海洋生态与环境科学重点实验室知识库

血卵涡鞭虫（Hematodinium spp.）是一类感染海洋甲壳动物的致病性寄生甲藻，在世界范围内危害了多种重要野生及养殖蟹、虾类的渔业生产。血卵涡鞭虫生活史主要包括在宿主体内的系列滋养体生长、增殖阶段以及释放至环境水体中的孢子阶段，孢子是血卵涡鞭虫传播扩散的关键生活史阶段。目前，关于孢子在环境水体中行为、活力及影响因素的研究较少，也缺乏评估孢子运动活力的方法。因此，本研究借助计算机图像分析技术，建立了血卵涡鞭虫孢子运动活力评估方法，解析了孢子在环境水体中的行为特征，并探讨了不同温度、盐度及常用水产养殖药物对孢子运动活力的影响。主要研究发现如下：

（1）通过监测天津厚蟹自然释放血卵涡鞭虫孢子，发现成年天津厚蟹可一次性释放出~1×10⁹大孢子或1×10¹⁰小孢子，大孢子14.0×8.5 µm呈椭圆形，小孢子6.2×3.5 µm呈螺旋形。建立了基于计算机图像分析的血卵涡鞭虫孢子运动轨迹、运动面积检测方法，包括运动视频采集、目标区段提取、细胞与背景分离、最近邻算法检测等步骤，可有效获取血卵涡鞭虫孢子运动轨迹及运动活力。血卵涡鞭虫大孢子运动轨迹呈最大螺旋直径6 µm、最大速度22 µm/s、左手旋向的圆柱螺旋形，血卵涡鞭虫小孢子运动轨迹呈最大螺旋直径50 µm、最大速度130 µm/s、左手旋向的圆柱螺旋形。

（2）在自然海水条件下，释出的血卵涡鞭虫孢子逐渐失去活力，大孢子运动逐渐停止、形态由椭圆形变为圆形，而小孢子运动变缓慢、停止，最终破碎消散。低盐（22）条件下2-6 h，大孢子运动活力下降89.4-91.1 %，小孢子运动活力下降74.7-81.2 %。高温（30 °C）条件下2-6 h，大孢子运动活力下降55.2-88.1 %，小孢子运动活力下降54.1-93.2 %。较低盐度（22盐度）和较高温度（30 °C）显著抑制大、小孢子的运动活力，温度和盐度对两种孢子运动活力影响均具有显著的交互作用，两种孢子保持最佳运动活力的条件为15 °C，32盐度。温度、盐度对血卵涡鞭虫孢子的影响，在一定程度上解释了美国蓝蟹和挪威龙虾等宿主种群中流行病的季节性和地区性爆发过程。

（3）苯扎溴铵、戊二醛、辛硫磷、聚维酮碘对大孢子运动活力的2 h EC₅₀（半数效应浓度）分别为0.380 mg·L^-1、1.411 mg·L^-1、1.808 mg·L^-1、108.694 mg·L^-1，苯扎溴铵、戊二醛、辛硫磷对小孢子运动活力的2 h EC₅₀分别为0.172 mg·L^-1、0.038 mg·L^-1、0.101 mg·L^-1。苯扎溴铵抑制两种孢子运动活力的效果明显优于其余3种药物，可作为水产养殖中防控血卵涡鞭虫流行病的候选药物。将2 h内使孢子运动活力降低50 %-90 %时的药物浓度确定为有效使用浓度，防治大孢子和小孢子的有效使用浓度分别为0.38 mg·L^-1-1.72 mg·L^-1和0.17 mg·L^-1-0.35 mg·L^-1，具体浓度应视养殖生物以及需防治的血卵涡鞭虫孢子类型而定。

本研究论文建立了基于计算机图像分析的血卵涡鞭虫孢子运动轨迹、运动面积检测方法，探究了不同温度、盐度以及常用水产养殖药物对孢子运动活力的影响，研究成果将有助于丰富血卵涡鞭虫孢子活力评估方法，揭示血卵涡鞭虫孢子在不同地区及季节的传播过程，为筛选水产养殖中防控血卵涡鞭虫流行病的潜在药物提供科学依据。

Hematodinium is a pathogenic parasitic dinoflagellate that spreads among marine crustaceans, affecting the production of several important wild and aquaculture crabs and shrimps worldwide. The life cycle of Hematodinium mainly includes a series of trophon growth and proliferation stages in the host, and dinospore stages released into the aquatic environment. Dinospores were the key life cycle stage for the spread of Hematodinium. At present, there is limited research on the behavior, motility, and influencing factors of dinospores in aquatic environment, and there is a lack of methods to evaluate dinospore motility. Therefore, this study used computer image analysis technology to establish an evaluation method for the motility of Hematodinium dinospores, analysed the behavioral characteristics of dinospores in aquatic environment, and investigated the effects of different temperatures, salinities, and commonly used aquaculture drugs on dinospore motility. The major findings were listed as following:

(1) By observing the natural release of Hematodinium dinospores from Helice tientsinensis, it was found that the microspores were 6.2×3.5 µm of spiral shaped, and the macrospores were 14.0×8.5 µm of elliptical. Adult H. tientsinensis can release ~1×10¹⁰ microspores or 1×10⁹ macrospores at a time. Methods for detecting the movement trajectory and area of dinospores based on computer image analysis were established, including steps such as motion video collection, target segment extraction, cell and background separation, and nearest neighbor algorithm detection. This method can effectively obtain the movement trajectory and motility of dinospores. Hematodinium microspores moved in a left-handed cylindrical spiral shape, with a maximum spiral diameter of 50 µm and a maximum velocity of 130 µm/s; Hematodinium macrospores moved in a left-handed cylindrical spiral shape, with a maximum spiral diameter of 6 µm and a maximum velocity of 22 µm/s.

 (2) Under the natural seawater conditions, the released Hematodinium dinospores gradually lost motility, the movement of macrospores gradually stopped, the shape changed from elliptical to circular, and the movement of microspores became slow, stopped, and eventually fragmented and disintegrated. Under low salt (22) treatment, the motility of macrospores decreased by 89.4-91.1%, and the motility of microspores decreased by 74.7-81.2%. Under high temperature (30 °C) treatment, the motility of macrospores decreased by 55.2-88.1%, and the motility of microspores decreased by 54.1-93.2%. Lower salinity (22) and higher temperature (30 °C) significantly inhibited the motility of macrospores and microspores, and the effects of temperature and salinity on the motility of macrospores and microspores were significantly interactive. The optimal conditions for maintaining the optimum motility of both dinospores were temperature 15 °C and salinity 32. The effects of temperature and salinity on the dinospores partly explain the seasonal and regional outbreak of epidemics in the hosts such as the American blue crabs and the Norway lobsters.

(3) The 2 h EC₅₀ (median effective concentration) for benzalkonium bromide, glutaraldehyde, phoxim, and povidone-iodine on macrospores were 0.380 mg·L^-1、1.411 mg·L^-1、1.808 mg·L^-1、108.694 mg·L^-1, respectively. Additionally, the 2 h EC₅₀ for benzalkonium bromide, glutaraldehyde, and phoxim on microspores were 0.172 mg·L^-1、0.038 mg·L^-1、0.101 mg·L^-1, respectively. The inhibitory effect of benzalkonium bromide on the motility of two dinospores was notably superior to the other three drugs, making it a promising candidate for the prevention and control of the epidemic caused by Hematodinium in aquaculture. The effective concentration was determined as the drug concentration that reduced the motility of dinospores by 50% -90% at 2h. The effective concentrations for the prevention and treatment of macrospores and microspores of Hematodinium were 0.38 mg·L^-1-1.72 mg·L^-1 and 0.17 mg·L^-1-0.35 mg·L^-1, respectively, while the specific concentration should depend on the aquaculture organisms and the type of Hematodinium dinospores that need to be controlled.

This study established a method based on computer image analysis for detecting the trajectory and area of movement of Hematodinium dinospores, and investigated the effects of different temperatures, salinities, and commonly used aquaculture drugs on dinospores motility. The research results will help enrich the evaluation methods for the motility of Hematodinium dinospores, elucidate the transmission process of Hematodinium dinospores in different regions and seasons, and provide scientific basis for screening potential drugs to prevent and control the epidemic of Hematodinium in aquaculture.

第1章 绪论    1
1.1 海洋寄生甲藻血卵涡鞭虫    1
1.1.1 血卵涡鞭虫分类地位及遗传多样性    1
1.1.2 血卵涡鞭虫生活史    2
1.1.3 血卵涡鞭虫孢子传播途径    6
1.1.4 血卵涡鞭虫检测方法    8
1.1.5 血卵涡鞭虫流行病分布    9
1.1.6 环境因子对血卵涡鞭虫的影响    11
1.2 计算机图像分析在细胞运动研究中的应用    13
1.2.1 微生物运动    13
1.2.2 计算机图像分析追踪细胞运动    14
1.3 本研究的研究目的、内容与意义    17
1.3.1 研究目的与内容    17
1.3.2 研究意义    18
第2章 血卵涡鞭虫孢子运动活力评估方法的建立    19
2.1 前言    19
2.2 实验仪器与材料    19
2.2.1 主要实验仪器    19
2.2.2 主要实验材料    19
2.3 实验方法    20
2.3.1 血卵涡鞭虫孢子的获取    20
2.3.2 血卵涡鞭虫孢子运动视频录制及预处理    21
2.3.3 血卵涡鞭虫孢子的运动轨迹检测    22
2.3.4 血卵涡鞭虫孢子的运动面积检测    24
2.4 结果    25
2.4.1 天津厚蟹释放血卵涡鞭虫孢子    25
2.4.2 血卵涡鞭虫孢子的运动轨迹特征    26
2.4.3 血卵涡鞭虫孢子的运动面积分析    28
2.5 讨论    29
2.6 小结    30
第3章 温度、盐度对血卵涡鞭虫孢子运动活力的影响    31
3.1 前言    31
3.2 实验仪器与材料    31
3.2.1 主要实验仪器    31
3.2.2 主要实验材料    32
3.3 实验方法    32
3.3.1 温度、盐度实验设计    32
3.3.2 血卵涡鞭虫孢子运动活力测定及计算    32
3.3.3 数据分析    33
3.4 结果    33
3.4.1 温度、盐度对血卵涡鞭虫大孢子的影响    33
3.4.2 温度、盐度对血卵涡鞭虫小孢子的影响    35
3.5 讨论    38
3.6 小结    39
第4章 水产药物对血卵涡鞭虫孢子运动活力的影响    41
4.1 前言    41
4.2 实验仪器与材料    42
4.2.1 主要实验仪器    42
4.2.2 主要实验材料    42
4.3 实验方法    42
4.3.1 药物实验设计    42
4.3.2 血卵涡鞭虫孢子运动活力测定及计算    43
4.3.3 数据分析    43
4.4 结果    44
4.4.1 水产药物对血卵涡鞭虫大孢子运动活力的影响    44
4.4.2 水产药物对血卵涡鞭虫小孢子运动活力的影响    45
4.5 讨论    47
4.6 小结    49
第5章 结论与展望    51
5.1 结论    51
5.2 创新点    51
5.3 不足与展望    51
参考文献    53
致  谢    63
作者简历及攻读学位期间发表的学术论文与其他相关学术成果    65