Population Growth And Interspecific Competition Of Marine Microalgae And Their Response To Enhanced UV-B Radiation: A Study Based On Flow Cytometry

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.