Study On The Immunological Fuction Of Different Types Of Hemocytes And Lymph Organ In Pacific White Shrimp Litopenaeus Vannamei

Hemocytes and lymphoid organ(named as Oka)are key immune tissues or organs in shrimp,and they play essential roles in innate imunity of shrimp.Till now,previous studies focused on function of all hemocytes in innate immunity and the morphological observation of Oka,but systematic knowledge of the immune relationship among different types of hemocytes and immunological function of Oka is very limited.Therefore,based on the separation technique of different types of hemocytes,we conducted transcriptome sequencing and bioinformatics analysis to systematically investigate the immunological function of different types of hemocytes in Pacific white shrimp Litopenaeus vannamei.Moreover,we filtered the specifically expressed genes of Oka using the differential expressed genes(DEGs)based on the transcriptome of different tissues,and further analyzed the immunological functions of these selected genes to systematically uncover the immune roles of Oka in the dissertation.This study provided important basis for systematic interpretation of innate immunity,and essential reference for effective stragy of disease prevention in shrimp.1.Comparative transcriptome analysis of hemocytes and lymphoid organ in shrimp.Four essential immune tissues of shrimp including hemocytes,lymphoid organ,hepatopancreas and gills were selected to conduct the transcriptome sequence and bioinformatics analysis.We analysed the annotations of genes highly expressed in hemocyte or Oka(RPKM > 50),and found that several immune genes including endocytosis,antibacterial peptides(AMP),phenoloxidase(PO)system and apoptosis were both highly expressed in hemocytes and Oka.Genes related to apoptosis,PO system and some AMP genes in hemocytes showed higher expression levels than those in Oka,while genes related to endocytosis and some ALF genes showed higher expression levels in Oka.Compared with the genes expressed in other immunological tissues like gill,hepatopancreas and Oka,several signaling pathways like NOD-like receptor,MAPK,JAK-STAT and Wnt signaling pathway were found to highly expressed in hemocytes.And several signaling pathways like TGF-beta,JAK-STAT,MAPK,Hedgehog and ECM-receptor interaction pathway were found to highly express in Oka compared to ther immunological tissues like gill,hepatopancreas and hemocytes.Oka and hemocytes showed great similarity and difference in the functions of shrimp innate immunity.2.Analysis of functional relationship among different types of hemocytes in shrimp.Total hemocytes were successfully separated into three types including hyalinocytes,semi-granulocytes and granulocytes by Percoll gradient centrifugation,and then were conducted transcriptome sequence and bioinformatics analysis.The data showed that different types of hemocytes showed significantly differential expression patterns at transcriptional level.The genes involved in phagocytosis like phagocytic receptors,lectins and actin cytoskeleton were highly expressed in hyalinocytes,while genes involved in the humoral immunity signaling pathways were highly expressed in semi-granulocytes,and genes involved in prophenoloxidase(proPO)system were highly expressed granulocytes.Flow cytometry analysis showed that hyalinocytes possessed higher capacity in phagocytizing the fluorescent beads than semi-granulocytes and granulocytes.Several genes related to phagocytosis including actin,cathepsin and phagocytic receptor like mannose receptorshowed higher expression levels in hyalinocytes via qPCR detection.It segguested that hyalinocytes were the main type of hemocytes responsive for ingesting fluorescent beads.Moreover,after treatment with different endocytic inhibitors,the uptake capability of fluorescent beads by hyalinocytes were significantly suppressed by macropinocytosis inhibitor like IPA-3 and NSC23766,suggesting that their ingestion process mainly depends on the endocytic way of macropinocytosis.3.Identification and functional study of E3 ubiquitin ligase TRIM9 genes in shrimp.Two TRIM9 genes(designated as Lv TRIM9-1 and LvTRIM9-2)were identified from the transcriptome of the Pacific whiteleg shrimp.The transcript of LvTRIM9-1 was specially detected in Oka,while the transcript of LvTRIM9-2 was detected in most tissues of the shrimp,with relatively higher levels in intestines and nerves.The expression levels of LvTRIM9-1 and LvTRIM9-2 were different post white spot syndrome virus(WSSV)infection,indicating that they played distinct roles in WSSV infection.Knockdown of two LvTRIM9 genes' expressions by double-strand RNA mediated interference could both lead to a decrease of virus copy number in WSSV-infected shrimp.Yeast two-hybrid analysis and gene regulation analysis showed that LvTRIM9-1 could positively regulate NF-?B pathway via directly interact with immune deficiency(Lv IMD),and Lv TRIM9-2 could negatively regulate NF-?B pathway via directly interact with beta-transducin repeat-containing protein of shrimp(Lv?-TrCP).These data indicated that LvTRIM9-1 and LvTRIM9-2 affected humoral immunity in different ways via distinct regulation on NF-?B pathway in shrimp.