海洋生态与环境科学重点实验室知识库

球形棕囊藻（Phaeocystis globosa）是一种具有复杂异型生活史的微藻，能够形成由薄而坚韧的多糖胶质膜包覆的巨大囊体。近年来，球形棕囊藻在我国沿海频繁暴发以囊体形态为主的有害藻华，不但对近海生态安全、沿海旅游养殖等产生重大危害，而且其囊体还会堵塞核电冷源系统，严重威胁滨海核电站的冷源安全，引起国内外的高度关注。改性粘土（MC）是一种在现场大规模应用的有害藻华应急处置方法，前期研究发现该方法不但可以通过絮凝作用快速消除水体中的藻华生物，而且还可以通过产生胁迫抑制未被絮凝的藻华生物继续生长。目前有研究表明，改性粘土也可以有效絮凝沉降球形棕囊藻单细胞和囊体，但对于残留在上层水体中的藻细胞生长和成囊的影响尚不清楚。为此，本文以不同浓度改性粘土处理后的残余球形棕囊藻细胞为对象，研究了改性粘土对其生长、成囊以及囊体发育的影响，并基于细胞生理学和分子生物学等方法，探讨了其影响机制。主要结果如下：

（1）通过浓度梯度控制实验，研究了不同浓度改性粘土对球形棕囊藻细胞生长、成囊和囊体发育的影响。结果表明，改性粘土可以有效抑制球形棕囊藻游离单细胞的生长，抑制效果随粘土用量增加而增强，0.3 g/L的改性粘土对游离单细胞生长的抑制率可达90%以上。对于残余藻细胞成囊的影响研究表明，当浓度低于0.2 g/L，改性粘土总体呈现促进囊体形成的作用，促进效果随改性粘土浓度升高呈现先增强、后减弱的变化趋势，在改性粘土浓度为0.075 g/L时促进效果最显著。当浓度高于0.2 g/L时，改性粘土会抑制囊体形成，抑制效果随改性粘土用量的增加而增强，0.5 g/L的改性粘土的抑制率可达90%以上。此外，改性粘土处理后囊体平均直径降低20%以上，而且囊体单位表面积上的细胞密度显著降低，说明改性粘土能够抑制囊体的正常发育。

（2）研究了低浓度（0.1 g/L）和高浓度（0.5 g/L）改性粘土去除球形棕囊藻后，对残余藻细胞内抗氧化应激、光合作用和多糖代谢等生理过程的影响。结果表明，低浓度改性粘土处理3 h后残余藻细胞的抗氧化酶活性、还原型谷胱甘肽（GSH）和丙二醛（MDA）含量均与对照组无显著差异，但随培养时间增加，抗氧化酶活性和GSH、MDA含量均不同程度升高，至实验结束时MDA含量显著高于对照组，说明残余藻细胞受到了一定程度的氧化胁迫。通过叶绿素a含量和叶绿素荧光动力学参数探究了低浓度改性粘土对残余微藻光合作用的影响，结果表明，整个培养过程中不论光合系统的反应中心活性还是整体光合效率均与对照组无显著差异，叶绿素a含量也没有显著降低，光合作用未受明显影响。多糖代谢方面，低浓度改性粘土处理后总多糖产量降低，但是囊被多糖比例升高，表明残余微藻内存在调控机制维持囊被多糖的合成。高浓度改性粘土处理后，残余藻细胞内抗氧化酶活性、GSH含量和MDA含量在3 h内均显著升高，且随培养时间的增加进一步升高，表明细胞受到严重的过氧化损伤。残余藻细胞内叶绿素a含量降低，叶绿素荧光参数表明光合系统II的部分反应中心失活、整体光合效率降低，表明高浓度改性粘土处理对残余微藻的光合作用产生明显的损伤。此外，残余微藻内总多糖产量降低，囊被多糖占比升高，而且葡萄糖、半乳糖、麦芽糖和海藻糖等单糖和二糖的含量显著降低，表明糖代谢受改性粘土影响严重。

（3）以低浓度（0.1 g/L）和高浓度（0.5 g/L）改性粘土处理后的残余球形棕囊藻细胞为对象，研究了残余微藻内重要细胞过程的转录表达变化。从整体上看，随着改性粘土浓度的升高和培养时间的推移，差异基因的数目增多，转录水平的差异逐渐扩大。低浓度改性粘土处理后，残余微藻的环境信号转导过程被激活，将胁迫信号通过级联反应传递至细胞内并引发了一系列应激反应；编码抗氧化酶、DNA损伤修复蛋白、光合结构蛋白、磷脂合成酶、呼吸代谢酶等蛋白的基因上调表达，表明残余藻细胞内存在积极的转录调控应对环境胁迫，提高了抗氧化能力，修复受损的细胞结构，同时提高能量代谢速率为细胞过程提供能量；大量参与多糖合成和转运的基因上调表达，促进囊被多糖的合成与分泌。总的来说，低浓度改性粘土处理后残余藻细胞受到一定程度的损伤并处于应激状态，胞外多糖的合成和分泌增强，通过促进囊体形成应对环境胁迫，同时，细胞内正常的生理过程未发生紊乱，为细胞损伤修复和成囊提供了物质和能量基础。高浓度改性粘土处理后，残余藻细胞内编码光合作用固碳反应关键酶的基因下调表达，表明光合作用的固碳过程被抑制，不但降低了光合效率，还会阻断电子传递链，加剧过氧化损伤；在细胞周期调控方面，编码检查点激酶的基因上调表达，编码细胞周期蛋白依赖性激酶的基因下调表达，将残余细胞限制在G1期，阻滞了细胞分裂；大量与多糖降解有关的基因显著上调表达，加速细胞多糖的分解。总的来说，高浓度改性粘土处理后残余藻细胞受到严重的细胞损伤，正常生理过程紊乱并发生了细胞周期阻滞，藻细胞的分裂受到抑制，囊体也难以形成。

总之，本文系统研究了改性粘土对残余球形棕囊藻生长和成囊的影响，基于细胞生理学和分子生物学等方法，探讨了其影响机制，为改性粘土有效治理球形棕囊藻藻华提供了重要参考。

Phaeocystis globosa is a phytoplankton species with a complex polymorphic lifecycle. The huge colonies, which are encased in a thin and tough polysaccharide gel matrix, are the most prominent feature of this species. Outbreaks of colonial P. globosa have frequently impacted Chinese coastal waters in recent years, threatening the marine ecosystem, coastal tourism industries, and aquaculture industries. In addition, the colonies can block the cooling systems of nuclear power plant, which seriously threatens the safety of nuclear power plants in China, and hence has received extensive attention. The application of modified clay (MC) is the most widely used method for the mitigation of harmful algae blooms (HABs) in the field. Specifically, MC can directly settle HAB organisms via flocculation and inhibit the growth of residual cells by inducing environmental stresses. Previous studies have found that MC could effectively flocculate both P. globosa solitary cells and colonies, but the effects of MC on residual P. globosa cells remained uncertain. Therefore, the residual P. globosa cells after treatment with MC were taken as the research object to investigate the effects of MC on their growth, colony formation and colony development. Meanwhile, the mechanism of MC affecting the colony formation was revealed by physiological and transcriptomic experiments. The main results are as follows:

(1) The effects of different concentrations of MC on residual P. globosa cells growth, colony formation and colony development were studied by a series of MC concentration gradient experiments. The results showed that MC could inhibit the growth of P. globosa solitary cells, with the inhibitory effect being enhanced with the increasing MC dosage and the inhibition efficiency of the 0.3 g/L MC treatment exceeding 90%. The formation of colonies was promoted under low concentrations of MC (<0.2 g/L), and the inhibitory effects initially increased and then decreased with increasing MC concentration, in which the 0.075 g/L MC induced the most obvious promoting effect. The formation of colonies was inhibited when the MC concentration was higher than 0.2 g/L, with the inhibitory effect being enhanced with increasing MC dosage and the inhibition efficiency could reach more than 90% after 0.5 g/L MC treatment. In addition, the mean diameter of colonies decreased by more than 20% after treatment with MC, and the colonial cell densities were also decreased, indicating that the development of the colony was also inhibited.

(2) After the application of low concentration (0.1 g/L) and high concentration (0.5 g/L) of MC to remove P. globosa cells, the changes of physiological processes were analyzed, including antioxidant response, photosynthesis, and metabolism of polysaccharides. The results showed that the low concentration of MC induced mild oxidative stress in residual cells. Specifically, there have been no significant differences in antioxidant enzyme activities, glutathione (GSH) content, and malondialdehyde (MDA) content between the low concentration of MC group and the control group since MC was added for 3 h. And then, the antioxidant enzyme activities, GSH content, and MDA content increased slightly with the increasing incubation time. At the end of the experiment, MDA content was significantly higher than that of the control group. The effects of 0.1 g/L MC on the photosynthesis of residual cells were investigated by analyzing chlorophyll a content and chlorophyll fluorescence parameters. The results showed that there were no significant changes in the photosystem Ⅱ (PSII) reaction centers activity, the photosynthetic efficiency, and the chlorophyll a content, indicating that photosynthesis was not significantly affected by 0.1 g/L MC. In terms of polysaccharide metabolism, the production of total polysaccharide (TPS) decreased in the low concentration MC group, but the percentages of bound extracellular polysaccharide (bEPS) to TPS per cell were increased, indicating that there was a response mechanism in the residual cells to maintain the biosynthesis of colonial polysaccharides. After being treated with the high concentration of MC, the antioxidant enzyme activities, GSH content and MDA content were significantly increased within 3 h, and increased continuously with increasing incubation time, indicating that the cells suffered significant oxidative stress. Moreover, photosynthesis was inhibited, including the decrease of chlorophyll a content, the inactivation of partial PSII reaction centers, and the decrease of the maximal photochemical efficiency of PSII (F_V/F_M). In addition, the TPS content decreased, the proportion of bEPS increased, and the contents of glucose, galactose, maltose, and trehalose were also decreased, indicating that sugar metabolism was seriously affected by the high concentration of MC.

(3) The effect of the low concentration (0.1 g/L) and high concentration (0.5 g/L) of MC on the transcriptional expression of P. globosa was investigated. On the whole, the number of differentially expressed genes (DEGs) increased, and the fold-changes in the expression of DEGs increased with increasing incubation time and MC concentration. After treatment with the low concentration of MC, the environmental information processing pathways were up-regulated, which can transduce stimulatory signals to the cell and regulate growth and stress resistance processing in cells. Multiple genes involved in antioxidant, DNA damage repair, photosynthesis, phospholipid biosynthesis, and respiratory metabolism were significantly up-regulated, indicating the existence of a positive transcriptional regulation in residual cells for cell damage repair, including enhancing antioxidative capacity, repairing cell damages, and enhancing energy metabolism. In addition, multiple genes involved in polysaccharide biosynthesis and transfer were also significantly up-regulated, promoting the biosynthesis of colonial polysaccharides. In summary, after treatment with the low concentration of MC, the residual P. globosa cells were slightly damaged and stressed, and the biosynthesis of polysaccharides was enhanced in the residual cells, which provided abundant materials for the colonial matrix to resist environmental stresses. Meanwhile, the normal cellular function of residual cells was only slightly damaged, providing the material and energy basis for colony formation, thereby stimulating the formation of P. globosa colonies. After treatment with the high concentration of MC, the genes encoding key enzymes of the dark reaction of photosynthesis were down-regulated in residual cells, indicating that the dark reaction of photosynthesis was inhibited, which not only reduced photosynthetic efficiency, but also enhanced the accumulation of ROS. In terms of cell cycle regulation, genes encoding checkpoint kinases were up-regulated, and genes encoding cyclin-dependent kinases were down-regulated, indicating that the cell cycle of residual cells was blocked, and the cell proliferation was inhibited. In addition, many genes related to polysaccharide degradation were significantly up-regulated, indicating that the degradation of polysaccharides was accelerated. Overall, treatment with the high concentration of MC disrupted the physiological processes of residual P. globosa cells and inhibited cell proliferation, which likely inhibited the formation of colonies.

In summary, the present study elucidated the concentration-dependent mechanism of MC affecting the growth and colony formation of residual P. globosa cells and provided a reference for the application of MC to control P. globosa blooms.