海洋生态与环境科学重点实验室知识库

        多环马格里夫藻（Margalefidinium polykrikoides）是一种世界性广泛分布的有毒有害藻华原因种。多环马格里夫藻最先由Margalef （1961）在加勒比海的波多黎各发现并描述为新种，随后在世界各地沿海陆续被发现和报道。在上个世纪90年代以前，仅有亚洲的韩国、日本、中国香港以及北美的东海岸等几个国家和地区有多环马格里夫藻及其藻华的报道。过去三十年里，多环马格里夫藻的分布范围呈现扩大的趋势，如亚洲的中国大陆、菲律宾、印度、印度尼西亚、新加坡和阿拉伯湾等国家和地区，美洲的美国、墨西哥和欧洲的西班牙等都是在过去的十来年内才见报道。1984-2016年间，我国的香港、广东、浙江、江苏、山东和天津等地的附近海域都有多环马格里夫藻小规模藻华的报道。多环马格里夫藻对鱼类、贝类和浮游动物都有很强的急性毒性，形成藻华时导致养殖区鱼类和其它动物大量死亡，由此给水产养殖业和渔业带来巨大的经济损失，且严重影响海洋生态系统健康。因此，多环马格里夫藻藻华已经成为了一个重要的海洋生态灾害，是少数几个全球性的最重要的藻华种类之一。

        过去研究发现多环马格里夫藻存在着不同的地理种群，而且不同地理来源的多环马格里夫藻在核糖体大亚基基因序列上有显著差别，并由此将其分成了4个不同的核糖体型（ribotype），即（1）东亚型（East Asian ribotype），（2）菲律宾型（Philippines ribotype），（3）美国/马来西亚型（American/Malaysian ribotype），和地中海型（Mediterranean ribotype）。虽然已知不同核糖体型种群的核糖体大亚基基因存在着较大差别，但是这些不同核糖体型的种群在形态和超微结构上是否也存在可辨识差异尚未见深入研究。为此，本研究对从中国青岛胶州湾、美国东海岸和马来西亚近海分离的三株多环马格里夫藻（中国株MPJZBC3，美国株CP1和马来西亚株MPCoKK23）的细胞表面形态和超微结构进行了详细的比较研究，并进一步基于核糖体大亚基序列对其分子系统学关系进行了分析。结果首先证明了分离自胶州湾的多环马格里夫藻属于东亚核糖体型，而美国株和马来西亚株均属美国/马来西亚核糖体型（中国株与美国株及马来西亚株在1523个碱基的核糖体大亚基序列上相似性为93.6%）。但是，在所有常用的具有分类学意义的细胞形态学和超微结构特征上（单细胞或群体及群体细胞数、上下锥形状、横沟位移度（绕细胞圈数）、纵沟形态、顶沟形态、细胞核位置与形状、染色体形状与数目、叶绿体的形状、结构、数目及分布、淀粉核形态等），中国株与美国株和马来西亚株（也即东亚核糖体型和美国/马来西亚核糖体型）都没有明显可区分的差别。因此，我们认为应该在生活史、休眠孢囊形态和生理生态学特征（如毒素种类及毒性强弱）以及性别相容性（sexual compatibility）等方面开展进一步比较研究，以获得对不同核糖体型种群的识别性状。在现阶段有害藻华的野外监测中，尤其是对于不同核糖体型种群可能造成的生物入侵的监测和追溯，多环马格里夫藻的鉴定尚必须以核糖体基因序列作为可靠的判决依据。

        已有众多研究表明甲藻孢囊在藻华的生消过程中起着重要的作用，即不仅孢囊的萌发为藻华的起始提供了种源，且藻华后期孢囊的形成在降低水体中营养细胞数量、预示藻华消亡的同时，也为种群的延续保存了种源。最近的研究发现多环马格里夫藻能形成休眠孢囊，但有关其孢囊形成和萌发的调控因素尚未被深入研究过。为此，本研究选取四种可能影响该藻孢囊形成的因素开展实验，包括氮限制、起始细胞密度、捕食者的存在和钙离子浓度。结果发现：1）氮元素限制（1N/100-1N）促进孢囊的形成，起始细胞密度、捕食者（卤虫）的存在和钙离子能够影响孢囊的形成；2）营养条件充足可以促进孢囊的萌发，25°Ϲ下的孢囊萌发率最高。研究结果获得了对多环马格里夫藻孢囊形成和萌发的影响因素更为全面的认识，为进一步研究多环马格里夫藻藻华的动力学过程等提供了有价值的研究基础，也为基于孢囊分布和丰度的藻华预测预警研究提供了一定的科学依据。

Margalefidinium polykrikoidesis an important toxic and harmful algal blooms (HABs) - forming dinoflagellate. M. polykrikoideswas initially described in1961 from Puerto Rico, Caribbean Sea, and was subsequently reported from many coastal waters around the world. Prior to 1990s, the presence of M. polykrikoides and its blooms were reported from several countries and regions only, such as South Korea, Japan, Hong Kong, and a limited area of the east coast of North America. In the past three decades, however, this species appeared to have expanded its geographic distribution greatly around the world and it has been reported from many new regions, such as the coastal waters of the mainland of China, the Philippines, India, Indonesia, Singapore, and the Arabian Gulf countries, the United States, Mexico, and Spain.In China, many small-scale blooms of M. polykrikoides were reported from the coastal waters of Hong Kong, Guangdong, Zhejiang, Jiangsu, Shandong, and Tianjin during 1984-2016. M. polykrikoidesexhibited acute toxicity to fish, shellfish, and zooplankton. Blooms of M. polykrikoideshave caused catastrophic mortalities of fish and other animals, and thus led to tremendous economic losses in aquaculture and fisheries and serious effects on marine ecosystems. Therefore, M. polykrikoides blooms have become an important marine ecological disaster and this species is regarded as one of the most important and most intensively investigated HAB species in the world.

Previous studies have shown that there are different geographical populations of M. polykrikoides around the world, which are in fact genetically different in the large subunit (LSU) rRNA gene, and thus they were divided into 4 different ribotypes on the basis of difference in LSU rDNA sequences, namely, (1) East Asian ribotype, (2) Philippine ribotype, (3) American/Malaysian ribotype, and (4) Mediterranean ribotype. Although the differences in the LSU rRNA sequences among different ribotypes are high enough to separate them into different species, it is not yet unclear whether these ribotype populations have discernible and stable morphological features. Therefore, in this study, we compared the cell morphology and ultrastructure among three strains of M. polykrikoides(the Chinese strain MPJZBC3, the USA strain CP1, and the Malaysia strain MPCoKK23) isolated from Jiaozhou Bay, China, the Paconic Bay of New York, the USA, and Malaysia. We confirmed at first that the strain MPJZBC3 from Jiaozhou Bay belongs to the East Asian ribotype, while both the American and Malaysian strains belong to the American/Malaysian ribotype (the identity between the Chinese strain and the USA/Malaysian strains is 93.6%, i.e., 1426 bp/1523 bp). Importantly, however, in all commonly-used diagnostic characteristics in morphology and ultrastructure (cell size, single cell or colony and number of cells in a colony, shapes of epicone and hypocone, cingular displacement (numbers of turns encircling the cell), shape of sulcus, shape of apical groove, position and shape of nuclei, shape and number of chromosomes, shape, structure, number and distribution of chloroplasts, and shape of pyrenoid, etc.), there was no discernible difference among the three strains, i.e. between the East Asian ribotype and American/Malaysia ribotype. Therefore, we believe that, in order to obtain identification traits of different ribotypes, further intensive comparative studies should be conducted among all ribotypes of the species in terms of their life histories, morphologies of resting cysts, and stable physiological characteristics such as toxin species and toxicity potency, and sexual compatibility. At present, in the field monitoring of harmful algal blooms, particularly the monitoring for alien species, a reliable identification of M. polykrikoides should be based on the LSU rDNA sequencing.

Numerous studies have shown that resting cyst plays an important role in the initiation and decline of algal blooms via germination and formation of cysts respectively. Recent studies have found that three ribotypes of M. polykrikoides can produce resting cyst, but the regulatory factors for cyst formation and germination have not been systematically investigated yet. In this study, four factors that have been shown to be important regulating factors for the formation of resting cyst or resting stage cells in other species of dinoflagellates and microalgae were investigated for the possible regulating effects on the cyst formation in M. polykrikoides, including nitrogen limitation, initial cell density, presence of predators, and calcium (Ca²⁺) concentration. Our results showed that: 1) Nitrogen-limited cultures (1N/100-1N) promotes the formation of cysts, initial cell density, the presence of predators (Brachionus plicatilis) and calcium ions can affect the formation of cysts. 2) Adequate nutritional conditions can promote cysts germination, the highest rate of cyst germination at 25 ℃. Our results summarized above provide important understandings about the regulating roles of the ambient factors affecting cyst formation and germination of M. polykrikoides, which may provide further insights into the bloom dynamics of M. polykrikoides. The results also provide a scientific basis for subsequent research on the prediction, prevention and control of algal blooms based on cyst distribution and abundance.

第1章 研究背景......................................................................... 1

1.1 多环马格里夫藻及其藻华.............................................................................. 1

1.1.1 多环马格里夫藻的分类学历史............................................................... 1

1.1.2 多环马格里夫藻的世界性分布与不同核糖体型................................... 1

1.1.3 多环马格里夫藻在中国的分布与地理扩散........................................... 3

1.1.4 多环马格里夫藻藻华、毒性及严重危害............................................... 3

1.1.5 多环马格里夫藻休眠孢囊的发现及重要意义....................................... 4

1.2 甲藻有害藻华与甲藻孢囊.............................................................................. 5

1.2.1 甲藻孢囊与甲藻藻华............................................................................... 5

1.2.2 甲藻孢囊的形态学特征........................................................................... 7

1.2.3 孢囊形成与萌发影响因素..................................................................... 10

1.2.4 国内甲藻孢囊的研究进展..................................................................... 12

1.3 研究目的和意义............................................................................................ 14

第2章 二种核糖体型的多环马格里夫藻（Margalefidinium polykrikoides）的形态和超微结构比较及分子系统学分析................................................................................................... 17

2.1 研究背景........................................................................................................ 17

2.2 材料与方法.................................................................................................... 17

2.2.1 藻种及培养条件..................................................................................... 17

2.2.2 培养基配制............................................................................................. 17

2.2.3 分子测序................................................................................................. 19

2.2.4 序列比对与系统学分析......................................................................... 19

2.2.5 光镜观察................................................................................................. 20

2.2.6 扫描电镜样品制备................................................................................. 20

2.3 实验结果........................................................................................................ 21

2.3.1 核糖体型的确定和系统进化分析......................................................... 21

2.3.2 细胞表面形态特征................................................................................. 23

2.3.3 细胞超微结构特征................................................................................. 26

2.4 讨论................................................................................................................ 28

2.5 小结................................................................................................................ 29

第3章 孢囊形成与萌发的调控研究............................................ 31

3.1 环境因素对多环马格里夫藻孢囊形成的影响............................................ 31

3.1.1 材料与方法............................................................................................. 32

3.1.2 数据处理................................................................................................. 35

3.1.3 实验结果................................................................................................. 35

3.2 环境因素对多环马格里夫藻孢囊萌发的影响............................................ 41

3.2.1 实验方法................................................................................................. 42

3.2.2 数据处理................................................................................................. 43

3.2.3 实验结果................................................................................................. 43

3.3 讨论................................................................................................................ 45

3.4 小结................................................................................................................ 46

3.4.1 环境因素对多环马格里夫藻孢囊形成的影响..................................... 47

3.4.2 环境因素对多环马格里夫藻孢囊萌发的影响..................................... 47

第4章 结论与展望.................................................................... 49

参考文献..................................................................................... 51

致 谢......................................................................................... 59

作者简历及攻读学位期间发表的学术论文与研究成果................... 61