Estimated Frequency And Identification Of Cadherin Alleles Conferring Resistance To Bt Toxin Cry1Ac In Field Populations Of Helicoverpa Armigera (Hübner)

The cotton bollworm, Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae), is oneof the most serious agricultural pests worldwide. There were serious outbreaks in the cottonarea of northern China in 1990s because of resistance to synthetic chemical insecticides.Transgenic cotton expressing Bacillus thuringiensis toxin Cry1 Ac has now been widelyadopted for insect pest control. Transgenic Bt cotton has planted in China since 1996 andplanting area is expanding rapidly. The extensive planting of Bt cotton will greatly intensifyselection pressure for Bt resistance in the target insects such as H. armigera. The potentialrisk of Bt resistance will be a limiting factor for the long-term usefulness of Bt cotton. Toaddress this concern, we need to assess the risk of Bt resistance development, investigatesusceptibility levels of field populations of H. armigera to Bt cotton and detect frequencyof resistance genes at low level in the field, which will provide an accurate informationabout whether the resistant management strategy is working correctly or not. Thedevelopment of DNA-based screening methods will greatly improve the efficiency ofresistance monitoring, but it has to depend on thorough understanding of molecularmechanisms of Bt-resistance. In present study, the frequency of resistance-associatedcadherin alleles was estimated in northern China, and new cadherin alleles associated withBt resistance from field populations of H. armigera were identified.1. Susceptibility levels of field populations of H. armigera to Btδ-endotoxin Cry1AcSusceptibility levels of five field populations of Helicoverpa armigera collected fromthe northern China cotton area and Xinjiang cotton area during 2004-2005 wereinvestigated using a surface contamination bioassay. Geographic variations (＜8 fold) werefound between these populations. No significant resistance to Cry1Ac was found in fieldpopulations of H. armigera in China.2. Estimated frequency of alleles conferring resistance to Bt toxin Cry1Ac in fieldpopulations of Helicoverpa armigera An improved F₁ screen technique was used to estimate the frequency of allelesconferring resistance to Bt cotton producing the Cryl Ac toxin in field populations of thecotton bollworm Helicoverpa armigera (Hubner) collected from Northern China in 2004and 2005. Eggs were collected from Bt cotton in Anyang of Henan Province and Cangxianof Hebei Province, respectively. The collected eggs were reared with artificial diet in thelaboratory. Second instar larvae were subjected to a primary selection with 1μg/cm² ofactivated Cry1Ac and survivors were individually mated with moths from alaboratory-selected resistant GYBT strain. After screening F₁ with a discriminating dose of2.5μg/cm² activated Cry1 Ac, we were able to estimate the field resistance frequency of5.6×10^-3 for Anyang population from Henan Province in 2004 and 1.4×10^-3 in 2005, and1.5×10^-3 for Cangxian population from Hebei Province in 2005. In general, fieldpopulations of H. armigera collected from Northern China have not evolved prominentresistance to Cry1Ac, the resistance frequency to Cry1 Ac is at its normal level.3. Identification of cadherin mutations from field populations of H. armigeraThere are two cadherin alleles in the F₁ resistant offspring from the seven single-pairfamilies identified above, one allele is from the resistant laboratory parent (rl, GenBankaccession no. AY647975) and the other allele is from field-collected parent. The full lengthcDNA of the cadherin gene Ha_BtR alleles of F₁ resistant offspring from the sevensingle-pair families were cloned with RT-PCR and 3'-RACE. Two new mutant alleles (r2,r3) of Ha_BtR were identified. Compared with the wild type cadherin amino acid sequenceof a susceptible GY strain (GenBank accession no. AY647974), the allele r2 is truncated bya premature stop codon TGA at the position 1113, and the truncated protein has only 371amino acids, and the allele r3 has a 507bp deletion that eliminates 169 amino acids.Identification of these two new alleles will facilitate developing DNA-based detectionmethods for cadherin mutations in H. armigera.