近年来,鲍鱼养殖业受到细菌病原体的威胁。在Haliotisdiscushannai中,血细胞吞噬的免疫反应机制尚不清楚。有必要研究应对这些细菌病原体挑战的免疫机制。在这项研究中,通过流式细胞术结合电子显微镜和转录组学分析检查了H.discushannai中血细胞的吞噬活性。副溶血性弧菌的结果,使用电子显微镜对溶藻弧菌和金黄色葡萄球菌的攻击显示了血细胞吞噬体形成过程。金黄色葡萄球菌的吞噬率(PP)高于其他五种异物颗粒,约为63%。哈维氏弧菌的PP约为43%,1.5h时血细胞中溶藻弧菌的PP峰值为63.7%。副溶血性弧菌和溶藻弧菌攻击后,酸性磷酸酶,碱性磷酸酶,总超氧化物歧化酶,溶菌酶,总抗氧化能力,过氧化氢酶,在不同时间测量血细胞中的一氧化氮合酶和谷胱甘肽过氧化物酶活性,通过定量转录组学分析鉴定差异表达基因(DEGs)。副溶血性弧菌攻击后鉴定的DEGs包括血凝素/细胞聚集因子样,超绒毛样亚型X4,钙调蛋白样和脊柱后凸肽酶样;溶藻弧菌攻击后鉴定的DEG包括白细胞介素6受体亚基β样,蛋白龟同源物B样,rhoGTP酶激活蛋白6样亚型X2,白细胞表面抗原CD53样,calponin-1-like,钙调素样,肌钙蛋白C,肌钙蛋白I样亚型X4,肌钙蛋白T样亚型X18,肿瘤坏死因子配体超家族成员10样,rho相关蛋白racA样和血凝素/变形细胞聚集因子样。一些免疫相关的KEGG通路在攻击后显著上调或下调,包括甲状腺激素的合成,Th17细胞分化信号通路,病灶粘连,黑色素生成,白细胞跨内皮迁移,TRP通道的炎症介质调节,ras信号通路,rap1信号通路。这项研究是了解H.discushannai免疫系统的第一步,方法是将几种工具应用于腹足动物,并对其血细胞进行首次详细的形态功能研究。
In recent years, the abalone aquaculture industry has been threatened by the bacterial pathogens. The immune responses mechanisms underlying the phagocytosis of haemocytes remain unclear in Haliotis discus hannai. It is necessary to investigate the immune mechanism in response to these bacterial pathogens challenges. In this study, the phagocytic activities of haemocytes in H. discus hannai were examined by flow cytometry combined with electron microscopy and transcriptomic analyses. The results of Vibrio parahaemolyticus, Vibrio alginolyticus and Staphylococcus aureu challenge using electron microscopy showed a process during phagosome formation in haemocytes. The phagocytic rate (PP) of S. aureus was higher than the other five foreign particles, which was about 63%. The PP of Vibrio harveyi was about 43%, the PP peak of V. alginolyticus in haemocyte was 63.7% at 1.5 h. After V. parahaemolyticus and V. alginolyticus challenge, acid phosphatase, alkaline phosphatase, total superoxide dismutase, lysozyme, total antioxidant capacity, catalase, nitric oxide synthase and glutathione peroxidase activities in haemocytes were measured at different times, differentially expressed genes (DEGs) were identified by quantitative transcriptomic analysis. The identified DEGs after V. parahaemolyticus challenge included haemagglutinin/amebocyte aggregation factor-like, supervillin-like isoform X4, calmodulin-like and kyphoscoliosis peptidase-like; the identified DEGs after V. alginolyticus challenge included interleukin-6 receptor subunit beta-like, protein turtle homolog B-like, rho GTPase-activating protein 6-like isoform X2, leukocyte surface antigen CD53-like, calponin-1-like, calmodulin-like, troponin C, troponin I-like isoform X4, troponin T-like isoform X18, tumor necrosis factor ligand superfamily member 10-like, rho-related protein racA-like and haemagglutinin/amebocyte aggregation factor-like. Some immune-related KEGG pathways were significantly up-regulated or down-regulated after challenge, including thyroid hormone synthesis, Th17 cell differentiation signalling pathway, focal adhesion, melanogenesis, leukocyte transendothelial migration, inflammatory mediator regulation of TRP channels, ras signalling pathway, rap1 signalling pathway. This study is the first step towards understanding the H. discus hannai immune system by adapting several tools to gastropods and providing a first detailed morpho-functional study of their haemocytes.