中国科学院海洋研究所机构知识库

    近几十年来，米氏凯伦藻藻华在我国近岸海域频频暴发，致使海水养殖生物，如鱼类与贝类大规模死亡，导致重大经济损失。其中，2012年福建省米氏凯伦藻藻华暴发，导致大量养殖贝类死亡，经济损失达20.11亿元，引起广泛关注。本实验室前期已经研究了该藻对受灾生物皱纹盘鲍的影响。但该藻藻华对海洋中其它生物的危害效应与原因目前并不清楚。本文选取了2012年福建省藻华区分离的一株米氏凯伦藻，在实验室条件下探究了该藻对几种典型海洋生物的危害效应与毒理机制，以求探讨福建省米氏凯伦藻藻华对海洋生态的影响与其原因。

    本文首先探究了米氏凯伦藻对几种典型海洋生物，包括褶皱臂尾轮虫、卤虫和黑褐新糠虾幼虾，黄东海浮游动物关键种中华哲水蚤，养殖生物南美白对虾幼虾和大菱鲆幼鱼的急性毒性。结果显示，该藻在藻华密度下（3×10⁴ cells/mL）对所有实验生物的存活率都有着极显著的影响，实验进行96 h后，各实验生物死亡率分别为100、23、20、97、33、53%，其中轮虫对该藻最为敏感，在藻密度约30 cells/mL时 24 h存活率为57%。米氏凯伦藻在溶解氧较高的情况下仍能导致实验生物死亡，而且非曝气条件能加剧该藻对大菱鲆的毒性效应。这说明米氏凯伦藻对海洋中不同营养级生物的存活都有着不利影响，而且这种影响来源于藻本身的毒性；在水体缺氧的条件下，米氏凯伦藻藻华可能会对生物产生更强烈的影响，从而破坏海洋生态环境，并严重威胁邻近海域的海水养殖业。

    本文进一步探究了米氏凯伦藻对褶皱臂尾轮虫行为、结构和酶活性的影响。显微观察发现，受到该藻影响，轮虫立即出现了剧烈的回避反应，游泳能力逐渐减弱，并产生粘液，最终纤毛停止摆动，失去活性。结合扫描电镜的观察结果显示，在米氏凯伦藻影响下，轮虫身体结构短时间内出现明显的变化，个体逐渐失水皱缩，纤毛明显脱落，轮盘出现囊泡与溃烂的情况。这表明米氏凯伦藻能够显著影响轮虫的行为，并在短时间内破坏其身体结构。米氏凯伦藻在低密度下（30、30、300、1000 cells/mL）就能够分别显著抑制轮虫酯酶（Esterase）、乙酰胆碱酯酶（Acetylcholinesterase）、总ATP酶活性与Na⁺-K⁺-ATP酶的活性。这说明米氏凯伦藻低密度下就能够显著影响轮虫体内多种酶的活性，从而可能对其体内渗透调节、能量代谢与物质转运等过程产生不利影响。

    为探究米氏凯伦藻的毒理机制，本文首先分析了米氏凯伦藻毒性的来源，探究了该藻不同组分对六种实验生物的毒性，同时利用隔离、冻干、重悬等手段进一步探究其对褶皱臂尾轮虫的毒性特性。结果显示，只有细胞重悬液对所有实验生物的存活有着显著影响，去藻过滤液与细胞破碎液对实验生物的存活都没有显著影响。利用3μm滤膜将藻细胞与轮虫隔离或是将藻细胞冻干后，该藻对轮虫存活并无显著影响。这表明米氏凯伦藻的毒性来自于存活的藻细胞，并与接触相关，其毒性物质可能附着在藻细胞膜上。离心重悬后米氏凯伦藻对轮虫毒性明显减弱，但会随时间逐渐恢复，这表明米氏凯伦藻毒性可能与藻细胞活性有关。

    本文还验证了活性氧在米氏凯伦藻毒理机制中的作用，结果显示，米氏凯伦藻中超氧阴离子（·O₂^-）与过氧化氢（H₂O₂）的含量分别为0.014±0.004 OD/(10⁴ cells)与3.00±0.00 nmol/(10⁴ cells)，但利用活性氧酶类将活性氧去除后，该藻对轮虫的毒性并没有显著变化。而且能显著影响轮虫存活的过氧化氢浓度是米氏凯伦藻含量的几十倍；受该藻影响的轮虫也没有表现出受到明显的氧化压力的情况。这说明活性氧并不是米氏凯伦藻导致轮虫死亡的主要原因。

    本文同时探究了米氏凯伦藻脂溶性毒素对生物的影响效应，结果显示，该藻氯仿甲醇提取物在藻华浓度下对六种实验生物的存活都没有显著影响，这说明米氏凯伦藻脂溶性毒素可能并不能够直接对生物产生影响。不同介质与手段提取的物质对轮虫的毒性有着明显的差异，其中低温氮吹法（0 ℃）提取物的毒性明显高于悬蒸法提取物（30-60 ℃），这表明米氏凯伦藻毒性物质可能不耐高温，在提取过程中有所损耗。

    以上实验结果表明，米氏凯伦藻能够显著抑制不同营养级生物，包括浮游生物与养殖生物的存活，这说明福建省米氏凯伦藻藻华能够对海洋生态系统产生不利影响，威胁当地海水养殖业的发展。这种影响来源与藻本身的毒性，并与存活的完整藻细胞相关。米氏凯伦藻还能够显著影响褶皱臂尾轮虫的行为与身体结构，并抑制轮虫体内多种酶的活性。米氏凯伦藻对轮虫的毒性与接触相关。藻细胞产生的活性氧与脂溶性毒素可能并不是米氏凯伦藻导致生物死亡的主要原因。

    In recent decades, harmful algal blooms have occurred frequently in coastal waters of China, and they have caused serious problems with significant economic, social, and human health consequences. Dinoflagellate Karenia mikimotoi is a typical HABs species. It has bloomed in large scales almost every year since 1998, which caused massive mortalities of cultured fish and shellfish, and led to huge economic losses. Blooms of K. mikimotoi were responsible for massive mortalities of abalones in Fujian coastal in 2012, with an economic loss of more than 2 billion yuan. However, little is known about the effects and mechanisms of these blooms on marine organisms. In this thesis, the toxic effects and the possible mechanisms of toxicity of K. mikimotoi from Fujian coastal waters on typical marine organisms at different trophic levels, including zooplankton (Brachionus plicatilis, Artemia salina, Neomysis awatschensis, and Calanus sinicus) and aquaculture species (Penaeus vannamei and Scophthalmus maximus) were investigated.

    The results of acute toxicity test showed that at a bloom density of 3×10⁴ cells/mL, the Fujian strain of K. mikimotoi significantly affected the tested organisms, which had mortality rates at 96 h of 100, 23, 20, 97, 33, and 53%, respectively. Rotifer B. plicatilis was the most sensitive species of the six test organisms to K. mikimotoi, for the survival rate of rotifer was inhibited to 57% by K. mikimotoi at a density of 30 cells/mL, and 0% at a density of 3200 cells/mL in 24 h. Those results indicate that HABs of K. mikimotoi may have negative impacts on marine organisms from different trophic levels, including wildlife and culture organisms.

    K. mikimotoi also affected biological behavior and body structure of marine organisms. Under the microscopic observation, the rotifers B. plicatilis had an immediate avoidance response to K. mikimotoi, and gradually lose their swimming ability in several minutes. After a 1 h treatment, we found that the cilia of rotifers stopped swing, and the body of rotifers dehydrated and shrank significantly. Several vesicles and ulcerations were also observed. Scanning electron microscopy observations also found out that rotifers affected by K. mikimotoi showed significant body shrinkage, and their cilia lose significantly compared to the control.

    The enzymes activities were significantly affected by K. mikimotoi. Results showed that algae significantly inhibited the activities of esterase, acetylcholinesterase, total ATPase, and Na⁺-K⁺-ATPase at densities of 30, 30, 300, 1000 cells/mL, respectively. After 3 h K. mikimotoi treatment at a density of 1000 cells/mL, their activities, compared to the controls, decreased 19.1, 34.0, 17.7, 31.4%, respectively. Results indicate that K. mikimotoi can affect energy metabolism and substance transport in organisms by inhibiting their enzymes activities.  

    In terms of the toxic mechanism of K. mikimotoi, it was found that the toxicity of K. mikimotoi to turbot S. maximus increased under non-aeration conditions, but we also found that dissolved oxygen in the water remained at high levels (5.53, 6.41 ppm in algae treatment at densities of 1×10⁴, 3×10⁴ cells/mL). Combined that turbot was able to tolerate 2.15 ppm of hypoxic conditions in blank, we believe that the toxicity of the algae is the main reason for fish mortalities, although hypoxia might contribute to it.  

    In this study, the Fujian strain of K. mikimotoi was tested for its ability to generate reactive oxygen species, including ·O₂^- and H₂O₂, and their concentrations were 0.014±0.004 OD/(10⁴ cells) and 3.00±0.00 nmol/(10⁴ cells), respectively. However, reactive oxygen species at that concentrations cannot harm rotifers, and there were no significant changes in the mortality rate of rotifers when reactive oxygen species were eliminated by enzymes. These results suggest that ROS are not the main mechanism of toxicity of K. mikimotoi.

    Lipophilic extracts, with hemolytic toxicity and cytotoxicity, from K. mikimotoi was tested as nontoxic to marine organisms at a bloom density of 3×10⁴ cells/mL. It is found that lipophilic extracts only affect the survival of rotifers at much higher densities than the bloom density of K. mikimotoi. Those results indicate that the toxins produced by K. mikimotoi may not directly affect marine organisms. We also extracted algae in different ways with different mediums, and found that K. mikimotoi may produce more toxins, which had high activities and decomposed easily during extracting.    

    Further investigation on the toxicity mechanism of K. mikimotoi had revealed that the toxicity of this species was associated with intact living algal cells, and it also associated the close contact of algal cells. The cell-free culture supernatant showed nontoxic to all test organisms, and toxicity also disappeared when algal cells were ruptured or separated with organisms by semipermeable membrane. We also found toxicity of the intact cell suspension significantly decreased compared with unfiltered cell culture, but gradually recovered over time. This result suggests that the toxic effect occurs on or near the algal cell membrane.   

    This Fujian strain of K. mikimotoi was proved to be toxic to all test species, including zooplankton and aquaculture species varying in size and feeding habit at or below the bloom density of 3×10⁴ cells/mL, these results indicate that HABs caused by the Fujian strain of K. mikimotoi can have detrimental effects on local ecology and lead to huge finical losses to the marine aquaculture (shrimp, fish and shellfish) industry along the Fujian coast. K. mikimotoi may affect marine organisms by contact, and was associated with intact living algal cells, while hypoxia might contribute to organisms’ mortalities, especially in still water. Neither reactive oxygen species nor the hemolytic lipophilic extract had a lethal effect on the test organisms at a bloom density. K. mikimotoi may produce more toxins with high activities, and toxic effect may occur on or near the algal cell membrane.