海洋生态与环境科学重点实验室知识库

血卵涡鞭虫（Hematodinium spp.）是一类主要感染海水甲壳类的致病性寄生甲藻，目前可感染全球范围内超过40种蟹、虾类，在全球多种重要经济甲壳类中频繁导致流行性病害，也严重影响我国沿海重要经济甲壳类（如三疣梭子蟹、拟穴青蟹等）的渔业生产，并对相关养殖产业造成了巨大经济损失。厘清血卵涡鞭虫的生活史可为有效防控其流行病的发生提供重要的理论依据。因此，本论文系统研究了从我国天津厚蟹（Helice tientsinensis）中分离的血卵涡鞭虫株系的生活史，以及关键环境因子-温度对其发展的影响，以期为经济甲壳动物渔业生产中血卵涡鞭虫流行病有效防控提供理论依据。本论文主要研究结果如下：

1. 通过血涂片法、H&E染色法研究了血卵涡鞭虫在宿主体内的生活史，刻画了血卵涡鞭虫在宿主体内（血淋巴、组织）的生长发育与增殖过程。研究发现血卵涡鞭虫在感染宿主期间会经历丝状滋养体、类变形虫滋养体、蛛网状滋养体、团块状聚合体、孢子母细胞、孢子前细胞和孢子等生活史阶段。在宿主感染初期血卵涡鞭虫以少量的丝状滋养体和类变形虫滋养体形式存在，在感染中期增殖形成大量类变形虫滋养体，并在宿主主要组织器官内形成蛛网状滋养体，虫体细胞聚合形成团块状聚合体，虫体数量逐渐增多，感染程度逐渐加重。在感染后期，滋养体逐渐发展成孢子母细胞、孢子前细胞，并进一步转化成孢子，最终大量孢子从宿主体内释放到水体中。感染实验结果显示，注射到宿主体内的类变形虫滋养体比孢子前细胞增殖速度更快，具有更强的感染能力。
2. 通过室内体外培养实验研究了血卵涡鞭虫在体外培养条件下的生活史，刻画了血卵涡鞭虫在体外培养中的生长发育过程以及血卵涡鞭虫的各个生活史阶段的细胞形态。研究发现血卵涡鞭虫在体外培养中经历了类变形虫滋养体、蛛网状滋养体、团块状聚合体、孢子母细胞、孢子前细胞和孢子等生活史阶段。在体外培养中血卵涡鞭虫经过类变形虫滋养体、蛛网状滋养体、团块状聚合体后能够重新形成类变形虫滋养体。蛛网状滋养体后期还能形成孢子母细胞，进一步发育成孢子前细胞，并最终形成孢子（包括大孢子和小孢子两种类型）；此外，体外培养产生的孢子具备感染甲壳类宿主的能力。
3. 明确了温度对血卵涡鞭虫生活史发展的影响。研究发现温度可显著影响血卵涡鞭虫在宿主体内的细胞生长发育与增殖过程；在低温（10 °C）和高温（30 °C）条件下，血卵涡鞭虫不能在宿主体内顺利完成生活史；在15~25 °C范围内，温度越高其细胞生长发育及细胞增殖速度越快，而且在15 °C和20 °C条件下，感染血卵涡鞭虫的天津厚蟹可产生并释放游动孢子。温度同样显著影响血卵涡鞭虫在体外培养中的细胞生长发育过程，在低温（10 °C）和高温（30 °C）条件下血卵涡鞭虫不能正常生长发育，而在15 °C、20 °C和25 °C条件下血卵涡鞭虫可正常生长发育，但在15 °C条件下生活史发展明显慢于20 °C、25 °C。

Hematodinium spp. is a type of parasitic dinoflagellate that mainly infects marine crustaceans. The parasite infects more than 40 species of crabs and shrimps worldwide, and causes epidemic diseases to multiple commercially valuable crustacean species. In recent years, Hematodinium epizootics have seriously impacted the fishery stocks of the importantly economic crustacean species, like Portunus trituberculatus and Scylla paramamosain, in the coastal waters of China and caused severe economic losses to the related cultures in China.The study on its life cycle is the fundamental basis to prevent and control Hematodinium diseases.Thus, this dissertation systematically explored the life cycle of H. perezi infecting the mudflat crab Helice tientsinensis in China and the effects of the key environmental factor- temperature on its development, so as to provide theoretical basis to prevent and control the epidemics caused by Hematodinium in the fishery stocks of the economic crustacean species. The major findings are listed as following:

1. The life cycle of H. perezi inside the hosts was explored by using a blood smear assay and H&E staining, and the growth and proliferation of the parasite in haemolymph and major tissues of the hosts were described meticulously. Cells of H. perezi inside the hosts underwent life stages including filamentous trophonts, amoeboid trophonts, arachnoid trophonts, clump colony, sporoblasts, prespores and dinospores. In the early stage of infection, the parasites existed in the forms of a few filamentous trophonts and amoeboid trophonts. In the middle stage of infection, the parasites proliferated and formed a large number of amoeboid trophonts, which aggregated to form clump colony, and form arachnoid trophonts in the main tissues and organs of the hosts. The number of cells of the parasites gradually increased, and the degree of infection gradually aggravated. In the late stage of infection, trophonts gradually developed into sporoblasts, prespores and dinospores, and finally a large number of dinospores were released from the host body into water column. In addition, the amoeboid trophonts were more infectious and proliferated rapidly compared to prespores when inoculated into host crabs.
2. In vitro life cycle of H. perezi was explored by establishing in vitro culture of the parasite starting with amoeboid trophonts, and the growth and cell morphology of various life stages of the parasite were described meticulously. Cells of H. perezi in in vitro culture underwent life stages including amoeboid trophonts, arachnoid trophonts, clump colony, sporoblasts, prespores and dinospores. After passing through amoeboid trophonts, arachnoid trophonts and clump colony, H. perezi reformed amoeboid trophonts. In the late stage of arachnoid trophonts, arachnoid trophonts could also develop into sporoblasts, prespores and dinospores (macrospores and microspores). Moreover, the dinospores in in vitro culture were capable of infecting the healthy H. tientsinensis.
3. The effects of temperature on the development of H. perezi were also investigated. Significant effects of temperature on the growth and proliferation of H. perezi were found in the in vivo studies. H. perezi could not complete its parasitism inside the hosts at the low (10 °C) and high temperature (30 °C). Within the temperature range of 15~25 °C, the growth and proliferation of H. perezi were positively related to the temperature. At 15 °C and 20 °C, the mudflat crabs experimentally infected with H. perezi were able to release dinospores at the late stage of infection. Significant effects of temperature on the growth of H. perezi were also found in the in vitro studies. H. perezi could not grow normally at the low (10 °C) and high temperature (30 °C) in in vitro culture, and at 15 °C, 20 °C and 25 °C H. perezi could grow normally. But the growth of H. perezi at 15 °C was slower than that at 20 °C and 25 °C.

第 1 章 引言... 1

1.1 血卵涡鞭虫... 1

1.1.1 系统分类地位... 1

1.1.2 宿主种类及分布... 1

1.1.3 生活史... 7

1.1.4 检测鉴定方法... 10

1.1.5 遗传多样性... 12

1.1.6 传播、扩散机制... 16

1.1.7 致病机理... 18

1.1.8 流行病爆发规律... 19

1.2 本论文的研究目标、内容和意义... 20

1.2.1 研究内容与目标... 20

1.2.2 研究意义... 21

第 2 章 血卵涡鞭虫在宿主-天津厚蟹体内的生活史过程研究... 23

2.1 前言... 23

2.2 实验材料与试剂... 24

2.2.1 主要实验仪器... 24

2.2.2 主要实验试剂... 24

2.2.3 实验所需其他试剂及具体配制方法... 24

2.2.4 实验天津厚蟹及病原采集... 24

2.3 实验方法... 25

2.3.1 血卵涡鞭虫感染检测... 25

2.3.2 分子测序和遗传分析... 26

2.3.3 注射感染实验... 27

2.4 结果... 28

2.4.1 血卵涡鞭虫的序列比对及分子系统发育分析... 28

2.4.2 血卵涡鞭虫在宿主血淋巴内的生活史... 29

2.4.3 血卵涡鞭虫在宿主组织内的生活史阶段... 32

2.4.4 温度对宿主体内血卵涡鞭虫生活史的影响... 34

2.4.5 不同生活史阶段细胞注射感染实验... 36

2.5 讨论... 37

2.6 小结... 40

第 3 章 血卵涡鞭虫体外培养生活史过程研究... 41

3.1 前言... 41

3.2 实验材料与试剂... 42

3.2.1 主要实验仪器... 42

3.2.2 主要实验试剂... 42

3.2.3 实验所需其他试剂及具体配制方法... 42

3.2.4 实验动物... 42

3.3 实验方法... 43

3.3.1 体外培养液的配制... 43

3.3.2 血卵涡鞭虫的体外培养连续观测... 43

3.3.3 温度梯度血卵涡鞭虫的体外培养... 43

3.3.4 血卵涡鞭虫在不同培养液中的体外培养... 44

3.3.5 体外培养孢子的感染能力... 44

3.4 结果... 45

3.4.1 血卵涡鞭虫在体外培养条件下的生活史及其形态学特征... 45

3.4.2 温度对体外培养条件下血卵涡鞭虫生活史的影响... 47

3.4.3 血卵涡鞭虫在不同培养液中的体外培养... 48

3.4.4 体外培养孢子的感染能力... 49

3.5 讨论... 49

3.6 小结... 52

第 4 章 结论与展望... 55

4.1 结论... 55

4.2 创新点... 56

4.3 不足与展望... 56

参考文献... 57

附 录... 65

致 谢... 69

作者简历及攻读学位期间发表的学术论文与研究成果... 71