【作者】 周斌；

【导师】 唐学玺；

【作者基本信息】 中国海洋大学 ， 生态学， 2010， 博士

【摘要】 臭氧层衰减而导致的UV-B(280-320 nm)辐射增强是全球性的重大环境问题之一,海洋生态系统不可避免会受到紫外辐射的明显影响。海洋微藻是海洋食物链的基础,是海洋的主要初级生产者,它不仅驱动着整个海洋生态系统的能流和物流,而且对调节全球变化起着重要的作用。海洋浮游植物的种群和群落的动态变化一旦受到破坏将会危及其它海洋生物及整个海洋生态系统。建立灵敏、快速的微藻生物分析方法将为预警性的指示和了解UV-B辐射增强对海洋生态系统的影响提供基础的依据,也为深入了解水生生态系统对UV-B辐射增强的响应过程与机制提供理论参考。流式细胞术(Flow Cytometry, FCM)是一种在功能水平上对单个细胞进行定量分析的检测技术,通过同步高速分析大量细胞的多个参数而对细胞生理状态进行评价,在藻类生态学和毒理学的研究领域具有广泛的应用前景。目前该技术主要用以指示污染的退化程度以及污染胁迫条件下藻类的生理响应等方面,在微藻种群增长、种间竞争及其胁迫-响应生物标志物筛选方面的研究还未见报道。本文因此以四种微藻：绿藻门的青岛大扁藻(Platymonas helgolandica)、金藻门的球等鞭金藻(Isochrysis galbana)、黄藻门的赤潮异弯藻(Heterosigma akashiwo)和甲藻门的米氏凯伦藻(Karenia mikimotoi)为研究目标,在实验生态条件下基于流式细胞术系统研究了微藻种群增长和种间竞争的变化对UV-B辐射增强的响应,探讨了可能的作用机制,并筛选了能灵敏指示微藻生理响应变化的生物标志物。结果如下：1.UV-B辐射增强对微藻的急性毒性作用研究(1)利用流式细胞术能灵敏区分四种微藻：青岛大扁藻、球等鞭金藻、赤潮异弯藻和米氏凯伦藻种群中微藻活体细胞、细菌、死亡微藻细胞碎片以及培养体系中的悬浮颗粒,从而对种群中微藻密度的变化进行准确判定。(2)根据荧光信号变化对细胞体积、内容物复杂程度、叶绿素a荧光、存活率、酯酶活性、活性氧含量和种群密度的同步检测,结果表明,不同微藻对UV-B辐射增强的响应存在较大的差别：在实验的剂量和时间范围内,a.四种微藻均呈现出活性氧含量不断增加、种群密度不断下降的变化趋势；米氏凯伦藻的存活率随辐射剂量的增加而不断下降,但对青岛大扁藻、球等鞭金藻细胞和赤潮异弯藻存活率的影响并不明显。b.UV-B辐射增强能降低四种微藻的叶绿素a荧光和酯酶活性,但对细胞体积、内容物复杂程度的影响因种而异：球等鞭金藻和米氏凯伦藻的体积大小和内容物的复杂程度不断下降,而在赤潮异弯藻中细胞体积下降、内容物复杂程度却有所增加,在青岛大扁藻中的变化不显著。(3)基于流式细胞术的检测研究发现不同种类微藻对UV-B辐射增强响应的敏感性指标之间存在差异：青岛大扁藻与球等鞭金藻的活性氧含量变化对胁迫最为敏感,其EC50分别为508.6 J·m-2和560.2 J·m-2；而酯酶活性则是赤潮异弯藻和米氏凯伦藻中对胁迫响应的最敏感的指标,其EC50分别为为433.3 J·m-2和455.1J·m-2。2.共培养体系中UV-B辐射增强对微藻的急性毒性作用研究(1)用流式细胞术能灵敏区分共培养体系中的两种不同种类的微藻；混合培养对微藻的生理指标产生了影响：与单独培养体系中的同种微藻相比,与球等鞭金藻共培养可引起青岛大扁藻和赤潮异弯藻存活率的降低,对其产生不利影响,却可导致米氏凯伦藻存活率升高,对其产生有益作用；共培养体系中的球等鞭金藻的存活率均未产生显著变化,但却出现酯酶活性下降而活性氧含量上升的变化趋势,说明混合培养虽然未对球等鞭金藻产生致死作用,但对其生理活动产生了不利影响。(2)UV-B辐射增强可抑制共培养体系中微藻的种群生长,与未经UV-B辐射处理的对照组相比,3种共培养体系中微藻密度均显著降低,但对微藻生理活动的影响效果存在种间差异：共培养体系中青岛大扁藻和米氏凯伦藻叶绿素a荧光降低、活性氧含量升高,表明其生理活动受到明显抑制,而赤潮异弯藻则表现出酯酶活性上升和活性氧含量下降的变化趋势；UV-B辐射对共培养体系中球等鞭金藻的作用效果与共培养体系的微藻种类有关：当与青岛大扁藻共培养时活性氧含量随UV-B辐射增强而不断升高,而与赤潮异弯藻和米氏凯伦藻共培养时则表现出相反的变化趋势,即活性氧含量随UV-B辐射的升高反而有所下降。(3)与单独培养体系相比,共培养体系中微藻对UV-B辐射响应的敏感性发生变化,指示指标也有所不同：球等鞭金藻-青岛大扁藻培养体系中2种微藻的生理敏感指标均为活性氧含量(EC50值分别为1187.1 J·m-2和694.6 J·m-2)；球等鞭金藻-米氏凯伦藻体系中微藻敏感生化指标均为酯酶活性,EC50值分别为612.6 J·m-2和391.0 J·m-2；球等鞭金藻-米氏凯伦藻体系中敏感指标分别活性氧含量和种群密度,EC50值分别为832.8 J·m-2和578.2 J·m-2。研究结果表明,活性氧含量和酯酶活性变化可作为灵敏指示UV-B辐射胁迫-响应的生物标志物。3.长时间、亚致死剂量的UV-B辐射对微藻种群生长的影响(1)运用实验生态学方法研究了球等鞭金藻、赤潮异弯藻和米氏凯伦藻种群生长对UV-B辐射的响应。长时间、亚致死剂量的UV-B辐射能明显影响3种微藻种群的生长：3种微藻的种群密度均表现出低剂量(2.064 J·m-2·d-1)上升而高剂量(≥8.256 J·m-2·d-1)下降的趋势；球等鞭金藻与赤潮异弯藻的环境容纳量(carrying capacity, K)表现出低剂量(2.064J·m-2·d-1)上升而高剂量(≥8.256 J·m-2·d-1)下降的趋势,种群生长拐点时间(Tp)缩短；米氏凯伦藻K值无显著变化,但Tp显著延长。(2)长时间、亚致死剂量的UV-B辐射条件均能导致3种微藻细胞体积增大；球等鞭金藻和赤潮异弯藻的叶绿素a荧光与存活率均表现为逐渐下降的变化趋势,但前者的内容物复杂程度升高,而后者则无显著变化；米氏凯伦藻内容物复杂程度下降,叶绿素a荧光与存活率无显著变化。4.长时间、亚致死剂量的UV-B辐射对共培养体系中微藻种群生长和种间竞争的影响(1)混合培养抑制了微藻种群生长,与单独培养体系中的对照组相比球等鞭金藻、赤潮异弯藻和米氏凯伦藻K值均显著降低；(2)长时间、亚致死剂量的UV-B辐射能对共培养微藻的微藻种群增长的动态变化产生影响：球等鞭金藻-米氏凯伦藻体系中球等鞭金藻种群密度、K值和Tp降低,而米氏凯伦藻种群密度、K值和Tp显著上升；球等鞭金藻-赤潮异弯藻体系中球等鞭金藻种群密度和K值降低,Tp显著延长,赤潮异弯藻种群密度与K值随UV-B辐射剂量的增加呈现先升高(2.064-66.048 J·m-2·d-1)后降低(≥132.096 J·m-2·d-1)的趋势。(3)长时间、亚致死剂量的UV-B辐射影响了共培养微藻的种间竞争作用,对UV-B辐射增强条件下共培养体系中微藻的种群密度变化进行数值模拟,结果显示：随辐射剂量的增加,球等鞭金藻-米氏凯伦藻体系中种群竞争状态由球等鞭金藻占据优势转变为不稳定平衡状态,最终转变为米氏凯伦藻占据竞争优势；球等鞭金藻-赤潮异弯藻体系中种群竞争状态由不稳定平衡转变为赤潮异弯藻占据竞争优势。更多还原

【Abstract】 Ozone-related increase in UV-B radiation (280-320 nm)can negatively influence aquatic ecosystems. Phytoplankton is the major biomass producer in the oceans, and forms the basis of the aquatic food webs. They play important role not only in material cycle and energy transportation but also in global climate change regulation. The destruction of phytoplankton population and community structure threaten other marine organisms and marine ecosystem. An algal bioassay method with rapid and sensitive endpoint will contribute to evaluating and predicting the influence of enhanced UV-B radiation on marine ecosystem, and is helpful to understanding the process and mechanism for marine ecosystem response to enhanced UV-B radiation.Flow cytometry is a rapid method for the quantitative measurement for individual cell in moving fluid.Measurements of multiple fluorescence properties of thousands of individual cells are collected simultaneously to detect physiological status of cells. Flow cytometry only recently been applied to ecotoxicological studies, but has aroused increasing interest among researchers.However, no study on the population growth and interspecific of marine microalgae based on flow cytometry has been conducted.In the present study, the response of population growth and interspecific competition of Platymonas helgolandica, Isochrysis galbana, Heterosigma akashiwo and Karenia mikimotoi were studied based flow cytometry to reveal its possible mechanism and choose biomarkers sensitive to enhanced UV-B radiation.1.Effect of acute exposure of enhanced UV-B radiation on microalgae(1)The four microalgae mentioned above can be distinguished from bacteria, cell debris and suspended particles.(2) Seven indexes-cell volume, cellular content complexity, chlorophyll a (Chl a) fluorescence, survival rate, esterase activity, content of reactive oxygen species (ROS) and population density-were detected simultaneously according to fluorescence properties. Results indicated that acute exposure of UV-B radiation showed negative effect on microalgae, and the action way was species-specific.Within the dose and time range investigated, a. the content of ROS of the four microalgae increased and their population density decreased;the survival rate of K.mikimotoi decreased with the increase in UV-B dose, but no significant changes were observed in survival rates of P. helgolandica, I. galbana and H. akashiwo.b.Chi a fluorescence and esterase activity of the four microalgae were obviously decreased when exposed to UV-B radiation, but variations of cell volume and cellular content complexity were species-specific:the cell volume and cellular content complexity decreased remarkably in cells of I.galbana and K. mikimotoi, while cell volume of H.akashiwo decreased, but cellular content incressed and no variation in P. helgolandica.(3)There are differences in sensitivity of the four microalgae to enhanced UV-B radiation. The content of ROS in cells of P. helgolandica and I. galbana was most sensitive to enhanced UV-B radiation (the value of EC50 was 508.6 and 560.2 J·m-2,respectively);esterase activity is the most sensitive index in cells of H. akashiwo and K.mikimotoi (the value of EC50 was 433.3 and 455.1 J·m-2, respectively).2. Effect of acute exposure of enhanced UV-B radiation on microalgae in bi-algal culture(1)Flow cytometry could be used to distinguish cells of one species of microalga from another one and detecting physiological and biochemical indexes. Mixed culture influenced physiological and biochemical processes of microalgae, and the effect was species-specific.In co-culture with I. galbana, survival rates of P. helgolandica and H. akashiwo decreased indicating that mixed culture showed negative effect on them, but the survival rate of K. mikimotoi increased indicating that it benefit from co-culture; survival rate of I. galbana in bi-algal culture had no significant variation, but esterase activity increased and the content of ROS increased.Results indicated that mixed culture was not lethal for I.galbana, but influenced its physiological activity.(2) Acute exposure to enhanced UV-B radiation showed inhibitive effect to the growth of microalgae, and compared with control group, microalgal population density decreased obviously. The effect to UV-B radiation was species-specific, P. helgolandica and K. mikimotoi showed increase in the content of ROS and decrease in Chi a; the response of I. galbana to UV-B radiation differed in different co-culture system:with increased in UV-B radiation, the content of ROS increased in bi-algal culture with P. helgolandica and decreased in bi-algal culture with H. akashiwo and K. mikimotoi.(3)Compared with microalgae in mono-cultures, microalgae in bi-algal cultures showed different sensitivity to enhanced UV-B radiation, and their sensitive index also changed:the most sensitive biochemical index of I.galbana and P. helgolandica in bi-algal cultures of them was the content of ROS (the value of EC50 was 1187.1 and 694.6J·m-2, respectively) esterase activity was the most sensitive index of microalgae in bi-algal culture of I.galbana and K. mikimotoi, and the value of EC50 was 612.6 and 391.0 J·m-2,respectively; the most sensitive index of I.galbana and K. mikimotoi in bi-algal cultures of them was the content of ROS (EC50=832.8 J·m-2) and the population density (EC5o=578.2·m-2), respectively. Responses of sensitive indexes to enhanced UV-B radiation were observed, and the content of ROS and esterase activity could be considered as sensitive stress-response biomarkers.3. Effect of long-term exposure to sublethal UV-B radiation enhancement on population growth of microalgae(1)The responses of population growth of I.galbana, H. akashiwo and K. mikimotoi to enhanced UV-B radiation were studied in laboratory. Population growth of microalgae mentioned above was obviously affected by long-term exposure to sublethal UV-B radiation: population densities of microalgae increased under low radiation dose (2.064 J·m-2·d-1) and decreased under high radiation dose (≥8.256 J·m-2·d-1);the carrying capacity (K) of I.galbana and H. akashiwo increased under low radiation dose (2.064 J·m-2·d-1) and decreased under high radiation dose (>8.256 J·m-2·d-1),and the time for reflection point in its population growth cure (Tp) was shortened;the K value of K. mikimotoi had no obvious variation and its Tp was prolonged.(2) Under long-term exposure to sublethal UV-B radiation, the cell value of the three microalgae mentioned above increased remarkably;the Chl a fluorescence and survival rate of I. galbana and H. akashiwo decreased with increase in radiation dose, but the cellular content complexity of I. galbana increased and that of H.akashiwo had no variation; for K. mikimotoi, the cellular content complexity decreased, but Chl a fluorescence and survival rate had no notable change. 4. Effect of long-term exposure to sublethal UV-B radiation enhancement on population growth and interspecific competition of microalgae in bi-algal culture(1)The population growth of microalgae was significantly inhibited in bi-algal culture, and the K value of I.galbana, H. akashiwo and K. mikimotoi decreased obviously.(2) Long-term exposure to sublethal UV-B radiation influenced population growth dynamics of microalgae:in bi-algal culture of I.galbana and K. mikimotoi, the population density and values of K and Tp of I.galbana decreased, and those of K. mikimotoi increased significantly; in bi-algal culture of I. galbana and H. akashiwo, for I.galbana the population density and the value of K decreased, whereas the value of Tp increased. The population density and value of K of H. akashiwo increased under low radiation dose (2.064-66.048 J·m-2·d-1) and decreased under high radiation dose (≥132.096 J·m-2·d-1).(3)Long-term exposure to sublethal UV-B radiation influenced the interactions of microalgae in bi-algal cultures, and the result of numerical simulation of effect of UV-B radiation on population density of microalgae showed that in bi-algal culture of I.galbana and K. mikimotoi, I. galbana could out-compete K. mikimotoi in control group, but with the increase in UV-B radiation, the two species might be coexistence although equilibrium is unstable, and finally K. mikimotoi could out-compete I.galbana; in bi-algal culture of I. galbana and H. akashiwo, fist the two species might be coexistence and then H. akashiwo could out-compete I.galbana with the increased in UV-B radiation.更多还原