The Role Of Diversity Of Midgut Microorganisms In Susceptibility Of Helicoverpa Armigera To Bt Cry1Ac Toxin

It was reported that the toxicity of Bacillus thuringiensis(Bt)toxins to insect depended on the enteric microorganisms that normally reside in the insect midgut.Gut microorganisms of insects could offer some kinds of digestive enzymes and detoxifying enzymes to their hosts,and it plays important role in Bt toxin-induced killing of insects.The gut microorganisms of insect might influence the toxicity of Bt and insect resistance to Bt might be partially caused by the effect of insect-gut microflora.Transgenic cotton expressing Bt toxin Cry1 Ac has been widely used for management of Helicoverpa armigera,but the resistance of H.armigera to Cry1 Ac has threatened the long-term benefits of this crop.Therefore,the aim of this study was to examine the symbiotic relationship between the insect-gut microbiota and pathogenicity of Cry1 Ac and roles of diversity of midgut microorganisms in resistance evolution of H.armigera to Bt.The main results were as followed:Exposure to each antibiotic as well as antibiotic cocktail significantly reduced the midgut microflora population in H.armigera.Pre-treatment of antibiotics(gentamicin,streptomycin,aureomycin and ampicillin)decreased larval mortality and increased weight gain due to Cry1 Ac in Btsusceptible strain(96S),Bt-resistant strain(BtR)and field-collected strain(FS).Significant effect of each antibiotic as well as antibiotic cocktail on percent mortality reduction and weight increase due to Cry1 Ac was observed among three strains,and the reduced mortality was proportional to the concentration of antibiotics in the artificial diet in all strains.The most remarkable synergetic effect between antibiotics and Cry1 Ac was observed in BtR as shown by the highest mortality reduction but this was not the same trend for larval weight increase.The gut microbiota composition of H.armigera was more complex and highly variable among different strains,after the diversity of midgut microorganisms was analysed by 16 S rRNA gene sequencing on Illumina MiSeq platform.The bacterial diversity was more abundant in larvae fed on normal diet in 96 S,followed by FS strain and BtR strain.Halomonas,Chloroplast_norank and Enterococcus were the most predominant genera that comprised 61.95% of total midgut microorganism in fifth instar larvae fed normal diet in 96 S strain,and Enterococcus was the most predominant genus in larval midgut that accounted for 92.78% and 96.62% in BtR and FS strain separately.After the larvae treated by antibiotics or Cry1 Ac or antibiotics + Cry1 Ac,the compositions of microorganism in larval midgut were obviously changed.Bacterial diversity was significantly difference across treatments in 96 S and FS strains by Shannon index estimator,while the effect of antibiotics and Cry1 Ac were apparent on bacterial sequences in BtR and FS strains by Simpson diversity analyzed.The differences of total number of bacterial abundance were significant among treatments in 96 S.In 10 most abundant genera,the Chloroplast_norank,Ralstonia and Mitochondria_norank were significant affected after treated by antibiotics and Cry1 Ac in 96 S strain.Enterococcus,Rhodanobacter,Lactobacillus,Pseudomanas and Bacteroides had obvious changed in BtR strain,and Enterococcus,Rhodanobacter,Lactobacillus,Pediococcus,Pseudomanas and Bacteria_unclassified were singnificant different among different samples in FS strain.The number of OTUs increased in treated samples by antibiotics regardless of treated by Cry1 Ac in BtR and FS strains.However,the number of OTUs reduced in treatments by antibiotics or treated by antibiotics-Cry1 Ac in 96 S strain.In comparison among strains,the most common dominant phyla(Proteobacteria,Cyanobacteria_norank and Firmicutes)and genera(Enterococcus,Halomonas and Methylobacterium)detection was obviously differed among tested strains across most treatments.The percentage of Enterococcus in total larval midgut microorganisms reduced after continuously treated by antibiotics or treated by antibiotics-Cry1 Ac compared with those larvae fed on normal diet in all three strains.Cry1 Ac alone treated had no significant direct adverse effects on Enterococcus in 96 S and FS strain,but the percentage of Enterococcus reduced after treated by Cry1 Ac in BtR strain.The results suggested that antibiotics could influence the toxicity of Cry1 Ac by eliminated gut microflora and the different effect in various strains might be due to the variation in diversity and composition of midgut microbial community,especially the common genus Enterococcus.Therefore,the microorganisms in insect midgut might be play essential roles in Bt-induced killing of H.armigera and in resistance evolution of H.armigera to Bt.