Studies On Early Immune Responses Of Different Immune-related Tissues To Vibrio And WSSV Immunostimulations In The Pacific White Shrimp Of Litopenaeus Vannamei

White spot syndrome virus(WSSV)and Vibrio parahaemolyticus(Vp)are two common pathogenic microorganisms causing shrimp epidemic diseases.Therefore,the systematic study on the immune response characteristics of shrimp against these two pathogens could pave the way to develop technologies for prevention and control of shrimp disease.In this dissertation,comparative transcriptome analysis was performed to investigate the features of the immune responses of shrimp primary immune tissues including hemocytes(He),lymphoid organ(Oka)and hepatopancreas(Hp),at the early stage of WSSV and Vp immunostimulations,with the aim to understand the mechanism of different immune tissues of Litopenaeus vannamei in response to different pathogens.The findings could enrich the fundamental theory of crustacean immunology and provide a new perspective for the healthy breeding and disease control of shrimp.The main results of this dissertation are as follows:(1)Transcriptome analysis of three primary immune tissues at the early stage of Vp and WSSV immunostimulations.A total of 1,288,985,148 clean reads were obtained by Illumina sequencing and 59,583 Unigenes were produced by de novo assembly with 17,721 annotated Unigenes(29.74%)."Metabolic process","cellular process" and "single-organism process" are three most enriched biological processes by GO enrichment analysis.KEGG pathway analysis found a large number of immune-related pathways,including "Endocytosis","Lysosome","Phagosome" and"Peroxisome",suggesting the biological functions of these tissues are closely related to immunity.All Unigenes in the transcriptome were divided into 17 modules through WGCNA analysis,which provided an important reference for the subsequent mining of hub genes.(2)Analysis of the early response characteristics of shrimp hemocytes to Vp and WSSV immunostimulations.2454 differentially expressed genes(DEGs)were obtained from the transcriptome of hemocytes before and after Vp stimulation(named VHc),including 1935 differentially up-regulated genes(DUGs)and 519 differentially down-regulated genes(DDGs).Accordingly,3444 DEGs were acquired from the transcriptome of hemocytes before and after WSSV stimulation(named WHc),including 3240 DUGs and 204 DDGs.GO enrichment analysis revealed that both pathogenic stimuli changed several biological processes of hemocytes,such as "RNA metabolism","amide biosynthetic process" and "gene expression".KEGG pathway analysis demonstrated that "Ribosome biogenesis in eukaryotes" and "Aminoacyl-tRNA biosynthesis" were significantly enriched in both pathogenic stimuli,indicating a change in the protein synthesis process of hemocytes.Further analysis and clustering of DEGs showed that Vp and WSSV stimulations caused changes in various physiological processes of hemocytes,including metabolism,translation,endocytosis,transport,immunity and neuroendocrine.In particular,WSSV infection significantly activated the hemocytes-mediated neuroendocrine-immune(NEI)system of shrimp.On the one hand,18 genes encoded putative neuropeptide precursors were significantly induced by WSSV infection.On the other hand,WSSV infection affected several typical processes in the NEI system,in which comprised proteolytic processing of prohormones,amino acid neurotransmitter pathways,biogenic amine biosynthesis and acetylcholine signaling pathway.Unlike WSSV,Vp stimulation had little extensive effect on the NEI system that only four neuropeptide precursors and their processing enzymes were changed.It is the first report in crustaceans on the involvement of the hemocytes-mediated NEI system in the immune response,providing a new idea for the prevention of shrimp diseases.(3)Analysis of the early response characteristics of shrimp lymphoid organ to Vp and WSSV immunostimulations.2127 DEGs were obtained from the transcriptome of lymphoid organ before and after Vp stimulation(named VOka),including 809 DUGs and 1318 DDGs.Correspondingly,1569 DEGs were acquired from the transcriptome of lymphoid organ before and after WSSV stimulation(named WOka),including 138 DUGs and 1431 DDGs.GO enrichment analysis showed that"aminoglycan metabolic process" was significantly enriched in both pathogenic stimuli and there were a large number of chitin-binding proteins(CBPs)found in the process.KEGG pathway analysis revealed that "Lysosome" were significantly enriched only in the VOka group,indicating that lysosomes played an important role in the clearance of Vp by shrimp lymphoid organ.And three pathways related to carbohydrate metabolism including "Inositol phosphate metabolism","Propanoate metabolism","Glycolysis/Gluconeogenesis" were significantly enriched only in the WOka group,suggesting that WSSV infection altered significantly the metabolic process of shrimp lymphoid organ.Further analysis and clustering of DEGs showed that immunostimulations apparently affected prophenoloxidase(proPO)activation system,lysosomes,extracellular matrix,cytoskeletal proteins and chitin-binding proteins in lymphoid organ.In addition,two key immune molecules that specifically responded to Vp stimulation,including cathepsin and VMO-I(vitelline membrane outer layer protein I),were identified,and their expression levels were clearly up-regulated upon Vp stimulation,suggesting their important roles in anti-bacterial immune response in lymphoid organ.(4)Analysis of the early response characteristics of shrimp hepatopancreas to Vp and WSSV immunostimulations.373 DEGs were obtained from the transcriptome of hepatopancreas before and after Vp stimulation(named VHp)whereas only 86 DEGs were acquired from the transcriptome of hepatopancreas before and after WSSV stimulation(named WHp).GO enrichment analysis found that the "pyruvate metabolic process" was significantly enriched under both treatments.KEGG pathway analysis showed that "Glycolysis/Gluconeogenesis" was significantly enriched both in VHp and WHp,further confirming the above results.Moreover,"Glutathione metabolism""Drug metabolism-cytochrome P450" and "Metabolism of xenobiotics by cytochrome P450" were also significantly enriched,indicating the critical detoxification of hepatopancreas.Further analysis and clustering of DEGs revealed that some immune-related genes,including pattern recognition receptors,serine proteases and serine protease inhibitors,were changed significantly caused by Vp stimulation.Besides,several transport and detoxification-related molecules,such as ABC transporters and cytochrome P450,were identified,indicating that they might play an important role in the immune system of shrimp hepatopancreas.(5)Identification of recombination activating genes(RAGs)and the molecules related to immune priming in shrimp.The homologous genes of RAGs involved in the gene rearrangement in vertebrates,were firstly identified from shrimp,namely LvRAG1L and LvRAG2L,which showed apparent similarity with higher invertebrates and cephalochordata RAGs in terms of protein sequence,expression pattern and alternative splicing,providing new molecular evidence for the origin and evolution of RAGs.Furthermore,the molecules related to immune priming like DSCAMs and FREPs in shrimp were identified,and their gene structure and function in response to pathogen infection were analyzed.The results showed that shrimp DSCAMs had multiple paralogs and FREPs might increase their diversities through multiple loci and alternative splicing.They were involved in the immune responses of Vp and WSSV immunostimulations.Our results would offer an important reference for the study of immune priming mechanism in shrimp.