The Functional Study Of Duox-ROS System And IMD Pathway In Bactrocera Dorsalis

There is a diversity relationship between the gut micriobes and the host.The gut symbionts affects host development,energy metabolism,harmful substance degradation,maturation and development of the immune system.Like most metazonans,the guts of insects are in permanent contact with the microbial realm that includes beneficial symbionts,nonsymbionts,food-borne microbes and life-threatening pathogens.Therefore,gut epithelia must be able to tolerate a certain amount of proliferation of commensal microbes for beneficial gut-microbe interactions to occur while still competently eliminating detrimental microbes.Based on the function of Duox-ROS system and immune deficiency pathway in the intestinal immune response,they should be involved in the regulation of gut microbiota homeostasis.However,the molecular mechanism that the host use to maintain the gut microbiota homeostasis is unclear.Bactrocera dorsalis is a devastating quarantine pest damaging 250 species and varieties of fruits and vegetables worldwide.In this study,we will employ RNAi,qRT-PCR and Illumina MiSeq bacterial 16 s rDNA highthroughput sequencing etc.molecular techniques to study the role of BdDuox gene in the gut immune response and regulation of the intestinal bacterial community homeostasis in B.dorsalis;study the effect of reactive oxygen species(ROS)on the fertility and longevity of B.dorsalis;clarify the role of the Imd pathway in the immune defences of B.dorsalis.1The role of Duox-ROS system in intestinal immune defence and regulation of the intestinal bacterial community homeostasis in B.dorsalisWe cloned the BdDuox cDNA and its coding region is 4584 bp long,encoding for a 1528 amino acid length protein.The protein contains seven transmembrane regions and harbors a characteristic peroxidase domain that is distinct from the Nox family.In this study,we found that B.dorsalis used the BdDuox-dependent immune response as a defense in the gut.The results showed that there was a significant increase of BdDuox gene expression and ROS production level at 6h and 12 h post oral infection(POI)with the Gram-negative bacteria,Escherichia coli DH5α,and Gram-positive bacteria,Staphylococcus aureus.There was no significant increase in BdDuox gene expression and the ROS production level when flies were fed with one of the five major symbiotic gut bacteria at 6h POI,except the minor bacteria Bacillus cereus.The Duox-ROS immune system could tolerate the commensal microbes,while eliminating the pathogens.We found that the level of BdDuox gene transcript was inhibited during 1-7 day post RNAi(DPR)treatment,and the reduction of BdDuox gene expression varied from 26% to 54%,the level of ROS followed the same pattern with a decrease of 31-44% as compared with the ds-egfp control;the BdDuox expression level was increased 1.40-1.68 times in ds-duox-treated flies from 9 to 17 DPR,the level of ROS increased 45-73% as compared with the ds-egfp control;the BdDuox expression level and the ROS production level returned to the basal expression level at 20 DPR.The results showed that knockdown of BdDuox led to an increased bacterial load,with a 1.68 and 1.49 increase at 5 and 15 DPR,and to a decrease in the relative abundance of Enterobacteriaceae and Leuconostocaceae bacterial symbionts in the gut.The resulting dysbiosis,in turn,stimulates an immune response by activating BdDuox and promoting ROS production that regulates the composition and structure of the gut bacterial community to normal status by repressing the overgrowth of minor pathobionts.Our results suggest that BdDuox gene plays a pivotal role in regulating the homeostasis of the gut bacterial community in B.dorsalis.2The effect of ROS on the fertility and longevity of B.dorsalisWe study the role of Bd Cat gene in maintaining the redox homeostasis,the fetility and longevity of B.dorsalis based on RNAi techniques.The results showed that the level of BdCat gene transcript was inhibited during 1-7 DPR treatment,with a 54.04-75.42% decrease,but the level of ROS increased 1.37-1.62 times as compared with the ds-egfp control;there were a 33.91% and 34.78% decrease of bacterial load at 5DPR and 10 DPR,respectively,when compared with the control.In the study of longevity of B.dorsali by silence of BdDuox and BdCat gene,the results showed there was no significant effect on the median longevity and the total suvival time of B.dorsali with the ds-duox and the ds-cat treatment.However,the knockdown of BdCat gene increased the median suvival time of B.dorsali.In addition,the knockdown of BdCat gene led to an decreased ovipostion amount and egg hatching,with a 31.01-57.09% decrease of the daily ovipostion amount in ds-cat treated flies at 14-20 DPR,with a 25.45% decrease of the egg hatching in ds-cat treated flies.While the silience of BdDuox gene had no significant effect on the ferility of B.dorsali.In the study of longevity of B.dorsali by feeding chemical substances,we found there was a dramatical decrease of longevity of B.dorsali after fed with paraquat and vitamin C,and all the flies died within 7 days.3The function of Imd pathway in immune defences of B.dorsalisTo study the function of Imd pathway in systemic immune and intestinal immune response to opportunity pathogens.The results showed that the Imd pathway was actived rapidly after injection of E.coli DH5α,and the expression levels of BdRel,BdDpt,BdPGRP-LB,BdPGRP-SB gene increased significantly,while the expression levels of BdPGRP-SC2 gene dramatical decreased,with a 55.63-90.73% reduction at 3-12 h PII.The short period oral infection by E.coli DH5α could only induce the intestinal immune response of Duox-ROS system,the expression levels of BdDuox gene increased 1.70-2.89 times at 3-12 h POI,the oral infeciton also induce the immune response of the key regulator BdRel,the PGRPs family gene BdPGRP-LB,BdPGRP-SB,BdPGRP-SC2 of the Imd pathway,but not the effector gene BdDpt.Then we studied the function of PGRPs,and we found the BdPGRP-LB,BdPGRP-SB,BdPGRP-SC2 gene were very important negative regulator of the Imd pathway,because the knockdown of BdPGRP-LB,BdPGRP-SB and BdPGRP-SC2 induced a derepression of immune response of the Imd pathway.In the long-time serial oral infection assay,we found the E.coli DH5α actived the Duox-ROS immune system at 6h and 12 h POI,but not the Imd pathway,similar as the results of the short-time assay.While at 3 day POI,we found E.coli DH5α induced a dramatical response of the Imd pathway,and the expreesion level of BdDpt gene increased 10.50-26.95 times at 3-15 day POI;but the Duox-ROS system was blunted in response to oral infection at 3-15 day POI.These results indicate there is a timing difference between intestinal Duox-ROS immune system and Imd patyway in response to oral infection.In addition,the long-time serial oral infection of E.coli DH5α also induced a higher expression of BdDpt gene in fatbody at 5-15 day POI,with a 4.08-7.25 times increase.And we found the increased ROS level by fed the flied with H2O2 and paraquat could induce the immune response of Imd pathway to E.coli DH5α in advance.The results indicate ROS has an important role in the immune coordination between the Duox-ROS immune system and Imd patyway in the gut immune response.