| The pest status of the cotton bollworm, Helicoverpa armigera (Hiibner) is enhanced by a suite of four physiological, behavioral, and ecological characteristics that enable the insect to develop resistance to most insecticides:polyphagy, high fecundity, high mobility and facultative diapause. Resistance to insecticides by the cotton bollworm developed rapidly due to the massive use of chemical pesticides in the1990s, eventually leading to severe outbreaks in the northern China cotton-growing areas, resulting in tremendous economic losses. In China, bioengineered Bt cotton producing Cry1Ac protein was commercialized from1997. The Bt cotton acreage accounted for71.5%of the total cotton acreage in2011. Vast plantings of Bt cotton have helped to control the damage of cotton bollworm effectively, and reduced the need for insecticide sprays, and also enhanced biocontrol services, bringing significant economic, environmental and social benefits. But there is a major potential risk of the development of resistance to the Bt toxin under the strong selection pressure in the field.Bt cotton producing Cry1Ac has been commercialized for15years in China, and non-Bt cotton as refugia has not been required. It is regarded as inevitable that resistance to Cry1Ac of H. armigera will increase under intensive selection of Bt cotton in China. Therefore it is important to investigate Bt resistance status and Bt resistance gene types in the field populations of H. armigera. In this study, the resistance levels to Cry1Ac toxin were surveyed, the Cry1Ac resistance alleles in field populations of H. armigera from northern China were characterized, and a mechanism conferring non-recessive resistance to CrylAc was studied by a cytosolic domain mutant of a cadherin molecule which is known to act as a binding site for the Bt protein in the cotton bollworm. The results here demonstrated that intensive planting of Bt cotton producing Cry1Ac resulted in field-evolved increases in resistance gene frequencies in some populations of H. armigera from northern China. These results will allow the development of adaptive resistance monitoring techniques, and facilitate the formulation of proactive countermeasures to Bt resistance in H. armigera in China.1. Detection of resistance to Bt toxin CrylAc in field populations of H. armigera.The baseline data from1994to1997, determined before Bt cotton planting in China, have showed that susceptibility to Cry1Ac was not different between field populations from northern China and northwestern China (Xinjiang). Bt cotton has been planted much more intensively in northern China than in northwestern China from1997to the present. The susceptibility to CrylAc was measured in fifteen populations of H. armigera collected from Xinjiang cotton planting area (2populations) and the northern China cotton planting area (13populations).The LC50values of CrylAc activated toxin did not differ significantly among the two field populations (Shawan and Shache) from Xinjiang and the laboratory susceptible strain SCD. The Anyang population from northern China had the highest LC50value for CrylAc activated toxin, which was16times higher compared to that of the Shawan population. The median LC50was2.8times higher for the13populations from northern China (52ng cm-2) compared with the median for the two populations from northwestern China (18.5ng cm-2). Similar to the results for Cry1Ac activated toxin, the data for Cry1Ac protoxin showed that the median LC50was3.0times higher for the13populations from northern China (80ng cm-2) compared with the median for the two populations from northwestern China (26.5ng cm-2), and the resistance ratio for CrylAc protoxin compared to the susceptible SCD standard, was10-fold for the Anyang population.We used1μg CrylAc activated toxin per cm2of artificial diet surface as a diagnostic concentration to screen the15field populations. Survival at the diagnostic concentration was significantly higher for the13populations from northern China pooled (1.3%,147of11,064) than for the two populations from northwestern China pooled (0%,0of1296) or the laboratory susceptible strain SCD (0%,0of168). The Anyang population, which had the highest LC50value for CrylAc activated toxin and CrylAc protoxin, also had the highest survival at the diagnostic concentration (2.6%,33of1248).The results indicate that intensive planting of Bt cotton has resulted in increased tolerance to CrylAc in some field populations of H. armigera in northern China. Although field control failures of Bt cotton have not been reported, we should consider and then implement proactive countermeasures to delay Bt resistance development in the field. 2. Diverse genetic basis of field-evolved resistance to Bt cotton in H. armigera.The F1screen method was employed to screen resistance alleles to CrylAc in the Xiajin population from Shandong Province collected in2009, and the dominance of the resistance alleles detected was evaluated. The F1screen results showed that among the146single-pair families between field-derived males and SCD-r1females (with recessive CrylAc resistance conferred by the r1allele of cadherin), the field-derived male adults of28single-pair families carried one resistance allele, and the male adults of3single-pair families carried two resistance alleles, so the resistance allele frequency was estimated to be0.116(95%CI:0.083-0.160). The dominance of the resistance alleles detected with the F1screen was determined by crossing survivors of the F1screen with the susceptible SCD strain and testing the progeny at the diagnostic concentration. The results showed that though the recessive cadherin alleles accounted for80%of resistance alleles detected.73%-84%of the resistant individuals carried at least one non-recessive resistance allele. Clearly, resistance management strategies will need to take non-recessive alleles into account.In order to further clarify the genetic diversity of Bt resistance in field populations of H. armigera, the survivors at the diagnostic concentration from the2010field populations were crossed with the susceptible SCD strain, and selected the progeny for one or two generations with CrylAc to establish resistant strains. Among the12resistant strains established,5strains carried cadherin-based recessive resistance alleles and7strains carried non-recessive resistance alleles. Among the7strains with non-recessive alleles,6carried cadherin-based resistance alleles,1carried a non-cadherin resistance allele. The results showed that field populations can evolve resistance to Bt toxins through a number of discrete mechanisms, and that many resistant individuals carried non-recessive Bt resistance alleles.Non-recessive resistance alleles will play a key role in the evolution of resistance to Bt cotton. Considering the status of Bt resistance genes in the field, it is suggested that both dual-Bt cotton and natural refugia should be combined to delay the evolution of resistance to Bt cotton in China.3. Non-recessive Bt toxin resistance conferred by an intracellular cadherin mutation in H. armigera.The cadherin mutations conferring Bt resistance in pests reported previously are all caused by truncations or deletions in extracellular domains, there are no reports of intracellular domain mutations involved in Bt resistance. Here, a novel cadherin resistance allele named r15, which had a55amino acids deletion in exon32of the cytosolic domain of HaCad, was discovered and characterized. To analyze the resistance conferred by r15, a resistant strain (XJ-r15), homozygous for the r15allele, was established. Allelism test showed XJ-r15was allelic with r1at the cadherin locus. Genetic linkage analysis demonstrated that resistance to Cry1Ac in the XJ-r15strain was non-recessive, and tightly linked with r15allele. Western-blot and ligand-receptor binding analysis indicated that the binding of BBMVs (brush-border membrane vesicles) of XJ-r15to Cry1Ac was as same as the ligand-receptor binding between BBMVs of SCD and Cry1Ac toxin, and that reduction in the binding was not therefore the mechanism of resistance in XJ-r15.Four types of cadherin alleles were expressed in heterologous Sf9cell cultures: susceptible (s), resistant with the intracellular domain mutation (r15), and two complementary chimeric alleles with (s/r15) and without the mutation (r15/S). Cells transfected with all four cadherin alleles were killed by Cry1Ac, but for cells transfected with alleles of HaCad, LC50values (95%FL) were significantly higher for alleles with the cytosolic domain of r15(r15:85[71-110] and s/r15:82[68-100]) than for alleles with the cytosolic domain of s (s:38[31-46] or r15/s:38[31-45]). LC50values did not differ significantly between Sf9cells transfected with alleles of HaCad that had the same cytosolic domain (r15and s/r15; and r15/s), implying that the intracellular domain of cadherin is involved in post-binding events that mediate toxicity of Cry1Ac.The mutation discovered here is not recessive and occurs in the intracellular region of cadherin, which differs from all previously characterized cadherin mutations that confer recessive resistance to Bt toxins and occur in the extracellular region of cadherin. The non-recessive resistance conferred by the novel mutation identified here has immediate implications for managing resistance in the field. Our data also show that the intracellular region of cadherin contributes to insect susceptibility, which clarifies how Bt toxins work and helps to resolve a controversy about the two main models proposed to explain the mode of action of Bt toxins. |
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