| Pearl culturing involves surgical implantation of a mantle graft(termed saibo)originating from a donor oyster,along with a spherical shell bead(nucleus)into the gonad of a second recipient oyster(host).The characteristics and quality of the produced pearl will be largely depended on the property of donor oyster from which the graft is taken.There are a variety of species of marine pearl oysters,including Pinctada fucata,Pinctada maxima,Pinctada margaritifera,Pteria penguin etc.They produce pearls of different sizes,colors,and luster.Among others,P.maxima and P.margaritifera could produce pearls of perfect colors and luster but large-scale production still cannot be achieved due to difficulties in nuclear insertion and high mortality in sea area cultivation.P.fucata is the common pearl oyster for producing marine pearls as the techniques of breeding,cultivation,nucleus insertion and pearl culture are in good command.The economic benefit of P.fucata culture will be substantially improved if the pearls of appropriate species can be cultured within the body of P.fucata using the mantle pieces of P.maxima or P.margaritifera,i.e.xenotransplantation.However,a transplanted mantle would be rejected by the immune system of the host oyster,especially in the case of xenotransplantation.Therefore,the investigations of immune reaction after mantle pieces transplantation,especially xenotransplantation is significant to improve and innovate the pearl culture techniques and also to create a technology of xenotransplanting a mantle piece of donor oyster into the viscera mass of host oyster.Thus the immune rejection of P.fucata to tissue transplantation was investigated in this study and the results are as following.1.Serum immune response of pearl oyster P.fucata to xenografts and allograftsThe humoral immune responses of P.fucata to allograft(mantle piece of P.fucata)and xenografts(mantle pieces of P.maxima and P.margaritifera,respectively)were studied in the first chapter.The oysters receiving no transplantations were served as the control group.The serum was collected from recipient P.fucata at 1 d,2 d,3 d,4 d,5 d,7 d,9 d,11 d,13 d,and 15 d,respectively after transplantation,and the serum antibacterial activity,lysozyme activity(LZM),alkaline phosphatase(AKP),acid phosphatase(ACP),total antioxidant capacity(TAC),and agglutination to rabbit red blood cells were investigated.The results indicated that serum of both the experimental groups and the control group can agglutinate rabbit red blood cells,with variation between groups and between time points,respectively.The antibacterial activity in the experimental group was significantly higher than that in the control group at 2-4 d,but lower at 5-11 d and returned back to normal at 15 d,with significant differences among experimental groups(P<0.05).The LZM in the experimental group was significantly higher than that in the control group at 3-7 d,with significant differences in bacteriolytic activity among various groups(P<0.05).Both the ACP and AKP activity levels in the experimental groups were higher than those in the control group at 2-9 d,with significant differences among various groups at 3-9 d(P<0.05).The TAC level in the experimental groups was higher than that in the control group at 1-7 d,with significant differences among various groups at 4-7 d(P<0.05).The above results indicated that all of the humoral immune factors investigated showed immune responses to both allografts and xenografts,with no specific to any of them.Thus,it is necessary to further screen immune rejection factors specific to xenografts,including cellular immune components.2.Immune reaction of pearl oyster P.fucata to xenograft from P.maximaThe immune responses of P.fucata hemocytes to allograft and xenograft were investigated after transplantation using transcriptome analysis.In total,107.93 Gb clean reads were produced and assembled using the reference genome of P.fucata.Gene Ontology Term enrichment and KEGG enrichment analyses indicated that apoptosis,hippo signaling pathway,oxidation-reduction,MAPK signaling pathway,ribosome,protein processing in endoplasmic reticulum,purine metabolism,NFkappa B signaling pathway,oxidative phosphorylation,Ras signaling pathway,and ubiquitin mediated proteolysis were involved in response to transplantation.Many genes related to oxidation-reduction reactions,the MAPK signaling pathway,and apoptosis were identified by comparison of the allograft group and the xenograft group at 0 h,6 h,12 h,24 h,48 h,72 h,and 96 h post-transplantation.Among them,the expression levels of NADH dehydrogenase,succinate dehydrogenase and other dehydrogenases were increased significantly in the xenograft groups compared with allograft groups at 0 h post transplantation,indicating that a respiratory burst of neutrophils occurred immediately after xenograft transplantation.Additionally,HSP70 was highly expressed from 0 h to 96 h in the xenograft groups,indicating an oyster immune response to the xenograft.The genes enriched in the ribosome and hippo-signaling pathways were also identified,and expression patterns of these DEGs were different as compared between transplantation and control groups.Finally,altered expression levels of 10 randomly selected immune-related DEGs were confirmed by quantitative real-time PCR.These results indicated that oxidation-reduction is likely the key factor responsible for immune rejection to xenotransplantation.The findings should provide some new insight into the molecular mechanism of immune rejection of the host against xenograft,and thus benefit to development of immunosuppressive reagents to facilitate effective xenograft pearling.3.Differentially expressed immune-related genes in hemocytes of the pearl oyster P.fucata against allograftHemocyte transcriptome analysis was performed to detect the immune responses to allograft in P.fucata at 0 h and 48 h after allograft transplantation.The sequencing reaction produced 92.5 million reads that were mapped against the reference genome sequences of P.fucata.The Gene Ontology(GO)annotation and the Kyoto Encyclopedia of Genes and Genomes(KEGG)were used to identify all immune-related differentially expressed genes(DEGs).Compared with patterns at 0 h,a total of 798 DEGs were identified,including 410 up-regulated and 388 down-regulated genes at 48 h.The expression levels of interleukin receptor and toll-like receptor in hemocytes were increased significantly 48 h post-transplant,indicating that the oyster immune response was induced.Finally,altered levels of 18 randomly selected immune-related DEGs were confirmed by quantitative real-time PCR(q RT-PCR).These results provide the basis for further analysis of the immune rejection of allotransplantation. |
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