Resistance Monitoring Of Helicoverpa Armigera To Bt Cotton In North China And Protein Utilization Of Resistant Bollworm

Transgenic cotton that expresses a gene derived from the bacterium Bacillus thuringiensis (Bt) has been widely adopted because of the economic advantages, reducing the use of conventional insecticides and regional suppression populations of a target pest. In China, Bt cotton was approved in1997for commercial use to control cotton bollworm, Helicoverpa armigera, and has steadily been planted by Chinese cotton growers. The widespread planting of transgenic Bt cotton imposes strong selection pressure on cotton bollworm in the field. Resistance to the Bt toxin Cry1Ac in H. armigera is considered to be a major threat for the long-term effectiveness of Bt cotton. In this study, we evaluated resistance to Bt Cry1Ac toxin by the bioassays of isofemale lines and diagnostic PCR in H. armigera populations in North China. Compared to the susceptible strain (96s and LF), loss of function of a trypsin protease in the resistant populations (LF20.0and LF120.0) was linked to high levels of Cry1Ac resistance, the4th instar larvae were restricted to diets varying in concentration of protein. Compare the differences of nutrition indicators and gut proteases activity in the various strains, to investigate the influence of protease changed on the adaptability in H. armigera.The main research results are as follows:1A total of1,694lines from Xiajin and933lines from Anci were screened for growth rate on normal artificial diet and the diet containing1.0μg of CrylA(c) toxin per ml. In2011-2013, the mean relative average development rates of H. armigera larvae in the Xiajin population was0.62,0.59,0.59,0.58and0.62, respectively; and the Anci population was0.54,0.58,0.60,0.53and0.62, respectively. Compared to previous results in2002, the RADR of H. armigera during2011-2013, with ratios of1.53-1.63and1.77-2.07in their respective Xiajin and Anci populations, however, the frequency of resistance alleles of H. armigera was remained low. The result suggested that resistance to Cry1Ac has increased in H. armigera populations in North China, but have not reached a high level of resistance.2According to the mutant allele of APN or cadherin conferring resistance to Bt toxin Cry1Ac, A DNA-based PCR method was developed to screen the mutation alleles from field populations of H. armigera collected from the main cotton planting areas of China in2009-2013, however, no mutation allele was detected from2,246moths.3We used four strains of H. armigera:susceptible strains (96s and LF) and Cry1Ac-resistant strains (LF20.0and LF120.0), the4th instar larvae were restricted to diets varying in concentration of protein (0%、5%、10%、20%and30%). As the protein content of the diet increased, the bigger mass of larval and pupal were observed when bollworm were reared on diet that protein concentration was0%-10%. When the concentration of protein in the diet was10%-30%, the mass of larval and pupal decreased with increasing concentration of protein. But the resistant strains didn’t follow this rule, The larvae mass of susceptible strains were significantly higher than that of resistant strains fed on any protein concentration of diets, and the resistant strains couldn’t pupate in the5kinds of diet. The results indicated that the ability of protein digestion and absorption of resistant strains was significantly lower than the susceptible strains.4The activities of midgut total protease, trypsin-like enzyme and chymotrypsin-like enzyme were estimated after larvae of resistant strains (LF20.0and LF120.0) and susceptible strains (96s and LF) fed on artificial diets containing different concentrations of protein. LF120.0strain feed on lower protein diets (0%and5%) after2days showed significantly higher total protease activity than other strains. However, there was no significant difference between the resistant strains and susceptible strains with the extension of feeding time. Larvae reared on high protein diets (10%and20%) after6d, Trypsin-like activity and chymotrypsin-like activity of the resistant strains were significantly higher than the susceptible strains. The maximum in trypsin-like activity of LF120.0strain feed on artificial diets contains10%concentrations of protein after2d, is significantly higher than other strains, however, as the feeding time was significantly reduced, until it is lower than the sensitive strains. The results showed that the resistant strains at the same time of altered protease lead to resistance to Cry1Ac, reduces the activities of midgut digesting enzyme, and hydrolysis of protein was affected in Cry1Ac-resistant strains.Compared with previous studies, resistance to Cry1Ac has increased in H. armigera populations in northern China, but not reached a high level of resistance. Although PCR diagnosis has not detected resistance mutations which have been reported, does’t excluded the possibility that there were other resistance alleles occured in field population. A laboratory-reared resistant strain that altered protease resulted in resistance to Cry1Ac, the digestion and absorption of protein was affected by digestive enzyme activity decreased significantly, and the adaptability of resistant strains was affected. The present study evaluated the level of Bt toxin susceptibility of cotton bollworm larvae from field populations, provide the basis for resistance management strategy formulation and implementation. Meanwhile, enhances the understanding of protein nutrition metabolism of Cry1Ac-resistant H. armigera, and providing leads that could be delay resistance to Bt cotton.