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寄生性甲藻血卵涡鞭虫流行病学及致病机理初探
王金凤
学位类型博士
导师李才文 研究员
2017-05-12
学位授予单位中国科学院大学
学位授予地点北京
学位专业海洋生态学
关键词血卵涡鞭虫 三疣梭子蟹 流行病学 生活史 组织病理学 基因表达
其他摘要        血卵涡鞭虫(Hematodinium)是一类感染多种海水甲壳动物的致病性寄生性甲藻。近年来,该藻导致的流行病频繁暴发,严重影响了全球多种野生经济甲壳动物的渔业生产,对我国海水养殖业的健康持续发展也造成了严重威胁。研究血卵涡鞭虫的发病规律、致病机制以及宿主对其免疫防御机制是防控该类病害的基础。因此,本论文分别从血卵涡鞭虫流行病学、生活史、致病机理及宿主免疫防御方面进行系统探究,以期为血卵涡鞭虫流行病的防控提供一定的数据参考和理论依据。本论文主要研究结果如下:
1.      采用血涂片和分子生物学方法对我国东部沿海地区野生和养殖蟹类感染血卵涡鞭虫状况进行调查,发现血卵涡鞭虫在我国分布十分广泛。在辽宁、山东、浙江地区的野生三疣梭子蟹和广东的野生拟穴青蟹(Scylla Paramamosain)中均发现了血卵涡鞭虫的感染;对山东青岛三疣梭子蟹重要养殖基地虾蟹混养池塘的连续监测发现,血卵涡鞭虫病在三疣梭子蟹整个养殖周期内持续发生,感染率呈现先上升后下降的变化趋势,最高值出现在7月末,可达90 %。伴随着三疣梭子蟹血卵涡鞭虫病的暴发,混养池塘中斑节对虾(Penaeus monodon)大批量死亡,经检测确定为血卵涡鞭虫感染所致,这在国内外尚属首次发现和报道。
2.      通过分子生物学和体外培养技术手段,研究了感染养殖三疣梭子蟹和斑节对虾的血卵涡鞭虫的生物学特征。成功建立了血卵涡鞭虫的体外培养,观测到其生活史过程中历经的丝状滋养体、类变形虫状滋养体、团块状聚合体、裂殖体、蛛网状滋养体、孢子体、孢子母细胞、孢子前细胞和孢子生活史阶段。分子遗传分析结果显示感染三疣梭子蟹和斑节对虾的血卵涡鞭虫的种群同源性高达99.2 %,与我国其他地区发现的血卵涡鞭虫属于同一类群(Hematodinium perezi II)。
3.      通过血涂片法和H&E染色法初步探究了血卵涡鞭虫感染三疣梭子蟹后的增殖过程和致病机理。发现血卵涡鞭虫主要寄生于宿主肝胰腺、心脏等主要器官的血腔和血窦内,其大量增殖可导致宿主肝胰腺、心脏、鳃和肌肉等重要器官、组织发生系统性病变,最终宿主消化、代谢、呼吸等功能的紊乱和丧失引发死亡。
4.      初步研究了三疣梭子蟹抵御血卵涡鞭虫侵染的免疫响应中发挥重要作用的抗氧化酶GPx。首次克隆获得三疣梭子蟹GPx基因全长cDNA序列,系统发育分析显示其优先与甲壳动物GPxs聚为一枝;三疣梭子蟹GPx蛋白序列的预测三维结构由3个α-螺旋和7个β-折叠片层组成,其催化位点为硒半胱氨酸(U),位于α1螺旋和β3折叠片层结构中;遭受血卵涡鞭虫侵染后,GPx基因在三疣梭子蟹血细胞、肝胰腺、心脏和肌肉组织中的转录表达水平发生显著性变化,表明GPx在三疣梭子蟹抵御血卵涡鞭虫的免疫响应过程中可通过缓解甲壳宿主体内的氧化应激反应从而对宿主产生重要的防护功能。
;         Parasitic dinoflagellates in the genus Hematodinium are serious infectious pathogens causing epidemic disease in a broad range of marine crustaceans. In recent years, frequent outbreaks of the disease had seriously impacted the wild stocks of many wild crustaceans around the world, as well as the sustainable development of marine crustacean cultures in China. The studies on its epidemology, the pathogenesis of Hematodinium sp. and the host immunologic responses against Hematodinium infection are the fundamental basis to find solution on how to prevent and control this infectious disease. Thus, this dissertation conducted three aspects of researches, including the epidemiology of Hematodinium sp. in wild and cultured stocks of major marine crustaceans along the coast of China, the developmental cycle of Hematodinium sp. and the immune response of P. trituberculatus responding to the Hematodinium parasite. The major findings are listed as following:
1.      Hematodinium sp. infections in wild and cultured crustaceans along the eastern coast of China were diagnosed by hemolymoph smear assay and molecular biological method. Hematodinium infections were widely distributed in China. Hematodinium infection was detected in wild Chinese swimming crabs Portunus trituberculatus in Liaoning, Shandong and Zhejiang provinces and mud crabs Scylla paramamosain in Guangdong province. In addition, Hematodinium infections were persistent in the polyculture ponds in Shandong during the culture period, with a peak prevalence (90 %) in late July. Furthermore, Hematodinium sp. infection was identified for the first time in the giant tiger prawn P. monodon co-cultured with P. trituberculatus in the polyculture system during outbreak of the disease.
2.      The biological characteristic of Hematodinium sp. infecting cultured P. trituberculatus and P. monodon was studied by molecular biological method and in vitro culture. Most typical life stages were observed in in vitro cultures, including filamentous trophont, ameboid trophont, arachnoid trophont, arachnoid sporont, schizonts, clump colony, sporoblast, prespores and dinospores. Phylogenetic analysis indicated the Hematodinium isolate infecting P. monodon was identical to the isolate infecting the co-cultured P. trituberculatus, and the isolates were grouped into the cluster (Hematodinium perezi II) together with the Hematodinium sequences obtained from other places in China.
3.      The pathogenic mechanism of Hematodinium sp. was explored with the hemolymoph smear assay and H&E assay. Histopathology indicated that Hematodinium sp. lives and proliferate in the hemolymph and hemocoel of major organs of infected hosts, caused systemic infections to its hosts and resulted in overt pathological alterations in hepatopancreas, heart, gills, and muscles. The systemic infections of Hematodinium sp. in P. trituberculatus led to malfunction or dysfunction of major organs, and eventually mortality of infected hosts.
4.     The immune response of host against Hematodinium was studied. A novel selenium-dependent glutathione peroxidase gene (PtGPx) was isolated and characterized from Portunus trituberculatus. The length of se-GPx gene cDNA was 696bp, including a 507bp open reading frame (ORF) encoding 168 amino acids. Amino acid comparison and phylogenetic analyses indicated that the PtGPx was closely related to and clustered with the crustacean SeGPxs. The PtGPx transcripts could be extensively and significantly induced during the immune response of P. trituberculatus responding to the Hematodinium parasite, implying a critical role of GPx in the crustacean innate immune defense against the parasitic infection.
学科领域海洋科学
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/136559
专题海洋生态与环境科学重点实验室
作者单位1.中国科学院海洋研究所
2.中国科学院大学
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王金凤. 寄生性甲藻血卵涡鞭虫流行病学及致病机理初探[D]. 北京. 中国科学院大学,2017.
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