海洋生态与环境科学重点实验室知识库

甲藻引起的藻华在全球有害藻华事件中占了绝大多数。甲藻有害藻华的发生是由外部原因和内部原因相互作用的结果。外部原因是自然环境和人类活动的影响, 而内部原因则是甲藻多样的生存策略。其中, 甲藻生活史（特别是形成休眠孢囊）作为重要的生存策略之一对有害藻华的形成具有非常重要的作用。休眠孢囊有诸多重要性, 如休眠孢囊在合适条件下萌发并且大量繁殖, 可为大规模藻华形成接种，休眠孢囊还可为甲藻提供一种地理扩散的方式, 使其从一个海域传播到另一个海域。因此, 研究某一甲藻是否会产休眠孢囊, 不仅丰富对藻华生物生活史的认识，也为藻华的发生和地理扩散提供一种机理性的解释, 还可能为后续基于孢囊分布和丰度的藻华预测和防控提供科学依据。 在众多藻华原因种中, 米氏凯伦藻（Karenia mikimotoi）、东海原甲藻（Prorocentrum donghaiense）和剧毒卡尔藻（Karlodinium veneficum）为我国重要的藻华种, 且常常造成养殖或渔业巨大的经济损失。然而, 在本研究之前, 尚未有文献报道上述三种甲藻产休眠孢囊的确凿证据。因此, 本论文基于三种甲藻的纯培养探索了它们的有性生活史，确认三者都可在实验室和自然水体产生休眠孢囊, 以此为基础，通过自行设计的特异性探针和引物检测三种甲藻休眠孢囊在中国近海沉积物中的分布和丰度。主要研究结果如下：

（1）三种甲藻基于纯培养的生活史观察。通过室内甲藻培养实验, 观察到三种甲藻的有性交配细胞对、具双鞭毛的游动合子以及有性休眠孢囊。关于三种甲藻休眠孢囊形态特征, 米氏凯伦藻休眠孢囊通常呈圆形, 表面光滑, 大小通常在15.9-28.9 μm, 壁薄，内含褐色或浅褐色的内含物；东海原甲藻休眠孢囊呈圆形或椭圆形, 表面光滑, 大小通常在10-30 μm, 具至少2层壁, 颜色较普通营养细胞深,内部可见褐色内含物；剧毒卡尔藻休眠孢囊呈圆形形态, 表面光滑且无特殊的表面结构, 大小在9.6-17.2 μm之间，具至少2层壁，呈深褐色或浅灰色，含橙色或红色内含物。三种休眠孢囊都可在实验室内被萌发。米氏凯伦藻休眠孢囊可在3-6天后萌发，东海原甲藻孢囊可在3天左右萌发，剧毒卡尔藻孢囊可在2-36天萌发，但前二者萌发率很低（< 20%）。

（2）海洋沉积物中孢囊检测方法的建立及目标休眠孢囊的检测。本论文应用普通PCR、荧光原位杂交（fluorescence in situ hybridization, FISH）和单细胞PCR相结合的技术手段对海洋沉积物中三种休眠孢囊进行检测。首先, 在28S rDNA D2区分别设计了米氏凯伦藻、东海原甲藻、剧毒卡尔藻的特异性引物, 提取沉积物样品的宏 DNA（曾经爆发过赤潮的区域或附近），经过普通PCR及其产物的克隆测序, 确定了沉积物中存在这三种甲藻的核酸信号。随后, 在28S rDNA D2区设计了三种甲藻的特异性FISH探针。探针5’端分别添加FITC（绿色荧光和Cy3（橙色荧光）两种荧光基团, 随后应用FISH技术在海洋沉积物中检测孢囊, 结合显微镜明场和荧光场寻找、观察到被双色探针标记的目标孢囊。最后, 应用单细胞PCR技术与单克隆测序手段确定被探针标记的孢囊的种类。根据形态特征，特别是测序序列的结果，确定了海洋沉积物中存在米氏凯伦藻、东海原甲藻、剧毒卡尔藻三种休眠孢囊。该研究结果填补了这三种甲藻生活史研究和生态学研究的重要空白, 为三种甲藻有害藻华的年际复发（藻华动力学）和全球地理扩散提供了一个合理的解释，为以后开展它们的藻华预警预测奠定了重要的基础。

（3）海洋沉积物中孢囊分布检测方法的建立及目标休眠孢囊在中国近海沉积物中的分布。选取了125个沉积物样品, 覆盖了渤海（8个）、黄海（22个）、东海（70个）和南海（25个），北起河北省北戴河沿岸，南至海南省三亚近岸，结合荧光定量PCR（qPCR, TaqMan法）和FISH技术绘制了米氏凯伦藻、东海原甲藻、剧毒卡尔藻休眠孢囊在中国近海沉积物中的分布图。其中, 荧光定量PCR（TaqMan法）根据三种甲藻单细胞rDNA拷贝数对沉积物中的米氏凯伦藻、东海原甲藻和剧毒卡尔藻的休眠孢囊进行定量，FISH法则是直接计数换算。由于DNA提取的过程中存在核酸损失，因此以三种甲藻营养细胞的提取效率为基准（未用孢囊是因为三种甲藻孢囊在实验室内产率都极低）对荧光定量PCR的结果进行了矫正。约三分之一的qPCR产物进行了克隆测序并分别确认为三种甲藻的特异性片段,保证了方法的特异性。同时, FISH技术作为另一种技术手段检测了40个样品（也覆盖了中国四大海域）中的米氏凯伦藻、东海原甲藻、剧毒卡尔藻的休眠孢囊。

米氏凯伦藻休眠孢囊在中国近海沉积物中的分布：125个样品中的58个沉积物样品中检测出米氏凯伦藻的休眠孢囊（46.4%）。应用荧光定量PCR定量的结果和孢囊的二倍体基因拷贝数换算后, 全部样品中休眠孢囊数量的范围为0-33 cysts /32g 湿重沉积物。应用FISH技术定量结果，休眠孢囊丰度范围为0-14 cysts /32g 湿重沉积物。结合两种定量方法的结果, 米氏凯伦藻休眠孢囊在东海的丰度明显高于黄渤海（P < 0.05），但与南海之间无显著差（P > 0.05）。就孢囊在样品中检出率而言, 东海最高，为65.7%（N=70），南海为44%（N=25），黄海为9%（N=22）, 渤海为25%（N=8）。两种方法定量结果之间存在显著线性关系（P < 0.05）, 但据线性方程斜率判断, 荧光定量PCR的结果总体低于FISH结果。尽管如此,两种方法的定量结果都表明米氏凯伦藻休眠孢囊在中国近海广泛分布, 但丰度很低。

东海原甲藻休眠孢囊在中国近海沉积物中的分布：125个样品中的36个沉积物样品中检测出东海原甲藻的休眠孢囊（28.8%）。应用荧光定量PCR定量的结果和孢囊的二倍体基因拷贝数换算后，全部样品中休眠孢囊数量的范围为0-62 cysts /32g 湿重沉积物。应用FISH技术定量结果，休眠孢囊丰度范围为0-8 cysts /32g 湿重沉积物。结合两种定量方法的结果, 东海海域的东海原甲藻休眠孢囊丰度明显高于其他海域丰度（P < 0.05）。最高孢囊丰度出现在东海。就孢囊检出率而言, 东海最高, 为42.8%（N=70），南海为20%（N=25），黄海为4.54%（N=22）, 渤海为0%（N=8）。东海海域孢囊检出率最高。两种方法定量结果之间存在明显的线性关系（P < 0.05），但根据线性方程斜率判断, 荧光定量PCR所获得的定量结果总体较高于FISH定量结果。尽管如此, 使用两种方法所获得的定量结果表明, 东海原甲藻休眠孢囊在中国东海海域, 南海和黄海都有分布, 且总体孢囊丰度极低。在渤海没有检测到其分布, 指示东海原甲藻的分布可能存在纬度边界。

剧毒卡尔藻休眠孢囊分布结果：125个样品中的94个沉积物样品检测出剧毒卡尔藻的休眠孢囊（75.2%）。应用荧光定量PCR定量的结果和孢囊的二倍体基因拷贝数换算后, 全部样品中休眠孢囊数量的范围为0-25 cysts /32g 湿重沉积物。应用FISH技术定量结果，休眠孢囊丰度范围为0-15 cysts /32g 湿重沉积物。结合两种方法的定量结果, 剧毒卡尔藻休眠孢囊丰度在各个海域之间无明显差别（P>0.05），但是各个样品间的丰度存在差异。最高孢囊的丰度出现东海。就孢囊检出率而言, 东海为76.05%（N=70）, 南海为76%（N=25）, 黄海为81.82%（N=22），渤海为75%（N=8），四者之间没有明显差别。两种方法定量结果之间存在明显的线性关系（P < 0.05）。但同样, 荧光定量PCR所获得的定量结果总总体较低于FISH定量结果。尽管如此, 使用两种方法所获得的定量结果表明, 剧毒卡尔藻休眠孢囊在中国近海沉积物中广泛分布, 但总体丰度极低。 上述三种有害藻华甲藻休眠孢囊在中国四大海域的检测表明它们不仅在自然水体产生休眠孢囊（相对于实验室而言），而且在中国海域的沉积物中广泛存在。虽然除局部海域外丰度普遍较低（相对于另一些常见孢囊种类），但其萌发仍可为有害藻华提供起始种群（“接种”）。因此, 上述检测结果对三种有害甲的藻华动力学具有重要意义。

Dinoflagellates are a group of protists that cause about 75% of all harmful algal bloom (HAB) events over the world. Both environmental factors and the biological characters of the HABs-causing species contribute to the occurrence of HABs of dinoflagellates. Among all contributing biological characters, the life history of dinoflagellates, especially the resting cyst formation in many species, plays a vital role in the initiation and geographic expansion of HABs because the germination of resting cysts under favorable conditions can inoculate ("seeding") an initial population in the pre-blooming seasons and the cysts can be more easily transferred, either via natural or anthropogenic (e.g. via ships' ballast water sediments) pathways, from one to another location. While about 200 among ca. 2400 species of dinoflagellates have been documented to be resting cysts producers, the majority, including many HABs-forming species, have not been proved to be capable of cyst producing. Therefore, proving whether or not a dinoflagellate can form resting cysts is of particular importance in terms of providing a mechanistic explanation for the recurrence and geographic spread of HABs, and even in providing a basis for the forecasting the occurrence and scale of HABs based on mapping the distribution of resting cyst in marine sediments. Among all HABs-forming dinoflagellate, Karenia mikimotoi, Prorocentrum donghaiense, and Karlodinium veneficum are three of the most important ones as they often form density blooms in many regions of the world, including China, cause massive kills of many marine animals or destructive effects on the coastal ecosystems, and consequent economic losses. Except for some fragmental evidences, however, whether or not these three species can form resting cysts have not be systematically investigated yet. Hence, this dissertation studied the life histories of three species based on laboratory cultures and confirmed these three species capable of producing resting cysts both in laboratory cultures and the field. We also mapped the distribution of resting cyst in the marine sediment of China coastal waters using multiple approaches. The major findings of the work are summarized as follows:

(1) Observations on the life histories of the three species based on laboratory cultures. We observed cell pairs mating, planozygotes, and resting cysts in all three dinoflagellates. The resting cyst of K. mikimotoi are characterized by a spherical shape, a smooth surface (without spiny process), a diameter ranging from 15.9 to 28.9 μm, an obvious brown or pale brown accumulation body, and a thin cyst wall. The resting cyst of P. donghaiense are spherical or elliptic, with a smooth surface of no spiny process, a diameter ranging from 10 to 30 μm, at least two layers of cyst wall, darker than the vegetative cells, and a brown accumulation body. K. veneficum cysts have a spherical shape, a smooth surface, a diameter ranging from 9.6 to 17.2 μm, an orange or red accumulation body, a brown or light gray color, and a thick cyst wall (at least two layers). The cysts of all three species can be germinated in the laboratory, with a germination time requirement for K. mikimotoi cyst 3-6 days, about 3 days for P. donghaiense, and 2-36 days for K. veneficum cyst, the first two have a low rate of germination (< 20%).

(2) Establishment of a protocol for the detection of resting cyst in marine sediments and the detection results. In this thesis, a method combining PCR, fluorescence in situ hybridization (FISH), single cell isolation, and single-cell PCR and clone sequencing was used to detect resting cyst in marine sediment samples. First, specific primers of K. mikimotoi, P. donghaiense and K. veneficum were designed and tested against the 28S rDNA D2 region, respectively. Second, the genomic DNA of the sediment samples collected from the locations where the blooms of the target species occurred was extracted, and PCR detections using the primers tested above were conducted to detect the presence of these three species in the sediments. Third, the respective FISH probes specifically designed against the 28S rDNA D2 region for three species, with the 5' end of each probe labeled with either the fluorochrome FITC (fluorescing green) or Cy3 (fluorescing orange) were applied to an optimized FISH protocol to detect the resting cysts from marine sediments and observed a epi-fluorescence light microscope. Finally, in order to further confirm the identity of cysts positively detected with both FISH probes labeled with FITC and Cy3, the fluorescing cysts were individually isolated and a single-cell PCR and subsequent sequencing was conducted. Based on the results of sequences, we confirmed that presence of resting cysts of K. mikimotoi, P. donghaiense and K. veneficum in the field marine sediments. The results fill up the vacancy of the life history and ecology in the three dinoflagellates, providing a reasonable explanation for the interannual recurrence and global geographical spread of the three dinoflagellates, and an important foundation for the early warning and prediction of the three dinoflagellates.

(3) Establishment of a protocol for mapping the resting cysts in marine sediments along the coast of China sea. Together, 125 sediment samples were selected for the detection, covering all four seas of China (Bohai Sea (BS), Yellow Sea (YS), East China Sea (ECS) and South China Sea (SCS)) and with 8, 22, 70, and 25 sediment samples from BS, YS, ECS, and SCS, respectively. Real-time PCR (TaqMan) and FISH were both used to map the distribution of resting cysts in sediments of China coastal waters. Real-time PCR was used to quantify the abundance of cysts after taking into consideration the 2N ploidy of cysts and the measurements of rDNA copy number of three dinoflagellates both in vegetative cells and cysts. Also, because of the DNA loss during the process of DNA extraction, the qPCR results were adjusted based on the DNA extraction efficiency of vegetative cells (due to the low availability of cysts for the three species measuring the efficiency). One third of the qPCR products amplified from all sediment samples were further cloned and sequenced to confirm the specificity of assays. FISH was conducted to enumerate the resting cysts in 40 sediment samples also covering all four seas of China and to compare with the results obtained via qPCR.

For K. mikimotoi, among the 125 sediment samples collected from all four seas of China, 58 sediment samples (46.4 %) were detected positive for the presence of K. mikimotoi cyst. The calculated abundance of K. mikimotoi cyst ranged from 0 to 33 cysts/32 g wet sediment. For FISH detection, the abundance of K. mikimotoi cysts varied from 0 to 14 cysts/32 g wet sediment across all 40 sample. Base on the results of the two methods, the abundance of K. mikimotoi cyst in ECS was the highest among all four seas, while no significant difference was found between ECS and SCS (P > 0.05). For the numbers of samples detected positive, ECS was 65.7% (N=70), SCS was 44% (N=25), YS was 9% (N=22), and BS was 25% (N=8). Regression analysis showed a significant correlation (P < 0.05) between the cyst abundance quantified using qPCR and that using FISH. According to the slope of the regression line, however, the cyst abundance obtained from FISH detection was generally higher than that from qPCR detection. Nevertheless, both methods (FISH and qPCR) detected a wide distribution of the K. mikimotoi cysts in the sediments of China seas but generally with a low abundance.

For P. donghaiense, 36 of the 125 sediment samples from all four seas of China were detected positive in the presence of P. donghaiense cyst (28.8%). The calculated abundance of P. donghaiense cysts ranged from 0 to 62 cysts/32 g wet sediment. For FISH method, the abundance of P. donghaiense cysts varied from 0 to 8 cysts/32 g wet sediment in 40 samples. Comparatively, the abundance of P. donghaiense cysts in ECS was the highest among all four sea areas (P < 0.05). For the numbers of samples detected positive, the detection rate in the ECS was 42.8% (N=70), the detection rate in the SCS was 20% (N=25), the detection rate in the YS was 4.54% (N=22), and the detection rate in the BS was 0% (N=8). The highest detection rate was found in ECS (42.8%). Regression analysis showed a significant correlation (P < 0.05) between the cyst abundance quantified by qPCR and that quantified by FISH. According to the slope of the regression line, indicating that cyst abundance obtained from FISH detections tended to be lower than that from qPCR detection. In spite of this, both methods (FISH and qPCR) showed an extremely low abundance of P. donghaiense cyst, a wide distribution in sediments of ECS, SCS, YS, and no distribution in BS, indicating the distribution of P. donghaiense cyst in China seas has a latitude boundary.

For K. veneficum, among the 125 sediment samples collected from all four seas of China, 94 sediment samples (75.2 %) were detected to be positive in the presence of K. veneficum cyst. The calculated abundance of K. veneficum cyst ranged from 0 to 25 cysts/32 g sediment (wet weight). For FISH method, the abundance of K. veneficum cysts varied from 0 to 15 cysts/32 g sediment (wet weight) across all 40 sampling sites. Base on the results of two methods, the abundance of K. veneficum cyst had no significant differences across all sites that were detected (P > 0.05), but significantly varied among different sampling regions. The highest abundance of cyst was observed in ECS via two methods. For the numbers of samples detected positive, the detection rate in the ECS was 76.05% (N=70), the detection rate in the SCS was 76% (N=25), the detection rate in the YS was 81.82% (N=22), and the detection rate in the BS was 75% (N=8).There are no significant differences among detection rate of sea area. Regression analysis showed a significant correlation (P < 0.05) between the cyst abundance quantified by qPCR and that quantified by FISH. According to the slope of the regression line, FISH detections generated higher levels of cyst abundance compared to qPCR detections. After all, both methods (FISH and qPCR) detected very low abundance but a wide distribution of K. veneficum cyst in the sediment samples of China seas. Our results summarized above demonstrated that the three HABs-forming dinoflagellates can produce resting cysts in the field (i.e. not only in laboratory) and the resting cysts widely distribute along the coastal sediment of all four China seas. Although the abundance of these cysts is generally very low in the sediments of most areas in comparison to those species well-known in having abundant cysts, their germinations are still enough to inoculate initial vegetative cell populations for bloom. Therefore, our detection results provide essential knowledge for the blooming dynamics of these HABs-forming dinoflagellates.