海洋生态与环境科学重点实验室知识库

剧毒卡尔藻（Karlodinium veneficum）是一种全球分布并产生毒素的有害藻华原因种，其有害藻华已经通过产生卡罗毒素在世界各地造成了很多鱼类的死亡。剧毒卡尔藻的环境适应能力和突出的繁殖能力与其多样化的生存策略是紧密相关的。本论文利用多种方法研究了剧毒卡尔藻的三种具有重要意义的生存策略，即毒性(对动物而言)、化感作用(对共存浮游植物而言)、和混合营养（主要是吞噬营养），以研究它们的作用机制以及对自身发展的生态学意义。我们研究了分别分离自中国（北戴河、宁德、珠江口）和美国（切萨皮克湾）的四株剧毒卡尔藻的毒性和化感作用以探究毒性和化感作用，并探讨了其生态学意义。毒性实验的方法为将三种毒型实验模式动物（海洋青鳉鱼Oryzias melastigma，卤虫Artemia salina，和褶皱臂尾轮虫Brachionus plicatilis）分别培养于四株不同浓度梯度的剧毒卡尔藻的活细胞培养液和超声细胞破碎液中作为实验组，将培养于f/2培养液和添加球等边金藻（Isochrysis galbana）作为饵料的f/2培养液中的相同个体数量的三种模式生物作为对照组，计算不同处理的四株剧毒卡尔藻分别对三种受试动物的致死率和半致死浓度（LC₅₀，Half lethal concentration）以比较毒性差异。同时，计算卤虫无节幼体和褶皱臂尾轮虫对剧毒卡尔藻的24 h摄食率用来研究剧毒卡尔藻毒性对动物摄食率的影响。化感作用实验的方法为以四株剧毒卡尔藻分别与受试微藻种（红色赤潮藻Akashiwo sanguinea，一种已被证明对化感作用敏感的裸甲藻）在不同浓度梯度下共培养作为实验组，以相应浓度和相同培养条件的所有株系的纯培养作为对照组。通过计算参数“相对于对照组细胞增长率%（G_rc，Growth relative to control %）”来定量比较剧毒卡尔藻不同株系间和不同细胞密度下的化感作用。同时我们也对一株分离自中国东海近岸（前述之福建宁德株）的剧毒卡尔藻的吞噬营养作用开展了系统研究。此部分主要通过加入不同种类的摄食对象对剧毒卡尔藻的摄食行为进行观察，以及通过用荧光探针对溶酶体/吞噬溶酶体进行染色的方法对剧毒卡尔藻在不同食物种类、生长周期和营养盐水平下的摄食能力进行比较。主要研究结果如下。

（1）对于剧毒卡尔藻的毒性，四株剧毒卡尔藻的毒性存在显著差异。其毒性从高到低的顺序总体为KVND-1（宁德株系，2016年分离）> KVBDH-1（北戴河株系，2014年分离）> KVPRE-1（珠江口株系，2011年分离）> KV7+8（美国切萨皮克湾株系，2006年分离）。我们发现毒性的减弱顺序与实验室的培养历史（即自分离建立纯培养的时间）明显相关，也即剧毒卡尔藻毒性表现为随在实验室长期培养过程中逐渐减弱。这进一步说明卡罗毒素的产生很可能是细胞对摄食压力和其它胁迫的响应。我们还发现卤虫对不同株系剧毒卡尔藻的摄食率明显受毒性强弱影响，即毒性更高的株系更不易被摄食，说明卡罗毒素一定程度上使剧毒卡尔藻避免了被捕食从而对种群增殖提供了优势。

（2）对于剧毒卡尔藻的化感作用，所有四株剧毒卡尔藻在高细胞密度下（>100,000 cells mL^-1）都不同程度且显著地抑制红色赤潮藻的生长，且不同株系的化感抑制作用由强到弱为KVBDH-1 >KV7+8 >KVND-1，KVPRE-1。但是在低密度下则随株系、共培养时间、受试种与目标种的起始细胞密度比等因素的变化而分别表现出或积极（促进）或消极（抑制）的化感作用。这些结果表明化感作用在剧毒卡尔藻藻华的起始可能并不发挥关键作用，但在藻华中、后期可能发生重要作用。不同株系的剧毒卡尔藻在化感作用和毒性的强度顺序并不是完全平行的，表明剧毒卡尔藻的化感物质与毒素可能不是相同的物质，或者虽然是相同的物质但对目标藻产生作用的方式不同。

（3）对于剧毒卡尔藻的吞噬营养，我们发现剧毒卡尔藻是杂食性的吞噬者，即可摄食受试的所有种类的有机体，包括活的或死的共培养的微藻细胞红色赤潮藻、多环马格里夫藻（Margalefidinium polykrikoides）、李氏亚历山大藻（Alexandrium leei）、盐生红胞藻（Rhodomonas salina）、球等鞭金藻、轮虫（褶皱臂尾轮虫）、卤虫、鱼类（海洋青鳉鱼），甚至本种的死细胞。尤其是剧毒卡尔藻可以摄食比他们自身细胞大很多的动物个体或藻类细胞，即所谓“微捕食”（micropredation）。剧毒卡尔藻具有灵活的摄食方式，其具体的表现为既可以直接吞噬小的摄食对象的整个细胞（即狭义的吞噬作用）也可以通过捕食茎（peduncle）摄取所有被提供的食物的细胞内容物（即捕食茎摄食myzocytosis），不管是浮游植物，浮游动物或鱼类，剧毒卡尔藻都倾向于摄食不能运动或刚死亡的对象。这一结果表明剧毒卡尔藻很有可能是通过趋化作用（chemoattraction）寻找食物和捕食。同种相食行为（cannibalism）即藻细胞摄食本种的其它不健康或死亡的细胞）在剧毒卡尔藻中为首次发现，这一行为利于在营养限制条件下帮助维持种群的生长和长期续存。剧毒卡尔藻的生长率与摄食率表现出显著的正相关，且在摄食不同的摄食对象时表现出差异，当添加盐生红胞藻作为摄食对象时生长率最高。剧毒卡尔藻的摄食能力可以在营养缺乏时得到激发。总之，我们认为杂食性吞噬营养（“无所不吃”）可能是剧毒卡尔藻扩展其营养生态位并成功形成世界性分布和频繁藻华的关键生态学机制。

Karlodinium veneficum, a cosmopolitan, toxic, and harmful algal bloom-forming dinoflagellate, has caused numerous blooms that killed massive fish by production of karlotoxins in many places of the world. The adaption to various environments and rapid reproduction of K. veneficum are attributed to its multiple survival strategies. This dissertation investigated three important survival strategies of K. veneficum through multiple approaches: toxicity to animals, allelopathy to co-cultured phytoplankton, and phagotrophy, to explore their mechanism and ecological implication. We examined the toxicity and the allelopathy of four strains of K. veneficum that were isolated from coastal waters of China (Beidaihe, Ningde, and the Pearl River Estuary) and the USA (Chesapeake Bay) and explored the ecological implications of toxicity and allelopathy. The toxicity bioassays were carried out by adding three species of test animals, namely, the finfish Oryzias melastigma, the brine shrimp Artemia salina, and the rotifer Brachionus plicatilis, into “whole live culture” and “sonicated culture” of the four strains of K. veneficum, respectively. The same numbers of three species of animals maintained in f/2 and Isochrysis galbana culture were set as controls, respectively. Half lethal concentrations (LC₅₀) were calculated to compare the toxicity among strains. The 24-h ingest rates of Artemia salina and Brachionus plicatilis on K. veneficum were calculated to evaluate the effects of toxicity on the animals’ ingest rates. The experiments for allelopathy were conducted by co-culturing four strains of K. veneficum with a selected target species (the naked dinoflagellate Akashiwo sanguinea, a species demonstrated being sensitive to allelopathy) in a series of density gradient. All monocultures in the same conditions and initial densities were used as controls. The parameter “Growth relative to control % (G_rc)” was quantified to compare the allelopathic effects among strains and different cell densities. We also launched an investigation on the phagotrophy of K. veneficum using the clonal culture isolated from the coastal water of East China Sea (Ningde, Fujian Province). We observed the feeding behavior of K. veneficum by adding different species or types of preys, and compared the ingestion ability of K. veneficum under different preys, growth stages, and nutrient levels by staining lysosomes or/and phagolysosomes with fluorescence probes. The major findings of our study are summarized as follows:

(1) Regarding the toxicity of K. veneficum, the four strains significantly varied and appeared a decreasing order as KVND-1(from Ningde, isolated in 2016) > KVBDH-1 (from Beidaihe, isolated in 2014) > KVPRE-1 (from the Pearl River Estuary, isolated in 2011) > KV7+8 (from Chesapeake Bay, isolated in 2006), which coincided with their culturing histories (i.e. duration of maintenance as clonal cultures) in the laboratory, suggesting K. veneficum may have gradually lost toxicity during laboratory culturing. This result further indicates that karlotoxins production may be a response of K. veneficum to grazing pressure or other stressors. Moreover, we found that the ingest rates of A. salina on different strains of K. veneficum were corresponded to the strength of toxicity and that A. salina avoided feeding on more toxic strains. This phenomenon suggests that karlotoxin(s) may help K. veneficum to avoid predation by zooplankton, which consequently provides an advantage for K. veneficum in terms of the population survival.

(2) As for the allelopathy of K. veneficum, all four strains of K. veneficum at high cell densities (>100,000 cells mL^-1) significantly inhibited the growth of A. sanguinea to different extents, and the inhibited ability from strong to weak was in the following order, KVBDH-1 >KV7+8 >KVND-1, KVPRE-1. However, due to the difference of strains, co-culturing time, and the initial cell density ratios of tested species and target species, they exhibited either positive (stimulative) or negative (inhibited) allelopathy at lower densities. These results suggest that allelopathy may not play an important role in initiating blooms of K. veneficum. The order of allelopathy among K. veneficum strains were not perfectly parallel to that for the toxic potency, indicating that either the allelochemicals and toxins of K. veneficum may be different chemicals, or the same chemicals functioned in different modes.

(3) As for the mixotrophy of K. veneficum, we found K. veneficum is an omnivorous phagotroph which can feed on all kinds of organisms tested, including both live and dead cells/bodies of co-cultivated microalgae Akashiwo sanguinea, Margalefidinium polykrikoides, Alexandrium leei, Rhodomonas salina, Isochrysis galbana, rotifer (Brachionus plicatilis), brine shrimp (Artemia salina), fish (Oryzias melastigma), and even its own dead species. Especially, the feeding behavior of K. veneficum on animals with sizes much larger than itself was called micropredation. Karlodinium veneficum exhibited flexible feeding modes, it can either engulf the whole cell of small cells directly (i.e. phagotrophy sensu stricto) or by sucking the cell contents of all species provided through a peduncle (i.e. myzocytosis). Karlodinium veneficum preferred to feed on preys which were non-motile or newly dead, no matter whether they were phytoplankton, zooplankton, or fish. This result suggested that K. veneficum may find and capture preys by chemoattraction. Cannibalism in K. veneficum, i.e. a cell feeds on other unhealthy or dead cells of the same species, was observed for the first time, which can contribute to the growth and long-term maintaining of the population under limited nutrient condition. The growth rate of K. veneficum exhibited significant positive correlation with the ingestion rate of K. veneficum, which varied with prey species, and the highest growth rate was observed when adding R. salina as prey. The ingest capacity of K. veneficum was stimulated by nutrient deficiency. In summary, the omnivorous mixotrophy is proposed to be a key autecological mechanism for K. veneficum to widen its ecological niche and succeed in forming a cosmopolitan distribution and frequent blooms.

目 录

第1章 引言............................................ 1

1.1 研究背景..................................................... 1

1.1.1 有害藻华.................................................. 1

1.1.2 甲藻的生存策略............................................ 2

1.1.3 剧毒卡尔藻................................................ 2

1.2 科学问题、研究目的与意义..................................... 9

1.2.1 科学问题.................................................. 9

1.2.2 研究目的................................................. 10

1.2.3 研究意义................................................. 11

第2章 四株不同地理来源和培养历史的剧毒卡尔藻的毒性比较研究..................................................... 13

2.1 实验材料.................................................... 13

2.1.1 藻种和实验动物........................................... 13

2.1.2 实验条件................................................. 13

2.2 实验方法..................................................... 14

2.2.1 实验设计................................................. 14

2.2.2 数据统计分析............................................. 16

2.3 结果......................................................... 16

2.3.1 不同株系剧毒卡尔藻的毒性比较............................. 16

2.3.2 卤虫和褶皱臂尾轮虫对四株剧毒卡尔藻摄食率的比较........... 20

2.4 讨论........................................................ 21

2.5 小结........................................................ 22

第3章 四株不同地理来源和培养历史的剧毒卡尔藻的化感作用比较研究................................................. 23

3.1 实验材料.................................................... 23

3.1.1 藻种..................................................... 23

3.1.2 实验条件................................................. 23

3.2 实验方法.................................................... 23

3.2.1 实验设计................................................. 23

3.2.2 数据统计分析............................................. 25

3.3 结果........................................................ 25

3.3.1 不同株系的剧毒卡尔藻的化感作用比较....................... 25

3.3.2 不同株系的剧毒卡尔藻对红色赤潮藻化感作用的响应........... 28

3.4 讨论........................................................ 29

3.4.1 四株剧毒卡尔藻的化感作用比较............................. 29

3.4.2 剧毒卡尔藻化感作用和毒性的生态意义....................... 31

3.5 小结........................................................ 32

第4章 剧毒卡尔藻的吞噬营养的研究...................... 33

4.1 实验材料.................................................... 33

4.1.1 藻种..................................................... 33

4.1.2 实验条件................................................. 34

4.2 实验方法.................................................... 35

4.2.1 实验设计................................................. 35

4.2.2 数据统计分析............................................. 40

4.3 结果........................................................ 40

4.3.1 剧毒卡尔藻是杂食性吞噬者................................. 40

4.3.2 剧毒卡尔藻的摄食胞器和摄食过程........................... 47

4.3.3 剧毒卡尔藻的摄食倾向性................................... 50

4.3.4 利用分子探针对溶酶体和吞噬溶酶体的染色................... 52

4.3.5 不同摄食对象或营养水平对营吞噬营养的剧毒卡尔藻生长的影响. 54

4.4 讨论......................................................... 58

4.4.1 剧毒卡尔藻的吞噬作用对象和机制........................... 58

4.4.2 剧毒卡尔藻的摄食能力和吞噬营养对剧毒卡尔藻生长的影响..... 61

4.4.3 剧毒卡尔藻的杂食性吞噬营养的意义......................... 63

4.5 小结......................................................... 64

第5章 结论、创新点与展望.............................. 63

5.1 主要结论.................................................... 65

5.2 创新点...................................................... 67

5.3 展望........................................................ 68

参考文献............................................... 71

附录 缩略语和专有名词中英文对照表....................... 83

致 谢................................................. 85

作者简历及攻读学位期间发表的学术论文与研究成果.......... 87