Study On The Mechanism Of Vitellogenin Receptor-Mediated Antimicrobial Immunity In Oocytes Of The Chinese Mitten Crab(Eriocheir Sinensis)

Reproduction is essential for the continuation of the species,and the gonads are important organs for sexual reproduction.In the face of a complex and changing external environment,how to effectively resist the infestation of pathogenic microorganisms in order to ensure their own health and reproduction is an important issue for all living organisms.The gonads are important organs for reproduction,and how to protect them efficiently after the body has been invaded by pathogens,so as to ensure that they are able to reproduce their offspring properly,is a scientific question of concern to many scholars.The Chinese mitten crab(Eriocheir sinensis)is a typical oviparous animal,and its eggs need to accumulate a large amount of vitellogenin(Vtg)during the development process,while the vitellogenin synthesised by the hepatopancreas needs to be transported to the ovary via the hemolymph,and then Vtg enters into the oocyte through endocytosis after being specifically recognised by vitellogenin receptor(VgR).However,recent studies have found that Vtg,in addition to its nutritional function,also has the functions of binding bacteria,antimicrobial and mediating the entry of microorganisms into the oocyte.So,when an organism is infected with bacteria,does Vtg in the hemolymph carry the bacteria along with it into the oocyte thereby impairing oogenesis? Based on the above considerations,the present study intends to investigate the self-protection mechanism of oocytes after pathogen infestation in E.sinensis in order to enrich the theoretical knowledge of crustacean reproductive immunology.In this study,we first obtained the full length of the ORF of EsVgR by gene cloning and performed bioinformatics analysis of the sequence,and revealed that EsVgR was highly conserved during the evolutionary process.It contained LBD1,LBD2,EGF1,EGF2,and transmembrane structural domains sequentially from the amino-terminal to the carboxyl-terminal.Tissue-specific distribution results showed that the mRNA forEsVgR was highly significantly expressed in the ovary compared to the other tissues examined,and the gene was most highly expressed during stage Ⅲ of ovarian development.The results of in vivo and in vitro experiments showed a significant downregulation of EsVgR expression and a significant increase in the expression of antimicrobial peptides(AMPs),which are highly effective effectors of humoral immunity,in ovarian tissues after Vibrio parahaemolyticus challenge.It was further determined by fluorescence in situ hybridisation experiments that both EsVgR and EsALF3(antimicrobial peptides)were localised to oocytes.It was also detected that the mRNA of EsRelish and EsDorsal were significantly induced to be highly expressed in the ovary after V.parahaemolyticus challenge.The above results suggest that EsVgR and AMPs in the ovaries of the Chinese mitten crab respond to the organism’s antimicrobial immune response.On this basis,the specific molecular mechanisms regulating EsVgR expression in V.parahaemolyticus-infected oocytes were further explored using relevant molecular biology tools.Firstly,four potential transcription factor genes forEsVgR were obtained using three transcription factor prediction software analyses.It was found that only EsGATA binding factor A responded to V.parahaemolyticus challenge,and the expression of EsVgR was also found to be significantly reduced after interfering with this transcription factor;Secondly,bioinformatics analysis of EsGATA binding factor A revealed that it is highly conserved in evolution and contains two zinc finger structural domains.Tissue-specific expression results showed that it was widely expressed in all tissues examined,but was highest in hepatopancreas,followed by testes and ovary;furthermore,the results of the expression of this gene at different stages of ovarian development showed that its highest expression was located in stage II of ovarian development,earlier than stage Ⅲ of ovarian development in EsVgR.Finally,EsGATA binding factor A was found to regulate EsVgR expression using Ch IP assay and dual luciferase reporter gene assay,and it was further confirmed by mutation experiments that the transcription factor binding site is a-495 bp to-484 bp gene sequence located in the promoter region of EsVgR.The above studies confirmed that EsGATA binding factor A is a transcription factor forEsVgR.Finally,the sites of interaction between vitellogenin and its receptor were identified by immunoprecipitation experiments as the LPD structural domain of EsVtg and the LBD1 structural domain of EsVgR,respectively.By constructing recombinant proteins rLPD,rVWD and rLPD-VWD of each structural domain of EsVtg,it was found that only recombinant proteins containing the LPD structural domain could enter the ovarian tissue by using WB experiments,which again proved that the LPD structural domain is the key to the entry of Vtg into the oocyte.In addition,using WB and immunofluorescence assays,it was found that the amount of rLPD,rLPD-VWD and rLPD-VWD recombinant proteins co-incubated with the bacteria into the ovarian tissues was significantly reduced in the experimental group infected by V.parahaemolyticus for 12 h,whereas the amount of the above recombinant proteins into the ovarian tissues of the experimental group not infected by V.parahaemolyticus was unaffected.To this end,the mRNA expression of EsVgR in the above bacterial infection and no bacterial infection groups was further examined using qRT-PCR,and it was found that there was a significant decrease in the expression of EsVgR in the group that was bacterially infected for 12 h.This confirms that the entry of the above proteins into the oocyte is through the EsVgR and that bacterial stimulation leads to the downregulation of expression of EsVgR,thus preventing the entry of EsVtg into the oocyte.In summary,this study reveals for the first time that Chinese mitten crab oocytes respond to V.parahaemolyticus challenge by regulating the expression of EsVgR and AMPs,and also reveals the specific molecular mechanisms regulating the expression of EsVgR as well as the specific reasons why oocyte uptake of EsVtg proteins is affected by bacterial infections,which greatly enriches the body of research on the antimicrobial immune response in invertebrates.