Study On The Functions Of STAT And MyD88in Penaeid Shrimp During WSSV Infection

Shrimp is the one of the major species in aquaculture. Disease is always aproblem which hinders the healthy development of shrimp aquaculture. Both virusand bacteria are dangerous pathogens to shrimp aquaculture. Shrimp defense againstpathogens mainly by initiating the innate immunue signal pathways. Toll andJAK/STAT signaling pathways play key roles in the antiviral immunity of mammals,fish and insect. However, limited knowledge is known about the function of MyD88and STAT in shrimp, which are regarded as an key component of Toll or JAK/STATsignaling pathway respectively, during WSSV infection. The present thesis mainlyfocused on the function of MyD88and STAT in Penaeid shrimp. The main progressesare as follows:1. The full length cDNA of a MyD88homologue (FcMyD88) was cloned frompenaeid shrimp Fenneropenaeus chinensis. The ORF of FcMyD88consisted of1434bp encoding477amino acids, which contains a death domain (DD), a typical TLRand IL-1R-related (TIR) domain and a C-terminal extension (CTE). Homologyanalysis revealed that the deduced amino acid sequence of FcMyD88shared morethan47%similarities with a variety of previously reported MyD88s. Thetime-dependent expression patterns of FcMyD88in cephalothoraxes of shrimpsinjected by heat killed Vibrio anguillarum (G-), Micrococcus luteus (G+) and livewhite syndrome spot virus (WSSV) were analyzed at transcription and protein levelby qPCR and Western Blot, respectively. The expression of FcMyD88mRNA wassignificantly up-regulated during24h after injection with both V. anguillarum and M.luteus. The expression level of FcMyD88protein was up-regulated at12hour postinjection (hpi) of V. anguillarum, while it didn’t change after M. lysodeikticu injectionfor12h. After WSSV injection, the expression level of FcMyD88mRNA wasrelatively constant, while the FcMyD88protein level was significantly up-regulated at12and24hpi. Further, dsRNA interfering technique was used to silence the expression of MyD88in Litopenaeus vannamei, and the mortality of shrimp wasreduced significantly compared to their controls during WSSV infection. These resultssuggested that the MyD88-dependent signaling pathway could be involved in thedefense of shrimp both to bacteria and WSSV infection, and shrimp MyD88mightbenefit to WSSV infection.2. In order to understand the function of JAK/STAT signaling pathway in theantiviral immunity of shrimp, dsRNA interfering technique was used to silence theexpression of STAT gene in Litopenaeus vannamei, and the mortality of shrimp wasdetected after WSSV infection. The WSSV copy number in STAT silenced shrimp was102-103copies/ng DNA which was much lower than those in the control and themortality in STAT silenced shrimp caused by WSSV infection reduced verysignificantly compared to their controls. The function of STAT was verified in vitrocultured cells of hematopoietic tissue of crayfish Cherax quadricarinatus by addingspecific inhibitor of STAT3(S3I-201), and the cultured cells treated with S3I-201showed much less WSSV copy number than their controls, which further suggestedthat STAT might be helpful for the replication of WSSV. Furthermore, the expressionprofile of some potential target genes regulated by STAT or genes related to RNAinterfering pathway were detected in STAT silenced shrimp during WSSV infection.Dicer2and AGO2were up-regulated in STAT silenced shrimp, which implied thatsilencing of STAT might enhance the expression of siRNA pathway related genes toresist the WSSV infection.3. The differentially expressed genes (DEGs) in STAT silenced shrimp wasscreened by high throughput RNA-Seq. The data showed that silencing of STATcaused transcription variations of genes related to energy metabolism, glucosemetabolism, composition of the exoskeleton, immune response and transcription.Among the DEGs, genes encoding enzymes related to energy metabolism such asadenylate cyclase and cytochrome c oxidase subunit I, genes encoding proteins relatedto exoskeleton compositions, such as cuticle protein, DD5and DD9A, and genesencoding transcription related factors such as ATP-dependent RNA helicase A,,transcription initiation factor TFIID etc. showed down-regulation. Genes related to glucose metabolism and immunity showed up or down regulations, which suggestedthat interference of STAT changed the expression of a lot of genes. The variations ofthese genes’ expression might not be benefical to the replication of WSSV in shrimp.,These information about the potential genes regualted by STAT will help us tounderstand the function of JAK/STAT pathway in the immunity of shrimp.