Study On The Interaction Between Bt GS57 With The Gut Microbes And Immune Genes Of Spodoptera Exigua Larvae

The Beet armyworm,Spodoptera exigua(Lepidoptera:Noctuidae,Spodoptera)is an important global polyphagous pest.Pathogen infection could destroy the immune system and intestinal microbial homeostasis of insects,leading to the death of the host.However,the effect of host intestinal microbial community on the insecticidal effect of Bacillus thuringiensis(Bt)is rarely studied.In this study,the role of gut microbes in the insecticidal activity of Bt GS57 against S.exigua were investigated using Illumina HiSeq high-throughput sequencing,RNAi,qRT-PCR and homologous recombination technology methods,the role of antimicrobial peptide-related genes(SePGRP-SA,Attacin,Defensin and Ceropin)in regulating intestinal immune and microbial homeostasis was investigated,and the role of uracil,a metabolite of Bt GS 5 7,in activating the Duox-ROS immune system and maintaining the stability of the intestinal microbes were clarified,thus providing a theoretical basis for the study of new insecticidal mechanisms of Bt.The main research results are as follows:1.The genome and biological activity of Bt GS57 was determined.The results showed that Bt GS57 genome consists of 61 million raw reads with an average G+C content of 35.66%and Subreads N50 value is 9,339 bp.It was found that Bt GS57 genome contains six cry genes:cry1Ca,cry1Da,cry2Ab,cry9Ea,cry1Ia,cry1Aa,and a vegetative insecticidal protein gene vip3Aa.Phylogenetic tree analysis shows that Bt GS57 strain was most closely related to B.thuringiensis.HD.29 strain.Observation of crystal morphology and biological activity measurement results show that Bt GS57 strain can produce biconical crystals,mainly expressing 70 kDa and 130 kDa crystal proteins.The LC50 values of the Bt GS57 strain against the S.exigua,H.armigera and H.cunea neonates were 0.339 mg/mL,1.340 mg/mL and 1.420 mg/mL,respectively.The physiological and biochemical reactions analysis showed that Bt GS57 belongs to B.t.var.thuringiensis.2.The effect of intestinal bacterial community of S.exigua after Bt GS57 treatment was analyzed.The results of using Illumina HiSeq high-throughput sequencing technology analysis showed that there are many specieses of intestinal microorganisms of S.exigua,mainly including Firmicutes and Proteobacteria,Enterococcus is the main genus of Firmicutes.The dilution method and streaking method were used to isolate cultivable intestinal bacteria from the intestinal microbes of S.exigua.The 16S rDNA sequencing and phylogenetic tree analysis showed that the two bacteria were named E.mundtii HB1 and B.cereus HB1,respectively.After treatment with Bt GS57 for 24 h,the structure of the intestinal community of S.exigua changed,the proportion of Firmicutes increased,while the proportion of Proteobacteria decreased.The corrected mortality of Bt GS57(4 mg/mL)on axenic S.exigua and non-axenic larvae at 72 h was 30.00%and 53.33%,respectively,indicating that the insecticidal activity of Bt requires the participation of insect gut microbes.TheBt GS57,Bt GS57+B.cereus HB1,Bt GS57+E.mundtii HB1 and Bt GS57+B.cereus HB1+E.mundtii HB1 were fed with 4th instar sterile larvae of S.exigua,and the corrected mortality of S.exigua after 72 h was 33.33%,63.33%,23.33%and 50.00%,respectively.The results showed that the presence of B.cereus HB1 increased the sensitivity of S.exigua to Bt GS57.3.The effect of Bt GS57 on the immunity of S.exigua was studied.qRT-PCR analysis showed that after the 4th instar larvae of S.exigua ingested Bt GS57,it can cause the host immune response.The expression level of intestinal antimicrobial peptide-related genes(SePGRP-SA,Attacin,Defensin and Ceropin)and the load of bacterial were significantly increased,and the SeDuox gene was not significantly changed at 24 h and 48 h after feeding on Bt GS57.While at 72 h the expression of antimicrobial peptide-related genes of S.exigua was down-regulated,the bacterial load increased,and the expression of SeDuox gene was significantly up-regulated.The bioassay results showed that the corrected mortality of Bt GS57 on 4th instar larvae of S.exigua was 23.33%,40.00%and 50.33%at 24 h,48 h and 72 h,which were consistent with the results of qRT-PCR.4.The Bt GS57 uracil auxotrophic strain Bt GS57ΔpyrE was successfully constructed.In order to clarify the role of uracil in the Duox-ROS system,the uracil auxotrophic strain Bt GS57ΔpyrE was obtained using homologous recombination technology.The analysis of biological characteristics and biological activity showed that there was no significant difference between Bt GS57ΔpyrE and Bt GS57.qRT-PCR analysis found that,after S.exigua feeding on Bt GS57ΔpyrE,the SeDuox expression and ROS levels were significantly down-regulated compared with Bt GS57(control).However,after the addition of uracil,the expression of SeDuox and the level of ROS were significantly higher than those in the Bt GS57ΔpyrE treatment group.In addition,when the S.exigua feeding on Bt GS57ΔpyrE for 12 h,the expression level of SePGRP-SA,Defensin,Ceropin and bacterial load was increased significantly with a trend of first increasing and then decreasing compared with Bt GS57(control).The results indicate thatthe uracil of Bt GS57 can activate the Duox-ROS system and affect the expression of antimicrobial peptide-related genes,which plays an important role in maintaining the homeostasis of intestinal microbes.5.The sensitivity of S.exigua larvae to Bt GS57 was investigated after SePGRP-SA gene silencing.RNAi results showed that compared with the injection of dsEGFP(control),the 4th instar larvae of S.exigua decreased the expression of SepGRP-SA gene by 95.26%when injected with dsSePGRP-SA for 72 h.After SePGRP-SA gene silencing,compared with the control group,the expression of antimicrobial peptide-related genes Cecropin,Attacin and Defensin were significantly down-regulation,whereas the load of intestinal microbial was significantly increased in S.exigua larvae.At the same time,the corrected mortality rate of S.exigua after 72 h treatment with Bt GS57+dsSePGRP-SA was 73.33%,and the corrected mortality of S.exigua was 50.00%in the Bt GS57+dsEGFP treatment group.The results of the study showed that the sensitivity of the larvae of S.exigua to Bt GS57 increased after gene silencing.