Function Studies Of Transcription Factor FOXO And Intestinal Microbiota Metabolite Linoleic And In Microbioti Homeostasis Regulation Of Shrimp

Shrimp aquaculture is one of the world’s fastest growing industries for producing animal proteins and has made a significant contribution to meeting the worldwide increased demand for animal proteins.The flora homeostasis of shrimp is closely related to the health and development of shrimp.However,the infection of pathogenic microorganisms(including bacterial and viral pathogenic microorganisms)in the shrimp aquaculture process has destroyed the flora homeostasis of shrimp,thereby affecting the healthy farming of shrimp and causing huge economic losses.Among them,Vibrio and white spot syndrome virus(WSSV)are the most representative bacterial and viral pathogens in shrimp aquaculture.Kuruma shrimp,Marsupenaeus japonicus is one of the aquaculture species with important economic value in the industry,it also can be used as a model to study the innate immunity in invertebrates.In the dissertation,taking M.japonicus as the experimental animal,the flora homeostasis regulation function of forkhead box transcription factor(FOXO)in normal shrimp,Vibrio and WSSV infected shrimp was studied.The changes of intestinal flora metabolites after WSSV infection and the antiviral function of metabolite linoleic acid in shrimp were also studied,trying to provide a new strategy for disease prevention and control in shrimp culture.The results are as follows:1.FOXO participates in the regulation of flora homeostasis in vivo of shrimpInvertebrates rely on the innate immune system,including humoral and cellular immunity to maintain the microbiota homeostasis and resist pathogen infection.In shrimp,the microbial flora of intestine and hemolymph is very important for shrimp health.Previous studies have shown that the subfamily O(FOXO)in Forkhead box(FOX)transcription factor family O is involved in mucosal immune response in mammals and intestinal humoral immune regulation in invertebrates.However,the function of FOXO is involved in systemic immunity regulation and homeostatic regulation of flora in invertebrates is still unclear.In this study,we identified a FOXO protein from M.japonicus.It was found that FOXO was expressed at the basic level in normal shrimp.FOXO played a critical role in maintaining hemolymph and intestinal microbiota homeostasis by promoting the expression of Relish,the transcription factor of the immune deficiency(IMD)signal pathway for expression of antimicrobial peptides(AMPs)in shrimp.The expression of FOXO was significantly up-regulated in shrimp infected with Vibrio anguillarum.The mechanism study found that V.anguillarum infection activated FOXO and induced its nuclear translocation by reducing serine/threonine kinase AKT activity.In the nucleus,activated FOXO directly regulated the expression of its target Amp and Relish genes against bacterial infection.Furthermore,FOXO was identified as being involved in cellular immunity by promoting the phagocytosis of hemocytes through upregulating the expression of the phagocytotic receptor scavenger receptor C(Src).Taken together,our results indicated that FOXO exerts its effects on homeostasis of hemolymph and the enteric microbiota by promoting AMP expression;FOXO can resist bacterial infection by promoting the phagocytosis of shrimp hemocytes to pathogens and promoting the expression of antimicrobial peptides.2.WSSV uses PP2A-FOXO signal axis to promote its replication in shrimpIn the "arms race"of virus host interaction,viruses have developed different strategies to escape host defenses or exploit host components and enable their infection.Studies have found that mammalian latent viruses use FOXO transcription factors in the process of reactivation,but if FOXOs is exploited by viruses during their infection remains unclear.In this study,we found that the FOXO of shrimp was hijacked by white spot syndrome virus(WSSV)during infection.Mechanistically,the expression of leucine carboxyl methyl transferase 1(LCMT1)was up-regulated during the early stages of WSSV infection,which promotes PP2A methylation to activated protein phosphatase 2A(PP2A),leading to dephosphorylation of FOXO and translocation into the nucleus.The FOXO directly promoted transcription of the immediate early(Ie)gene,Wsv079.Thus,WSSV utilized the host LCMT1-PP2A-FOXO axis to promote its replication during the early infection stage.We also found that,during the late stages of WSSV infection,the envelope protein of WSSV(VP26)promoted PP2A activity by directly binding to FOXO and the regulatory subunit of PP2A(B55),and thus it further facilitated WSSV production via the VP26-PP2A-FOXO axis in shrimp.Overall,this study reveals novel virus infection strategies by which WSSV hijacks host LCMT1-PP2A-FOXO or VP26-PP2AFOXO axes to promote its reproduction,and provides clinical targets for disease resistant strain cultivation in shrimp aquaculture.3.Linoleic acid,a metabolite of shrimp intestinal flora,has the function of resisting white spot syndrome virusIntestinal microbiota plays an important role in animal health and disease.Intestinal microbiota can produce metabolites through the changes of self-nutritional metabolism,so as to improve the immune response and nutritional absorption of the body.However,the changes of metabolites in the shrimp intestines after WSSV infection remain unclear.We established a model of shrimp infected with WSSV virus by oral feeding and analyzed the metabolites in intestinal content of shrimp infected with WSSV by HPLC and tandem mass spectrometry.Compared with normal shrimp,a total of 78 different metabolites and five different metabolic pathways were identified.Among them,we found that the content of linoleic acid,an unsaturated fatty acid,increased significantly after WSSV infection,indicating that linoleic acid might be involved in antiviral immunity in shrimp.Further study showed that,after oral administration of linoleic acid,WSSV proliferation decreased evidently in the shrimp,and survival rate of the shrimp increased significantly.Mechanical analysis showed that linoleic acid directly bound to WSSV virions and inhibited the viral replication.Linoleic acid also promoted the expression of antimicrobial peptides and IFN-like gene Vago5 by activating the ERK-NF-kappa B signaling pathway.Our results indicated that WSSV infection caused metabolomic transformation of intestinal microbiota and that the metabolite linoleic acid participated in the immune response against WSSV in shrimp.This study provides candidate drugs for disease prevention and control in shrimp culture.