Suceptibility Of Larvae And Adults To Cry1ac And Cry1ca In Spodoptera Exigua And Helicoverpa Armigera And The Possible Mechanisms

Helicoverpa armigera Hubner is a pest of cotton. It damages more than200plant species belonging to more than20families. It widely distributed in China and around the world, has occurred at China’s cotton-growing and vegetable growing areas, such as the Yellow River and the Yangtze River areas. In recent years, the pest has also occurred in Xinjiang. The Spodoptera exigua Hubner is a highly polyphagous pest and causes extensive damage to many field and truck crops, and even weeds and grasses. S. exigua has become a major economic pest of cotton across a wide distribution since the commercialization of Bt cottons in1997in China.Being delivered as sprays or expressed in plant, Bacillus thuringiensis (Bt) crystalline proteins (Cry toxins) display insecticidal activities against numerous Lepidopteran, Dipteran and Coleopteran larvae. However, the widespread and sustained use of Bt cotton and Bt sprays inevitably results in some negative effects. Comparative study of toxicities of Bt Cry toxins between larvae and adults may afford important new insights into the interactions of the toxins with receptor proteins in host insect, and represent intriguing targets for the control of insect pests.1. The toxicities of CrylAc and Cry1Ca to Helicoverpa armigera and Spodoptera exigua adultsThe effectiveness of CrylAc and CrylCa on lifespans and reproductive performance of H. armigera and S. exigua adults were evaluated by in vivo experiments, at the concentraion of500,100and20μg/ml in a10%sucrose aquous solution. At the highest concentration, Cry1Ac and Cry1Ca shortened48.1%and48.9%of H. armigera female lifespan, and43.5%and38.5%of S. exigua female lifespan, and they reduced37.8%and40.3%, and50.5%and47.4%of H. armigera and S. exigua male lifespans respectively. Bt toxins negatively affected copulation. Exposure to500μg/ml of Cry1Ac and Cry1Ca greatly reduced50.0%and46.8%, and58.7%and57.3%spermatophore acceptance by H. armigera and S. exigua females respectively. Similarly, CrylAc and Cry1Ca exposure decreased40.0%and50.3%, and61.3%and60.0%of spermatophore transfer by H. armigera and S. exigua males respectivly. Moreover, exposure females rather than males to500μg/ml of Cry1Ac and CrylCa significantly droped57.5%and57.5%of the number of eggs laid by H. armigera, and35.4%and45.8%of the number of egg masses deposited by S1. exigua. In contrast, both Cry1Ac and CrylCa did not negatively influence the egg hatchabilities. At the middle and the lowest concentrations, however, the biological effects of CrylAc and Cry1Ca on lifespans and reproductive performance partially or completely disappeared.2. Combined effects of three crystalline toxins with seven proteinase inhibitors on S. exiguaInteractions between the3Cry toxins and7proteinase inhibitors were investigated by monitoring larval growth. The proteinase inhibitors were phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, tannic acid, N-a-tosyl-L-phenylalanine chloromethyl ketone, N-a-tosyl-L-lysine chloromethyl ketone, elastatinal and ethylenediaminetetraacetic acid (EDTA). A6-day dietary exposure of the newly molted2nd instars to either the inhibitors (3test concentrations) or the toxins (CrylAc,31.30ng/cm2; Cry1Ab,3.2ng/cm2; Cry1Ca,0.6ng/cm2) alone only slightly affected larval growth. In contrast, exposure to the mixtures containing an inhibitor and a toxin, with the exception of those containing EDTA, synergistically reduced larval weight, In general, the synergisms were more obvious at higher inhibitor concentrations. These results indicated that trypsin-, chymotrypsin-. and elastase-like proteinases in S. exigua larval midgut were involved in proteolytical hydrolyzation of the3activated Cry toxins.3. Cloning and characterization of serine proteinase genesBased on the Spodoptera exigua transcriptome database and RT-PCR technology,6full-length cDNA sequences encoding serine proteinases were obtained from the midgut template of Spodoptera exigua. Sequence alignment and phylogenetic analysis showed that these six proteins showed high identify with corresponding serine protease-like proteins from Bombyx mori.4. Cadherin temporal expression and serine protease activity assay of H. armigera and S. exigua Cadherin gene expression level in different development stages was estimated by qPCR. It was found that cadherin protein mRNA level was higher in fifth and sixth instar larvae of H. armigera and S. exigua respectively. In contrast, cadherin genes did not expressed in pupa and adult stages. The intestinal serine protease activity in S. exigua larvae and adult was examined. Larvae serine protease activity was significantly stronger than the adult.