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三疣梭子蟹对寄生性病原-血卵涡鞭虫免疫响应的初步研究
李蒙
学位类型博士
导师李才文 研究员
2016-05-20
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
关键词三疣梭子蟹 血卵涡鞭虫 免疫响应
摘要三疣梭子蟹是我国重要的海产养殖经济蟹类。近年来,三疣梭子蟹养殖受到寄生性甲藻类病原-血卵涡鞭虫引发的流行病害“牛奶病”的严重危害,其可持续发展受到严重威胁。寄生性甲藻类病原-血卵涡鞭虫在世界范围内可寄生感染多种重要经济甲壳动物,其发生范围及感染宿主种类有逐渐扩大的趋势,不仅影响了重要经济甲壳动物的野生群体及其渔业产业,也开始危害重要经济甲壳动物养殖业的可持续发展。目前,针对该寄生性甲藻-血卵涡鞭虫的相关研究大多集中于物种鉴定、检测技术、流行病学等方面,尚缺乏病原宿主相互作用等基础研究工作。因此,本论文从宿主免疫响应的角度出发,系统研究了甲壳宿主-三疣梭子蟹与寄生性甲藻类病原-血卵涡鞭虫的相互作用过程,以期进一步丰富和完善甲壳动物免疫机制的基础理论,同时为寄生性甲藻类病原引发的养殖病害的防控提供理论依据。主要研究结果如下:
1.克隆获得三疣梭子蟹Toll基因的全长cDNA序列,全长为3745bp,开方阅读框为3012bp,编码1003个氨基酸,其编码氨基酸序列与拟穴青蟹和锯缘青蟹中的Toll序列相似性高达88%,并在系统发育树中优先与甲壳动物Tolls序列聚为一枝。Toll基因在三疣梭子蟹体内广泛表达且在血细胞中的表达丰度最高。在血卵涡鞭虫的侵染早期(侵染24 h内),Toll基因的转录表达水平在三疣梭子蟹的血细胞、鳃、心脏和肌肉组织中受到显著性抑制,反映出该寄生性病原可能对其甲壳宿主体内的Toll免疫信号传导途径具有一定的抑制作用,而该抑制过程也可能与该寄生性甲藻类病原在甲壳宿主体内的存活及寄生过程密切相关。之后,Toll基因的转录表达水平在受到侵染48 h时的血细胞和心脏组织以及受到侵染48 h 和96 h时的肝胰腺组织中显著上调,可能反映了甲壳动物Toll基因在相应时间阶段对血卵涡鞭虫的免疫防御作用。最后,经血卵涡鞭虫侵染192 h时,Toll基因在血细胞、肝胰腺和肌肉组织中的mRNA表达量显著下调,可能表明该寄生性病原在侵染后期已逐步破坏其宿主体内Toll受体介导的免疫信号途径的免疫防御功能。
2. 血卵涡鞭虫的侵入可显著影响三疣梭子蟹血细胞和肝胰腺组织中proPO基因的转录水平及酚氧化酶活力水平,并可诱导proPO基因表达水平和酚氧化酶活力水平显著上调,表明三疣梭子蟹proPO系统在对血卵涡鞭虫的免疫反应中发挥重要作用。同时,血卵涡鞭虫可显著抑制三疣梭子蟹proPO系统中重要诱导和激活因子(LGBPPPAF)以及proPO基因的转录表达,反映出该寄生性病原可能对三疣梭子蟹的proPO系统的激活过程具有一定的免疫抑制作用。另外,血卵涡鞭虫可导致三疣梭子蟹体内血细胞数量(THCs和DHCs)的显著下降,并可在侵染后期(侵染16 d和24 d)造成肝胰腺组织的显著病理学变化。
3. 血卵涡鞭虫侵染后,三疣梭子蟹体内3个重要clip-domain丝氨酸蛋白酶基因(PTcSP1-3)以及2个重要蛋白酶抑制剂基因(a2mserpin)的转录表达均发生显著性变化,反映出三疣梭子蟹体内的SPs级联反应受到血卵涡鞭虫入侵的严重干扰,并可能在甲壳宿主-三疣梭子蟹应对该寄生性病原的免疫响应过程中发挥重要作用。PTcSP1-3基因呈现不同的表达变化模式,反映出它们可能在应对血卵涡鞭虫免疫响应过程中执行不同的生物学功能,同时结合proPO基因表达水平和酚氧化酶活力的变化结果进行分析发现,PTcSP1和PTcSP3蛋白酶可能是三疣梭子蟹proPO系统中的重要组成成员,参与调控proPO系统的激活过程。
4. 克隆获得三疣梭子蟹的NO/O2·-合成关键酶基因NOSNOX以及三疣梭子蟹的抗氧化基因GPx的cDNAs序列。其中,三疣梭子蟹NOS基因cDNA序列全长为4002bp,开方阅读框为3612bp,编码1203个氨基酸,其编码氨基酸序列与甲壳动物NOS序列具有较高的相似性(>79%),并在系统发育分析树中优先与甲壳动物NOS序列聚在一起。组织表达分析结果表明,NOSNOXGPx基因在三疣梭子蟹的血细胞和肝胰腺组织中的表达量较高。对三疣梭子蟹进行血卵涡鞭虫人工侵染0-192 h后,NO/O2·-合成和清除相关基因的转录表达水平(NOSNOXCuZnSODCATGPx)及对应的酶活力水平(NOS、NOX、SOD、CAT和GPx)在血细胞和肝胰腺组织中均发生显著性变化,反映了NO/O2·-合成系统和抗氧化系统在甲壳宿主-三疣梭子蟹对血卵涡鞭虫免疫反应中的重要作用。自由基NO/O2·合成系统可能参与并促进了甲壳宿主对血卵涡鞭虫的免疫防御过程,而抗氧化系统在免疫响应过程中的ROS水平调控及宿主自我防护过程中发挥重要作用。
综上所述,本论文以三疣梭子蟹与寄生性甲藻类病原-血卵涡鞭虫的侵染体系为研究对象,首次从宿主免疫响应的角度出发,初步探究和阐述了甲壳动物宿主与寄生性甲藻类病原-血卵涡鞭虫的宿主病原相互作用过程。本论文有助于丰富甲壳动物免疫防御机制的基础理论研究,并为预防和控制寄生性甲藻类病原引发的海产虾、蟹类流行性疾病提供理论依据。
其他摘要The Chinese swimming crab, Portunus trituberculatus is an important commercial aquaculture crab species in China. In recent years, the aquaculture of the P. trituberculatus was seriously damaged by the epidemic disease “milk disease” caused by the parasitic dinoflagellate Hematodinium and the sustainable development of the crab aquaculture industry is under serious threat. The parasitic dinoflagellate Hematodinium could parasitize and infect many important economically crustacean species around the world with an expansion of the scope of its occurrence and the number of the host species, which affected not only the wild population of commercial valuable crustacean species, but also the sustainable aquaculture of major crustacean species. To date, most studies were focused on the species identification, detection technology and epidemiology of Hematodinium, while the basic research on the host-pathogen interaction between Hematodinium and its crustacean hosts is still very scarce. Thus, this dissertation systematically explored the interaction between the Hematodinium and its crustacean host P. trituberculatus from the perspective of the host immune responses so as to enrich the basic research of crustacean immune mechanism and provide theoretical basis for the prevention and control of the diseases caused by the pathogen of parasitic dinoflagellates in crab aquaculture. The major findings are listed as following:
1. A novel Toll gene was firstly isolated and characterized from P. trituberculatus, with the full-length cDNA of 3745bp and a 3012bp open reading frame (ORF) encoding 1003 amino acid (aa). The amino acid sequence of P. trituberculatus Toll shared high similarity to Scylla paramamosain and Scylla serrata Tolls (88%), and was clustered with the counterparts of crustaceans in the phylogenetic tree. The Toll transcripts were extensively expressed in various tissues of P. trituberculatus, with the highest expression in hemocytes. During the early period (within 24 h) of the Hematodinium challenge, the Toll transcripts were inhibited significantly in hemocytes, gill, heart, and muscles of P. trituberculatus, suggesting that the parasite might suggest a transient inhibition of the Toll-mediated immune response in the crab host, which were potentially associated with the survival and parasitism of parasites in crustacean hosts. Then, the Toll transcripts were significantly up-regulated in hemocytes and heart at 48 h, and in hepatopancreas at 48 and 96 h post the parasitic challenge, which suggested an important role of Toll in crustacean immune defense against the parasites. Finally, the Toll transcripts were decreased significantly in hemocytes, hepatopancreas and muscles by 192 h post Hematodinium challenge, implying that the parasite might gradually destroy the defense function of the Toll-mediated signaling immune pathways during the host immune responses in late infection.
2. The Hematodinium intrusion significantly influenced the host proPO system and could induce the increase of the proPO transcripts as well as the PO activity, which suggested the crustacean proPO system played an important role in the immune responses to the parasitic infection. Besides, the suppressed transcripts of the LGBP and PPAF genes by the Hematodinium challenge might imply an immunosuppressive effect to inhibit the function of hosts’ proPO system at the transcriptional level. In addition, the Hematodinium parasite could significantly decrease the number of hemocytes (THCs and DHCs) and result in obvious pathological alterations in hepatopancreas at 16 d and 24 d post infection.
3. After the Hematodinium challenge, significant transcriptional changes of three important clip-SPs genes (PTcSP1-3) and two key proteinase inhibitors (a2m, serpin) was observed in P. trituberculatus, which might reflect that the serine proteinase cascade reactions were disturbed severely and potentially played vital roles in the crustacean immune responses to the Hematodinium parasite. Besides, distinct expression profiles of PTcSP1-3 transcripts suggested their diverse functions in P. trituberculatus immune responses to the parasitic challenge. In addition, combined with results of the changes of the proPO transcripts and PO activities, PTcSP1 and PTcSP3 were suggested to be important members of the P. trituberculatus proPO system and involved in regulating the activation of proPO system.
4. Three novel genes (NOS, NOX and GPx) were firstly isolated from P. trituberculatus. Thereinto, the full-length cDNA of NOS was 4002 bp with a 3612 bp ORF encoding 1203 aa. The amino acid sequence of P. trituberculatus NOS shared high similarity to the counterparts of crustaceans (>79%) and was clustered with the crustacean NOS members in the phylogenetic tree. And the results of tissue distributions showed that all of the three novel genes showed high mRNA transcripts in hemocytes and hepatopancreas of P. trituberculatus. After challenged with the Hematodinium parasite (0-192 h), significant changes of the transcripts of the critical NO/O2·- generating/scavenging related genes (NOS, NOX, CuZnSOD, CAT, GPx) as well as the corresponding enzymatic activities of NOS, NOX, SOD, CAT and GPx were observed in hemocytes and hepatopancreas of P. trituberculatus, which suggested that the NO/O2·- generating and the antioxidant systems played vital roles in the crustacean innate immune responses to the parasitic intrusion. The NO/O2·- generating system was likely to participate in supplementing the crustacean immune responses against Hematodinium. Meanwhile, the antioxidant system was importantly involved in regulating the levels of ROS and benefiting the host self-protection from oxidative damages during immune responses to the parasite.
In conclusion, this dissertation shed the first and preliminary light on the host-parasite interaction between crustacean host P. trituberculatus and the parasitic dinoflagellate Hematodinium from the perspective of the hosts’ immune responses. The major finding of this dissertation will contribute to better understanding crustacean innate immune mechanisms and provide theoretical basis for the prevention and control of the diseases caused by the pathogen of parasitic dinoflagellates in aquaculture of the economic crustaceans.
语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/112497
专题海洋生态与环境科学重点实验室
作者单位中国科学院大学
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李蒙. 三疣梭子蟹对寄生性病原-血卵涡鞭虫免疫响应的初步研究[D]. 北京. 中国科学院大学,2016.
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