Resistance to Bacillus thuringiensis Mediated by an ABC Transporter Mutation Increases Susceptibility to Toxins from Other Bacteria in an Invasive Insect

Evolution of pest resistance reduces the efficacy of insecticidal proteins from the gram-positive bacterium Bacillus thuringiensis (Bt) used widely in sprays and transgenic crops. Recent efforts to delay pest adaptation to Bt crops focus primarily on combinations of two or more Bt toxins that kill the same pest, but this approach is often compromised because resistance to one Bt toxin causes cross-resistance to others. Thus, integration of Bt toxins with alternative controls that do not exhibit such cross-resistance is urgently needed. The ideal scenario of negative cross-resistance, where selection for resistance to a Bt toxin increases susceptibility to alternative controls, has been elusive. Here we discovered that selection of the global crop pest, Helicoverpa armigera, for >1000-fold resistance to Bt toxin Cry1Ac increased susceptibility to abamectin and spineotram, insecticides derived from the soil bacteria Streptomyces avermitilis and Saccharopolyspora spinosa, respectively. Resistance to Cry1Ac did not affect susceptibility to the cyclodiene, organophospate, or pyrethroid insecticides tested. Whereas previous work demonstrated that the resistance to Cry1Ac in the strain analyzed here is conferred by a mutation disrupting an ATP-binding cassette protein named ABCC2, the new results show that increased susceptibility to abamectin is genetically linked with the same mutation. Moreover, RNAi silencing of HaABCC2 not only decreased susceptibility to Cry1Ac, it also increased susceptibility to abamectin. The mutation disrupting ABCC2 reduced removal of abamectin in live larvae and in transfected Hi5 cells. The results imply that negative cross-resistance occurs because the wild type ABCC2 protein plays a key role in conferring susceptibility to Cry1Ac and in decreasing susceptibility to abamectin. The negative cross-resistance between a Bt toxin and other bacterial insecticides reported here may facilitate more sustainable pest control.     

Disruption of a Cadherin Gene Associated with Resistance to Cry1Ac δ-Endotoxin of Bacillus thuringiensis in Helicoverpa armigera

A laboratory strain (GY) of Helicoverpa armigera (Hübner) was established from surviving larvae collected from transgenic cotton expressing a Bacillus thuringiensis var. kurstaki insecticidal protein (Bt cotton) in Gaoyang County, Hebei Province, People's Republic of China, in 2001. The GYBT strain was derived from the GY strain through 28 generations of selection with activated Cry1Ac delivered by diet surface contamination. When resistance to Cry1Ac in the GYBT strain increased to 564-fold after selection, we detected high levels of cross-resistance to Cry1Aa (103-fold) and Cry1Ab (>46-fold) in the GYBT strain with reference to those in the GY strain. The GYBT strain had a low level of cross-resistance to B. thuringiensis var. kurstaki formulation (Btk) (5-fold) and no cross-resistance to Cry2Aa (1.4-fold). Genetic analysis showed that Cry1Ac resistance in the GYBT strain was controlled by one autosomal and incompletely recessive gene. The cross-resistance pattern and inheritance mode suggest that the Cry1Ac resistance in the GYBT strain of H. armigera belongs to “mode 1,” the most common type of lepidopteran resistance to B. thuringiensis toxins. A cadherin gene was cloned and sequenced from both the GY and GYBT strains. Disruption of the cadherin gene by a premature stop codon was associated with a high level of Cry1Ac resistance in H. armigera. Tight linkage between Cry1Ac resistance and the cadherin locus was observed in a backcross analysis. Together with previous evidence found with Heliothis virescens and Pectinophora gossypiella, our results confirmed that the cadherin gene is a preferred target for developing DNA-based monitoring of B. thuringiensis resistance in field populations of lepidopteran pests.     

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.     

High Susceptibility to Cry1Ac and Low Resistance Allele Frequency Reduce the Risk of Resistance of Helicoverpa armigera to Bt Soybean in Brazil

The Old World bollworm, Helicoverpa armigera (Hübner), was recently introduced into Brazil, where it has caused extensive damage to cotton and soybean crops. MON 87701 × MON 89788 soybean, which expresses the Bt protein Cry1Ac, was recently deployed in Brazil, providing high levels of control against H. armigera. To assess the risk of resistance to the Cry1Ac protein expressed by MON 87701 × MON 89788 soybean in Brazil, we conducted studies to evaluate the baseline susceptibility of H. armigera to Cry1Ac, in planta efficacy including the assessment of the high-dose criterion, and the initial resistance allele frequency based on an F₂ screen. The mean Cry1Ac lethal concentration (LC₅₀) ranged from 0.11 to 1.82 μg·mL⁻¹ of diet among all H. armigera field populations collected from crop seasons 2013/14 to 2014/15, which indicated about 16.5-fold variation. MON 87701 × MON 89788 soybean exhibited a high level of efficacy against H. armigera and most likely met the high dose criterion against this target species in leaf tissue dilution bioassays up to 50 times. A total of 212 F₂ family lines of H. armigera were established from field collections sampled from seven locations across Brazil and were screened for the presence of MON 87701 × MON 89788 soybean resistance alleles. None of the 212 families survived on MON 87701 × MON 89788 soybean leaf tissue (estimated allele frequency = 0.0011). The responses of H. armigera to Cry1Ac protein, high susceptibility to MON 87701 × MON 89788 soybean, and low frequency of resistance alleles across the main soybean-producing regions support the assumptions of a high-dose/refuge strategy. However, maintenance of reasonable compliance with the refuge recommendation will be essential to delay the evolution of resistance in H. armigera to MON 87701 × MON 89788 soybean in Brazil.     

Transgenic cotton co-expressing chimeric Vip3AcAa and Cry1Ac confers effective protection against Cry1Ac-resistant cotton bollworm

Wide planting of transgenic Bt cotton in China since 1997 to control cotton bollworm (Helicoverpa armigera) has increased yields and decreased insecticide use, but the evolution of resistance to Bt cotton by H. armigera remains a challenge. Toward developing a new generation of insect-resistant transgenic crops, a chimeric protein of Vip3Aa1 and Vip3Ac1, named Vip3AcAa, having a broader insecticidal spectrum, was specifically created previously in our laboratory. In this study, we investigated cross resistance and interactions between Vip3AcAa and Cry1Ac with three H. armigera strains, one that is susceptible and two that are Cry1Ac-resistant, to determine if Vip3AcAa is a good candidate for development the pyramid cotton with Cry1Ac toxin. Our results showed that evolution of insect resistance to Cry1Ac toxin did not influence the sensitivity of Cry1Ac-resistant strains to Vip3AcAa. For the strains examined, observed mortality was equivalent to the expected mortality for all the combinations of Vip3AcAa and Cry1Ac tested, reflecting independent activity between these two toxins. When this chimeric vip3AcAa gene and the cry1Ac gene were introduced into cotton, mortality rates of Cry1Ac resistant H. armigera larvae strains that fed on this new cotton increased significantly compared with larvae fed on non-Bt cotton and cotton producing only Cry1Ac. These results suggest that the Vip3AcAa protein is an excellent option for a “pyramid” strategy for pest resistance management in China.     

Functional validation of cadherin as a receptor of Bt toxin Cry1Ac in Helicoverpa armigera utilizing the CRISPR/Cas9 system

Cadherins have been identified as receptors of Bacillus thuringiensis (Bt) Cry1A toxins in several lepidopteran insects including the cotton bollworm, Helicoverpa armigera. Disruption of the cadherin gene HaCad has been genetically linked to resistance to Bt toxin Cry1Ac in H. armigera. By using the CRISPR/Cas9 genome editing system (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9), HaCad from the Cry1Ac-susceptible SCD strain of H. armigera was successfully knocked out. A single positive CRISPR event with a frame shift deletion of 4 nucleotides was identified and made homozygous to create a knockout line named SCD-Cad. Western blotting confirmed that HaCad was no longer expressed in the SCD-Cad line while an intact HaCad of 210 kDa was present in the parental SCD strain. Insecticide bioassays were used to show that SCD-Cad exhibited 549-fold resistance to Cry1Ac compared with SCD, but no significant change in susceptibility to Cry2Ab. Our results not only provide strong reverse genetics evidence for HaCad as a functional receptor of Cry1Ac, but also demonstrate that the CRISPR/Cas9 technique can act as a powerful and efficient genome editing tool to study gene function in a global agricultural pest, H. armigera.     

Production and Characterization of Bacillus thuringiensis Cry1Ac-Resistant Cotton Bollworm Helicoverpa zea (Boddie)

Laboratory-selected Bacillus thuringiensis-resistant colonies are important tools for elucidating B. thuringiensis resistance mechanisms. However, cotton bollworm, Helicoverpa zea, a target pest of transgenic corn and cotton expressing B. thuringiensis Cry1Ac (Bt corn and cotton), has proven difficult to select for stable resistance. Two populations of H. zea (AR and MR), resistant to the B. thuringiensis protein found in all commercial Bt cotton varieties (Cry1Ac), were established by selection with Cry1Ac activated toxin (AR) or MVP II (MR). Cry1Ac toxin reflects the form ingested by H. zea when feeding on Bt cotton, whereas MVP II is a Cry1Ac formulation used for resistance selection and monitoring. The resistance ratio (RR) for AR exceeded 100-fold after 11 generations and has been maintained at this level for nine generations. This is the first report of stable Cry1Ac resistance in H. zea. MR crashed after 11 generations, reaching only an RR of 12. AR was only partially cross-resistant to MVP II, suggesting that MVP II does not have the same Cry1Ac selection pressure as Cry1Ac toxin against H. zea and that proteases may be involved with resistance. AR was highly cross-resistant to Cry1Ab toxin but only slightly cross-resistant to Cry1Ab expressing corn leaf powder. AR was not cross-resistant to Cry2Aa2, Cry2Ab2-expressing corn leaf powder, Vip3A, and cypermethrin. Toxin-binding assays showed no significant differences, indicating that resistance was not linked to a reduction in binding. These results aid in understanding why this pest has not evolved B. thuringiensis resistance, and highlight the need to choose carefully the form of B. thuringiensis protein used in experiments.     

棉铃虫田间种群Bt毒素Cry1Ac抗性基因频率的估算

采用改进的F₁筛查法检测了2005年采自华北地区的棉铃虫Helicoverpa armigera (Hübner)田间种群对Bt毒素Cry1Ac的抗性基因频率。2005年从河南安阳和河北沧县转Bt基因抗虫棉上采集二代棉铃虫卵,在室内用人工饲料饲养至2龄幼虫,用1 μg/cm² 的Cry1Ac活化毒素进行初筛,将初筛存活成虫与室内筛选的GYBT抗性品系成虫进行单对杂交,并用区分剂量(2.5 μg/cm²)对F₁代进行检测。经检测,2005年河南安阳棉铃虫种群和河北沧县棉 铃虫种群对Cry1Ac抗性基因频率基本一致,分别为1.4×10^-3和1.5×10^-3。用毒素涂表法测定了2004、2005年采自河南安阳、河北高阳、河北沧县、新疆阿克苏和新疆沙湾棉铃虫田间种群对Cry1Ac活化毒素的敏感性水平,结果表明华北棉区与新疆内陆棉区棉铃虫种群对Cry1Ac的敏感性存在一定的地区性差异（<8倍）。总体上,我国华北棉区棉铃虫种群对Cry1Ac还未产生明显抗性,抗性基因频率处于正常水平。棉铃虫对转Bt基因抗虫棉的抗性风险依然存在,需要尽快启动全国性的早期抗性检测和预警工作。     

Binding of Cry δ-endotoxins to brush border membrane vesicles of Helicoverpa armigera and Helicoverpa punctigera （Lepidoptera： Noctuidae）

The relatively low susceptibility ofHelicoverpa armigera to CrylAc, its history of resistance to chemical insecticides and the seasonal decline in expression of CrylAc in transgenic cotton necessitated the development of cotton expressing two insecticidal proteins to provide sustainable control of this multinational pest. To manage the resistance issue, it was essential that the second insecticidal protein have a significantly different mode of action to CrylAc. A common feature of resistance to CrylA proteins in several species as well as H. armigera has been a change in the binding site. A study of binding sites for some Cry proteins in the brush border membrane vesicles （BBMV） ofH. armigera and Helicoverpa punctigera was undertaken. The binding affinity for CrylAc was higher than for CrylAb, matching their relative toxicities, and CrylAc and CrylAb were found to share at least one binding site in both I-1. armigera and I-1. punctigera. However Cry2Aa did not compete with CrylAc for binding and so could be used in transgenic cotton in combination with CrylAc to control H. armigera and manage resistance. Variation in the susceptibilities of three different H. armigera strains to CrylAc correlated with the parameter Bmax/Kcom.     

Non-Recessive Bt Toxin Resistance Conferred by an Intracellular Cadherin Mutation in Field-Selected Populations of Cotton Bollworm

Transgenic crops producing Bacillus thuringiensis (Bt) toxins have been planted widely to control insect pests, yet evolution of resistance by the pests can reduce the benefits of this approach. Recessive mutations in the extracellular domain of toxin-binding cadherin proteins that confer resistance to Bt toxin Cry1Ac by disrupting toxin binding have been reported previously in three major lepidopteran pests, including the cotton bollworm, Helicoverpa armigera. Here we report a novel allele from cotton bollworm with a deletion in the intracellular domain of cadherin that is genetically linked with non-recessive resistance to Cry1Ac. We discovered this allele in each of three field-selected populations we screened from northern China where Bt cotton producing Cry1Ac has been grown intensively. We expressed four types of cadherin alleles in heterologous cell cultures: susceptible, resistant with the intracellular domain mutation, and two complementary chimeric alleles with and without the mutation. Cells transfected with each of the four cadherin alleles bound Cry1Ac and were killed by Cry1Ac. However, relative to cells transfected with either the susceptible allele or the chimeric allele lacking the intracellular domain mutation, cells transfected with the resistant allele or the chimeric allele containing the intracellular domain mutation were less susceptible to Cry1Ac. These results suggest that the intracellular domain of cadherin is involved in post-binding events that affect toxicity of Cry1Ac. This evidence is consistent with the vital role of the intracellular region of cadherin proposed by the cell signaling model of the mode of action of Bt toxins. Considered together with previously reported data, the results suggest that both pore formation and cell signaling pathways contribute to the efficacy of Bt toxins.     

Fipronil resistance in the diamondback moth (Lepidoptera: Plutellidae): inheritance and number of genes involved

Bioassays (at generation 1, G1) using fipronil, spinosad, indoxacarb, and Bacillus thuringiensis toxins Cry1Ac and Cry1Ca with a newly collected field population of Plutella xylostella (L.) from farmers fields in the Cameron Highlands, Malaysia, indicated a resistance ratio of approximately 400-, 1,170-, 330-, 2,840-, and 1,410-fold, respectively, compared with a laboratory-susceptible population of P. xylostella (ROTH). At G3, the field-derived population was divided into two subpopulations, one was selected (G3 to G7) with fipronil (fip-SEL), whereas the second was left unselected (UNSEL). Bioassays at G8 found that selection with fipronil gave a resistance ratio of approximately 490 compared with UNSEL and approximately 770 compared with ROTH. The resistance ratio for fipronil, spinosad, indoxacarb, Cry1Ac, and Cry1Ca in the UNSEL population declined significantly by G8. Logit regression analysis of F1 reciprocal crosses between fip-SEL (at G8) and UNSEL indicated that resistance to fipronil in the fip-SEL population was inherited as an autosomal, incompletely recessive (D(LC) = 0.37) trait. At the highest dose of fipronil tested, resistance was completely recessive, whereas at the lowest dose it was incompletely recessive. A direct test of monogenic inheritance based on a backcross of F1 progeny with fip-SEL suggested that resistance to fipronil was controlled by a single locus. The fip-SEL population at G8 showed little change in its response to spinosad and indoxacarb compared with G1, whereas its susceptibility to Cry1Ac and Cry1Ca increased markedly over the selection period. This suggests that there may be some low level of cross-resistance between fipronil, spinosad, and indoxacarb.     

Resistance of Trichoplusia ni Populations Selected by Bacillus thuringiensis Sprays to Cotton Plants Expressing Pyramided Bacillus thuringiensis Toxins Cry1Ac and Cry2Ab

Two populations of Trichoplusia ni that had developed resistance to Bacillus thuringiensis sprays (Bt sprays) in commercial greenhouse vegetable production were tested for resistance to Bt cotton (BollGard II) plants expressing pyramided Cry1Ac and Cry2Ab. The T. ni colonies resistant to Bacillus thuringiensis serovar kurstaki formulations were not only resistant to the Bt toxin Cry1Ac, as previously reported, but also had a high frequency of Cry2Ab-resistant alleles, exhibiting ca. 20% survival on BollGard II foliage. BollGard II-resistant T. ni strains were established by selection with BollGard II foliage to further remove Cry2Ab-sensitive alleles in the T. ni populations. The BollGard II-resistant strains showed incomplete resistance to BollGard II, with adjusted survival values of 0.50 to 0.78 after 7 days. The resistance to the dual-toxin cotton plants was conferred by two genetically independent resistance mechanisms: one to Cry1Ac and one to Cry2Ab. The 50% lethal concentration of Cry2Ab for the resistant strain was at least 1,467-fold that for the susceptible T. ni strain. The resistance to Cry2Ab in resistant T. ni was an autosomally inherited, incompletely recessive monogenic trait. Results from this study indicate that insect populations under selection by Bt sprays in agriculture can be resistant to multiple Bt toxins and may potentially confer resistance to multitoxin Bt crops.     

in silico identification of cross affinity towards Cry1Ac pesticidal protein with receptor enzyme in Bos taurus and sequence,structure analysis of crystal proteins for stability

Any novel protein introduced into the GM crops need to be evaluated for cross affinity on living organisms. Many researchers are currently focusing on the impact of Bacillus thuringiensis cotton on soil and microbial diversity by field experiments. In spite of this, in silico approach might be helpful to elucidate the impact of cry genes. The crystal a protein which was produced by Bt at the time of sporulation has been used as a biological pesticide to target the insectivorous pests like Cry1Ac for Helicoverpa armigera and Cry2Ab for Spodoptera sp. and Heliothis sp. Here, we present the comprehensive in silico analysis of Cry1Ac and Cry2Ab proteins with available in silico tools, databases and docking servers. Molecular docking of Cry1Ac with procarboxypeptidase from Helicoverpa armigera and Cry1Ac with Leucine aminopeptidase from Bos taurus has showed the 125^th amino acid position to be the preference site of Cry1Ac protein. The structures were compared with each other and it showed 5% of similarity. The cross affinity of this toxin that have confirmed the earlier reports of ill effects of Bt cotton consumed by cattle.     

Impact of Water Content and Temperature on the Degradation of Cry1Ac Protein in Leaves and Buds of Bt Cotton in the Soil

Determining the influence of soil environmental factors on degradation of Cry1Ac protein from Bt cotton residues is vital for assessing the ecological risks of this commercialized transgenic crop. In this study, the degradation of Cry1Ac protein in leaves and in buds of Bt cotton in soil was evaluated under different soil water content and temperature settings in the laboratory. An exponential model and a shift-log model were used to fit the degradation dynamics of Cry1Ac protein and estimate the DT₅₀ and DT₉₀ values. The results showed that Cry1Ac protein in the leaves and buds underwent rapid degradation in the early stage (before day 48), followed by a slow decline in the later stage under different soil water content and temperature. Cry1Ac protein degraded the most rapidly in the early stage at 35°C with 70% soil water holding capacity. The DT₅₀ values were 12.29 d and 10.17 d and the DT₉₀ values were 41.06 d and 33.96 d in the leaves and buds, respectively. Our findings indicated that the soil temperature was a major factor influencing the degradation of Cry1Ac protein from Bt cotton residues. Additionally, the relative higher temperature (25°C and 35°C) was found to be more conducive to degradation of Cry1Ac protein in the soil and the greater water content (100%WHC) retarded the process. These findings suggested that under appropriate soil temperature and water content, Cry1Ac protein from Bt cotton residues will not persist and accumulate in soil.     

Control of Resistant Pink Bollworm (Pectinophora gossypiella) by Transgenic Cotton That Produces Bacillus thuringiensis Toxin Cry2Ab

Crops genetically engineered to produce Bacillus thuringiensis toxins for insect control can reduce use of conventional insecticides, but insect resistance could limit the success of this technology. The first generation of transgenic cotton with B. thuringiensis produces a single toxin, Cry1Ac, that is highly effective against susceptible larvae of pink bollworm (Pectinophora gossypiella), a major cotton pest. To counter potential problems with resistance, second-generation transgenic cotton that produces B. thuringiensis toxin Cry2Ab alone or in combination with Cry1Ac has been developed. In greenhouse bioassays, a pink bollworm strain selected in the laboratory for resistance to Cry1Ac survived equally well on transgenic cotton with Cry1Ac and on cotton without Cry1Ac. In contrast, Cry1Ac-resistant pink bollworm had little or no survival on second-generation transgenic cotton with Cry2Ab alone or with Cry1Ac plus Cry2Ab. Artificial diet bioassays showed that resistance to Cry1Ac did not confer strong cross-resistance to Cry2Aa. Strains with >90% larval survival on diet with 10 μg of Cry1Ac per ml showed 0% survival on diet with 3.2 or 10 μg of Cry2Aa per ml. However, the average survival of larvae fed a diet with 1 μg of Cry2Aa per ml was higher for Cry1Ac-resistant strains (2 to 10%) than for susceptible strains (0%). If plants with Cry1Ac plus Cry2Ab are deployed while genes that confer resistance to each of these toxins are rare, and if the inheritance of resistance to both toxins is recessive, the efficacy of transgenic cotton might be greatly extended.     

Feeding and Dispersal Behavior of the Cotton Leafworm,Alabama argillacea (Hübner) (Lepidoptera: Noctuidae), on Bt and Non-Bt Cotton: Implications for Evolution and Resistance Management

The host acceptance of neonate Alabama argillacea (Hübner) (Lepidoptera: Noctuidae) larvae to Bt cotton plants exerts a strong influence on the potential risk that this pest will develop resistance to Bt cotton. This will also determine the efficiency of management strategies to prevent its resistance such as the “refuge-in-the-bag” strategy. In this study, we assessed the acceptance of neonate A. argillacea larvae to Bt and non-Bt cotton plants at different temperatures during the first 24 h after hatching. Two cotton cultivars were used in the study, one a Bt DP 404 BG (Bollgard) cultivar, and the other, an untransformed isoline, DP 4049 cultivar. There was a greater acceptance by live neonate A. argillacea larvae for the non-Bt cotton plants compared with the Bt cotton plants, especially in the time interval between 18 and 24 h. The percentages of neonate A. argillacea larvae found on Bt or non-Bt plants were lower when exposed to temperatures of 31 and 34°C. The low acceptance of A. argillacea larvae for Bt cotton plants at high temperatures stimulated the dispersion of A. argillacea larvae. Our results support the hypothesis that the dispersion and/or feeding behavior of neonate A. argillacea larvae is different between Bt and non-Bt cotton. The presence of the Cry1Ac toxin in Bt cotton plants, and its probable detection by the A. argillacea larvae tasting or eating it, increases the probability of dispersion from the plant where the larvae began. These findings may help to understand how the A. argillacea larvae detect the Cry1Ac toxin in Bt cotton and how the toxin affects the dispersion behavior of the larvae over time. Therefore, our results are extremely important for the management of resistance in populations of A. argillacea on Bt cotton.     

Mutated Cadherin Alleles from a Field Population of Helicoverpa armigera Confer Resistance to Bacillus thuringiensis Toxin Cry1Ac

The cotton bollworm Helicoverpa armigera is the major insect pest targeted by cotton genetically engineered to produce the Bacillus thuringiensis toxin (transgenic Bt cotton) in the Old World. The evolution of this pest's resistance to B. thuringiensis toxins is the main threat to the long-term effectiveness of transgenic Bt cotton. A deletion mutation allele (r₁) of a cadherin gene (Ha_BtR) was previously identified as genetically linked with Cry1Ac resistance in a laboratory-selected strain of H. armigera. Using a biphasic screen strategy, we successfully trapped two new cadherin alleles (r₂ and r₃) associated with Cry1Ac resistance from a field population of H. armigera collected from the Yellow River cotton area of China in 2005. The r₂ and r₃ alleles, respectively, were created by inserting the long terminal repeat of a retrotransposon (designated HaRT1) and the intact HaRT1 retrotransposon at the same position in exon 8 of Ha_BtR, which results in a truncated cadherin containing only two ectodomain repeats in the N terminus of Ha_BtR. This is the first time that the B. thuringiensis resistance alleles of a target insect of Bt crops have been successfully detected in the open field. This study also demonstrated that bollworm larvae carrying two resistance alleles can complete development on Bt cotton. The cadherin locus should be an important target for intensive DNA-based screening of field populations of H. armigera.     

Reduction of Bacillus thuringiensis Cry1Ac toxicity against Helicoverpa armigera by a soluble toxin-binding cadherin fragment

A cadherin-like protein has been identified as a putative receptor for Bacillus thuringiensis (Bt) Cry1Ac toxin in Helicoverpa armigera and plays a key role in Bt insecticidal action. In this study, we produced a fragment from this H. armigera Cry1Ac toxin-binding cadherin that included the predicted toxin-binding region. Binding of Cry1Ac toxin to this cadherin fragment facilitated the formation of a 250-kDa toxin oligomer. The cadherin fragment was evaluated for its effect on Cry1Ac toxin-binding and toxicity by ligand blotting, binding assays, and bioassays. The results of ligand blotting and binding assays revealed that the binding of Cry1Ac to H. armigera midgut epithelial cells was reduced under denaturing or native conditions in vitro. Bioassay results indicated that toxicities from Cry1Ac protoxin or activated toxin were reduced in vivo by the H. armigera cadherin fragment. The addition of the cadherin fragment had no effect on Cry2Ab toxicity.