Effects Of Gut Bacteria To The Insecticidal Activity Of Bacillus Thuringiensis Toward Helicoverpa Armigera

Bacillus thuringiensis always expresses insecticidal crystal proteins largely during formation of spore and its crystal proteins have efficient specific toxicity against many kinds of insects. Researchers proposed some kinds of insecticidal mechanism of Bacillus thuringiensis. Recently some researchers believe insect midgut symbiotic bacteria are necessary for Bacillus thuringiensis insecticidal activity, but some others debate with it. This work explored the role of midgut bacteria in Helicoverpa armigera when faced to Bacillus thuringiensis toxin and provide a view for studying the mechanism of Bacillus thuringiensis toxicity.Firstly,9 kinds of midgut bacteria strains were isolated from ordinary H. armigera midgut, including 8 kinds of Gram-positive cocci and 1 kind of Gram-negative cocci. According to bacteria quantity and distribution characteristics in different instar, we could infer No.2 strain, No.3 strain and No.7 strain were dominant intestinal flora in the midgut of H. armigera.Secondly, obtained aseptic H. armigera by amending ampicillin, streptomycin, rifamycin and gentamicin with a final concentration of 500μg/mL into sterile ordinary diet. None colony was detected on NA plates after spread gut anatomy liquid of aseptic H. armigera. However, yellow, white or red colonies appeared on NA plates after spread gut anatomy liquid of ordinary H. armigera. Average OD600 value of ordinary H. armigera gut anatomy second time enrichment liquid was 1.773; meanwhile, the OD600 value of aseptic H. armigera was 0.004. These dates showed no culturable bacteria existed in aseptic H. armigera. Extracted 1st to 3rd instar larvae of ordinary and aseptic H. armigera intestinal DNA, and amplified 16S rDNA of both of them.1.5 kb bands can be found in agarose gel electrophoresis of ordinary H. armigera 16S rDNA. No bands appeared in agarose gel electrophoresis of aseptic H. armigera 16S rDNA. All these results suggest our method can eliminate bacteria in the gut of H. armigera.At the same time, comparing the growth state and survival rate of two groups H. armigera, we found that two groups H. armigera had close weight and survival rate in the same batches reared under same conditions. The weights of each ordinary 3rd instar and 6th instar larvae were 38.75 mg and 223.25 mg. The weights of each aseptic 3rd instar and 6th instar larvae of H. armigera were 37.50 mg and 231.00 mg. The survival rates of ordinary 3rd instar and 6th instar larvae of H. armigera were 90.25% and 86.00% respectively. Similarly, the survival rates of aseptic 3rd instar and 6th instar larvae of H. armigera were 89.50% and 87.00%.Then tested the toxicities of CrylAc10 against ordinary H. armigera and aseptic H. armigera. Their LC50 values were 0.0579±0.0389 mg/mL and 0.0143±0.0054 mg/mL respectively. Cry1Ac10 had high toxicity against both group insect, but Cry1Ac10 toxicity was 4 times higher against aseptic H. armigera than ordinary H. armigera.Subsequently, amend No.3 bacteria, one domenate gut bacteria, with aseptic diet to test the toxicity of Cry1Ac10. Results showed No.3 bacteria could reduce the sensitivity of aseptic H. armigera toward Cry1Ac10 toxin, and reduced the morality from 36.94% to 26.92%. Hence, gut bacteria was not required for Bt insecticidal activity to H. armigera. In contrast, an intestinal microbiota can protect H. armigera from toxin attack.