| Adaptability is the basic characteristic of living organisms,which means that the organism can adjust internal function and maintain homeostasis to better survive according to the change of internal and external environment.The organisms can show adaptation to the environmental change in the morphological structure or physiological function,of which immune adaptation is an important aspect.Studies have shown that there is a widespread immune adaptation in animals,which can memory their past infection experiences and increase their immune resistance and survival opportunity when meet the similar pathogen again,the "adaptive immunity" in vertebrates as a good example.Owing to the lack of cellular(B/T lymphocytes)and molecular(Ig antibody)bases of classical "adaptive immunity",the specific mechanisms of immune adaptation in invertebrate(also known as immune priming)are still unclear,which severely prevented the immune prevention and cure of the diseases of important aquaculture invertebrates.This dissertation explores the detail mechanism of the immune adaptation in Pacific oyster caused by continuous stimulation of Vibrio splendidus at the cellular and molecular levels,with the main conclusion obtained as below:Firstly,the oyster granulocytes are the main immunocompetent hemocytes and the core hemocytes population in the immune adaptation.Three types of circulating hemocytes were morphologically identified and separated in oyster as agranulocytes,semi-granulocytes and granulocytes.Among of those hemocytes,granulocytes possessed the strongest activities of phagocytosis,encapsulating and bactericidal factors(the lysosome activity,the productions of ROS and NO).Besides,compared to agranulocytes and semi-granulocytes,the RNA transcripts of immune related genes(CgTLR,CgClathrin,CgATPeV,CgLysozyme,CgDefensin and CgIL-17)were the highest expressed in granulocytes.Therefore,the granulocytes were characterized as the main immunocompetent hemocytes in oysters.In addition,after two consecutive stimulations of Vibrio splendidus,the total hemocytes counts and the proportion of granulocytes both increased significantly,compared with that after first stimulation.Meanwhile,the anti-apoptosis capacity,phagocytic activity and bactericidal activity of hemocytes were also enhanced remarkably,displaying a characteristic of immune adaptation.These results collectively suggested that the granulocytes were also the core cell population in immune adaptation of oysters.Secondly,the processes of hemocytes phagocytosis and anti-apoptosis play a key role in the immune adaptation of oyster.After two consecutive stimuli of Vibrio splendidus,a total of 1800 differentially expressed genes were screened from hemocytes transcriptom,which were simultaneously responding to the two times immune stimulation.Among of those genes,1037 differentially expressed were obtained at 0 h after challenging primarily and secondarily,which indicated those genes might be related with faster immune response in the secondary challenge.And 996 genes were differentially expressed at 6 h after challenging primarily and secondary,which suggested that those genes might be involved in the stronger immune response in the secondary challenge.Besides,among all of those differentially expressed genes,the genes in the process such as recognition,cell deformation,endocytosis,phagolysosome maturation and anti-apoptosis were all up-regulated significantly,which suggesting that hemocytes phagocytosis and anti-apoptosis might play indispensable role in the immune adaptation of oysters.Thirdly,it was found that the mTOR regulating anaerobic glycolysis was the key energy metabolism pathway in the immune adaptation of Pacific oyster.After two consecutive stimulations,the expression of glucose transporter in hemocytes increased significantly,the key speed limiting enzyme in the process of glycolysis,such as hexokinase,phosphoglucomutase and glucokinase were also up-regulated significantly.Meanwhile,the continued immune challenge could also induce an increase of glucose consumption,the enhancement of anaerobic respiration and glycolysis,and the up-regulation of secondary metabolite NAD+/NADH and lactic acid level.Besides,the expression of key factors in the mTOR pathway such as mTOR and HIFα also increased significantly.As expected,when the phosphorylation of mTOR(p-mTOR)was inhibited,the glucose consumption,the anaerobic glycolysis,the phagocytosis and ROS level in oyster hemocytes were all decreased significantly,blocking the resonse of immune adaptation in oyster.Therefore,the results suggested that anaerobic glycolysis mediated by mTOR is the key energy metabolism pathway in the immune adaptation of oysters.Forthly,it is preliminary revealed that histone modifications may play a regulatory role in the immune adaptation of Pacific oysterIn oyster,the histone methyl-transferase CgHMT646 gene and histone acetyl-transferase CgHAT009 gene were expressed mainly in the hemocytes,which can enhance the intracellular histone methylation and acetylation modification levels when transferred to HEK293 cells,respectively.After two consecutive stimuli by Vibrio splendidus,the expression of CgHMT646 genes in hemocytes increased markedly,along with elevated histone H3K4 methylation level of hemocytes,and the expression of CgHAT009 gene decreased markedly,accompanied by reduced histone acetylation level of hemocytes.All the results suggested that histone modifications might play a regulatory role in the immune adaptation of Pacific oyster.To sum up,among the three kinds of circulating hemocytes of Pacific oysters,granulocytes are the main immunocompetent hemocytes,which play fundamental role in immune adaptation induced by the two consecutive stimulations of Vibrio splendidus.Moreover,phagocytosis and anti-apoptotic processed play a key role in immune adaptation,mTOR regulates anaerobic glycolysis is the key energy metabolic pathway,and histone modifications may play a regulatory role in immune adaptation.The results will further establish the basis of the immune adaptation mechanism in Pacific oyster at both the cellular and molecular level,promote our understanding of immune adaptation mechanism in invertebrate,and provide some new ideas for the prevention and cure of diseases of important aquaculture invertebrates. |
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