca-resistantP. xylostellacolony was collected from fieldsin South Carolina. Laboratory selection using first Cry1Ca protoxin, and in latergenerations transgenic broccoli expressing Cry1Ca, increased Cry1Ca resistanceto 12400-fold (136).



A P. xylostellacolony (SERD3) with considerable levels of resistance to bothBtkandBtahas been reported from Malaysia (134). After rearing in the lab for sevengenerations without selection, resistance toBtkandBtawas 330-fold and 160-fold,respectively. Selection during three generations withBtkincreased resistance toBtkbut only marginally toBtaand vice versa. AnotherP. xylostellacolony (UNSEL-MEL) that developed field resistance toBtkproducts in Malaysia (Melaka region)showed resistance to Cry1Ac, Cry1Ab,Btk, andBta (93). Selection with Cry1Ac(1AcSEL-MEL) during 5 generations resulted in a more than 95-fold increasein resistance to this toxin, whereas selection with Cry1Ab (1AbSEL-MEL),Btk(BtkSEL-MEL), orBta (BtaSEL-MEL) increased resistance to these toxins orBtproducts only tenfold or less.

High levels of resistance to Cry1Ac (over 10,000-fold) were obtained in aHeliothis virescenscolony by selection with Cry1Ac protoxin (33). This colony(YHD2) was highly cross-resistant to Cry1Ab and Cry1Fa, only moderately cross-resistant to Cry2Aa, and almost nonresistant to Cry1Ca and Cry1Ba. Followingcontinued selection on Cry1Ac, the colony (YHD21000MVP) became more than230,000-fold resistant to this toxin (J.L. Jurat-Fuentes, F. Gould & M.J. Adang,personal communication). Cross-resistance to Cry2Aa remained low (9.5-fold)(53). In aH. virescenscolony (SEL) selected on Cry1Ab, resistance to Cry1Ab was20-fold after 14 generations (100) and further increased to 71-fold by 4 additionalgenerations of selection with Dipel (97). Selection of another colony ofH. virescenswith Cry1Ac resulted in a 50-fold resistance to Cry1Ac, 13-fold to Cry1Ab, and53-fold to Cry2Aa (34). Cross-resistance in this colony (CP73-3) also extended toCry1Aa, Cry1Ba, and Cry1Ca. Further selection with Cry2Aa resulted in higherresistance levels to both Cry1Ac and Cry2Aa. Although larvae from this colony(CxC1000IIA) were more than 330-fold resistant to Cry2Aa, they suffered 100%mortality on leaves from tobacco plants expressing this protein (53).

Selection of aSpodoptera exiguacolony with Cry1Ca resulted in significantlevels of resistance to Cry1Ca (850-fold) and cross-resistance to Cry1Ab, Cry2Aa,and Cry9Ca (78). Selection experiments onSpodoptera littoralislarvae usingspore-crystal mixtures of a recombinantBt strain expressing only Cry1Ca gen-erated a resistant colony with greater than 500-fold resistance to Cry1Ca (81).The resistant colony was fully susceptible to Cry1Fa but exhibited limited cross-resistance to Cry1Ab and Cry1Da and a somewhat higher level of cross-resistanceto Cry1Ea.

Decreased susceptibility to Dipel was observed in all the five colonies ofOstrinia nubilalisselected in the laboratory with thisBt formulation (47). Upon3 generations of selection with Dipel, one colony (KS-SC-R) displayed a 36-folddecrease in susceptibility; further selection for 4 generations increased the resis-tance ratio to 73-fold. Although neonates of KS-SC-R were observed to inflictmore damage on certainBt-maize hybrids than susceptible larvae, it was not re-ported whether the larvae could survive and develop on such plants (46). Cry1Acselection on otherO. nubilaliscolonies led to increased resistance to this toxinwith resistance levels peaking at about 160-fold after 8 generations of selection.



Continued selection resulted in a decrease rather than further increase of Cry1Acresistance (10).

Selection ofPectinophora gossypiellalarvae with Cry1Ac protoxin resultedin a 300-fold-resistance level. High levels of cross-resistance were observed forCry1Aa and Cry1Ab protoxin and only low levels for Cry1Bb protoxin. The colonyshowed no cross-resistance to Cry1Ca, Cry1Da, Cry1Fa, Cry1Ja, and Cry2Aa pro-toxins or to Cry9Ca toxin. Larvae from this colony (AZP-R) showed 40% adjustedsurvival (percentage of survival onBt plants divided by percentage of survival onnon-Bt plants) on Cry1Ac-expressing cotton compared with 1.6% adjusted sur-vival for susceptible larvae (111). Following three rounds of Cry1Ac selection ofAPHIS-BTX, anotherP. gossypiellacolony previously exposed to artificial dietcontaining leaf powder from Cry1Ac-cotton, resistance to Cry1Ac increased tomore than 100-fold (colony APHIS-98R) in comparison with a susceptible colony(APHIS-S) (61). APHIS-98R showed a narrow spectrum of cross-resistance, sim-ilar to colony AZP-R, with limited cross-resistance to Cry1Ja protoxin (111).

Diptera

Cyt1A plays an important role in the (lack of) development of resistance inmosquitoes towardBt var. israelensis(Bti). Indeed, there are no reported casesof field or significant laboratory resistance toBti (132). In selection experimentsusing individual toxins or combinations of individual toxins fromBti on a labo-ratory colony ofCulex quinquefasciatus, the rate and final level of resistance (atleast at the LC95 level, concentration required to kill 95% of insects) was inverselycorrelated with the number of constituent toxins in the selecting agent. Whereas allselected lines displayed varying levels of resistance to all selecting agents not con-taining Cyt1Aa, none of the selected lines showed significant levels of resistanceto the Cry4A/Cry4B/Cry11A/Cyt1A mixture, which suggests that the presenceof Cyt1Aa suppresses the development of resistance toward Cry4/Cry11 toxins(27, 131). It is interesting that combining Cyt1Aa with Cry4 or Cry11 proteinsrestored the toxicity of Cry4 and Cry11 againstCx. quinquefasciatuscolonies re-sistant against the latter proteins (132). It is possible that the presence of Cyt1Afacilitates the binding and/or membrane insertion of Cry4/Cry11 proteins in re-sistant larvae. In this context, it is noteworthy that the level of synergy betweenCyt1Aa and Cry4 and/or Cry11 was higher in resistant larvae than in susceptiblelarvae. A similar synergic effect of Cyt1A has also been observed with the binarytoxin of Bacillus sphaericus(130, 133).

Coleoptera

A colony of Leptinotarsa decemlineata, established from adults collected fromfields sprayed withBt formulations containing Cry3Aa, was subjected to labora-tory selection with Cry3Aa formulations for 29 generations, resulting in 293-foldresistance (88, 128). No reduction in feeding, compared with susceptible larvaeon nontreated plants, was observed on potato foliage treated with the Cry3Aa



formulation. However, foliage from transgenic potatoes expressing Cry3Aa virtu-ally eliminated feeding by resistant larvae within 4 h (2).

Selection with Cry3Aa elicited high levels of resistance in another coleopteranspecies,Chrysomela scripta(9). Cross-resistance to Cry1Ba (400-fold), but not toCyt1Aa, was observed in this colony (19). The observation of coleopteran activityof Cyt1Aa, although to a much lower extent than Cry3Aa, on the susceptible colonyis remarkable because this toxin had been considered to be strictly dipteran-specificon ingestion. Although Cyt1Aa suppresses Cry resistance in Diptera (132) and canovercome Cry resistance in Coleoptera (19), suppression of resistance to Cry1Aproteins by Cyt1Aa did not occur in resistant colonies from two lepidopteranspecies,P. xylostellaandP. gossypiella(77).

BIOCHEMICAL BASIS OF RESISTANCE

In principle, the mechanism of insect resistance toBt could be located at each ofthe various steps in the mode of action ofBt Cry proteins (solubilization, prote-olytic processing, passage through the peritrophic membrane, receptor binding,membrane insertion, pore formation, and osmotic lysis of midgut cells) (39, 124).Whereas different mechanisms have been observed in resistant colonies selectedunder laboratory conditions, only one major mechanism has been reported so farfor resistance developed under field conditions (Table 2).

Altered Proteolytic Processing

P. interpunctellacolony 198r displays resistance to the selecting agent,BteHD-198,and to purified Cry toxins, including Cry1Ac. Midgut extracts from the resistantinsects had lower proteolytic activity toward severalp-nitroanilide substrates thanextracts from susceptible insects and had a reduced capacity to activate Cry1Acprotoxin (84). A subsequent study demonstrated a genetic linkage between de-creased susceptibility to Cry1Ac and the absence of a major gut protease (83).Moreover, the involvement of changes in midgut proteases in resistance was fur-ther corroborated by the observation of 11-fold higher resistance levels for Cry1Abprotoxin than for Cry1Ab toxin in this colony (45). Forcada et al. (22) reporteda slower activation of Cry1Ab protoxin and a faster degradation of Cry1Ab toxinin midgut extracts from larvae of theH. virescensCP73-3 colony compared withextracts from susceptible larvae. In the NO-95C colony ofP. xylostella, resis-tance levels for crystalline Cry1Ca protoxin were about 2.5-fold higher than forCry1Ca toxin. Thus, reduced conversion to toxin is a minor mechanism of re-sistance in this colony (60). The resistance mechanism in the 343-R colony ofP. interpunctellawas not related to altered proteolytic processing (50). Similarly,gut protease activity was not altered in theH. virescensSEL colony (68), andit probably does not contribute to resistance in theP. xylostellacolony NO-QA(58, 105).


TA

BL

E2

Sele

cted

inse

ctsp

ecie

san

dst

rain

sth

atha

vede

velo

ped

resi

stan

ceto

B.t

huri

ngie

nsis

Det

ails

onre

sist

ance

Sele

ctin

gag

ent

Sele

ctio

nE

SdF

orm

ulat

ion/

RR

eR

esis

tanc

eSp

ecie

sO

rigi

nT

ypea

For

mb

envi

ronm

ent

Nam

ecTo

xin

mec

hani

smf

Ref

.

P.in

terp

unct

ella

Okl

ahom

aD

ipel

FSC

Bin

s/L

ab34

3-R

36D

ipel

>25

0P

50,7

2C

ry1A

b(8

77)

↓B12

7

P.in

terp

unct

ella

Kan

sas

Bte

HD

-198

SCL

ab19

8r19

HD

-198

32Ñ

75Ñ

Cry

1Ac

(PT

)12

8↓A

85Ñ

Cry

1Ab

251

B45

ÑC

ry1A

cÑ

B45

P.in

terp

unct

ella

Kan

sas

Dip

elFS

CL

abD

plr

24D

ipel

70Ñ

74Ñ

Cry

1Ab

290

↓B45

P.xy

lost

ella

Phili

ppin

esB

tkFS

CFi

eld

BL§

ÑD

ipel

1Ñ

21Ñ

Cry

1Ab

>20

0↓B

21

P.xy

lost

ella

Phili

ppin

esB

tkFS

CFi

eld

ÑD

ipel

0.4

Ñ7

ÑC

ry1A

b23

6Ñ

7A

bove

colo

nyC

ry1A

b/T

/CC

Lab

PHI

ÑC

ry1A

a,C

ry1A

c>

1B

8,11

3Ñ

Cry

1Ab

>1

↓B11

3

P.xy

lost

ella

Haw

aii

Btk

/Dip

elFS

CFi

eld/

Lab

NO

-QA

ÑD

ipel

3300

P58

,105

,108

ÑC

ry1A

a>

100

1B

8,11

3,11

5Ñ

Cry

1Ab

>10

0↓B

113,

115

ÑC

ry1A

c>

100

↓B10

3,11

3,11

5

P.xy

lost

ella

Haw

aii

Btk

/Bta

/Cry

1Ca

FSC

/CC

/TFi

eld/

Lab

NO

-95C

11C

ry1C

a19

B60

11C

ry1C

a(P

T)

48Ñ

60

P.xy

lost

ella

Flor

ida

Btk

FSC

Fiel

dL

oxa

AÑ

Jave

lin30

0Ñ

121

ÑC

ry1A

b>

200

↓B12

1

P.xy

lost

ella

Sout

hB

tk/B

ta/C

ry1C

aFS

C/P

T/C

ry1C

-Fi

eld/

Lab

Cry

1C-S

elÑ

Cry

1Ca

1240

01

B13

6C

arol

ina

broc

coli

Ñ≡ ≡ ≡ ≡ ≡

Cry

1Ac-

1Ab


P.xy

lost

ella

Penn

sylv

ania

Btk

/Cry

1Ac,

FSC

Fiel

d/L

abPE

N6

Cry

1Aa

Hig

h1

B8,

113

Cry

1Ca

6C

ry1A

b,C

ry1A

cH

igh

↓B11

3

P.xy

lost

ella

Mal

aysi

aB

tk/B

taFS

CFi

eld

SER

D3

ÑD

ipel

330

Ñ13

4Ñ

Flor

bac

160

Ñ13

4Ñ

Cry

1Ab

Ñ↓B

134

ÑC

ry1A

a,C

ry1A

cÑ

B13

4

P.xy

lost

ella

Mal

aysi

aB

tk/C

ry1A

cFS

C/T

Fiel

d/L

ab1A

cSE

L-

5C

ry1A

c10

500

↓B93

ME

L§

5C

ry1A

b26

4↓B

93

H.v

ires

cens

Nor

thC

ry1A

c/C

ry1A

cT

/CC

Lab

YH

D2

ÑC

ry1A

a↓B

33,5

5C

arol

ina

ÑC

ry1A

b>

2300

33,5

519

Cry

1Ac

>10

000

33,5

5Ñ

Cry

1Fa

(CC

)37

00Ñ

33H

.vir

esce

nsN

orth

Cry

1Ab/

Dip

elC

C/F

SCL

abSE

L22

Cry

1Ab

(CC

)71

1B

,P

65,6

8C

arol

ina

22C

ry1A

c16

1B

,P

65,6

8

H.v

ires

cens

Nor

thC

ry1A

cT

Lab

CP7

3-3

17C

ry1A

b13

B,

↓A22

,23,

34,6

7C

arol

ina

↑D,

↑CP

17C

ry1A

c50

B,

↑CP

34,6

7

S.ex

igua

Ala

bam

aC

ry1C

aIB

/TL

ab25

Cry

1Ca

850

↑NS

B78

a Bta

(Btv

ar.a

izaw

ai)

and

Btk

(Btv

ar.k

urst

aki)

refe

rto

com

mer

cial

form

ulat

ions

ofB

.thu

ring

iens

is.D

ipel

and

Jave

linar

etr

aden

ames

for

com

mer

cial

form

ulat

ions

ofB

tk.F

lorb

acis

atr

aden

ame

for

aco

mm

erci

alfo

rmul

atio

nof

Bta

.Bte

=B

tvar

.ent

omoc

idus

.b Fo

rmul

ated

spor

e-cr

ysta

lpre

para

tions

(FSC

),sp

ore-

crys

talp

repa

ratio

ns(S

C),

mic

roen

caps

ulat

edre

com

bina

ntP.

fluo

resc

ens

cells

expr

essi

nga

cry

gene

(CC

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Binding Site Modification

Receptor-binding studies using brush border membrane vesicles (BBMV) preparedfrom midguts of resistantP. interpunctella343-R larvae showed a 50-fold reductionin binding affinity (Kd) but showed no change in the number of binding sites (Rt)for Cry1Ab (127). TheKd value for Cry1Ca, which binds to another high-affinitysite, was similar in both the resistant and susceptible colonies, but theRt valuewas significantly higher (threefold) in the resistant colony. These data providedevidence that resistance was due to an alteration only in the binding site for Cry1Ab.The increase in the number of binding sites for Cry1Ca could explain the highersusceptibility of the selected colony toward this crystal protein.

Similar to colony 343-R, Cry1Ab resistance in the Dplr P. interpunctellacolonywas associated with a 60-fold reduction in binding affinity (45). In contrast, Cry1Acbinding in the latter colony did not differ from the susceptible colony, althoughCry1Ac shares at least one binding site with Cry1Ab. Perhaps the alteration inthis Cry1A binding site has an impact on binding affinity, in case of Cry1Ab, andonly on post-binding events, such as membrane insertion, in case of Cry1Ac. Incontrast to colonies 343-R and Dplr, colony 198r showed only a slight reductionin binding of Cry1Ab (fivefold higherKd and threefold lowerRt) (45).

High levels of resistance to Cry1Ac in the YHD2H. virescenscolony werenot correlated with altered binding of Cry1Ac, nor of Cry1Ab, to which it washighly cross-resistant. However, binding of Cry1Aa, to which the colony wasmoderately cross-resistant, to BBMV of resistant larvae was dramatically reduced(55). Competition-binding studies (55, 126) had led to the following model forCry1A binding sites inH. virescens: Cry1Aa binds to receptor A; Cry1Ab bindsto this receptor and also to receptor B; and Cry1Ac recognizes both of thesesites, as well as receptor C. Thus, Cry1Ac and Cry1Ab also bind to the Cry1Aabinding site. Consequently, it was proposed that the altered Cry1Aa binding sitecauses resistance to all three Cry1A proteins and that the additional binding sitesrecognized by Cry1Ab and Cry1Ac may not be involved in toxicity (55). Recently,it was demonstrated that Cry1Fa, as well as Cry1Ja, share the “A” binding site withCry1A toxins inH. virescens(51). BBMV from colony YHD21000MVP, obtainedfollowing continued selection on Cry1Ac, no longer bound any of the three Cry1Atoxins nor Cry1F. It is not a surprise that none of these toxins could permeate theseBBMV, as assessed by a pore-formation assay using a light-scattering technique,whereas all toxins permeated BBMV from the susceptible colony (J.L. Jurat-Fuentes, F. Gould & M.J. Adang, personal communication).

In contrast to the YHD2 colony, binding experiments in the SEL and CP73-3H. virescenscolonies showed either small and compensatory changes inKd andRt values (SEL) (65) or no differences for binding of Cry1Ab and Cry1Ac (CP73-3) (34) compared with the respective susceptible colonies. However, it shouldbe pointed out that binding of Cry1Aa was not studied in these two colonies.Therefore, if the mechanism observed in colony YHD2 was also present in thesecolonies, it would have been overlooked.



Figure 1 Proposed model for binding ofBt Cry proteins to the brush border mem-brane of midgut cells ofP. xylostellalarvae.

Cry1Ac resistance inP. xylostellacolony NO-QA was demonstrated to be dueto dramatically reduced binding (103). Cross-resistance in this colony extendsto Cry1Aa, Cry1Ab, Cry1Fa, and Cry1Ja (113). At first this seemed remarkablein view of the limited amino acid sequence homology between Cry1A proteinsand Cry1Fa and Cry1Ja. However, taking into account the model for the Cryprotein–binding sites inP. xylostella(Figure 1), these results can be easily under-stood. Compilation of results from homologous and heterologous competition–binding experiments in susceptibleP. xylostellacolonies (7, 8, 21, 35; S. Herrero,J. Gonzalez-Cabrera, B. Tabashnik & J. Ferr´e, unpublished information) indicatesthe presence of (at least) four Cry binding sites: One (site 1) is recognized only byCry1Aa; another (site 2) is shared among Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa, andCry1Ja; and two additional sites bind Cry1Ba and Cry1Ca (sites 3 and 4, respec-tively). Thus, the absence of cross-resistance to Cry1Ba and Cry1Ca and unalteredbinding of Cry1Ca (103, 113) in NO-QA is in agreement with this model. LikeCry1Ac, binding of Cry1Ab was virtually absent to BBMV from resistant insects(113). In contrast, Cry1Aa bound equally well to BBMV from resistant and suscep-tible larvae (8, 113). From the binding and cross-resistance data, Cry1Aa can stillbind to site 1 in NO-QA, and this additional Cry1Aa binding site is apparently notinvolved in toxicity. In conclusion, if we consider only sites 2, 3, and 4 to be func-tional Cry receptors, patterns of cross-resistance correspond to patterns of receptorspecificity. Indeed, while Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa, and Cry1Ja competefor the altered site 2, Cry1Ba and Cry1Ca recognize the unaltered sites 3 and 4



and remain fully effective toward resistant larvae. Moreover, patterns of receptorspecificity inP. xylostellacorresponded to levels of amino acid sequence homologyin domain II (115), known to play a crucial role in receptor binding (94). Surpris-ingly, binding of Cry1A proteins to the resistant larvae could be demonstratedin alternative binding assays [surface plasmon resonance–based binding analysis(70), binding to tissue sections (16), or to purified aminopeptidase (41)], whichsuggests that there is a difference in the accessibility of the common Cry1A bindingsite in the different assay systems or that new binding sites become accessible.

A P. xylostellacolony (PEN) from Pennsylvania with a similar spectrum ofcross-resistance also showed decreased binding of Cry1Ab and Cry1Ac but notof Cry1Aa (113). The mechanism of resistance in theP. xylostellaLoxa colonywas similar. Indeed, a virtually complete lack of Cry1Ab binding, but unalteredCry1Ba and Cry1Ca binding, was demonstrated in the resistant colony (121).

Likewise, whereas BBMV from the resistantP. xylostellaBL colony and thesusceptible colony did not show any significant difference in binding of Cry1Baand Cry1Ca, specific binding of Cry1Ab could be obtained only with BBMV fromsusceptible larvae (21). Lack of binding of Cry1Ab to the resistant colony was alsoconfirmed by a histological study of resistant larvae intoxicated with Cry1Ab (12).Furthermore, there was no damage to midgut epithelial cells from these larvae,whereas cells from susceptible larvae showed clear binding and damage. In contrastto Cry1Ab, Cry1Ba bound to and damaged midgut cells in both resistant andsusceptible larvae. These data clearly demonstrated a causal relationship betweendecreased binding and decreased susceptibility (resistance).

P. xylostellacolony PHI was partially resistant to Cry1Aa, Cry1Ab, and Cry1Acand still susceptible to Cry1Ca, Cry1Fa, and Cry1Ja (113). Binding of Cry1Ab,but not of Cry1Aa and Cry1Ac, was dramatically reduced in this colony (8, 113).Thus, an alteration in site 2 may affect binding of Cry1Ab without affecting Cry1Aaand Cry1Ac binding, which suggests that the binding epitopes of these three Cryproteins are not identical. Therefore, an additional mechanism of resistance, otherthan a reduction in binding, must be involved in this colony.

A large decrease in binding of Cry1Ab, but not of Cry1Aa, Cry1Ac, or Cry1Ca,was observed inP. xylostellaSERD3 larvae from Malaysia (134). Taking intoaccount the binding-site model forP. xylostella(Figure 1), these data illustrateanother example where modification of a shared binding site (site 2) affects onlyone Cry protein recognizing this site, which suggests that the binding epitopes ofthe different toxins only partially overlap. In contrast, in a different colony fromMalaysia (1AcSEL-MEL), resistance to both Cry1Ac and Cry1Ab was correlatedwith a dramatic reduction in binding of both toxins (93).

Thus, we can discriminate two types of binding-site alterations inP. xylostella.Colony PHI is an example of a resistant colony containing a type I binding sitealteration: Here, a change in binding site 2 affects binding of Cry1Ab but notof Cry1Aa or Cry1Ac. Type II binding-site alterations affect binding of Cry1Aband Cry1Ac owing to an alteration in site 2; this type of alteration also affectsbinding of Cry1Aa to site 2, but this effect is masked by unaltered binding ofCry1Aa to site 1. This type of binding-site alteration has been observed in colonies



NO-QA and PEN. Because these colonies are cross-resistant to Cry1Fa and Cry1Jaand because these toxins share binding sites with Cry1Ab/Cry1Ac toxins, it istempting to speculate that the alteration in site 2 in the above colonies also reducesbinding of the former Cry proteins.

Even if resistance is controlled by one locus and is due to a change in a receptorshared among various toxins, the resistance ratios for these various toxins candiffer significantly. For example, in theBtkSEL-MEL colony, where resistance toBtk is monogenic (92), cross-resistance to Cry1Ac (10,700-fold) was significantlyhigher than to Cry1Ab (900-fold) (93). Cry1Ac and Cry1Ab resistance is likely dueto the same single locus as resistance toBtk. Assuming that both toxins recognizeidentical epitopes on site 2, it is possible that one particular mutation in site 2affects binding of Cry1Ac more than binding of Cry1Ab. Alternatively, the bindingepitopes of site 2 may overlap only partially for Cry1Ab and Cry1Ac. The existenceof type I binding-site alterations would seem to favor the latter hypothesis.

Reduction of binding does not play a (major) role in Cry1Ca resistance inP.xylostellacolonies NO-95C (60) or Cry1C-Sel (136). High levels of resistance toCry1Ca in aS. exiguacolony could not be explained by dramatic alterations inbinding: Only a fivefold increase inKd and a higher level of nonspecific bindingwere observed in comparison with the susceptible colony (78).

Other Mechanisms

Ingestion of a sublethal dose of Cry1Ac by fourth instar CP73-3H. virescenslarvae resulted in similar histopathological changes in columnar gut cells com-pared with cell damage in susceptible larvae (67). Likewise, larvae from both asusceptible colony and another resistantH. virescenscolony (KCB) showed com-parable midgut epithelium damage following Cry1Ac ingestion (23). Therefore,it is possible that resistance in CP73-3 and KCB is due to a more efficient repair(or replacement) of damaged midgut cells.

GENETICS OF RESISTANCE

Intraspecific Variation in Baseline Susceptibility

One approach to estimate the potential of insect populations to evolve resistance toBt is to determine differences in susceptibility within and among populations. Intwo studies carried out to determine susceptibility of two lepidopteran forest pests,Choristoneura fumiferana(11 populations) andLymantria dispar(4 populations),to Btk HD-1, intrapopulation variation was higher than interpopulation variation(91, 123).

Studies measuring variation in susceptibility among, but not within, populationsof the same species are more abundant. In a study with Spanish populations ofcorn borers, small differences in susceptibility to Cry1Ab were found among fourpopulations ofSesamia nonagroides(threefold), but no differences between twopopulations ofO. nubilalis(29). In another study withO. nubilalis, the variation in



susceptibility to Dipel among five colonies of insects collected from three differentU.S. states was also low (1.9-fold) (47). Despite the low level of natural variation,all five colonies responded rapidly to intense selection pressure. This indicates thatlow variability in baseline susceptibility among populations does not necessarilyimply low potential to respond to selection pressure because variability withinpopulations can still be high.

Higher differences in baseline susceptibility were found in heliothine species.In one study with 12 populations ofH. virescensand 15 populations ofHelicoverpazea, the range of LC50among populations of a given species varied up to 16-fold forCry1Ac and up to 13-fold for Dipel (99). In another study testing Cry1Ab, Cry1Ac,and threeBt products (Javelin, Dipel, and Condor), variability was maximal forDipel (71-fold) among 16 colonies ofH. virescensand for Cry1Ac (441-fold)among 11 colonies ofH. zea(64). In studies withHelicoverpa armigerain China,variation of LC50 for Cry1Ac among 23 populations was 100-fold (135) and forBtkHD-1 among 8 populations was 4-fold (137).

Significant differences in baseline susceptibility toBtkproducts were also foundin P. interpunctella(42-fold among 13 populations) (52),S. exigua(554-fold toDipel among 8 colonies) (64), and severalP. xylostellapopulations (8- to 18-fold)(15, 80, 86, 96, 102).

Variability for Resistance Genes as Detectedby Laboratory Selection

The heritability (h2) is the proportion of phenotypic variation for a given trait ac-counted for by additive genetic variation (18). Measurements of theh2 in laboratoryselection experiments can be used to estimate the variability for resistance genes inpopulations. No assumptions about mode of inheritance are made to estimateh2.In selection experiments, realizedh2 is estimated as the ratio between the responseto selection (R) and the selection differential (S). Tabashnik (101) estimated real-izedh2 of resistance toBt products and Cry1A toxins for 27 selection experimentsand found a relatively highh2 in P. interpunctella(0.22–0.61 in 11 experiments,mean= 0.33) compared with 6 other moth species (0.04–0.20 in 10 experiments,mean= 0.13), the coleopteranL. decemlineata(0.09 and 015 in two experiments),and with the dipteranAedes aegypti(0.01–0.10 in four experiments, mean= 0.04).Relatively high realizedh2 (up to 0.44) was found inP. xylostellain two popula-tions from Malaysia (93, 134). The relatively highh2 in P. interpunctellaand inP.xylostellafrom Malaysia indicates high additive genetic variation for susceptibilityto Cry proteins in those populations.

Estimation of Resistance Gene Frequency

The initial frequency of an insecticide-resistance allele is one of the key ele-ments for predicting the rate of evolution of resistance in a population subjected toperiodic insecticide treatments. In spite of the obvious interest to obtain estimatesof initial frequencies of resistance alleles, only a few studies have been carried outto estimate the frequency of major resistance genes in field populations conferring



resistance toBt products or toxins. An indirect estimate of the frequency of suchalleles is provided by laboratory selection experiments that have succeeded in se-lecting for resistance toBt. Assuming no resistant mutants arose after selectionbegan, at least one copy of the resistance allele was present when selection started.Given that successful selection experiments withBt in Lepidoptera used a smallsample of insects from the field population (from 100 to 700) (33, 34, 74), thefrequency of resistance alleles in those populations must have been relatively high(from∼1× 10−3 to 5× 10−3). However, this value can be overestimated if we donot consider other selection attempts in these same populations leading to unsuc-cessful results (33). Another bias in these type of estimates is when there has beena history of previous exposure toBt, either as a natural infestation or to inadvertentbioinsecticide treatments (17, 74).

A more direct approach to estimate the frequency of a majorBt-resistance allelehas been applied to field populations ofH. virescensmaking use of a practicallyhomozygous resistant strain for a recessive resistance allele (32). Over 2000 maleswere collected in four states of the United States and were individually mated tofemales of the resistant strain. By testing the F1 and F2 offspring from over 1000 ofthose single pair matings, the authors estimated a frequency of resistance allelesin the field sample of 1.5× 10−3, in close agreement with a preliminary estimateobtained from a selection experiment (33). One main disadvantage of the abovedirect approach is that the estimate applies only to recessive alleles of the locusfor which the laboratory strain is homozygous for resistance. Frequencies of otherrecessive alleles at different loci escape detection.

Andow & Alstad (4) proposed a method to detect and estimate frequencies ofresistance alleles in field populations based on an F2-screening procedure. The ad-vantages over other methods are that it does not require use of a resistant laboratorystrain and that it is far more sensitive (more than 10 times) than a discriminating-dose assay for detection of recessive traits. When this method was applied to twofield populations ofO. nubilalis, no resistant homozygotes were found for majorresistance genes, and the estimated frequency of Cry1Ab-resistance alleles was<0.013 for a Minnesota population (5) and<0.0039 for an Iowa population (6).In a study with populations from Australia, this method detected a frequency of4× 10−3 for low-level resistance alleles but<10−3 for high-level resistance inP. xylostella, and<7× 10−4 for any type of resistance alleles inH. armigera(1).These figures are in strong contrast with the extremely high frequency of resistancealleles (namely 0.12) reported for a susceptible laboratory colony ofP. xylostellafrom Hawaii (112) or in field populations ofP. gossypiellasampled in 1997 incotton fields in Arizona (average frequency of 0.16) (116). It is remarkable thatin the latter study the frequency of resistance alleles in samples collected in 1998and 1999 from the same cotton fields was much lower (<8× 10−4).

Mode of Inheritance of Resistance

Bioassays to determine the dominance level have been carried out in three typesof environments (Table 3). Those with transgenic plants expressingBt toxins are



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the most instructive with respect to implications for resistance management forBtcrops because they best represent the situation that insects are going to face whenexposed to such crops. Leaf-dip bioassays simulate the situation rather well inthe field because insects ingestBt toxins or spores/crystal mixtures along with thefresh vegetable material. Finally, bioassays on artificial diets are the least similar tothe field situation. Because different concentrations of the insecticidal agent can beused in leaf-dip and in artificial-diet bioassays, the dominance based on mortalityat single concentrations of toxin varies substantially because it depends on theconcentration tested. Alternatively, if dose-mortality curves are used to determinethe dominance level, the value obtained will indicate the relationship among theLC values (normally LC50) of the susceptible homozygous, resistant homozygous,and heterozygous individuals. However, the dominance level obtained in this waymay have little relevance to resistance management because the practical point instrategies to delay resistance is to determine whether it is technically feasible tofind a dose that kills all heterozygous insects.

Bourguet et al. (11) have pointed out the differences among the dominancevalues obtained from single-dose mortality tests (DML, dominance of survival at agiven insecticide dose or “effective dominance”), from LC values (normally LC50

values) of dose-mortality curves (DLC, dominance of insecticide resistance), andfrom the fitness of the three genotypes in insecticide-treated areas (DWT, dominanceof relative fitness in the treated area). Of the three, the latter is the most relevantparameter to resistance management, although it is the most difficult to estimate.The range for all three parameters is the same: from 0 for complete recessivity to1 for complete dominance. The only straightforward relationship among them iswhenDML = 0, thenDWT equalsDML, and no heterozygote survives treatment.However,DWT can still be 0 even ifDML > 0 (11). In contrast, the range forD,Stone’s degree of dominance (98), is from−1 to 1. DLC is related toD by theexpressionDLC = (D + 1)/2 (11).

Survival of heterozygotes on transgenic plants has been measured withP. xy-lostellain transgenic broccoli expressing eithercry1Ac(76, 118) orcry1Ca(136),and withP. gossypiellain transgenic cotton expressingcry1Ac(59). An importantconclusion from these studies is that in all three cases, the effective dominance ofresistance was 0, which is one of the requirements of the high-dose/refuge strategy.

Of the differentDML values reported forP. xylostellaandP. gossypiella, Table 3shows the lowest value obtained for a givenBt product or Cry protein, which rep-resents the highest level of recessivity that was obtained with the concentrationstested. The overall conclusion is that, in general, resistance toBt products or tox-ins can behave as completely to partially recessive (57, 61, 92, 93, 113, 116, 136).Only for one colony (PHI) did resistance to Cry1Aa behave more dominant thanrecessive, and resistance to Cry1Ac behaved as codominant (113).

DLC values in Table 3 were calculated from LC50 values, and contrarily toDML,the former are absolute values because they do not depend on the concentrationof the insecticidal agent used. MostDLC values in Table 3 fall within the rangeof 0.24–0.47 (13 values out of 22), which indicates that resistance toBt products



and toxins, at the LC50 level, is in general partially recessive, although closer tocodominance than to complete recessivity (13, 33, 34, 48, 66, 71, 72, 92, 93, 136).In four cases, resistance was partially recessive, although closer to complete reces-sivity (DLC values from 0.09 to 0.16) (38, 71, 117, 120). Finally, there are five casesof DLC> 0.50, which indicates that resistance was more dominant than recessive.High dominance levels were obtained inO. nubilalis with Dipel (46) and inL.decemlineatawith Cry3Aa (88). More moderate dominance levels were obtainedin the NO-95C colony ofP. xylostellawith Cry1Ca (57), in the SEL colony ofH.virescenswith Cry1Ab (97), and in the YHD2 colony ofH. virescenswith Cry2Aa(33).

In general, tests to determine the number of loci involved in resistance indicatedthat backcross data fitted fairly well to a single locus model (or a set of tightlylinked loci) (Table 3). Notable exceptions to this are the resistance to Cry1Cain the Cry1C-Sel colony ofP. xylostella(136), resistance to Cry1Ab in the SELcolony ofH. virescens(97), resistance to Cry1Ca inS. littoralis(13), and resistanceto Cry3Aa inL. decemlineata(88). It is then likely that the values of dominanceobtained in these cases represent the combined result of the interaction of resistancealleles from the various loci involved.

At least in two cases the dominance of resistance has been approached froma biochemical perspective. In the BL colony ofP. xylostella, F1 insects showedbinding of this toxin, and thus, lack of binding was inherited as an autosomalrecessive trait (66). Crosses between susceptible insects and insects from the 198r

colony ofP. interpunctellaindicated that, similarly to the susceptible insects, F1

insects possessed the major gut protease T1, whose absence is characteristic of theresistant insects (83). Thus, the absence of this protease is inherited as a recessivetrait.

In all cases of resistance toBt studied so far, resistance was autosomally in-herited. However, there are a few examples where the sex of the resistant parentshad a significant influence on the survival of the F1, such as in the BL and theCry1Ac-SEL colonies ofP. xylostella(66, 93) and a colony ofS. littoralis (13).

Complementation tests to determine if resistance in different populations isdue to mutations at the same locus or at different loci have only been carriedout with three colonies ofP. xylostellafrom distant geographic areas (113, 114).The F1 progeny resulting from the cross NO-QA×PEN showed the same patternof resistance to Cry1A and Cry1F toxins as the parental colonies (see Table 2).Furthermore, the F1 progeny of PHI×NO-QA and PHI×PEN were resistant toCry1Ab (Cry1Ab is the only toxin to which PHI showed recessive inheritance).The results suggested that the three colonies shared a genetic locus that controlledresistance to Cry proteins, that the same or a different mutation in NO-QA andPEN conferred resistance to Cry1A and Cry1F toxins, and that a different mutationin this locus in PHI conferred resistance to Cry1Ab. As already indicated, the PHIcolony also differed from the two other colonies at the biochemical level (8, 113).Additional work supported the hypothesis that the mutation conferring resistanceto Cry1A and Cry1F toxins also confers resistance to Cry1Ja (110).



Stability of Resistance

A crucial requisite in management strategies based on rotations of insecticides orof crops expressing different Cry proteins is that the resistance level decreases onceselection pressure is discontinued. In most studies on resistance toBt products andtoxins, resistance reverted once selection ceased, most likely owing to fitness costsassociated with resistance genes or with other loci closely linked to these. However,there are a few examples in which resistance levels remained stable after selectionwas discontinued. In the 343-R colony ofP. interpunctella, resistance to Dipeldid not decline even after 29 generations on untreated diet (72). No fitness costseemed to be associated to resistance in this colony either on untreated diet or ontreated diet (82).P. xylostellaprovides three additional examples where resistanceremained high and stable after discontinuing selection (62, 86, 107).

In the studies discussed below, resistance is unstable. To measure the rate atwhich resistance declines, Tabashnik et al. proposed to use the genetic parameterR, which is usually used to measure response to selection (109).R, or the averagerate of decline in resistance per generation, can be calculated as log (final LC50)—log (initial LC50) divided by the number of generations without exposure to theinsecticide, or by an equivalent equation in which the above numerator is substi-tuted by log (final resistance ratio/initial resistance ratio) (109). The inverse of theabsolute value ofR is the number of generations required for a tenfold change inLC50. Because of its intuitive value, we use this parameter in our review, and inthe studies in which this parameter was not given, we have estimatedR from theLC50 values or resistance ratios.

In P. xylostellafrom Hawaii, a rapid decline in resistance to Dipel (R rangedfrom −0.26 to−0.30) was found in three resistant laboratory-selected coloniesderived from the NO field–resistant population (103, 109). The LC50 of the mostresistant colony (NO-Q, 2800-fold resistant) declined to a similar value to that ofa susceptible colony in 13 generations without selection. The decline was muchslower in an unselected colony, with 22-fold resistance, for whichR = −0.06.The revertant NO-Q colony was reselected with Dipel and the NO-QA colony wasderived. Using cabbage leaves, decreased fitness was found in the NO-QA colonycompared with the revertant NO-Q colony (36, 37, 103). However, no differencein fitness was found in NO-QA compared with a susceptible colony (LAB-PS)on nontransgenic canola plants, or for NO-QA insects between nontransgenic andtransgenic canola expressing thecry1Acgene (89).

In the Loxa A colony ofP. xylostella, derived from field-resistant insects, resis-tance to Javelin was unstable from the second to the third generation (R = −0.50),but thereafter it reached a plateau (150- to 300-fold resistance) and remained prac-tically stable for at least 7 generations (120). Fast reversal of resistance was foundin a number of other populations ofP. xylostella(38, 92, 134, 136).

Similar studies were conducted on other insect species. InH. virescens(SELcolony), resistance to Cry1Ab protoxin declined from 69-fold to 13-fold in 5generations of nonselection (R = −0.14) and seemed to remain stable thereafter



(97). No differences in fitness were detected in a colony derived from these insects(31). When selection with Cry1C was discontinued in a colony ofS. littoralis(resistance>500-fold), resistance declined in one generation, remained relativelystable in the subsequent five generations, and then declined again in the next twogenerations (R< −0.21 for the eight generations) (81). There were no significantdifferences between the resistant colony and a susceptible colony in sex ratio andpupal weights; however, development time was increased in the resistant insects. Inthree different experiments with a resistant colony ofL. decemlineata(80- to 223-fold resistant), resistance to Cry3A declined to a stable 30- to 80-fold level in fourto eight generations after selection was stopped (R = −0.08 to−0.11) (88, 129).Significant reduction in several fitness components, relative to susceptible insects,was detected in the resistant colony (3, 122). Resistance to Cry1Ac was also foundto be unstable in a laboratory-selected colony ofO. nubilalis(R = −0.31) (10).

Simultaneous Occurrence of Different Resistance Genes

For the sake of simplicity, most theoretical models describing the evolution of resis-tance in natural populations are developed considering monogenic inheritance ofresistance. However, results obtained by different approaches indicate that insectpopulations may contain more than one gene conferring resistance to Cry pro-teins. A first line of evidence comes from backcross experiments, which showedthat resistance to Cry proteins in some laboratory-selected colonies did not fit amonofactorial pattern of inheritance, which suggests that resistance was caused bymore than one gene (13, 88, 97, 136; Table 3). InH. virescens, genetic analysis ofYHD2 with marker loci (see next section) revealed that different linkage groupscontributed to resistance to Cry1Ac in this colony (43).

A more direct line of evidence comes from experiments in which resistance toone type of Cry protein (orBtproduct) segregates independently from resistance toa second type. In the SERD3 population ofP. xylostella, the response to selectionwith differentBt products and the cross-resistance pattern indicated that resistanceto Dipel segregated independently from resistance to Florbac (a commercial prod-uct based onBta) (134). In another study, split brood bioassays from single-paircrosses showed that resistance to Cry1Ab and Cry1Ca segregated independentlyin colony NO-95C ofP. xylostella(57). A similar approach in this same insectspecies revealed that in the colonies NO-QA, PEN, and PHI, there was at leastan additional locus for resistance to Cry1Aa segregating independently from themultitoxin resistance locus (112–114).

Reversion of resistance in selection experiments also provides indirect evidencefor the simultaneous occurrence of more than oneBt-resistance gene in a popu-lation. In the Loxa A colony ofP. xylostella, resistance to Javelin was extremelyhigh when the insects were first brought to the laboratory (>1500-fold) but thenrapidly declined in the absence of selection, stabilizing at a resistance ratio around300-fold (120). This observation, together with results from selection experimentson Loxa A, suggests that the field population contained at least two alleles (from



either the same or different genes) conferring resistance to Javelin. Results fromexperiments of prolonged selection of the NO-Q colony ofP. xylostellaand subse-quent relaxation of selection of the resistant line and six isofemale lines suggestedthat resistance to Dipel was not controlled solely by one locus with two alleles(107).

A different indirect line of evidence comes from the biochemical approach. InP. interpunctella, selection of a colony with differentBt serovars gave rise, amongothers, to a colony highly resistant toBtk HD-1 (Dplr) and to a colony (198r)moderately resistant toBteHD-198 (74). Resistance in colony 198r to HD-198 wasaccounted for in part by loss of a major gut protease (83) and in part by a reductionin Cry1Ab binding to the insect midgut (45). The Dplr colony has a differentalteration affecting Cry1Ab binding (45). These results suggest the presence of atleast three different mutations in genes involved in resistance toBt in the initialcolony. In the YHD2 colony ofH. virescens, there is a major partially recessive geneconferring resistance to structurally related toxins (Cry1Aa, Cry1Ab, Cry1Ac, andCry1Fa) by apparently modifying a membrane receptor (33, 55) and another gene(or genes) conferring low to moderate resistance to Cry1B, Cry1C, and Cry2Aa.This latter genetic mechanism may be the same as the one found in the CP73-3colony, responsible for broad-spectrum resistance and unlikely to be related toreceptor alteration (34).

Identification of Resistance Genes

The identification ofBt-resistance genes has proved elusive. Only in one case thebiochemical approach has given strong evidence of the identity of the resistancegene: Genetic linkage between resistance to Cry1Ac and a locus coding for amidgut protease was found in the 198r colony ofP. interpunctella(83). However,reduced binding of Cry proteins to membrane target sites is the best known, if notthe most common, mechanism of resistance toBtCry proteins. Cry1A-binding pro-teins, most of them belonging to the aminopeptidase N family, have been isolatedand characterized from several insect species (94). In spite of the sound evidencethat these putative membrane receptors specifically bind Cry1A proteins in vitro,there is so far no conclusive evidence that a modification affecting their primarystructure is responsible for reduced binding of Cry1A proteins or has any effecton the resistance level in the resistant insects (63, 79, 138).

The identity ofBt-resistance genes has also been approached by genetic analy-sis. Classical genetic linkage analysis using isozyme polymorphisms was appliedto the YHD2 colony ofH. virescens. The backcross design with 10 marker loci (on10 of the 31 chromosomes ofH. virescens) revealed the existence of a major locus,namedBtR-4, on linkage group 9 (marker locusmannose-6-phosphate isomerase),responsible for as much as 80% of to the total resistance to Cry1Ac (43). Addi-tionally, linkage group 11 (marker locusguanine deaminase) also made a smallcontribution to Cry1Ac resistance. The isozyme-linkage approach was applied tothe PHI colony ofP. xylostella, and a strong correlation was also found between



Cry1A resistance and two mannose-6-phosphate isomerase isozymes (44). It isnotable that linkage of a Cry1A-resistance gene to the same isozyme marker hasbeen observed in two Lepidoptera species. This suggests that YHD2 and PHI sharea common genetic mechanism of resistance involving a major gene and supportsthe hypothesis that some resistant strains in different lepidopteran species mayhave a common genetic mechanism of resistance to Cry1A proteins (114).

Linkage analysis ofBt-resistance genes inH. virescens, H. armigera, andP.xylostellahas also used DNA markers obtained by the restriction fragment lengthpolymorphism (RFLP), randomly amplified polymorphic DNA (RAPDs), andamplified fragment length polymorphism (AFLP) methods (40–42). TheBtR-1-resistance locus in the NO-QA colony ofP. xylostella, most likely correspondingto the multitoxin resistance gene, has been mapped in a 47 cM region of linkagegroup 7 at 8.4 cM of the closest AFLP marker (41). One of the linked markers, at18.5 cM from theBtR-1 locus, has been cloned and sequenced, and represents asequence-tagged site (STS) that can be used in linkage tests of candidate resistancegenes in this or other resistant colonies.

A different approach has made use of the nematodeCaenorhabditis elegans,in which ethyl-methane sulfonate mutagenesis allowed the isolation of ten reces-sive mutants resistant to Cry5B (69). Based on complementation tests, some ofthese mutations were allelic and defined five genes distributed in three chromo-somes. None of the genes mapped to areas with genes similar to those codingfor aminopeptidase N or cadherin-like proteins. In addition to the interest of thisstudy for the resistance management ofBt as a nematocide, it opens the possi-bility of C. elegansto serve as a model system to identifyBt-resistance genes ininsects.

CONCLUSIONS

Three different biochemical mechanisms of resistance toBt have been observed sofar: proteolytic processing of protoxins, improved repair of damaged midgut cells,and modification of a Cry protein–binding site. However, only for the binding-reduction mechanism has a causal link been observed between the biochemicalmodification and decreased susceptibility (resistance).

In all cases of binding site modification, resistance is due to a recessive or par-tially recessive mutation in a major autosomal gene, and strong cross-resistanceextends only to Cry proteins sharing binding sites. Such Cry proteins do not nec-essarily display high levels of sequence similarity.

A reduction in binding is a major mechanism of resistance in all cases of field-evolved resistance toBt products or Cry proteins inP. xylostella, except for resis-tance against Cry1Ca. This observation, together with the variety of Cry-bindingsites existing in insects, indicates that Cry proteins with different binding sitespecificity should be considered for use in “pyramiding” strategies for resistancemanagement.



Important variability in insect populations regarding genes conferring resistancetoBthas been shown in studies of baseline susceptibility and in laboratory selectionexperiments. There have been few studies addressed to obtain estimates of thefrequency ofBt-resistance genes. The results indicate that these genes may not beextremely rare, with values above 10−3 in some populations.

In all cases studied, resistance toBt is autosomally inherited, although in somecases the sex of the parents had some influence on the resistance level of theoffspring. Regarding the number of loci involved in resistance, in most casesinheritance is monofactorial. In the only study that included allelism tests amongdifferent resistant colonies of the same species, the results showed that threeP.xylostellacolonies from geographically distant areas carried mutations in the samelocus conferring resistance toBt.

Estimates of dominance levels of resistance depended on the experimentalprocedure utilized. In three cases that used transgenic plants, resistance to theexpressed Cry protein was completely recessive, an important observation withrespect to the efficiency of the high-dose/refuge resistance–management strategy(30). In most experiments usingBt products or Cry proteins, resistance behavedas completely to partially recessive (depending on the dose of toxin), althoughexamples exist of partially dominant resistance.

In the vast majority of cases, resistance toBtproducts and Cry proteins has beenfound to be unstable. Instability is most likely caused by fitness costs associatedwith resistance genes or with other loci closely linked to them. However, thereare a few examples in which resistance levels did not decline once selection wasdiscontinued.

Besides the cases where inheritance of resistance follows a multifactorial pat-tern, studies on the biochemical basis of resistance in some colonies uncoveredthe presence of more than one resistance mechanism, which suggests the in-volvement of more than one gene. Also, studies on stability of resistance andmode of inheritance indicate that it is not uncommon to find more than one resis-tance gene contributing to the overall resistance of a strain. In some cases, geneslinked toBt resistance have been genetically mapped, but none of such genes havebeen cloned yet. When such genes would become available, they would not onlyallow more detailed analysis of biochemical and genetic aspects of resistance,but they might also provide interesting tools to monitor development of (field)resistance.

It is expected that insect control strategies based on the insecticidal crystal pro-teins ofBt are going to increase in the future, especially with the wide adoptionof transgenic crops. Detailed understanding of resistance mechanisms, togetherwith increasing knowledge of pest biology and plant molecular biology (in orderto tailorcry gene expression in the case ofBt crops), and the possibility to experi-mentally evaluate resistance-management methods on a small scale should allowfor fine-tuning of existing management tactics and design alternative options. Thecareful implementation of such tactics should safeguard the value ofBt for insectcontrol.



ACKNOWLEDGMENTS

We thank B.E. Tabashnik, J. González-Cabrera, S. Hernández, B. Escriche, and S.Herrero for their critical reading of the manuscript, and to the latter for his helpin drawing the figure. This work was supported, in part, by grants 1FD97-0917-C02-01 and AGL2000-0840-C03-01 from the Ministerio de Ciencia y Tecnolog´ıa,Spain.

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NOTE ADDED IN PROOF

Two recent papers have identifiedBt resistance genes. InC. elegans, loss or re-duction of function of a putativeβ-1,3-galactosyltransferase is responsible forCry5B resistance (Griffitts JS, Whitacre JL, Stevens DE, Aroian RV. 2001. Bttoxin resistance from loss of a putative carbohydrate-modifying enzyme.Science293:860–64), while inH. virescens, Cry1Ac resistance is associated with disrup-tion of a cadherin-superfamily gene in a locus previously referred to asBtR-4(Gahan LJ, Gould F, Heckel DG. 2001. Identification of a gene associated with Btresistance inHeliothis virescens. Science293:857–60).

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