Relative fitness of Cry1A-resistant and -susceptible Helicoverpa armigera (Lepidoptera: Noctuidae) on conventional and transgenic cotton

Glasshouse and laboratory experiments were conducted to evaluate the relative fitness of Cry1A-susceptible and laboratory-selected resistant strains of Helicoverpa armigera (Hübner). Life history parameters of H. armigera larvae feeding on young cotton plants showed a significant developmental delay of up to 7 d for the resistant strain compared with the susceptible strain on non-Bacillus thuringiensis (Bt) cotton. This fitness cost was not evident on artificial diet. There was no developmental delay in the F1 hybrid progeny from the reciprocal backcross of the resistant and susceptible strains, indicating that the fitness cost is recessive. In two cohorts tested, survival to pupation of resistant larvae on Bt cotton expressing Cry1Ac was 54 and 51% lower than on non-Bt cotton, whereas all susceptible and F1 larvae tested on Cry1Ac cotton were killed. Mortality of susceptible larvae occurred in the first or second instar, whereas the F1 larvae were able to develop to later instars before dying, demonstrating that resistance is incompletely recessive. The intrinsic rate of increase was reduced by >50% in the resistant strain on Cry1Ac cotton compared with the susceptible strain on non-Bt cotton. There was a significant reduction in the survival of postdiapausal adults from the resistant strain and the F1 strains, indicating that there is a nonrecessive overwintering cost associated with Cry1A resistance in H. armigera.     

Altered Glycosylation of 63- and 68-kilodalton microvillar proteins in Heliothis virescens correlates with reduced Cry1 toxin binding,decreased pore formation,and increased resistance to Bacillus thuringiensis Cry1 toxins

The binding and pore formation abilities of Cry1A and Cry1Fa Bacillus thuringiensis toxins were analyzed by using brush border membrane vesicles (BBMV) prepared from sensitive (YDK) and resistant (YHD2) strains of Heliothis virescens. 125I-labeled Cry1Aa, Cry1Ab, and Cry1Ac toxins did not bind to BBMV from the resistant YHD2 strain, while specific binding to sensitive YDK vesicles was observed. Binding assays revealed a reduction in Cry1Fa binding to BBMV from resistant larvae compared to Cry1Fa binding to BBMV from sensitive larvae. In agreement with this reduction in binding, neither Cry1A nor Cry1Fa toxin altered the permeability of membrane vesicles from resistant larvae, as measured by a light-scattering assay. Ligand blotting experiments performed with BBMV and 125I-Cry1Ac did not differentiate sensitive larvae from resistant larvae. Iodination of BBMV surface proteins suggested that putative toxin-binding proteins were exposed on the surface of the BBMV from resistant insects. BBMV protein blots probed with the N-acetylgalactosamine-specific lectin soybean agglutinin (SBA) revealed altered glycosylation of 63- and 68-kDa glycoproteins but not altered glycosylation of known Cry1 toxin-binding proteins in YHD2 BBMV. The F1 progeny of crosses between sensitive and resistant insects were similar to the sensitive strain when they were tested by toxin-binding assays, light-scattering assays, and lectin blotting with SBA. These results are evidence that a dramatic reduction in toxin binding is responsible for the increased resistance and cross-resistance to Cry1 toxins observed in the YHD2 strain of H. virescens and that this trait correlates with altered glycosylation of specific brush border membrane glycoproteins.     

Integrative model for binding of Bacillus thuringiensis toxins in susceptible and resistant larvae of the diamondback moth (Plutella xylostella)

Insecticidal crystal proteins from Bacillus thuringiensis in sprays and transgenic crops are extremely useful for environmentally sound pest management, but their long-term efficacy is threatened by evolution of resistance by target pests. The diamondback moth (Plutella xylostella) is the first insect to evolve resistance to B. thuringiensis in open-field populations. The only known mechanism of resistance to B. thuringiensis in the diamondback moth is reduced binding of toxin to midgut binding sites. In the present work we analyzed competitive binding of B. thuringiensis toxins Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F to brush border membrane vesicles from larval midguts in a susceptible strain and in resistant strains from the Philippines, Hawaii, and Pennsylvania. Based on the results, we propose a model for binding of B. thuringiensis crystal proteins in susceptible larvae with two binding sites for Cry1Aa, one of which is shared with Cry1Ab, Cry1Ac, and Cry1F. Our results show that the common binding site is altered in each of the three resistant strains. In the strain from the Philippines, the alteration reduced binding of Cry1Ab but did not affect binding of the other crystal proteins. In the resistant strains from Hawaii and Pennsylvania, the alteration affected binding of Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F. Previously reported evidence that a single mutation can confer resistance to Cry1Ab, Cry1Ac, and Cry1F corresponds to expectations based on the binding model. However, the following two other observations do not: the mutation in the Philippines strain affected binding of only Cry1Ab, and one mutation was sufficient for resistance to Cry1Aa. The imperfect correspondence between the model and observations suggests that reduced binding is not the only mechanism of resistance in the diamondback moth and that some, but not all, patterns of resistance and cross-resistance can be predicted correctly from the results of competitive binding analyses of susceptible strains.     

Fitness of Cry1A-resistant and -susceptible Helicoverpa armigera (Lepidoptera: Noctuidae) on transgenic cotton with reduced levels of Cry1Ac

The performance of Helicoverpa armigera (Hübner) on 15-wk-old cotton plants was compared for a susceptible strain, a near-isogenic laboratory-selected strain, and F1 progeny of the two strains. Glasshouse experiments were conducted to test the three insect types on conventional plants and transgenic plants that produced the Bacillus thuringiensis (Bt) toxin Cry1Ac. At the time of testing (15 wk), the Cry1Ac concentration in cotton leaves was 75% lower than at 4 wk. On these plants, < 10% of susceptible larvae reached the fifth instar, and none survived to pupation. In contrast, survival to adulthood on Cry1Ac cotton was 62% for resistant larvae and 39% for F1 larvae. These results show that inheritance of resistance to 15-wk-old Cry1Ac cotton is partially dominant, in contrast to results previously obtained on 4-wk-old Cry1Ac cotton. Growth and survival of resistant insects were similar on Cry1Ac cotton and on non-Bt cotton, but F1 insects developed more slowly on Cry1Ac cotton than on non-Bt cotton. Survival was lower and development was slower for resistant larvae than for susceptible and F1 larvae on non-Bt cotton. These results show recessive fitness costs are associated with resistance to Cry1Ac.     

Integrative Model for Binding of Bacillus thuringiensis Toxins in Susceptible and Resistant Larvae of the Diamondback Moth (Plutella xylostella)

Insecticidal crystal proteins from Bacillus thuringiensis in sprays and transgenic crops are extremely useful for environmentally sound pest management, but their long-term efficacy is threatened by evolution of resistance by target pests. The diamondback moth (Plutella xylostella) is the first insect to evolve resistance to B. thuringiensis in open-field populations. The only known mechanism of resistance to B. thuringiensis in the diamondback moth is reduced binding of toxin to midgut binding sites. In the present work we analyzed competitive binding of B. thuringiensis toxins Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F to brush border membrane vesicles from larval midguts in a susceptible strain and in resistant strains from the Philippines, Hawaii, and Pennsylvania. Based on the results, we propose a model for binding of B. thuringiensis crystal proteins in susceptible larvae with two binding sites for Cry1Aa, one of which is shared with Cry1Ab, Cry1Ac, and Cry1F. Our results show that the common binding site is altered in each of the three resistant strains. In the strain from the Philippines, the alteration reduced binding of Cry1Ab but did not affect binding of the other crystal proteins. In the resistant strains from Hawaii and Pennsylvania, the alteration affected binding of Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F. Previously reported evidence that a single mutation can confer resistance to Cry1Ab, Cry1Ac, and Cry1F corresponds to expectations based on the binding model. However, the following two other observations do not: the mutation in the Philippines strain affected binding of only Cry1Ab, and one mutation was sufficient for resistance to Cry1Aa. The imperfect correspondence between the model and observations suggests that reduced binding is not the only mechanism of resistance in the diamondback moth and that some, but not all, patterns of resistance and cross-resistance can be predicted correctly from the results of competitive binding analyses of susceptible strains.     

Altered Glycosylation of 63- and 68-Kilodalton Microvillar Proteins in Heliothis virescens Correlates with Reduced Cry1 Toxin Binding, Decreased Pore Formation, and Increased Resistance to Bacillus thuringiensis Cry1 Toxins

The binding and pore formation abilities of Cry1A and Cry1Fa Bacillus thuringiensis toxins were analyzed by using brush border membrane vesicles (BBMV) prepared from sensitive (YDK) and resistant (YHD2) strains of Heliothis virescens. ¹²⁵I-labeled Cry1Aa, Cry1Ab, and Cry1Ac toxins did not bind to BBMV from the resistant YHD2 strain, while specific binding to sensitive YDK vesicles was observed. Binding assays revealed a reduction in Cry1Fa binding to BBMV from resistant larvae compared to Cry1Fa binding to BBMV from sensitive larvae. In agreement with this reduction in binding, neither Cry1A nor Cry1Fa toxin altered the permeability of membrane vesicles from resistant larvae, as measured by a light-scattering assay. Ligand blotting experiments performed with BBMV and ¹²⁵I-Cry1Ac did not differentiate sensitive larvae from resistant larvae. Iodination of BBMV surface proteins suggested that putative toxin-binding proteins were exposed on the surface of the BBMV from resistant insects. BBMV protein blots probed with the N-acetylgalactosamine-specific lectin soybean agglutinin (SBA) revealed altered glycosylation of 63- and 68-kDa glycoproteins but not altered glycosylation of known Cry1 toxin-binding proteins in YHD2 BBMV. The F1 progeny of crosses between sensitive and resistant insects were similar to the sensitive strain when they were tested by toxin-binding assays, light-scattering assays, and lectin blotting with SBA. These results are evidence that a dramatic reduction in toxin binding is responsible for the increased resistance and cross-resistance to Cry1 toxins observed in the YHD2 strain of H. virescens and that this trait correlates with altered glycosylation of specific brush border membrane glycoproteins.     

Dual resistance to Bacillus thuringiensis Cry1Ac and Cry2Aa toxins in Heliothis virescens suggests multiple mechanisms of resistance

One strategy for delaying evolution of resistance to Bacillus thuringiensis crystal (Cry) endotoxins is the production of multiple Cry toxins in each transgenic plant (gene stacking). This strategy relies upon the assumption that simultaneous evolution of resistance to toxins that have different modes of action will be difficult for insect pests. In B. thuringiensis-transgenic (Bt) cotton, production of both Cry1Ac and Cry2Ab has been proposed to delay resistance of Heliothis virescens (tobacco budworm). After previous laboratory selection with Cry1Ac, H. virescens strains CXC and KCBhyb developed high levels of cross-resistance not only to toxins similar to Cry1Ac but also to Cry2Aa. We studied the role of toxin binding alteration in resistance and cross-resistance with the CXC and KCBhyb strains. In toxin binding experiments, Cry1A and Cry2Aa toxins bound to brush border membrane vesicles from CXC, but binding of Cry1Aa was reduced for the KCBhyb strain compared to susceptible insects. Since Cry1Aa and Cry2Aa do not share binding proteins in H. virescens, our results suggest occurrence of at least two mechanisms of resistance in KCBhyb insects, one of them related to reduction of Cry1Aa toxin binding. Cry1Ac bound irreversibly to brush border membrane vesicles (BBMV) from YDK, CXC, and KCBhyb larvae, suggesting that Cry1Ac insertion was unaffected. These results highlight the genetic potential of H. virescens to become resistant to distinct Cry toxins simultaneously and may question the effectiveness of gene stacking in delaying evolution of resistance.     

Resistance to the Cry1Ac delta-endotoxin of Bacillus thuringiensis in the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae)

Three laboratory strains of Helicoverpa armigera (Hübner) were established by mating of field-collected insects with an existing insecticide-susceptible laboratory strain. These strains were cultured on artificial diet containing the Cry1Ac protoxin of Bacillus thuringiensis using three different protocols. When no response to selection was detected after 7-11 generations of selection, the three strains were combined by controlled mating to preserve genetic diversity. The composite strain (BX) was selected on the basis of growth rate on artificial diet containing Cry1Ac crystals. Resistance to Cry1Ac was first detected after 16 generations of continuous selection. The resistance ratio (RR) peaked approximately 300-fold at generation 21, after which it declined to oscillate between 57- and 111-fold. First-instar H. armigera from generation 25 (RR = 63) were able to complete their larval development on transgenic cotton expressing Cry1Ac and produce fertile adults. There appeared to be a fitness cost associated with resistance on cotton and on artificial diet. The BX strain was not resistant to the commercial Bt spray formulations DiPel and XenTari, which contain multiple insecticidal crystal proteins, but was resistant to the MVP formulation, which only contains Cry1Ac. The strain was also resistant to Cry1Ab but not to Cry2Aa or Cry2Ab. Toxin binding assays showed that the resistant insects lacked the high affinity binding site that was detected in early generations of the strain. Genetic analysis confirmed that resistance in the BX strain of H. armigera is incompletely recessive.     

Efficacy of Genetically Modified Bt Toxins Alone and in Combinations Against Pink Bollworm Resistant to Cry1Ac and Cry2Ab

Evolution of resistance in pests threatens the long-term efficacy of insecticidal proteins from Bacillus thuringiensis (Bt) used in sprays and transgenic crops. Previous work showed that genetically modified Bt toxins Cry1AbMod and Cry1AcMod effectively countered resistance to native Bt toxins Cry1Ab and Cry1Ac in some pests, including pink bollworm (Pectinophora gossypiella). Here we report that Cry1AbMod and Cry1AcMod were also effective against a laboratory-selected strain of pink bollworm resistant to Cry2Ab as well as to Cry1Ab and Cry1Ac. Resistance ratios based on the concentration of toxin killing 50% of larvae for the resistant strain relative to a susceptible strain were 210 for Cry2Ab, 270 for Cry1Ab, and 310 for Cry1Ac, but only 1.6 for Cry1AbMod and 2.1 for Cry1AcMod. To evaluate the interactions among toxins, we tested combinations of Cry1AbMod, Cry1Ac, and Cry2Ab. For both the resistant and susceptible strains, the net results across all concentrations tested showed slight but significant synergism between Cry1AbMod and Cry2Ab, whereas the other combinations of toxins did not show consistent synergism or antagonism. The results suggest that the modified toxins might be useful for controlling populations of pink bollworm resistant to Cry1Ac, Cry2Ab, or both.     

The HevCaLP protein mediates binding specificity of the Cry1A class of Bacillus thuringiensis toxins in Heliothis virescens

Retrotransposon-mediated disruption of the BtR-4 gene encoding the Heliothis virescens cadherin-like protein (HevCaLP) is linked to high levels of resistance in the YHD2 strain to Cry1Ac toxin from Bacillus thuringiensis. This suggests that HevCaLP functions as a Cry1Ac toxin receptor on the surface of midgut cells in susceptible larvae and that the BtR-4 gene disruption eliminates this protein in resistant larvae. However, Cry1Ac toxin binding to HevCaLP is yet to be reported. We used the polymerase chain reaction and immunoblotting as tools to discriminate between individual H. virescens larval midguts from susceptible (YDK) and resistant (CXC, KCBhyb, and YHD2-B) strains according to their BtR-4 gene disruption genotype and phenotype. This approach allowed us to test the correlation between BtR-4 gene disruption, lack of HevCaLP, and altered Cry1A toxin binding. Toxin-binding assays using brush border membrane vesicles revealed that a wild-type BtR-4 allele is necessary for HevCaLP production and Cry1Aa toxin binding, while most of Cry1Ab and Cry1Ac binding was independent of the BtR-4 genotype. Moreover, toxin competition experiments show that KCBhyb midguts lacking HevCaLP are more similar to midguts of the original YHD2 strain than to the current YHD2-B strain. This resolves discrepancies in published studies of Cry1A binding in YHD2 and supports our earlier suggestion that a separate genetic change occurred in YHD2 after appearance of the cadherin disruption, conferring even higher resistance in the resulting YHD2-B strain as well as a large reduction in Cry1Ab and Cry1Ac binding.     

A proteomic approach to study Cry1Ac binding proteins and their alterations in resistant Heliothis virescens larvae

Binding of the Bacillus thuringiensis Cry1Ac toxin to specific receptors in the midgut brush border membrane is required for toxicity. Alteration of these receptors is the most reported mechanism of resistance. We used a proteomic approach to identify Cry1Ac binding proteins from intestinal brush border membrane (BBM) prepared from Heliothis virescens larvae. Cry1Ac binding BBM proteins were detected in 2D blots and identified using peptide mass fingerprinting (PMF) or de novo sequencing. Among other proteins, the membrane bound alkaline phosphatase (HvALP), and a novel phosphatase, were identified as Cry1Ac binding proteins. Reduction of HvALP expression levels correlated directly with resistance to Cry1Ac in the YHD2-B strain of H. virescens. To study additional proteomic alterations in resistant H. virescens larvae, we used two-dimensional differential in-gel electrophoresis (2D-DIGE) to compare three independent resistant strains with a susceptible strain. Our results validate the use of proteomic approaches to identify toxin binding proteins and proteome alterations in resistant insects.     

Inheritance of resistance to Bacillus thuringiensis Cry1Ac toxin in a greenhouse-derived strain of cabbage looper (Lepidoptera: Noctuidae)

A population of cabbage looper, Trichoplusia ni (Hübner), collected from commercial greenhouses in the lower mainland of British Columbia, Canada, in 2001 showed a resistance level of 24-fold to Dipel, a product of Bacillus thuringiensis (Bt) subspecies kurstaki. This population was selected with Cry1Ac, the major Bt Cry toxin in Dipel, to obtain a homogenous population resistant to Cry1Ac. The resulting strain of T. ni, named GLEN-Cry1Ac, was highly resistant to Cry1Ac with a resistance ratio of approximately 1000-fold. The larvae from the GLEN-Cry1Ac strain could survive on Cry1Ac-expressing transgenic broccoli plants that were highly insecticidal to T. ni and diamondback moth, Plutella xylostella (L.). The inheritance of Cry1Ac resistance in this T. ni strain was autosomal and incompletely recessive. The degree of dominance of the resistance was -0.402 and -0.395, respectively, for the neonates in reciprocal crosses between the GLEN-Cry1Ac and a laboratory strain of T. ni. Using chi2 goodness-of-fit test, we demonstrated that the inhibition of larval growth resulting from testing 12 toxin doses in the progeny of the backcross fit the predicted larval responses based on a monogenic inheritance model. Therefore, we conclude that the inheritance of the resistance to Cry1Ac in the T. ni larvae is monogenic.     

Susceptibility of dipel-resistant and -susceptible Ostrinia nubilalis (Lepidoptera: Crambidae) to individual Bacillus thuringiensis protoxins

Dipel-resistant and -susceptible strains of Ostrinia nubilalis (Hübner) were evaluated for larval mortality and growth inhibition when fed diets containing individual Bacillus thuringiensis protoxins. Resistance ratios for four of the protoxins in Dipel (Cry1Aa, Cry1Ab, Cry1Ac, and Cry2Aa) were 170-, 205-, 524-, and > 640-fold, respectively, considerably higher than the 47-fold resistance to Dipel. The Dipel-resistant strain was 36-fold resistant to Cry1Ba, a protoxin not present in Dipel. Another non-Dipel protoxin, Cry1Ca, did not cause significant mortality for either resistant or susceptible larvae with doses as high as 1.0 mg/ml. In an evaluation of larval growth inhibition, resistance to Cry1Aa, Cry1Ab, Cry1Ac, and Cry1Ba was significant at concentrations of 0.054 and 0.162 microg/ml. However, growth inhibition with Cry2Aa was not significant at either dose. These data provide information on the spectrum of resistance and cross-resistance to individual Cry protoxins in this strain.     

Dual Resistance to Bacillus thuringiensis Cry1Ac and Cry2Aa Toxins in Heliothis virescens Suggests Multiple Mechanisms of Resistance

One strategy for delaying evolution of resistance to Bacillus thuringiensis crystal (Cry) endotoxins is the production of multiple Cry toxins in each transgenic plant (gene stacking). This strategy relies upon the assumption that simultaneous evolution of resistance to toxins that have different modes of action will be difficult for insect pests. In B. thuringiensis-transgenic (Bt) cotton, production of both Cry1Ac and Cry2Ab has been proposed to delay resistance of Heliothis virescens (tobacco budworm). After previous laboratory selection with Cry1Ac, H. virescens strains CXC and KCBhyb developed high levels of cross-resistance not only to toxins similar to Cry1Ac but also to Cry2Aa. We studied the role of toxin binding alteration in resistance and cross-resistance with the CXC and KCBhyb strains. In toxin binding experiments, Cry1A and Cry2Aa toxins bound to brush border membrane vesicles from CXC, but binding of Cry1Aa was reduced for the KCBhyb strain compared to susceptible insects. Since Cry1Aa and Cry2Aa do not share binding proteins in H. virescens, our results suggest occurrence of at least two mechanisms of resistance in KCBhyb insects, one of them related to reduction of Cry1Aa toxin binding. Cry1Ac bound irreversibly to brush border membrane vesicles (BBMV) from YDK, CXC, and KCBhyb larvae, suggesting that Cry1Ac insertion was unaffected. These results highlight the genetic potential of H. virescens to become resistant to distinct Cry toxins simultaneously and may question the effectiveness of gene stacking in delaying evolution of resistance.     

Mechanism of resistance to Bacillus thuringiensis toxin Cry1Ac in a greenhouse population of the cabbage looper, Trichoplusia ni

The cabbage looper, Trichoplusia ni, is one of only two insect species that have evolved resistance to Bacillus thuringiensis in agricultural situations. The trait of resistance to B. thuringiensis toxin Cry1Ac from a greenhouse-evolved resistant population of T. ni was introgressed into a highly inbred susceptible laboratory strain. The resulting introgression strain, GLEN-Cry1Ac-BCS, and its nearly isogenic susceptible strain were subjected to comparative genetic and biochemical studies to determine the mechanism of resistance. Results showed that midgut proteases, hemolymph melanization activity, and midgut esterase were not altered in the GLEN-Cry1Ac-BCS strain. The pattern of cross-resistance of the GLEN-Cry1Ac-BCS strain to 11 B. thuringiensis Cry toxins showed a correlation of the resistance with the Cry1Ab/Cry1Ac binding site in T. ni. This cross-resistance pattern is different from that found in a previously reported laboratory-selected Cry1Ab-resistant T. ni strain, evidently indicating that the greenhouse-evolved resistance involves a mechanism different from the laboratory-selected resistance. Determination of specific binding of B. thuringiensis toxins Cry1Ab and Cry1Ac to the midgut brush border membranes confirmed the loss of midgut binding to Cry1Ab and Cry1Ac in the resistant larvae. The loss of midgut binding to Cry1Ab/Cry1Ac is inherited as a recessive trait, which is consistent with the recessive inheritance of Cry1Ab/Cry1Ac resistance in this greenhouse-derived T. ni population. Therefore, it is concluded that the mechanism for the greenhouse-evolved Cry1Ac resistance in T. ni is an alteration affecting the binding of Cry1Ab and Cry1Ac to the Cry1Ab/Cry1Ac binding site in the midgut.     

Cross-resistance and mechanism of resistance to Cry1Ab toxin from Bacillus thuringiensis in a field-derived strain of European corn borer, Ostrinia nubilalis

The cross-resistance spectrum and biochemical mechanism of resistance to the Bacillus thuringiensis Cry1Ab toxin was studied in a field-derived strain of Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) that was further selected in the laboratory for high levels (>1000-fold) of resistance to Cry1Ab. The resistant strain exhibited high levels of cross-resistance to Cry1Ac and Cry1Aa but only low levels of cross-resistance (<4-fold) to Cry1F. In addition, there was no significant difference between the levels of resistance to full-length and trypsin-activated Cry1Ab protein. No differences in activity of luminal gut proteases or altered proteolytic processing of the toxin were observed in the resistant strain. Significantly reduced binding of radiolabeled Cry1Aa was observed in the resistant strain whereas binding of Cry1Ab and Cry1Ac was practically the same in both resistant and susceptible strains. The interpretation of the overall data seems to suggest the involvement of an alteration in the binding of Cry1A toxins to a common receptor, which is more clearly revealed by the binding assays using radiolabeled Cry1Aa.     

Different cross-resistance patterns in the diamondback moth (Lepidoptera: Plutellidae) resistant to Bacillus thuringiensis toxin Cry1C.

Two strains of the diamondback moth, Plutella xylostella (L.), were selected using Cry1C protoxin and transgenic broccoli plants expressing a Cry1C toxin of Bacillus thuringiensis (Bt). Both strains were resistant to Cry1C but had different cross-resistance patterns. We used 12 Bt protoxins for cross-resistance tests, including Cry1Aa, Cry1Ab, Cry1Ac, Cry1Bb, Cry1C, Cry1D, Cry1E, Cry1F, Cry1J, Cry2Ab, Cry9Aa, and Cry9C. Compared with the unselected sister strain (BCS), the resistance ratio (BR) of one strain (BCS-Cry1C-1) to the Cry1C protoxin was 1,090-fold with high level of cross-resistance to Cry1Aa, Cry1Ab, Cry1Ac, Cry1F, and Cry1J (RR > 390-fold). The cross-resistance to Cry1A, Cry1F, and Cry1J in this strain was probably related to the Cry1A resistance gene(s) that came from the initial field population and was caused by intensive sprayings of Bt products containing Cry1A protoxins. The neonates of this strain can survive on transgenic broccoli plants expressing either Cry1Ac or Cry1C toxins. The other strain (BCS-Cry1C-2) was highly resistant to Cry1C but not cross-resistant to other Bt protoxins. The neonates of this strain can survive on transgenic broccoli expressing Cry1C toxin but not Cry1Ac toxin. The gene(s) conferring resistance to Cry1C segregates independently from Cry1Ac resistance in these strains. The toxicity of Cry1E and Cry2Ab protoxins was low to all of the three strains. The overall progress of all work has resulted in a unique model system to test the stacked genes strategy for resistance management of Bt transgenic crops.     

New resistance mechanism in Helicoverpa armigera threatens transgenic crops expressing Bacillus thuringiensis Cry1Ac toxin

In Australia, the cotton bollworm, Helicoverpa armigera, has a long history of resistance to conventional insecticides. Transgenic cotton (expressing the Bacillus thuringiensis toxin Cry1Ac) has been grown for H. armigera control since 1996. It is demonstrated here that a population of Australian H. armigera has developed resistance to Cry1Ac toxin (275-fold). Some 70% of resistant H. armigera larvae were able to survive on Cry1Ac transgenic cotton (Ingard) The resistance phenotype is inherited as an autosomal semidominant trait. Resistance was associated with elevated esterase levels, which cosegregated with resistance. In vitro studies employing surface plasmon resonance technology and other biochemical techniques demonstrated that resistant strain esterase could bind to Cry1Ac protoxin and activated toxin. In vivo studies showed that Cry1Ac-resistant larvae fed Cy1Ac transgenic cotton or Cry1Ac-treated artificial diet had lower esterase activity than non-Cry1Ac-fed larvae. A resistance mechanism in which esterase sequesters Cry1Ac is proposed.     

Association of Cry1Ac toxin resistance in Helicoverpa zea (Boddie) with increased alkaline phosphatase levels in the midgut lumen

Resistance to Bacillus thuringiensis Cry1Ac toxin was characterized in a population of Helicoverpa zea larvae previously shown not to have an alteration in toxin binding as the primary resistance mechanism to this toxin. Cry1Ac-selected larvae (AR1) were resistant to protoxins and toxins of Cry1Ab, Cry1Ac, and the corresponding modified proteins lacking helix α-1 (Cry1AbMod and Cry1AcMod). When comparing brush border membrane vesicles (BBMVs) prepared from susceptible (LC) and AR1 larval midguts, there were only negligible differences in overall Cry1Ac toxin binding, though AR1 had 18% reversible binding, in contrast to LC, in which all binding was irreversible. However, no differences were detected in Cry1Ac-induced pore formation activity in BBMVs from both strains. Enzymatic activities of two putative Cry1Ac receptors (aminopeptidase N [APN] and alkaline phosphatase [ALP]) were significantly reduced (2-fold and 3-fold, respectively) in BBMVs from AR1 compared to LC larvae. These reductions corresponded to reduced protein levels in midgut luminal contents only in the case of ALP, with an almost 10-fold increase in specific ALP activity in midgut fluids from AR1 compared to LC larvae. Partially purified H. zea ALP bound Cry1Ac toxin in ligand blots and competed with Cry1Ac toxin for BBMV binding. Based on these results, we suggest the existence of at least one mechanism of resistance to Cry1A toxins in H. zea involving binding of Cry1Ac toxin to an ALP receptor in the larval midgut lumen of resistant larvae.