ABSTRACT Title of Document: THE EFFECT OF AMBIENT N:P RATIO AND LIGHT ON THE NITROGEN UPTAKE AND GROWTH OF SELECT ESTUARINE AND OCEANIC DINOFLAGELLATES Ji Li, Doctor of Philosophy, 2011 Directed By: Patricia M. Glibert, Professor Marine Estuarine Environmental Science Increasing frequency of harmful algal blooms (HABs) have been observed in eutrophic coastal ecosystems. The relationship between environmental factors (nutrients and light) and bloom-forming dinoflagellates were explored in this dissertation by both historical data analysis and laboratory experiments. The growth and nitrogen (N) acquisition of the HAB dinoflagellates Prorocentrum minimum, P. donghaiense, Karlodinium veneficum and Karenia brevis, were studied. It is hypothesized that estuarine species Prorocentrum spp. develops blooms in relative high N:P ratio water, while K. veneficum blooms near or lower than the Redfield ratio; these species will grow faster in the N:P ratio in which they develop blooms, even when these nutrients are not at limiting levels; Prorocentrum spp. preferentially take up more DIN in high DIN:DIP ratio water, while Karlodinium can better use other source of N in the low DIN water; low-light-adapted nitrogen acquisition by Prorocentrum spp. serves as an adaptive advantage to grow in low light waters. Historical data analysis showed that P. minimum generally develops blooms in high DIN, high N:P ratio, but turbid water in Chesapeake Bay, while K. veneficum blooms near or lower than the Redfield ratio, when DIN was depleted, but organic N sources were still available. Following these results, the effects of ambient N:P ratio and light on the growth and N acquisition of P. minimum and P. donghaiense were studied in both batch and continuous culture (turbidistat). Prorocentrum spp. were grown in a wide range of N:P ratios, and across a wide range of light intensities in turbidistat. Experiments to determine rates of N acquisition of different N sources were conducted using 15N tracer techniques at each N:P ratio and light treatment. However, in culture, the growth of the Prorocentrum species was not regulated by the ambient N:P ratio. When nutrients were sufficient, light, instead of ambient N:P ratio, regulated the algal ability to acquire N. The adaptive strategies of the two types of dinoflagellates, Prorocentrum spp. and Karlodinium/Karenia spp., are different. Prorocentrum minimum was shown to take up N in the dark. This light independent N uptake allows it to be more competitive in the relative low light near- shore water. Karlodinium/Karenia spp. apparently only takes up N in the light phase, but it can be mixotrophic and directly use organic sources, and thus may be more competitive after DIN was depleted. The Droop model, which describes the growth rate regulated by the cell quota, was used to interpret the relationship between N acquisition and the growth rate over the diel cycle of growth. Prorocentrum spp. continuously take up nitrogen at night to supplement the cell quota, and reaches the maximum cell quota at the beginning of light phase, when they reach the higher growth rate in a diel cycle. In eutrophic coastal systems (e.g., Chesapeake Bay), the ambient N:P ratio, as well as light may be critical factors for HAB growth. The dinoflagellates studied here have different adaptive strategies to grow in low light and to take advantage of high nutrients in the eutrophic waters. Prorocentrum spp. may dominant the high DIN water, while Karlodinium/Karenia spp. prefers organic nutrients. THE EFFECT OF AMBIENT N:P RATIO AND LIGHT ON THE NITROGEN UPTAKE AND GROWTH OF SELECT ESTUARINE AND OCEANIC DINOFLAGELLATES By Ji Li Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2011 Advisory Committee: Professor Patricia M. Glibert, Chair Research Associate Professor Jeffrey C. Cornwell Professor Ming Li Dr. Peter Tango Professor Daniel Terlizzi, Dean?s Representative ? Copyright by Ji Li 2011 ii Dedication I dedicate this dissertation to my loving and supportive family. iii Acknowledgements I would like to thank all people who have helped and inspired me during my study. I especially want to thank my advisor, Dr. Patricia M. Glibert, for her hard work and guidance throughout this entire study and for believing in my abilities. Her perpetual energy and enthusiasm in research had greatly motivated me. In addition, she was always accessible and willing to help my research. I would like to express my gratitude to Dr. Jeffrey Cornwell, Dr. Ming Li, Dr. Peter Tango and Dr. Daniel Terlizzi as my academic committee members and advisors. Their suggestions and encouragement helped me throughout the research and writing of this thesis. I like to express my thanks to Jeff Alexander, Cindy Shoemaker and Caroline Solomon. Without their support in both lab and life, this dissertation would not have been written. I am appreciative for the support from Horn Point Laboratory fellowship and student small grant. Especially, I would like to give my special thanks to my wife Yonghui whose patient love enabled me to complete this work. iv Table of Contents Dedication ..................................................................................................................... ii? Acknowledgements ...................................................................................................... iii? Table of Contents ......................................................................................................... iv? List of Tables .............................................................................................................. vii? List of Figures ............................................................................................................ viii? Chapter 1: Introduction ............................................................................................... 1? Eutrophication and Harmful Algal Blooms .......................................................... 1? N:P Ratio and Nutrient Limitation ........................................................................ 2? N:P Ratio Regulating Dinoflagellate Blooms ....................................................... 4? Light Dependence of Photosynthesis and Nutrient Uptake .................................. 6? Perspective of N:P ratio and Light - the Matrix ................................................... 8? Hypothesis............................................................................................................... 10? Research Objectives ................................................................................................ 10? References ............................................................................................................... 12? Figures..................................................................................................................... 19? Chapter 2: An Exploratory Analysis of Nutrients and Other Related Variables Related to Select Harmful Algal Bloom Events, in the Chesapeake Bay 1991-2008 .............. 21? Abstract ................................................................................................................... 21? Introduction ............................................................................................................. 22? Methods................................................................................................................... 25? Results ..................................................................................................................... 25? Eutrophic Level and Bloom Events .................................................................... 25? Spatial Distribution ............................................................................................. 27? Salinity ................................................................................................................ 28? Seasonal Records and Temperature .................................................................... 28? Secchi Depths and TSS ....................................................................................... 29? Dissolved Oxygen ............................................................................................... 30? Dissolved Carbon and Nutrients ......................................................................... 31? Components of Particulate Matter ...................................................................... 33? Discussion ............................................................................................................... 34? A Comparison with Ecosystems with Similar Nutrient Dynamics ..................... 34? A Comparison with Spatial Historical Data ........................................................ 36? The Spatial Variation of Nutrients and HAB Events .......................................... 39? The Environmental Factors and HAB Species ................................................... 41? References ............................................................................................................... 45? Figures..................................................................................................................... 54? Chapter 3: Effects of Ambient DIN : DIP Ratio on the Nitrogen Uptake of Harmful Dinoflagellate Prorocentrum minimum and Prorocentrum donghaiense in Turbidistat* ................................................................................................................. 90? Abstract ................................................................................................................... 90? Introduction ............................................................................................................. 91? v Materials and Methods ............................................................................................ 93? Algae Culture ...................................................................................................... 93? Continuous Culture Design ................................................................................. 94? Experimental Design and Algal Growth ............................................................. 95? Nitrogen Uptake and Assimilation Experiments ................................................ 96? Analytical Methods ............................................................................................. 97? Data Analysis ...................................................................................................... 98? Results ..................................................................................................................... 98? Ambient Parameters and Cell Counts ................................................................. 98? Culture Growth in the Turbidistat ....................................................................... 99? Nitrogen Uptake Experiments ........................................................................... 100? Application of Droops Model ........................................................................... 103? Discussion ............................................................................................................. 104? Value of Tubidostat Approach .......................................................................... 105? Value of Droop?s Model Approach .................................................................. 108? The N:P Ratios in the Coastal Ecosystems ....................................................... 109? References ............................................................................................................. 112? Tables .................................................................................................................... 120? Figures................................................................................................................... 122? Chapter 4: Effects of Light on Growth and Nitrogen Uptake of Prorocentrum minimum and Prorocentrum donghaiense ................................................................ 133? Abstract ................................................................................................................. 133? Introduction ........................................................................................................... 134? Materials and Methods .......................................................................................... 136? Algal Culture ..................................................................................................... 136? Continuous Culture Design ............................................................................... 137? Experimental Design and Algal Growth ........................................................... 138? Nitrogen Uptake and Assimilation Experiments .............................................. 139? Analytical Methods ........................................................................................... 140? Data Analysis .................................................................................................... 141? Results ................................................................................................................... 142? Ambient Parameters .......................................................................................... 142? Growth Rate in the Turbidistat ......................................................................... 142? Nitrogen Uptake Experiments ........................................................................... 143? Discussion ............................................................................................................. 145? Growth Regulation in Turbidistat ..................................................................... 145? N Uptake versus Irradiance ............................................................................... 149? Light Condition and Prorocentrum Blooms ...................................................... 151? References ............................................................................................................. 153? Tables .................................................................................................................... 161? Figures................................................................................................................... 163? Chapter 5: Growth and Competition of Several Harmful Dinoflagellates under Different Nutrient and Light Condition .................................................................... 173? Abstract ................................................................................................................. 173? Introduction ........................................................................................................... 174? Materials and Methods .......................................................................................... 177? vi Algae Cultures and Methods of Growth ........................................................... 177? Experimental Design ......................................................................................... 178? Analytical Protocols .......................................................................................... 180? Data Analysis and Simulation ........................................................................... 181? Results ................................................................................................................... 181? Experiment 1 ..................................................................................................... 181? Experiment 2 ..................................................................................................... 183? Experiment 3 ..................................................................................................... 183? Experiment 4 ..................................................................................................... 184? Experiment 5 ..................................................................................................... 185? Experiment 6 ..................................................................................................... 185? Discussion ............................................................................................................. 186? Growth Rate ...................................................................................................... 186? Nutrient Strategy ? Autotrophy and Mixotrophy.............................................. 188? References ............................................................................................................. 196? Tables .................................................................................................................... 205 Figures................................................................................................................... 208? Chapter 6: Research Conclusions ............................................................................. 222? Dinoflagellate Blooms and the Matrix of Nutrients and Light ............................. 222? The Application of Cell Quota Model .................................................................. 226? The Improvement of Cell Quota Model ................................................................ 229? References ............................................................................................................. 230? Figure .................................................................................................................... 232? Complete References ................................................................................................ 233? vii List of Tables Table 3-1 Ambient nutrient concentrations in 4 different tubidistat systems supplied by medium at different DIN:DIP ratios. ..................................................... 120? Table 3-2 Calculated parameters of uptake kinetics of nitrogen sources (NH4+, urea, and glycine) by Prorocentrum minimum at different ambient DIN:DIP ratio in turbidistat based on the Michaelis-Menten equation. ............................. 121 Table 4-1 Ambient nutrient concentrations in 4 different tubidistat systems supplied by medium at different DIN:DIP ratios. ..................................................... 120? Table 4-2 Calculated parameters of uptake kinetics of nitrogen sources (NH4+, urea, and glycine) by Prorocentrum minimum at different ambient DIN:DIP ratio in turbidistat based on the Michaelis-Menten equation. ............................. 121? Table 5-1 Monoculture exponential growth rates of Prorocentrum minimum and Karlodinium veneficum .............................................................................. 198? Table 5-2 Exponential growth rates (? day-1) of Prorocentrum minimum and Karlodinium veneficum in mixed culture experiments. .............................. 206? Table 5-3 Exponential growth rates (? day-1) of Prorocentrum minimum and Karlodinium veneficum with Rhodomonas in mixed culture experiments. 207? Table 5-4 Monoculture exponential growth rates of Prorocentrum donghaiense and Karlodinium brevis ..................................................................................... 208? Table 5-5 Exponential growth rates of Prorocentrum donghaiense and Karlodinium brevis in mixed culture experiments ........................................................... 209? Table 5-6 Exponential growth rates of Prorocentrum donghaiense and Karlodinium brevis with Synechococcus in mixed culture experiments ......................... 210? Table 5-7 Summary of the growth rates of 4 dinoflagellates reported in laboratory experiments. ................................................................................................ 211? viii List of Figures Figure 1-1 Comparison of the range in ambient nitrogen: phosphorus ratios during different phases of the blooms in the East China Sea and in different phytoplankton assemblages among the southwest Florida shelf during May 2005.. ............................................................................................................ 19? Figure 1-2 Predominant dinoflagellate life-form Types associated with the turbulence-nutrient matrix along an onshore?offshore continuum characterizing pelagic habitats . ................................................................... 20? Figure 2-1 The routinely sampled stations in the Chesapeake Bay and its tributaries by Maryland Departments of Natural Resources ......................................... 54? Figure 2-2 a. samples of different type of eutrophic level chlorophyll a recorded at Chesapeake Bay area (1991-2008); b, samples of different type of Prorocentrum minimum cell density level recorded; c, samples of different type of Karlodinium veneficum cell density level recorded; d, the number of samples of different type of cyanobacteria cell density level recorded ........ 55? Figure 2-3 The spatial distribution of Chl a samples in the Chesapeake Bay and its tributaries 1991-2008. ................................................................................... 56? Figure 2-4 The spatial distribution of Prorocentrum minimum in the Chesapeake Bay and its tributaries 1991-2008. ....................................................................... 57? Figure 2-5 The spatial distribution of Karlodinium veneficum in the Chesapeake Bay and its tributaries 2003-2008. ....................................................................... 58? Figure 2-6 The spatial distribution of cyanobacteria in the Chesapeake Bay and its tributaries 1991-2008. ................................................................................... 59? Figure 2-7 The salinity of samples with different eutrophic levels of Chl a and harmful algae densities: Prorocentrum minimum, Karlodinium veneficum and cyanobacteria in the Chesapeake Bay .................................................... 60? Figure 2-8 The ambient parameters of Prorocentrum minimum (PM), Karlodinium veneficum (KV) and cyanobacteria (Cyn) in blooms in the Chesapeake Bay ...................................................................................................................... 61? Figure 2-9 The month number of samples with different eutrophic levels of Chl a and harmful algae densities: Prorocentrum minimum, Karlodinium veneficum and cyanobacteria in the Chesapeake Bay .................................................... 62? Figure 2-10 The water temperature of samples with different eutrophic levels of Chl a and harmful algae densities: Prorocentrum minimum, Karlodinium veneficum and cyanobacteria in the Chesapeake Bay .................................. 63? Figure 2-0-11 The secchi depth of samples with different eutrophic levels of Chl a and harmful algae densities: Prorocentrum minimum, Karlodinium veneficum and cyanobacteria in the Chesapeake Bay .................................. 64? Figure 2-12 The total suspended solids (TSS) of samples with different eutrophic levels of Chl a and harmful algae densities: Prorocentrum minimum, Karlodinium veneficum and cyanobacteria in the Chesapeake Bay ............. 65? Figure 2-13 The dissolved oxygen (DO) of samples with different eutrophic levels of Chl a and harmful algae densities: Prorocentrum minimum, Karlodinium veneficum and cyanobacteria in the Chesapeake Bay .................................. 66? ix Figure 2-14 The dissolved organic carbon (DOC) of samples with different eutrophic levels of Chl a and harmful algae densities: Prorocentrum minimum, Karlodinium veneficum and cyanobacteria in the Chesapeake Bay ............. 67? Figure 2-15 The dissolved organic nitrogen (DIN) concentration of samples with different eutrophic levels of Chl a and harmful algae densities: Prorocentrum minimum, Karlodinium veneficum and cyanobacteria in the Chesapeake Bay ............................................................................................ 68? Figure 2-16 The phosphate (PO43-) concentration of samples with different eutrophic levels of Chl a and harmful algae densities: Prorocentrum minimum, Karlodinium veneficum and cyanobacteria in the Chesapeake Bay ............. 69? Figure 2-17 The dissolved inorganic nitrogen (DIN) to dissolved inorganic phosphorus (DIP) ratios of samples with different eutrophic levels of Chl a and harmful algae densities: Prorocentrum minimum, Karlodinium veneficum and cyanobacteria in the Chesapeake Bay .................................. 70? Figure 2-18 The ambient carbon, nitrogen and phosphorus rates of Prorocentrum minimum (PM), Karlodinium veneficum (KV) and cyanobacteria (Cyn) in blooms in the Chesapeake Bay ..................................................................... 71? Figure 2-19 The particle carbon (PC) concentration of samples with different eutrophic levels of Chl a and harmful algae densities: Prorocentrum minimum, Karlodinium veneficum and cyanobacteria in the Chesapeake Bay ...................................................................................................................... 72? Figure 2-20 The particle nitrogen (PN) concentration of samples with different eutrophic levels of Chl a and harmful algae densities: Prorocentrum minimum, Karlodinium veneficum and cyanobacteria in the Chesapeake Bay ...................................................................................................................... 73? Figure 2-21 The particle phosphorus (PP) concentration of samples with different eutrophic levels of Chl a and harmful algae densities: Prorocentrum minimum, Karlodinium veneficum and cyanobacteria in the Chesapeake Bay ...................................................................................................................... 74? Figure 2-22 The particle carbon (PC) to particle phosphorus (PP) ratios of samples with different eutrophic levels of Chl a and harmful algae densities: Prorocentrum minimum, Karlodinium veneficum and cyanobacteria in the Chesapeake Bay ............................................................................................ 75? Figure 2-23The particle carbon (PC) to particle phosphorus (PP) ratios of samples with different eutrophic levels of Chl a and harmful algae densities: Prorocentrum minimum, Karlodinium veneficum and cyanobacteria in the Chesapeake Bay ............................................................................................ 76? Figure 2-24 The particle nitrogen (PN) to particle phosphorus (PP) ratios of samples with different eutrophic levels of Chl a and harmful algae densities: Prorocentrum minimum, Karlodinium veneficum and cyanobacteria in the Chesapeake Bay ............................................................................................ 77? Figure 2-25 Comparison of the range in ambient N:P ratios during different phase of the blooms in the East China Sea (ECS) during May 2005, in different phytoplankton assemblages along the southwest Florida Shelf (SFS) during May 2003 and different phase of blooms in the Chesapeake Bay from 1991 to 2008. ......................................................................................................... 78? x Figure 2-26 The spatial distribution of salinities in surface waters along the length of Chesapeake Bay major axis in May and August in the time range of 2000- 2008, 1991-1999, and 1982-1987. ................................................................ 79? Figure 2-27 The spatial distribution of dissolved inorganic nitrogen (DIN) in surface waters along the length of Chesapeake Bay major axis in May and August in the time range of 2000-2008, 1991-1999, and 1982-1987 ........................... 80? Figure 2-28 The spatial distribution of phosphate (PO43-) in surface waters along the length of Chesapeake Bay major axis in May and August in the time range of 2000-2008, 1991-1999, and 1982-1987 ................................................... 81? Figure 2-29 The spatial distribution of dissolved organic nitrate: dissolved organic phosphate (N:P) ratio in surface waters along the length of Chesapeake Bay major axis in May and August in the time range of 2000-2008, 1991-1999, and 1982-1987. ............................................................................................. 82? Figure 2-30 The spatial distribution of chlorophyll a in surface waters along the length of Chesapeake Bay major axis in May and August in the time range of 2000-2008, 1991-1999, and 1982-1987. .................................................. 83? Figure 2-31. The summer surface water salinity in the Chesapeake Bay ................... 84? Figure 2-32 The seasonal variation of monthly average dissolved inorganic nitrogen (DIN) concentrations in 3 zones of Chesapeake Bay. .................................. 85? Figure 2-33 The seasonal variation of monthly average phosphate (PO43-) concentrations in 3 zones of Chesapeake Bay. ............................................. 86? Figure 2-34 The seasonal variation of ambient dissolved inorganic nitrogen (N): phosphate (P) ratios in 3 zones of Chesapeake Bay. .................................... 87? Figure 2-35 The seasonal variation of monthly average chlorophyll a concentrations in 3 zones of Chesapeake Bay. ..................................................................... 88? Figure 2-36 The seasonal variation of monthly maximum cell densities of cyanobacteria in Zone I and Prorocentrum minimum and Karlodinium veneficum in Zone II of Chesapeake Bay. .................................................... 89? Figure 3-1 The setup of a turbidistat system ............................................................ 122? Figure 3-2 Diurnal growth rates of Prorocentrum donghaiense in turbidistat system for 4 constant 12 h: 12 h light dark cycles (96 hrs). ................................... 123? Figure 3-3 a. Diurnal growth rates of Prorocentrum minimum in turbidistat system at 14: 10 h light dark cycle. b. Diurnal growth rates of Prorocentrum donghaiense in turbidistat system at 12: 12 h light dark cycle ................... 124? Figure 3-4 a. Average growth rates of Prorocentrum minimum in a 14h: 10h light dark cycle at different ambient N:P ratios. b. Average growth rates of P. minimum in the light phase of 14h: 10h light dark cycle at different ambient N:P ratios. c. Average growth rates of P. donghaiense in a 12h: 12h light dark cycle at different ambient N:P ratios. d. Average growth rates of P. donghaiense in the light phase of 12h: 12h light dark cycle at different ambient N:P ratios ...................................................................................... 125? Figure 3-5 Kinetic relationships between nitrogen specific uptake rates V (h-1, upper panels) and assimilation rates A (h-1, lower panels) of NH4+, urea, and glycine with 15N substrate concentration (?M-N) by Prorocentrum minimum at different DIN:DIP ratio cultures. ............................................................ 126? xi Figure 3-6 Nitrate specific uptake rates V (h-1) and assimilation rates A (h-1) of NH4+, urea, and glycine with 15N substrate concentration (?M-N) by Prorocentrum minimum grown at different DIN:DIP ratios. ............................................. 127? Figure 3-7 a. The NO3? specific uptake rates (V, h-1) of Prorocentrum donghaiense grown at different ambient N:P ratios; b. The NO3? specific assimilation rates (A, h-1) of P. donghaiense grown at different ambient N:P ratios. c. The NH4+ uptake rates of P. donghaiense grown at different ambient N:P ratios; d. The NH4+ assimilation rates of P. donghaiense grown at different ambient N:P ratios. ................................................................................................... 128? Figure 3-8 The assimilation efficiencies % (assimilation rate/ uptake rate) of NO3? by Prorocentrum donghaiense grown at different ambient N:P ratios; b. The assimilation efficiencies % (assimilation rate/ uptake rate) of NH4+ by P. donghaiense grown at different ambient N:P ratios ................................... 129? Figure 3-9 The ratio of specific N assimilation rate/ growth rate (A/?) of Prorocentrum minimum for major N sources at different DIN:DIP ratios 130? Figure 3-10 The ratio of specific N uptake rate/ growth rate (V/?) of Prorocentrum donghaiense for NO3- (a) and NH4+ (b) at different ambient N:P ratios; the ratio of specific N assimilation rate/ growth rate (A/?) of P. donghaiense for NO3- (c) and NH4+ (d) at different ambient N:P ratios ............................... 131? Figure 3-11 The conceptual model of the diel cell nutrient quota variation in a light- dark cycle. ................................................................................................... 132? Figure 4-1 Diurnal growth rates of Prorocentrum donghaiense in turbidistat system for 4 constant 12 h: 12 h light dark cycles (96 hrs).. .................................. 163? Figure 4-2 a. Diurnal growth rates based on changes in optical signals of Prorocentrum minimum in turbidistat system at 14 h: 10 h light dark cycle. b. Diurnal growth rates of Prorocentrum donghaiense in turbidistat system at 14 h: 10 h light dark cycle. ..................................................................... 164? Figure 4-3 a. Average growth rates of Prorocentrum minimum in a 14 h: 10 h light dark cycle at different ambient N:P ratios. b. Average growth rates of P. minimum in the light phase of 14 h: 10 h light dark cycle at different ambient N:P ratios. ................................................................................................... 165? Figure 4-4 Diurnal growth rates based on changes in optical signals of Prorocentrum donghaiense in turbidistat systems in 12 h: 12 h light dark cycles at different light intensities ............................................................................................ 166? Figure 4-5 Average diurnal growth rates (?) and average growth rates based on changes in optical signals of Prorocentrum donghaiense in the light phase ( ?) in a 12 h: 12 h light dark cycle at different ambient light irradiance. .. 167? Figure 4-6 The relationship of N (Nitrate (NO3-), ammonium (NH4+), urea and glycine) uptake rates of Prorocentrum minimum at different irradiances. . 168? Figure 4-7 Average specific uptake rates V (h-1) of 4 nitrogen substrates (Nitrate (NO3-), ammonium (NH4+), urea and glycine) near ambient concentration by Prorocentrum donghaiense in high light and low light incubation at different ambient N:P ratios. ..................................................................................... 169? Figure 4-8 Kinetic relationships between nitrogen specific uptake rates V (h-1) of NO3- and NH4+ with 15N substrate concentration (?M-N) by Prorocentrum donghaiense in different light irradiance. ................................................... 170? xii Figure 4-9 Average specific uptake rates V (h-1) and assimilation rates A (h-1) of nitrogen substrates nitrate (NO3-) and ammonium (NH4+) near ambient concentration by Prorocentrum donghaiense growing in high light and low light treatments ........................................................................................... 171? Figure 4-10 Ratio of assimilation rates A : uptake rates V of nitrogen substrates nitrate (NO3-) and ammonium (NH4+) by Prorocentrum donghaiense growing in high light and low light treatments. ......................................... 172? Figure 5-1 Time course of growth of Prorocentrum minimum (a) and Karlodinium veneficum (b) in monoculture with 2 nitrogen sources NO3- (a, c) and urea (b, d) at high and low N:P ratios (HNP, LNP) and high and low light intensities (HL, LL) .................................................................................... 212? Figure 5-2 Time course of growth of Prorocentrum minimum and Karlodinium veneficum in mixed culture with 2 nitrogen sources at 2 N:P ratios and 2 light intensities ............................................................................................ 214? Figure 5-3 Time course of growth of Prorocentrum minimum and Karlodinium veneficum with Rhodomonas in mixed culture with NO3- at 2 N:P ratios and 2 light intensities ......................................................................................... 215? Figure 5-4 Time course of growth of Prorocentrum donghaiense (a., b.) and Karenia brevis (c., d.) in monoculture with 2 nitrogen sources NO3- (a., c.) and urea (b., d.) at high and low N:P ratios (HNP, LNP) and high and low light intensities (HL, LL) .................................................................................... 216? Figure 5-5 Time course of growth of Prorocentrum donghaiense and Karenia brevis in mixed culture with 2 nitrogen sources at 2 N:P ratios and 2 light intensities .................................................................................................... 218? Figure 5-6 Time course of growth of Prorocentrum donghaiense, Karenia brevis and Synechococcus in mixed culture at 2 N:P ratios and 2 light intensities ..... 219? Figure 5-7 The exponential grow rates of 4 harmful dinoflagellates Prorocentrum minimum (PM), P. donghaiense (PD), Karlodinium veneficum (KV) and Karenia brevis (KB) in 3 sets of culture setting with treatments of high light and low light; low N:P ratio and high N:P ratio ......................................... 221? Figure 6-1 The conceptual model of the bloom development in the Chesapeake Bay: Prorocentrum minimum developed blooms on DIN in the spring; Karlodinium veneficum developed blooms on DON or directly feeding on other microalgae as food sources. .............................................................. 232? 1 Chapter 1: Introduction Eutrophication and Harmful Algal Blooms There has been a global increase in nutrient input to the coastal marine ecosystem as a result of increasing population in coastal areas, more urbanization, and increasing use of fertilizer for agriculture (Smith 2003, Smith et al. 2006, Andersen et al. 2006, Glibert et al. 2008). Eutrophication, defined as ?the enrichment of water by nutrients, especially nitrogen (N) and/or phosphorus (P), causing an accelerated growth of algae and higher forms of plant life to produce an undesirable disturbance to the balance of organisms present in the water and to the quality of water concerned? by European Commission Nitrates Directive (http://www.cearac- project.org/2ndCEAWS/presen_PDF /Ulrich_CLAUSSEN.pdf), has been considered as one of the major threats to health of marine ecosystems (Smith et al. 1999, Smith 2003, Andersen et al. 2006, Smith et al. 2006). Using this definition, eutrophication is mostly concerned with the macronutrients (N and P) and the undesirable increases of phytoplankton biomass. Human activities have been one of the most important contributors to the increase in N and P loading to the coastal system. In the United States, N fertilizer consumption has a four-fold increase from the 1960s and phosphate (PO43-) fertilizer consumption also doubled (USDA 2008). As a result, N and P enrichment to the coastal ecosystem by human activities has been increasing (Howarth et al. 2002). One prominent example is that N fluxes have increased 10 to 15 fold or more in coastal areas in past few decades (Jaworski et al. 1997, Howarth 2008). 2 In the past several decades, there have also been global increases in harmful algal blooms (HABs) in frequency, intensity and geographic distribution. HABs have caused many ecological, economic and human health problems in coastal areas (Hallegraeff 1993, Smayda 1997, Anderson et al. 2002, Glibert et al. 2005a, Glibert et al. 2008a, Heisler et al. 2008). At least in part, the increases of HABs have been strongly correlated to the global increase in eutrophication level (Paerl 1997, Anderson et al. 2002, Anderson et al. 2008, Glibert et al. 2008a, Heisler et al. 2008). Eutrophication caused by the high terrestrial nutrients input is one of the most important mechanisms of HABs events (Hallegraeff 1993, Anderson et al. 2002, Smayda 2002, Glibert and Burkholder 2006). N:P Ratio and Nutrient Limitation Even though phytoplankton biomass has shown strong positive relationships with N and P enrichment all over the world (Anderson et al. 2002, Smith 2006, Anderson et al. 2008, Glibert et al. 2008a, Heisler et al. 2008), the linkages between bulk quantities of nutrients (N, P) and algal blooms are complex (Anderson et al. 2008, Glibert et al. 2008a). The ratios of these nutrients could be very critical to regulate phytoplankton communities (Smayda 1990, Hodgkiss and Ho 1997, Flynn 2002, Li et al. 2009b). There have been many studies on N:P ratios and their effect on the phytoplankton biomass (Radach et al. 1990, Smayda 1990, Riegman 1995, Burkholder and Glasgow 1997). N:P ratios have been used as an important determinant in the question of whether N or P is the more limiting nutrient. The Redfield ratio (N:P = 16:1, Redfield 1934, 1958) has been used successfully to define 3 the limiting nutrient in many ecosystems (Hecky et al. 1993, Doering et al. 1995, Justic et al. 1995, Koerselman and Meuleman 1996, Lee et al. 1996). However, N and P load in the coastal water is complicated and does not always keep a close stoichometric balance in most ecosystems. While the total N and P load to the coastal environments has increased in the past several decades, the increases of N and P to coastal ecosystems have not necessarily been proportionate. For example, while both N and P fertilizer consumption has increased in USA since the 1960s, N fertilizer consumption has increased over 4 fold , but P fertilizer consumption only increased 80% up to 2006 (USDA 2008). Actually, the consumption of P fertilizer reached its peak in 1979 and has slightly decreased after that year (USDA 2008). One of reasons is that the principal source of effluent P was from P in laundry detergent, and the use of P detergent has been gradually replaced by P-free detergent since the late 1970s (Doemel and Brooks 1975, Sharfstein et al. 1977, Hartig and Horvath 1982, Berthouex et al. 1983, Maki et al. 1984, Hoffman and Bishop 1994, Lee and Joneslee 1995). This unbalanced increase of N and P nutrients loading could change the N: P ratio in the coastal water and subsequently change the ecosystem structure. This shift could be from seasonal to decades. A shift in the N:P ratios in coastal waters could result in an alteration in dominant species in the phytoplankton community (Phillips and Tanabe 1989, Bulgakov and Levich 1999, Stelzer and Lamberti 2001, Lagus et al. 2004, Paerl et al. 2004, Vrede et al. 2009). For example, the phytoplankton community of downstream Neuse River, USA, shifted from an N-limited cyanobacteria dominated freshwater phytoplankton community to a P-limited community after the P detergent ban in 1988 in this area (Paerl et al. 2004). As a 4 result of P reduction, N substrates taken up by phytoplankton in the downstream decreased and more N was delivered to the lower estuary and stimulated HABs comprised of dinoflagellates, cryptomonads, and diatoms there (Paerl et al. 2004). N:P Ratio Regulating Dinoflagellate Blooms Changes in the N:P ratio in inorganic and organic forms were suggested to be important in the bloom succession in the Chanjiang River estuary and East China Sea (ECS, Li et al. 2009). The Changjiang River is the third longest river of the world. Annual freshwater input is 9.32?1011 m3, and annual input of N is 6.3?106 tonne (t) and P is 0.13?106 t (Shen et al. 2003), representing roughly a doubling of nutrient input in the past 20 years (Shen et al. 2003). While both N and P input by human usage have increased, the recent increases have been far greater in N. In 2005, China nitrogenous fertilizer production reached 36 ?106 t, including 20 ?106 t of urea fertilizer, a 5-fold increase since 1989 (Shen et al. 2003). The annual dissolved inorganic nitrogen (DIN) load to the coastal water from the Changjiang River has reached 1.4 ?106 t, which is higher than the Mississippi River and the Amazon River (Goolsby and Battaglin 2000, Duan et al. 2008). The total N off Changjiang River estuary has been shown to be about 70 to 110 ?M-N, and is mostly in the form of NO3--N (Shen et al. 2003). The total P is about 2 to 25 ?M-P, mostly as particulate P; PO43--P is only about 10% to 20% of the total P (Yan and Zhang 2003). As a result, the N:P ratio is typically > 100 near the river mouth (Chai et al. 2006, Zhang et al. 2007), which is much higher than the Redfield ratio and suggests P limitation. Large scale dinoflagellate blooms in these areas in late spring and early summer have been recorded in the past decade (Zhou et al. 2003). These more 5 frequent and persistent algal blooms are thought to result from increasing nutrient inputs to the ECS (Anderson et al. 2002, Zhou et al. 2003, Glibert et al. 2006). The dinoflagellate Prorocentrum donghaiense has been found to be the dominant species in this region since large-scale field studies began in 2000. During late spring and early summer of 2005, large scale (> 15,000 km2) mixed dinoflagellate blooms developed in the coastal ECS. Karenia mikimotoi was the dominant HAB species in the first stage of the bloom from late May and was succeeded by P. donghaiense approximately 2 weeks later. Samples were taken and experiments were conducted in the bloom region during 3 cruises of the Chinese Ecology and Oceanography of Harmful Algal Blooms (CEOHAB) Program during the bloom progression (Li et al. 2009). Experiment results supported that there was P limitation in these regions and suggested that the dominant bloom species and their temporal and spatial distribution were related to the ambient N:P ratios (Li et al. 2010). The uptake rates of N nutrients were also related to the ambient N:P ratios (Li et al. 2009). These results suggested ambient N:P ratio might be an important factor regulating the succession of dominant blooms species from K. mikimotoi to P. donghaiense. Ambient N:P ratio was also suggested to drive the phytoplankton community in the southwest Florida Shelf (Heil et al. 2007), where the spatial distribution of dinoflagellates (Karenia brevis and Prorocentrum minimum), cyanobacteria and diatom dominated communities reflected the variation of N:P ratios. Therefore, the tolerance and preference to certain N:P ratios might be an important regulator for the bloom expansion in these areas. Furthermore, a direct comparison of the ambient N:P ratios during both the Florida and East China Sea blooms shows considerable correspondence (Fig. 1-1). In 6 both systems, the diatom dominant community was found associated with a high ambient DIN:DIP ratio (> 45), and the Karenia spp. dominant communities were found associated with DIN:DIP ratios that were below the Redfield ratio (< 16). The P. donghaiense community in the ECS and the mixed dinoflagellate assemblage off Florida were both observed to be associated with DIN: DIP ratios that more closely approximated the Redfield ratio. This comparison suggested there might be interesting similar ambient N:P preference for similar species: P. donghaiense and P. minimum or K. mikimotoi and K. brevis, and different N:P ratio preference between Prorocentrum and Karenia spp. Light Dependence of Photosynthesis and Nutrient Uptake Light is a very important environmental parameter for the phytoplankton community. Light is the essential source for the phytoplankton photosynthesis. Light energy is utilized by the algae cell during the photosynthesis process, distributed to ATP storage, and produced reductants for carbon fixation and nutrient reduction. Photosynthesis is a highly integrated metabolic process which could be regulated by both light and other environmental factors including nutrients. There is always a periodic diel cycle in nature, and the photosynthesis variation on the diel cycle among different phytoplankton species could be wide (Behrenfeld et al. 2004). In coastal areas and adjacent oceans, the irradiance available to phytoplankton is varying over a wide range, due to possible high turbidity and/or high biomass in the water column. The vertical migration of many dinoflagellate species also allows cells to live in different depth where the light irradiance is different. In many cases, light is limiting factor of phytoplankton growth (Goldman et 7 al. 1979, Tett et al. 1985). Therefore, those bloom forming dinoflagellate species might not only dominate and bloom in their favorable nutrient condition (N:P ratio), but also have special strategy to dominate and bloom in their favorable light condition. Nitrogen uptake and assimilation are energy consuming process, and can be regulated by light factors. Uptake rates varys over diel cycles, and the maximum uptake rates occurred at noon for some species (Anderson and Roels 1981). The light dependence of nutrient uptake has been studied in both laboratory cultures and in field communities, and the response to light is varied among different species. Numerous studies have shown that uptake of nutrient responding to light are different among taxonomic groups (Eppley et al. 1971, Wheeler et al. 1983). For example, the diatom Skeletonema costatum has lower dark uptake rates of NO3- and NH4+, while the coccolithophore Emiliania huxleyi was more uniform over a night range (Eppley et al. 1971). The light dependence of N uptake rates also varies for different nutrients. The ability of some cyanobacteria to uptake NH4+, a reduced N source, was relatively unaffected by darkness (Mulholland et al. 1999). The NO3- uptake rates appeared to be more limited at night than uptake of NH4+ (Cochlan et al. 1991). NO3- needs to be reduced before it can be assimilated by the algae, and it is an energy consuming process. NO3- uptake generally shows stronger light dependence than reduced N sources (NH4+ etc.) (Cochlan et al. 1991). However, for dinoflagellates, the light dependence of NO3- uptake still varied. P. minimum can continuously take up NO3- in the dark; while K. mikimotoi has been found to show almost no NO3- uptake in the 8 dark (Paasche et al. 1984). The light dependence might be weakened with increasing nutrient limitation (Healey 1977). Irradiance-induced variation in nutrient uptake can alter the competitive abilities of species: those capable of maintaining high uptake in the dark may have a competitive advantage in light limiting environment. The different nutrient uptake rates in the dark might be an ecological strategy. If a species can continuously take up and assimilate N at high rates in darkness, it may be advantageous in low ambient light. The bloom regions of ECS, west Florida Shelf and Chesapeake Bay area are under strong interaction of relative turbid terrestrial runoff and clear sea water, during a HAB progression, the available light is limited by both turbidity and biomass, therefore, the strategy of bloom forming dinoflagellates in different light condition is also very important to be considered for bloom development. Perspective of N:P ratio and Light - the Matrix Smayda has suggested dinoflagellates have diverse habitat preferences and adaptive strategies and characterized the dinoflagellate bloom species in coastal areas as 5 types which fit to a nutrient-turbulence matrix (Smayda and Reynolds 2001, Smayda 2002). Each dinoflagellate species has its specific niche for the bloom expansion, and these niches are described by available nutrients and turbulence/mixing intensity (Fig. 1-2). Based on Smayda?s model, Prorocentrum spp. (length 15~22 ?m width 9~14 ?m) are described as Type II species which are small, rapidly growing competitors blooming in nutrient rich near-shore regions. Conversely, Karenia (length 18-37 ?m, width 14-35 ?m) are relatively slower 9 growing, larger, motile Type IV, V, VI species, which are generally considered to be off-shore species. These 2 groups of algae have different adaptive strategies to the ambient environment and have their own niches, described by different nutrient and turbulence preferences. The ambient N:P ratio has been suggested to be an important nutrient parameter regulating the Prorocentrum and Karenia spp. bloom progression (Li et al. 2009, Li et al. 2010). Ambient N:P ratio could the key factor which make the niches of these two type of algae different, as distinct N:P ratio variation distributed between the near-shore and off-shore water in the high frequent bloom area of these species (Heil et al. 2007, Li et al. 2009, Li et al. 2010). Moreover, near- shore regions are generally considered higher turbidity and less light available for the phytoplankton than clearer off-shore water. The light strategy of these 2 types of algae might also be different. The physiology differences of these 2 types of algae might determine their success in water column with different ambient N:P ratios and light condition. Among these 2 types of algae, K. mikimotoi bloomed with P. donghaiense in the ECS (Li et al. 2009), K. brevis bloomed with P. minimum in the southwest Florida coast (Heil et al. 2007), and Karlodinium veneficum bloomed with P. minimum in the Chesapeake Bay (Zhang et al. 2008). Either temporal or spatial gradient existed when two bloom species developed the blooms. The range of N:P ratios for bloom expansion was different between Prorocentrum spp. and other species. The dependence of light might also be different among these species. Therefore, the range of N:P ratio and light irradiance preferred by each dinoflagellate group could be critical for bloom development. In this dissertation, the 10 research is focused on, but not limited to, P. minimum and K. veneficum in Chesapeake Bay area. Historical observational data were analyzed to obtain the linkage between bloom-forming dinoflagellates and their ambient N:P ratios and light condition. Laboratory experiments were conducted to test physiological characteristics at specific ambient N:P ratios and light condition. Hypothesis The following hypotheses were tested. ? In Chesapeake Bay, P. minimum develops blooms in relative high DIN:DIP ratio water; while K. veneficum blooms near or lower than the Redfield ratio. ? These species will grow faster in the N:P ratio in which they develop blooms, even when these nutrients are not at limiting levels. ? Prorocentrum spp. preferentially take up more DIN in high DIN:DIP ratio water, while Karlodinium can better use other source of N in the low DIN water (e.g. DON, particulate N). ? Low-light-adapted nitrogen acquisition by Prorocentrum spp. serves as an adaptive advantage to grow in low light waters Research Objectives Historical observational data were analyzed and laboratory experiments were conducted to test the hypotheses and accomplish the following objectives: 11 1. Undertake a comprehensive analysis on decadal observational data of major dinoflagellate bloom species and cyanobacteria with the changes of ambient nutrient parameters (i.e., ambient N:P ratio) and related physical variables (i.e., light) in the Chesapeake Bay. (Chapter II) 2. Identify the quantitative relationship between growth and physiological characteristics of selected dinoflagellate, Prorocentrum minimum and P. donghaiense, with ambient N:P ratio in laboratory experiments; identify related parameters (growth rate, N uptake rate, etc.), and compare the results between these two species. (Chapter III) 3. Evaluate the importance of light on the nitrogen uptake and growth of Prorocentrum minimum and P. donghaiense and compare the difference. (Chapter IV) 4. Simulate the interspecies competition between three near-shore harmful dinoflagellates, Prorocentrum minimum, P. donghaiense and Karlodinium veneficum, and one off-shore dinoflagellate, Karenia brevis, at different ambient N:P ratio and light condition. (Chapter V) 5. Synthesize the findings in an overall conceptual framework, leading to suggestions to further study. (Chapter VI) The experimental results were combined with the long term observational environmental data with statistical and mathematical approaches to understand the ambient N:P ratio and light regulation on these harmful algal bloom dynamics. 12 References Andersen JH, Schluter L, ?rtebjerg G (2006) Coastal eutrophication: recent developments in definitions and implications for monitoring strategies. 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Journal Water Pollution Control Federation 49:2017-2021 Shen ZL, Liu Q, Zhang SM, Miao H, Zhang P (2003) A nitrogen budget of the Changjiang River catchment. Ambio 32:65-69 Smayda T (1990) Novel and nuisance phytoplankton blooms in the sea: Evidence for a global epidemic. In: Gran?li E, Sundstrom B, Edler L, Anderson DM (eds) Toxic Marine Phytoplankton. Elsevier, New York 17 Smayda TJ (1997) Harmful algal blooms: Their ecophysiology and general relevance to phytoplankton blooms in the sea. Limnology and Oceanography 42:1137- 1153 Smayda TJ (2002) Adaptive ecology, growth strategies and the global bloom expansion of dinoflagellates. Journal of Oceanography 58:281-294 Smayda TJ, Reynolds CS (2001) Community assembly in marine phytoplankton: application of recent models to harmful dinoflagellate blooms. Journal of Plankton Research 23:447-461 Smith VH (2003) Eutrophication of freshwater and coastal marine ecosystems - A global problem. Environmental Science and Pollution Research 10:126-139 Smith VH (2006) Responses of estuarine and coastal marine phytoplankton to nitrogen and phosphorus enrichment. Limnology and Oceanography 51:377- 384 Smith VH, Joye SB, Howarth RW (2006) Eutrophication of freshwater and marine ecosystems. Limnology and Oceanography 51:351-355 Smith VH, Tilman GD, Nekola JC (1999) Eutrophication: impacts of excess nutrient inputs on freshwater, marine, and terrestrial ecosystems. Environmental Pollution 100:179-196 Stelzer RS, Lamberti GA (2001) Effects of N : P ratio and total nutrient concentration on stream periphyton community structure, biomass, and elemental composition. Limnology and Oceanography 46:356-367 Tett P, Heaney SI, Droop MR (1985) The Redfield ratio and phytoplankton growth- rate. Journal of the Marine Biological Association of the United Kingdom 65:487-504 USDA (2008) Fertilizer consumption and use - By year. United States Department of Agriculture: http://www.ers.usda.gov/Data/FertilizerUse/ Vrede T, Ballantyne A, Mille-Lindblom C, Algesten G, Gudasz C, Lindahl S, Brunberg AK (2009) Effects of N:P loading ratios on phytoplankton 18 community composition, primary production and N fixation in a eutrophic lake. Freshwater Biology 54:331-344 Wheeler PA, Olson RJ, Chisholm SW (1983) Effects of photocycles and periodic ammonium supply on 3 marine-phytoplankton species .2. ammonium uptake and assimilation. Journal of Phycology 19:528-533 Yan WJ, Zhang S (2003) The composition and bioavailability of phosphorus transport through the Changjiang (Yangtze) River during the 1998 flood. Biogeochemistry 65:179-194 Zhang H, Litaker W, Vandersea MW, Tester P, Lin SJ (2008) Geographic distribution of Karlodinium veneficum in the US east coast as detected by ITS-ferredoxin real-time PCR assay. Journal of Plankton Research 30:905-922 Zhang J, Liu SM, Ren JL, Wu Y, Zhang GL (2007) Nutrient gradients from the eutrophic Changjiang (Yangtze River) Estuary to the oligotrophic Kuroshio waters and re-evaluation of budgets for the East China Sea Shelf. Progress in Oceanography 74:449-478 19 Figures Figure 1-1 Comparison of the range in ambient nitrogen: phosphorus ratios during different phases of the blooms in the East China Sea and in different phytoplankton assemblages among the southwest Florida shelf during May 2005. The southwest Florida Shelf data are derived from Heil et al. (2007). 20 Figure 1-2 Predominant dinoflagellate life-form Types associated with the turbulence-nutrient matrix along an onshore?offshore continuum characterizing pelagic habitats (From Smayda 2001). Type I = gymnodinioids; Type II = peridinioids and prorocentroids; Type III = ceratians; Type IV = frontal zone species; Type V = upwelling relaxation taxa; Type VI = coastal current entrained taxa; Type VII = dinophysoids; Type VIII = tropical oceanic flora; Type IV = tropical shade flora. Consult text for Type species. 21 Chapter 2: An Exploratory Analysis of Nutrients and Other Related Variables Related to Select Harmful Algal Bloom Events in the Chesapeake Bay 1991-2008 Abstract An exploratory analysis was conducted relating Chesapeake Bay nutrient concentrations and other related water quality variables to the harmful algal bloom events of select species: the dinoflagellates Prorocentrum minimum, Karlodinium veneficum, and a composite density of freshwater cyanobacteria species. Chesapeake Bay, especially the upper bay and its tributaries, is characterized as a turbid, low light available, highly eutrophic system. Prorocentrum minimum, K. veneficum and cyanobacteria blooms can further decreased the light availability, enrich particle organic matter to the water column and carry the potential to be toxic. Prorocentrum minimum and K. veneficum generally develop blooms in the mesohaline religions, while freshwater cyanobacteria mostly bloomed in the tidal freshwater or oligohaline endmembers of tributaries. In contrast to P. minimum concentrations that correlates with high nitrogen (N) water, K. veneficum is associated with high phosphate (P) concentrations and low DIN:DIP ratio water in the summer. K. veneficum may be less limited by P in the low DIN:DIP ratio water after P. minimum blooms. The nutrients ratios during dinoflagellates events were comparable to those previous reported for analogous blooms in the East China Sea and southwest Florida Shelf. 22 Introduction Chesapeake Bay is the largest estuarine system of the USA. Chesapeake Bay extends over 300 km from the Susquehanna River at its north head, to the Atlantic Ocean at its mouth, with an average depth of 8 m. The majority of freshwater flow (64%) and nutrient-loading of Chesapeake Bay is from the Susquehanna River (Boynton et al. 1995, Kemp et al. 2005), while there are several major tributaries on both sides of the Chesapeake Bay, including the Patuxent River, Potomac River, Rappahannock River and Choptank River. Extensive studies of nutrient loading and its processing by primary producers have been conducted on the Chesapeake Bay ecosystem (Fisher et al. 1990, Fisher et al. 1992, Boynton et al. 1995, Glibert et al. 1995, Cornwell et al. 1996, Malone et al. 1996, Harding and Perry 1997, Glibert et al. 2001, Glibert and Magnien 2004, Glibert et al. 2005b, Fisher et al. 2006). Massive terrestrial loading results in excessive phytoplankton production in the Chesapeake Bay (Malone et al. 1988, Malone et al. 1996, Glibert and Magnien 2004, Adolf et al. 2006c, Fisher et al. 2006). Generally, the peak biomass of Chesapeake Bay occurs during the spring diatom blooms, while peak production occurs in the summer when summer temperature reaches its maximum (Malone et al. 1988, Malone et al. 1996, Harding et al. 2002). Even though there is a large amount of nutrient loading into the Chesapeake Bay, phosphorus (P) and silica still tend to be limiting nutrients in spring, while nitrogen (N) is the primary limiting nutrient in summer (Fisher et al. 1992, Glibert et al. 1995, Malone et al. 1996, Fisher et al. 1999). The Chesapeake Bay region has been subject to eutrophication which is linked to the pressures of increasing human population, urbanization (e.g. Washington DC, 23 Baltimore area), development of animal and plant agriculture and non-point nutrient pollution in its watershed (Glibert and Magnien 2004, Glibert et al. 2005b, Hagy et al. 2004, Kemp et al. 2005, Fisher et al. 2006). As a consequence of this eutrophication, Chesapeake Bay has suffered from major harmful algal bloom problems for decades (Glibert et al. 2001, Goshorn et al. 2004, Marshall et al. 2004, Tango et al. 2004, Tango et al. 2005, Tango and Butler 2008). In the Chesapeake Bay, harmful algal blooms have caused environmental damage including hypoxia, mortalities of fish and shellfish, decline of submerged aquatic vegetation, illness and death of various invertebrates, seabirds, and marine mammals (Deeds et al. 2002, Gallegos and Bergstrom 2005, Heil et al. 2005, Tango et al. 2005, Adolf et al. 2006a, Tango and Butler 2008). Prorocentrum minimum is one of the major bloom forming harmful dinoflagellates, both globally distributed in coastal waters (Heil et al. 2005, Glibert et al. 2008b) and common in Chesapeake Bay (Glibert et al. 2001, Tango et al. 2005). Karlodinium veneficum, formerly recorded as Gyrodinium galatheanum, Gymnodinium galatheanum and Karlodinium micrum (Li et al. 2000, Adolf et al. 2006b, Deeds 2009), is also a widespread mixotrophic dinoflagellate which has caused HABs since 1950 in coastal waters of Southwest Africa, Europe, United States, Western Australia, and other temperate coastal environments, including Chesapeake Bay (Li et al. 2000, Zhang et al. 2008, Deeds 2009). Karlodinium veneficum is a toxigenic dinoflagellate, which can produce Karlotoxin and has been implicated to cause fish-kill events in the Chesapeake Bay area (Deeds et al. 2006, 24 Adolf et al. 2008). High biomass blooms of K. veneficum have been recorded in middle and upper Chesapeake Bay between May and September (Li et al. 2000). Some toxic species of cyanobacteria, including Microcystis spp. and Anabaena spp., have also been reported to cause cyanobacterial HAB events in the Chesapeake Bay area (Marshall et al. 2005). Diverse toxic activity of cyanobacteria has been recorded in the tidal waters of Chesapeake Bay and has caused environmental problems in recent years (Tango and Butler 2008). In this study, the occurrences of these two major bloom developing algae (dinoflagellates P. minimum, K. veneficum) and a composite density of freshwater cyanobacteria in Chesapeake Bay regions are described in terms of their relationship with eutrophication indicators. This study provided interpretations of the relationships between these HAB species and water quality and enabled consideration of questions such as: ? did the scale and frequency of HAB species in the Chesapeake Bay estuary change over time? ? how were biological changes related to water quality? ? what water quality conditions best explain the success of HAB species that developed blooms? By addressing these questions, this study is helpful in gaining a better understanding of the complex mechanisms that influence the development of HABs in eutrophic ecosystem. 25 Methods Long-term observational data on the surface dinoflagellate and cyanobacteria abundance and water quality were acquired from the Chesapeake Bay Program (http://www.chesapeakebay. net). These data were derived from routinely sampling in the Chesapeake Bay and its tributaries for water chemistry and phytoplankton community composition since the mid-1980s (http://www.chesapeakebay.net/data_waterquality.aspx, Fig. 2-1). The dinoflagellate data were derived from phytoplankton samples which were preserved with Lugol?s, and subsequently counted by conventional light microscopy techniques. The cyanobacteria data were also derived from phytoplankton samples which were preserved with Lugol?s, and subsequently counted by conventional light microscopy techniques. Picocyanobacteria (e.g. Synechococcus), which are generally smaller than the resolution limit of light microscopy, were not identified or counted, and are not involved in this anaylsis. Water quality and chemical analysis included ambient parameters (salinity, temperature, secchi depth, particulate and dissolved nitrogen/ phosphorus and carbon) and biological variables (chlorophyll). Data are available from samples which were conducted from biweekly to monthly. Results Eutrophic Level and Bloom Events From 1991 to 2008, there were ~ 2830 records of P. minimum and ~ 4755 records of cyanobacteria sampled in the Chesapeake Bay and its tributaries. However, 26 the records of K. veneficum (~ 4755 records) were only available from 2003 in the available Chesapeake Bay Program data sets. Each species was either documented as mixed with other species or as population dominant in bloom conditions. Cell densities varied across wide range. According to United States National Estuarine Eutrophication Assessment (Bricker et al. 1999), Chlorophyll (Chl) a can be used as an indicator of algal blooms to define estuarine eutrophic condition. The thresholds and ranges are defined as: hypereutrophic: > 60 ?g Chl a L-1; high eutrophic: > 20 but ? 60 ?g Chl a L-1; medium eutrophic: > 5 but ? 20 ?g Chl a L-1; low eutrophic: > 0 but ? 5 ?g Chl a L-1. Of the Chesapeake Bay records, over half of the samples (3350) were medium eutrophic when P. minimum, K. veneficum and cyanobacteria were observed. Over 1000 samples were in hyper and high eutrophic condition (Fig. 2-2 a). For this analysis, the bloom status of the two dinoflagellate species in the water column was defined according to Tango et al (2005) and Maryland Eco-check (http://www.eco-check.org/), and the thresholds are set as: Type I: bloom condition, cell density > 5,000,000 cell L-1; Type II, sub-bloom condition, cell density > 1,000,000 but ? 5,000,000 cell L-1, dinoflagellates could be dominant species to develop bloom, but have not reached bloom condition; Type III, cell density >1,000 but ? 1,000,000 cell L-1; Type IV, cell density > 0 but ? 1,000 cell L-1; Type V, cell density = 0, dinoflagellates were not observed at this station. Similar types were defined on cyanobacteria, however, the thresholds of each type are 10 times of dinoflagellates, as cyanobacteria have much smaller cell size (e.g. Microcystis aeruginosa cell size 2-3 ?m; P. minimum 14-22 ?m). 27 A total of 48 P. minimum Type I samples were recorded, with a maximum cell density of 60,915,000 cell L-1, in the lower Potomac River database (Fig. 2-2b). There were 133 Type II samples. Most of the records of P. minimum (2157) were Type III level. Cell abundance data of K. veneficum were only available from 2003 in the available Chesapeake Bay Program data sets. Since 2003, there were 10 Type I records of K. veneficum and 38 Type II records, with a maximum cell density of ~16,000,000 cell L-1 in the lower Patuxent River (Fig. 2-2c). There were 196 Type III samples recorded. For the composite density of freshwater cyanobacteria, there were 329 Type I samples recorded, with a maximum cell density of 632,000,000 cell L-1 in the upper Sassafras River (Fig. 2-2d). There were 717 Type II records. Over 70% of the cyanobacteria records (3521) were Type III level. Spatial Distribution Hyper and high eutrophic level Chl a samples were documented all over the Chesapeake Bay (Fig. 2-3). However, most of these observations were from in the upper part of the Chesapeake Bay and major tributaries. The Type I and II levels of P. minimum have been also recorded throughout the Chesapeake Bay (Fig. 2-4), but especially in the lower Potomac River, Patuxent River, Choptank River and the upper Bay. Karlodinium veneficum were only recorded to have reached Type I and II level in upper Chesapeake Bay and lower Potomac River and Patuxent River (Fig. 2-5). High biomass of the cyanobacteria considered here were mostly found in the upper river of major tributaries (Fig. 2-6). 28 Salinity Approximately 80% of hyper and high eutrophic samples had salinities less than 10, which represents the tidal freshwater and oligohaline regions of Chesapeake Bay (Fig. 2-7). Over 50% of the Type I and II P. minimum samples were collected in a narrow range of salinity of 6 - 10. Prorocentrum minimum cell densities decreased to Type IV as salinity increased to 20. Over 50% of the Type I and II K. veneficum samples were collected in a narrow range of salinity of 7.5 - 11. Of the K. veneficum samples, 80% were observed in the salinity range of 5 - 13. 90% Type I and 75% Type II levels of the cyanobacteria were recorded in salinity less than 10. There was significant difference between the salinity where the dinoflagellate and the cyanobacteria blooms formed (p < 0.001) (Fig. 2-8). More than 80% of the P. minimum blooms (Type I) were developed in salinities of 5.5 and 11, while 80% of the K. veneficum blooms developed in salinities between 7 and 12.7, and 75% of the cyanobacterial blooms developed at stations where salinities were lower than 5.5. Dinoflagellate blooms developed in the tidal mixing area, generally in the lower part of the tributaries (Fig. 2-4 and 5), while cyanobacteria blooms developed in the non- tidal and tidal freshwater area, generally in the upper part of the tributaries (Fig. 2-6). Seasonal Records and Temperature More than 50% of the hyper, high and medium eutrophic samples were collected between May to August, and about 80% of these samples were collected between April and October (Fig. 2-9). More than 50% of the hyper, high and medium eutrophic conditions occurred when water temperature was in the range of 14.2 ?C to 26.6 ?C with a median over 21 ?C (Fig. 2-10). Levels of Chl a dropped to Type IV 29 conditions when median temperatures were lower than 15 ?C. Most of the Type I and II P. minimum samples were recorded in April and May, when water temperatures were in the range of 13.7 ?C to 19.1 ?C. Karlodinium veneficum reached Type II levels mostly in May, June and July, when temperatures were higher than 17.1?C. However, 50% of the K. veneficum blooms developed in July, August and September. Blooms developed when temperature were between 21 ?C and 29.5 ?C. Cyanobacteria also bloomed mostly in July, August and September when water temperatures were higher than 20 ?C. Cyanobacteria reached Type II level from May, when water temperatures were higher than 15 ?C. More than 80% of the P. minimum blooms developed when temperatures were between 12 ?C and 21.4 ?C. 80% of the K. veneficum blooms developed in the temperature range between 20.7 ?C and 30 ?C. The water temperature ranges of the two dinoflagellates blooms were significantly different (p < 0.001). Therefore, even though these two dinoflagellates typically bloom in similar area in the Chesapeake Bay (Fig. 2-4 and 5), these two species bloomed at different time of the year. Prorocentrum minimum blooms started from late spring, while K. veneficum bloomed in summer and autumn. Secchi Depths and TSS The median secchi depths in the hyper and high eutrophic were less than 0.5 m (Fig. 2-11), which is lower than the thresholds (0.65 m) for underwater sea grass growing in oligohaline and tidal freshwater of Chesapeake Bay area (Carter et al. 1994). Even though freshwater carries massive sediments and particles to the bay and its tributaries, secchi depths in low eutrophic stations were still less than 1.5 m. For P. 30 minimum, K. veneficum and cyanobacteria, the median secchi depths decreased from over 1 m to less than 0.5 m as the cell densities increased to bloom level (Fig. 2-11). The median total suspended solids (TSS) increased from 10 mg L-1 in low and medium eutrophic stations to about 26 mg L-1 in high and hyper eutrophic stations (Fig. 2-12). TSS values were mostly around than 10 mg L-1, when the two dinoflagellates were observed, except during P. minimum blooms, when the median TSS was about 18 mg L-1. The median TSS also increased from 12 mg L-1 in stations with Type IV cyanobacteria level to 22 mg L-1 in stations with Type I cyanobacteria level. Dissolved Oxygen Dissolved oxygen in surface water was replete in most of the reported data. Over 80% of the observations had DO higher than 6 mg L-1, and the median was over 8 mg L-1 (Fig. 2-13). This is likely due to the fact that all the samples were taken from less than 0.5 m and wind influenced air-water exchange and the photosynthesis activities kept the surface DO at a high level. All observations were also made during the midday. The hyper eutrophic samples had even higher DO, as the results of high photosynthesis activities of the blooming algae. When P. minimum was present, the median DO was higher than 8 mg L-1. However, the median DO decreased to 5.5 mg L-1, when K. veneficum bloomed. The median DO in Type I and II cyanobacteria- dominated stations was about 7.5 mg L-1. 31 Dissolved Carbon and Nutrients The median of dissolved organic carbon (DOC) in hyper and high eutrophic samples was about 400 ?M-C (Fig. 2-14), and 50% of the samples were in the range of 310 - 450 ?M-C, while the median of the low eutrophic samples decreased to 250 ?M-C. The DOC concentration decreased from 400 ?M-C to 230 ?M-C as cell densities of P. minimum and cyanobacteria species decreased from Type I to Type IV level, but this decrease was not significant (p>0.05). The DOC concentrations of K. veneficum were steady in a narrow range around 320 ?M-C. Dissolved inorganic nitrogen (DIN, sum of NO2-+ NO3- and NH4+) concentrations were higher in low eutrophic stations. The median was about 36 ?M-N in low eutrophic water, and 50% of the samples were in the range of 5 - 70 ?M-N (Fig. 2-15). DIN concentrations were lower in the samples characterized as more eutrophic; more DIN was incorporated into biomass. The median decreased to 10-16 ?M-N in medium and high eutrophic water, and 75% of the samples were less than 40 ?M-N. When P. minimum was observed, the median DIN concentrations decreased from ~ 20 ?M-N in Type I and II level to 10 ?M-N in Type III level, then further decreased to less than 5 ?M-N in Type IV level samples, which was significantly lower than the Type VI condition (p<0.001). However, an opposite trend was observed as K. veneficum cell densities increased, NO3- concentrations also increased. The median DIN concentration increased from less than 5 ?M in Type I and II level samples to 10 ?M-N in Type III, and continuously increased to over 40 ?M in Type IV level, which was significantly higher than the Type V condition 32 (p<0.001). There was no significant difference in change of DIN when cyanobacteria cell densities changed. Dissolved inorganic phosphorus (DIP, ~PO43-) concentrations did not show a significant trend among stations characterized as different eutrophic levels. About 70% of the samples had DIP concentrations less than 0.5 ?M-P (Fig. 2-16). The median concentration of hyper eutrophic stations was 0.4 ?M-P, similar to the median of low eutrophic stations. DIP concentrations were around 0.1 ?M-P in Type I and II P. minimum samples. In the K. veneficum samples, the median DIP concentrations were also in the range of 0.1 - 0.2 ?M-P. The DIP concentrations were higher when cyanobacterial bloom developed. The median values were about 0.3 - 0.4 ?M-P in the Type I, II and III samples, which were significantly higher than the values of Type V samples (0.2 ?M-P, p<0.05). Both P. minimum and cyanobacteria had significantly higher ambient DIN concentrations than K. veneficum when blooms occurred (p<0.05). However, both K. veneficum and cyanobacteria had significantly higher ambient DIP concentrations than P. minimum when blooms occurred (p<0.05). The DIN:DIP ratios of all eutrophic level samples were mostly higher than the Redfield ratio (16), and the median values were in the range 40 to 60. The DIN:DIP ratios varied in a much larger range for the dinoflagellate-dominated samples (Fig. 2- 17). The median values of Type I and Type II P. minimum samples were ~170, and 50% of DIN:DIP ratios were in the range of 10 - 370. The median value of Type III samples decreased to ~50, and 50% of DIN:DIP ratios were in the range of 10 - 180. The median value of Type IV samples decreased to around 16. An opposite trend was 33 observed in K. veneficum, as K. veneficum cell densities increased, DIN:DIP ratios decreased to the Redfield ratio instead. The DIN:DIP ratios at which P. minimum blooms were documented were significantly higher than those at which K. veneficum and cyanobacteria blooms were documented (p<0.001), which had median DIN:DIP values around the Redfield ratio. The median DIN:DIP ratio was about 16 and all the values were under 110 in bloom condition. The median DIN:DIP ratios slightly increased to 20-30 in Type II and III sample, and the upper range of 50% values increased to 200. The median DIN:DIP ratio increased to 360 in Type IV sample, and 50% of the values were in the range of 160 - 500. When cyanobacteria were observed, the DIN:DIP ratios were relatively stable with median value around 16 and 75% of the ratios were less than 70 in all types of samples. Components of Particulate Matter Particulate carbon (PC) in the water samples increased with the eutrophic level. The median PC concentrations significantly increased from 65 ?M-C under low eutrophic conditions to 380 ?M-C (P<0.001) under hyper eutrophic conditions (Fig. 2-19). The values varied in a narrow range at each defined eutrophic level. Similar trends were observed for all 3 target species or species groups. Particulate nitrogen (PN) also increased with the eutrophic level. The median PN concentrations increased from 12 ?M-N under low eutrophic conations to 24 ?M-N under hyper eutrophic conditions (Fig. 2-20). There were no significantly different changes of PN for the HABs, considered here. Particulate phosphorus (PP) also increased with the eutrophic level. The median PP concentrations increased from 1.4 ?M-P to 2.7 ?M-P (Fig. 2- 34 21). However, the increase of PP in relation to cell density for each species or species groups was not significant. The PC:PN ratios of the particulate matter increased from near Redfield ratio (6.6) in middle and low eutrophic observations to a median value of 15 in hyper eutrophic observations (Fig. 2-22). The PC:PP values increased to over 200 in hyper eutrophic samples (Fig. 2-23). A similar trend was observed for P. minimum. When this species bloomed, the median of PC:PN increased to 20. Median PC:PP ratios also increased to over 200, when P. minimum and K. veneficum bloomed. The PC:PP ratios of the particulate matter were generally around 50 in low eutrophic water which was lower than Redfield ratio (106). The PN:PP ratios were in similar range in all eutrophic level samples. Most of the samples had ratios lower than Redfield ratio (16) (Fig. 2-24). The median number of PN:PP were only higher than 16 in Type I, II and III K. veneficum samples. Discussion A Comparison with Ecosystems with Similar Nutrient Dynamics In a healthy Chesapeake Bay ecosystem, the most important event for the primary production of the bay is the spring diatom bloom, when the N nutrients accumulate during the winter and spring are consumed to support the production (Malone et al. 1988, Glibert et al. 1995, Malone et al. 1996, Harding et al. 2002). Due to the extensive terrestrial nutrient input in the past several decades (Glibert and Magnien 2004, Hagy et al. 2004, Kemp et al. 2005), the upper bay and its tributaries has been in high eutrophic condition indicated by Chl a level. Not only nutrient 35 concentrations, but also the nutrients ratios could be important for the succession of phytoplankton assemblages (Smayda 1997, Bulgakov and Levich 1999, Stelzer and Lamberti 2001, Vrede et al. 2009). Surveys conducted on the nutrients and phytoplankton in the East China Sea (Li et al. 2009) and southwest Florida Shelf (Heil et al. 2007) have suggested that ambient nutrients ratios may regulate the dominant species of the phytoplankton community. In both systems, the diatom dominated community was found associated the highest ambient DIN:DIP ratios, and the Prorocentrum spp. (P. donghaiense in the East China Sea and P. minimum in southwest Florida Shelf) dominant communities were associated with DIN:DIP ratios approximated higher than, but still close to, Redfield ratio. The Karenia spp. (K. mikimotoi in the East China Sea and K. minimum in southwest Florida Shelf) dominant communities were found in association with DIN:DIP ratio less than Redfield ratio. Remarkable correspondence of community succession was also observed in the Chesapeake Bay (Fig. 2-25). Diatoms bloomed in the spring when the ambient DIN:DIP ratios were the highest, followed by the P. minimum blooms when the ambient DIN:DIP ratios began to decrease. K. veneficum bloomed in the late summer when ambient DIN:DIP ratios decreased to the Redfield ratio. All three areas appeared to display similar patterns in the species dominance relative to DIN:DIP ratios, except the DIN:DIP ratios during the diatom and P. minimum blooms were higher in the Chesapeake Bay, which was due to the relatively higher DIN concentrations (compared to the southwest Florida Shelf) and lower PO43- concentrations (compared to the East China Sea) in the Chesapeake Bay. 36 A Comparison with Spatial Historical Data Fisher et al. (1992) presented the evidence of seasonal shifts from P to N as the limiting nutrient of phytoplankton biomass based on the data collected in the major axis of the Chesapeake Bay from 1982 to1987. Although the major axis is not a high frequent HAB area compared to the near-shore and tributary areas, there were still data collected along the stations in the same month along the major axis which were associated with the HAB species in the 1990s and 2000s. The decadal variation of salinity, nutrient and chlorophyll data from the 1980s (Fisher et al. 1992 ) were directly compared with the data in the 1990s and 2000s, along the major axis of the Chesapeake Bay. The seasonal variation of salinity is significant. The fresh water river flow in the Chesapeake Bay is dominated by the Susquehanna River. A salinity gradient developed from the Susquehanna River mouth to the lower Chesapeake Bay. The river flow in the late spring (May) is higher than late summer (August), and as a result, the surface salinities in May are 3-5 lower than August. The salinity distribution was almost the same over the past 3 decades, except the samples from the 2000s (Fig. 2-26), when the salinities of 2000-2008 in May and August dropped about 10 at stations near the Potomac River mouth. The samples were likely taken after large scale of rainfalls in the nearby watershed, which caused the dilution. River flow from the major tributaries on both sides of the Chesapeake Bay also had significantly dilution effects on the major axis. The dilution impact on the water column was not only on salinity, but also on the water quality which is discussed below. 37 The seasonal variation of DIN concentrations along the major axis of the Chesapeake Bay is also significant (p < 0.05) and well documented (Fisher 1992). In May, the DIN is highest near the head of the Chesapeake Bay (60-100 ?M-N) and gradually decreases to 10 ?M-N at the mouth of the bay (Fig. 2-27). In August, DIN decreases to 10 ?M-N from 80 km away from the head of the bay, and is depleted throughout the bay. However, decadal comparisons suggest that the DIN concentrations in the upper river have increased, as previous study suggested (Hagy et al. 2004, Kemp et al. 2005), which increased almost 20 ?M-N every decade in May (Fig. 2-27). In August, the DIN concentrations were 30-50 ?M-N higher in recent decades than 1980s. DIN concentration peaks match with the low salinity stations in the 2000s data. The N input from the major tributaries on both sides of the Chesapeake Bay could be important complement for the main channel. The PO43- concentrations were slightly higher in the August than in the May. Near the head of the Chesapeake Bay, the PO43- concentrations reached 0.7 ?M-P in August, but only 0.4 ?M in May. In the lower part of the Chesapeake Bay, the PO43- concentrations were around 0.2 ?M-P in August, but only slightly higher than 0.1 ?M-P in May (Fig. 2-28). There was no significant difference among PO43- concentrations in three decades (p > 0.05), except near the head of the Chesapeake Bay, where the peak value of PO43- concentrations increased since 1980s. Due to the different pattern of the DIN and PO43- concentrations, the seasonal ambient DIN:DIP ratio variation is significant (Fig. 2-29). The DIN:DIP ratios are mostly higher than 100 in May, but lower than 100 in August. The ambient DIN:DIP ratios from the 1990s were significantly higher than those of 1980s (p < 0.01) in 38 August. In May, the DIN:DIP ratios only decreased to near Redfield ratio in the lower bay in the 1980s. In August, most of the DIN:DIP ratios were low all three decades, except the first 50 km near the head of the Chesapeake Bay. The DIN:DIP ratios decreased to less than 10 and even less than 1 in the lower bay in the 1980s. However, the DIN:DIP ratios in the past 2 decades only decreased to near Redfield ratio, not as low as the 1980s. In May, the Chl a concentrations were highest in the 1980s, when Chl a peaks (~40 ?g L-1) were observed in the stations about 40 km to the head of the bay and near the mouth of the bay (Fig. 2-30). However, the Chl a concentrations were generally lower than 40 ?g L-1 in the recent two decades. In August, high Chl a concentrations were only observed in the upper bay, and there was no significant difference among the three decades. As shown here and elsewhere (Hagy et al. 2004, Kemp et al. 2005), the DIN concentrations have increased since 1980s, especially in the upper bay. As a result, the overall ambient DIN:DIP ratios increased, which suggests that Chesapeake Bay might be more P limited in the later spring and early summer than the 1980s. The spring blooms depleted DIN (<1 ?M-N) in summer in the 1980s, however, in recent decades, DIN in the upper and middle bay remains high (20-50 ?M-N). High PO43- concentrations were also observed in the upper bay. These PO43- could be supplied by P released from the hypoxia zone which has been increased in recent decades (Kemp et al. 2005). Therefore, historical data comparison suggested the Chesapeake Bay is more eutrophic now than the 1980s in the upper and middle bay (Hagy et al. 2004, Kemp et al. 2005), which could provide more nutrients sources available for dinoflagellate species to develop blooms in the summer and early autumn. 39 The Spatial Variation of Nutrients and HAB Events Based on the ambient parameters (i.e., salinity and eutrophic level) and spatial distributions of 3 types of harmful algal blooms, the Chesapeake Bay and its tributaries can be divided to 3 zones (Fig. 2-31). Zone I: The tidal fresh water area of the Chesapeake Bay, where surface salinity is generally less than 5, very eutrophic. Zone II: the lower part of the tributaries, upper and middle Chesapeake Bay, where surface salinity is generally between 5 and 15, and eutrophic. Zone III: the middle- lower bay, where surface salinity is higher than 15, but low eutrophic. Zone I is the area where most of the cyanobacteria blooms developed. This area is characterized by significantly higher nutrient levels than the other 2 zones, due to the terrestrial nutrients input. In winter and early spring, the DIN concentrations accumulates to over 100 ?M-N (Fig. 2-32), and the DIP concentration accumulates to over 0.5 ?M-P (Fig. 2-33). The ambient DIN:DIP ratios are over 200, suggesting P limitation (Fig. 2-34). When the phytoplankton biomass begins to develop from March (Fig. 2-35, 36), the ambient DIN is consumed and starts to decease, while DIP concentrations are stable, and even slightly increase. As a result, the ambient DIN:DIP ratios start to decrease. When cyanobacterial blooms develop in the summer (Fig. 2-36), the ambient DIN:DIP ratios decrease to median value less than 40, which is a factor of 2 of the Redfield ratio. Cyanobacterial blooms generally last until October. Zone II is the area where most of the P. minimum and K. veneficum blooms were documented. This area has lower nutrients compared to Zone I. In winter and early spring, the DIN concentrations are generally less than 50 ?M-N (Fig. 2-32), and 40 the DIP concentrations accumulate to around 0.2 ?M-P (Fig. 2-33). The ambient DIN:DIP ratios can reach over 500, suggesting P limitation (Fig. 2-34). The peak of P. minimum blooms occurred in April and May in both frequency and biomass, when the ambient DIN:DIP ratios dropped to 200. When P. minimum biomass accumulates in these high N low P environments, the ambient DIN further drops to ~ 20 ?M-N in June, but PO43- increases. When K. veneficum blooms develop in summer and autumn (June to September), the ambient DIN:DIP ratios drop to near the Redfield ratio. These changes are not only caused by the decrease of DIN, but also by the significantly increased DIP concentrations, which increases from ~ 0.2 ?M-P to ~ 1 ?M-P. Zone III is the area mostly under the influence of oceanic water of all 3 zones. It is characterized as low DIN (mostly less than 10 ?M-N), and low DIP (~ 0.1 ?M- P), even in the early spring (Fig. 2-32, 33). The Chl a concentrations are less than 20 ?g L-1 most times of the year and there are generally no significant blooms of the species groups considered here. The ambient DIN:DIP ratios drop from over 100 in the spring to near the Redfield ratio in July. The low DIN:DIP ratios last until November, even though there is no significant decrease in DIN concentrations. The decrease of NO3- concentrations from spring to late summer was caused by the consumption by phytoplankton communities. However, the maxima of DIP were observed in the late summer. It has been suggested that the DIP is from the remineralization of organic matter (Taft and Taylor 1976, Boynton and Kemp 1985, Jordan et al. 2008). Spring and summer blooms and terrestrial input accumulate massive organic matter in the bottom layer of the Chesapeake Bay and its tributaries. 41 DIP released from the remineralization may still not available to the primary producer due to absorption of ferric (Fe3+) oxides (Jordan et al. 2008). Decomposition of organic matter consumes the oxygen in the bottom layer after stratification develops in the water column in summer, and hypoxia develops in the bottom layer. DIP escapes precipitation when ferric oxides were reduced in the anoxic condition (Boynton and Kemp 1985). This DIP can be vertically transported to the euphotic layer and be available for the phytoplankton, or can be accessed by dinoflagellates which can vertically immigrate to the deeper layer of the water column. The Environmental Factors and HAB Species In a eutrophic estuary like Chesapeake Bay, the potential relationship between the frequency of HABs and increased anthropogenic nutrient inputs to coastal waters is a particular concern (Glibert et al. 2005a), as the phytoplankton assemblages will respond to excessive nutrient inputs with increased biomass and decreased diversity, and nutrient enrichment generally promotes a shift in dominance from diatoms to dinoflagellates and cyanobacteria (Pinckney et al. 2001, Anderson et al. 2008). The variations in nutrient loadings and supply ratios can regulate algal growth rates and community composition (Rudek et al. 1991, Smayda 1997, Heil et al. 2007). Herein, species specific correspondences were found between selected HAB species and nutrients and other related environmental variables. These correspondences are consistent with the physiological characters of each species, which are reported in previous field and laboratory studies. Prorocentrum minimum blooms have been often linked to eutrophication and occur in waters influenced by freshwater inputs (coastal waters and estuaries) and/or 42 anthropogenic loads (Heil et al. 2007, Glibert et al. 2001). In the lower part of the tributaries and the upper Chesapeake Bay, nutrients accumulate in the winter and early spring and DIN concentration can reach over 50 ?M-N (Fig. 2-32). Prorocentrum minimum may have competitive advantages over other phytoplankton populations in such nutrient-enriched, but very turbid environments. Prorocentrum minimum can survive in dark environment for months (Harding and Coats 1988), and grow well under low light conditions (Grzebyk and Berland 1996). Prorocentrum minimum also shows light independent N uptake ability (Fan and Glibert 2005). Prorocentrum minimum appears to be a good competitor in such high turbidity waters. Although the DIP concentrations are generally very low (~ 0.1 ?M-P, Fig. 2- 16) during the P. minimum blooms, P. minimum is capable to induce alkaline phosphatases and utilize organic phosphorus (Dyhrman 2005). In contrast to P. minimum which bloomed in high DIN water, Karlodinium veneficum were generally associated with high DIP concentrations and low DIN:DIP ratio (lower than Redfield ratio) water in the summer. Karlodinium veneficum developed blooms, even when ambient DIN has been depleted. Karlodinium veneficum, known as a mixotrophic dinoflagellate, may have competitive advantages in such waters. It can take up organic nitrogen to support growth, and ingest bacteria and cryptophytes (e.g. Rhodomonas salina) (Nygaard and Tobiesen 1993, Li et al. 1996, Li et al. 1999, Li et al. 2000, Adolf et al. 2003, Adolf et al. 2006a, Adolf et al. 2007, Adolf et al. 2008). Growth rates of mixotrophic K. veneficum (0.52 - 0.75 day-1) have been found to be comparable to or greater than maximum growth rate in phototrophic mode (0.55 day-1) (Adolf et al. 2006b). Phagotrophy of K. veneficum has 43 been intensively studied in Chesapeake Bay (Li et al. 1996, Li et al. 1999, Li et al. 2000, Adolf et al. 2003, Adolf et al. 2006a, Adolf et al. 2007, Adolf et al. 2008). From field and laboratory data, phagotrophy of K. veneficum was positively correlated with prey density (Li et al. 2000). N and/or P deficiency, or N:P ratios that were substantially higher or lower than the optimum N:P ratio (~10), also increased phagotrophic activity, suggesting feeding as a supplement for major nutrients (N, P) (Li et al., 2000). Phagotrophic activity might also supplement organic carbon sources or assist in acquiring trace growth factors (Li et al., 2000). High cryptophyte abundance in the summer has been suggested to trigger K. veneficum blooms as additional nutrient source in the Chesapeake Bay area (Adolf et al. 2008). Cyanobacterial blooms (not including the picocyanobacteria species) are most common in the tidal freshwater of the Chesapeake Bay (Marshall et al. 2006), which is spatial difference from the regions of maximum abundance of the two dinoflagellates, which generally did not bloom in such fresh water. High frequent cyanobacterial blooms are generally observed in the summer and associated with high DIP, low DIN:DIP ratio water. Some cyanobacteria (e.g. Anabaena spp.) have been reported to be capable of fixing N2 as a new N source in relative low N waters in the Chesapeake Bay (Burns et al. 2002, Moisander et al. 2007). This N2-fixation ability may an important competitive advantage for these cyanobacteria to develop blooms. Cyanobacterial blooms are also associated with very turbid water (secchi dept ~ 0.5 m). Cyanobacteria can possess gas vesicles in the cell, or/and form filaments to keep high buoyancy and stay in the surface water (Sellner 1997), where they can 44 reach light and N2. These characters make cyanobacteria excellent competitors in the low N, high turbidity freshwater. In summary, P. minimum blooms generally develop in high DIN water in the spring and early summer in mesohaline water of Chesapeake Bay, while K. veneficum blooms develop after DIN was depleted in similar areas. These trends were consistent with East China Sea and west Florida shelf for analogous species. Cyanobacterial blooms (not including the picocyanobacteria species) generally develop in the tidal freshwater of the Chesapeake Bay in the summer. Each species has corresponding physiological strategies that may provide completive advantages to the environments. The eutrophic areas in the Chesapeake Bay are the high frequent bloom areas. 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Journal of Phycology 19:528-533 Yan WJ, Zhang S (2003) The composition and bioavailability of phosphorus transport through the Changjiang (Yangtze) River during the 1998 flood. Biogeochemistry 65:179-194 Zhang H, Litaker W, Vandersea MW, Tester P, Lin SJ (2008) Geographic distribution of Karlodinium veneficum in the US east coast as detected by ITS-ferredoxin real-time PCR assay. Journal of Plankton Research 30:905-922 Zhang J, Liu SM, Ren JL, Wu Y, Zhang GL (2007) Nutrient gradients from the eutrophic Changjiang (Yangtze River) Estuary to the oligotrophic Kuroshio waters and re-evaluation of budgets for the East China Sea Shelf. Progress in Oceanography 74:449-478 54 Figures -77.5 -77 -76.5 -76 37 37.5 38 38.5 39 39.5 Figure 2-1 The routinely sampled stations (all the triangles) in the Chesapeake Bay and its tributaries by Maryland Departments of Natural Resources. Black triangles: the stations where Prorocentrum minimum, Karlodinium veneficum and cyanobacteria were recorded (cyanobacteria only include the species which could be counted in the resolution limit of light microscopy). 55 a. b. Hyper 91 High 844 Medium 3350 Low 1625 Type I 46 Type II 133 Type III 2157 Type IV 494 c. d. Type 1 10 Type II 38 Type III 196 Type IV 6 Type I 329 Type II 717 Type III 3521 Type IV 188 Figure 2-2 a. the number of samples of different type of eutrophic level chlorophyll a recorded at Chesapeake Bay area (1991-2008); b, the number of samples of different type of Prorocentrum minimum cell density level recorded; c, the number of samples of different type of Karlodinium veneficum cell density level recorded; d, the number of samples of different type of cyanobacteria cell density level recorded. Type I: hypereutrophic; type II: high eutrophic; Type III: medium eutrophic; Type IV: low eutrophic. 56 -77 -76.5 -76 37 37.5 38 38.5 39 39.5 20 ug Chl a L-1 60 ug Chl a L-1 200 ug Chl a L-1 Figure 2-3 The spatial distribution of Chlorophyll a samples (> 10 mg L-1) in the Chesapeake Bay and its tributaries 1991-2008. Data were derived from routinely sampling by Maryland Department of Natural Resources. Each circle stands for one sample, and the diameters of the circles are propositional to the Chlorophyll a concentrations according to the scale in the inset. The circles overlap at stations where high Chlorophyll a concentrations were frequently recorded. 57 -77 -76.5 -76 37 37.5 38 38.5 39 39.5 1.0E+6 cells per L 1.0E+7 cells per L 3.0E+7 cells per L Figure 2-4 The spatial distribution of Prorocentrum minimum (cell density > 1,000,000 cells L-1) in the Chesapeake Bay and its tributaries 1991-2008. Data were derived from routinely sampling by Maryland Department of Natural Resources. Each circle stands for one sample, and the diameters of the circles are propositional to the cell densities according to the scale in the inset. The circles overlap at stations where cells were frequently recorded. 58 -77 -76.5 -76 37 37.5 38 38.5 39 39.5 1.0E+6 cells per L 5.0E+6 cells per L 1.0E+7 cells per L Figure 2-5 The spatial distribution of Karlodinium veneficum (cell density > 1,000,000 cells L-1) in the Chesapeake Bay and its tributaries 2003-2008. Data were derived from routinely sampling by Maryland Department of Natural Resources. Each circle stands for one sample, and the diameters of the circles are propositional to the cell densities according to the scale in the inset. The circles overlap at stations where cells were frequently recorded. 59 -77 -76.5 -76 37 37.5 38 38.5 39 39.5 1.0E+7 cells per L 1.0E+8 cells per L 5.0E+8 cells per L Figure 2-6 The spatial distribution of cyanobacteria (cell density > 10,000,000 cells L-1) in the Chesapeake Bay and its tributaries 1991-2008. Data were derived from routinely sampling by Maryland Department of Natural Resources. Each circle stands for one sample, and the diameters of the circles are propositional to the cell densities according to the scale in the inset. The circles overlap at stations where cells were frequently recorded. 60 Chl a Hyper High Medium Low S a l i n i t y 0 5 10 15 20 25 30 35 P. minimum I II III IV S a l i n i t y 0 5 10 15 20 25 30 35 K. veneficum I II III IV S a l i n i t y 0 5 10 15 20 25 30 35 Cyanobacteria I II III IV S a l i n i t y 0 5 10 15 20 25 30 35 Figure 2-7 The salinity of samples with different eutrophic levels of Chl a and harmful algae densities: Prorocentrum minimum, Karlodinium veneficum and cyanobacteria in the Chesapeake Bay. Box plots - line: median; box: 25th to 75th percentiles; whiskers: 10th and 90th percentiles; dots: outliers. Hypereutrophic: > 60 ?g Chl a L-1; high eutrophic: > 20 but ? 60 ?g Chl a L-1; medium eutrophic: > 5 but ? 20 ?g Chl a L-1; low eutrophic: > 0 but ? 5 ?g Chl a L-1. For P. minimum and K. veneficum,Type I: > 5,000,000 cell L-1; Type II: > 1,000,000 but ? 5,000,000 cell L-1; Type III: >1,000 but ? 1,000,000 cell L-1; Type IV: > 0 but ? 1,000 cell L-1. For cyanobacteria, Type I: > 50,000,000 cell L-1; Type II: > 10,000,000 but ? 50,000,000 cell L-1; Type III: >10,000 but ? 10,000,000 cell L-1; Type IV: > 0 but ? 10,000 cell L-1 61 PM KV Cyn S a l i n i t y 0 5 10 15 20 PM KV Cyn W a t e r T ( o C ) 0 5 10 15 20 25 30 35 PM KV Cyn D I N ( ? M - N ) 0 20 40 60 80 100 120 140 160 180 200 PM KV Cyn D I P ( ? M - P ) 0.0 0.5 1.0 1.5 2.0 2.5 Figure 2-8 The ambient parameters (salinity, water temperature, dissolved inorganic nitrogen and phosphorus) of Prorocentrum minimum (PM), Karlodinium veneficum (KV) and cyanobacteria (Cyn) in blooms in the Chesapeake Bay. Box plots - line: median; box: 25th to 75th percentiles; whiskers: 10th and 90th percentiles; dots: outliers. Hypereutrophic: > 60 ?g Chl a L-1; high eutrophic: > 20 but ? 60 ?g Chl a L-1; medium eutrophic: > 5 but ? 20 ?g Chl a L-1; low eutrophic: > 0 but ? 5 ?g Chl a L-1. For P. minimum and K. veneficum,Type I: > 5,000,000 cell L-1; Type II: > 1,000,000 but ? 5,000,000 cell L-1; Type III: >1,000 but ? 1,000,000 cell L-1; Type IV: > 0 but ? 1,000 cell L-1. For cyanobacteria, Type I: > 50,000,000 cell L-1; Type II: > 10,000,000 but ? 50,000,000 cell L-1; Type III: >10,000 but ? 10,000,000 cell L-1; Type IV: > 0 but ? 10,000 cell L-1 62 Chl a Hyper High Medium Low M o n t h 0 2 4 6 8 10 12 P. minimum I II III IV M o n t h 0 2 4 6 8 10 12 K. veneficum I II III IV M o n t h 0 2 4 6 8 10 12 Cyanobacteria I II III IV M o n t h 0 2 4 6 8 10 12 Figure 2-9 The month number of samples with different eutrophic levels of Chl a and harmful algae densities: Prorocentrum minimum, Karlodinium veneficum and cyanobacteria in the Chesapeake Bay. Box plots - line: median; box: 25th to 75th percentiles; whiskers: 10th and 90th percentiles; dots: outliers. Hypereutrophic: > 60 ?g Chl a L-1; high eutrophic: > 20 but ? 60 ?g Chl a L-1; medium eutrophic: > 5 but ? 20 ?g Chl a L-1; low eutrophic: > 0 but ? 5 ?g Chl a L-1. For P. minimum and K. veneficum,Type I: > 5,000,000 cell L-1; Type II: > 1,000,000 but ? 5,000,000 cell L-1; Type III: >1,000 but ? 1,000,000 cell L-1; Type IV: > 0 but ? 1,000 cell L-1. For cyanobacteria, Type I: > 50,000,000 cell L-1; Type II: > 10,000,000 but ? 50,000,000 cell L-1; Type III: >10,000 but ? 10,000,000 cell L-1; Type IV: > 0 but ? 10,000 cell L-1 63 Chl a Hyper High Medium Low W a t e r T ( o C ) 0 10 20 30 40 P. minimum I II III IV W a t e r T ( o C ) 0 10 20 30 40 K. veneficum I II III IV W a t e r T ( o C ) 0 10 20 30 40 Cyanobacteria I II III IV W a t e r T ( o C ) 0 10 20 30 40 Figure 2-10 The water temperature of samples with different eutrophic levels of Chl a and harmful algae densities: Prorocentrum minimum, Karlodinium veneficum and cyanobacteria in the Chesapeake Bay. Box plots - line: median; box: 25th to 75th percentiles; whiskers: 10th and 90th percentiles; dots: outliers. Hypereutrophic: > 60 ?g Chl a L-1; high eutrophic: > 20 but ? 60 ?g Chl a L-1; medium eutrophic: > 5 but ? 20 ?g Chl a L-1; low eutrophic: > 0 but ? 5 ?g Chl a L-1. For P. minimum and K. veneficum,Type I: > 5,000,000 cell L-1; Type II: > 1,000,000 but ? 5,000,000 cell L-1; Type III: >1,000 but ? 1,000,000 cell L-1; Type IV: > 0 but ? 1,000 cell L-1. For cyanobacteria, Type I: > 50,000,000 cell L-1; Type II: > 10,000,000 but ? 50,000,000 cell L-1; Type III: >10,000 but ? 10,000,000 cell L-1; Type IV: > 0 but ? 10,000 cell L-1 64 Chl a Hyper High Medium Low s e c c h i ( m ) 0 1 2 3 4 P. minimum I II III IV s e c c h i ( m ) 0 1 2 3 4 K. veneficum I II III IV s e c c h i ( m ) 0 1 2 3 4 Cyanobacteria I II III IV s e c c h i ( m ) 0 1 2 3 4 Figure 2-11 The secchi depth of samples with different eutrophic levels of Chl a and harmful algae densities: Prorocentrum minimum, Karlodinium veneficum and cyanobacteria in the Chesapeake Bay. Box plots - line: median; box: 25th to 75th percentiles; whiskers: 10th and 90th percentiles; dots: outliers. Hypereutrophic: > 60 ?g Chl a L-1; high eutrophic: > 20 but ? 60 ?g Chl a L-1; medium eutrophic: > 5 but ? 20 ?g Chl a L-1; low eutrophic: > 0 but ? 5 ?g Chl a L-1. For P. minimum and K. veneficum,Type I: > 5,000,000 cell L-1; Type II: > 1,000,000 but ? 5,000,000 cell L-1; Type III: >1,000 but ? 1,000,000 cell L-1; Type IV: > 0 but ? 1,000 cell L-1. For cyanobacteria, Type I: > 50,000,000 cell L-1; Type II: > 10,000,000 but ? 50,000,000 cell L-1; Type III: >10,000 but ? 10,000,000 cell L-1; Type IV: > 0 but ? 10,000 cell L-1 65 Chl a Hyper High Medium Low T S S ( m g L - 1 ) 0 20 40 60 80 100 P. minimum I II III IV T S S ( m g L - 1 ) 0 20 40 60 80 100 K. veneficum I II III IV T S S ( m g L - 1 ) 0 20 40 60 80 100 Cyanobacteria I II III IV T S S ( m g L - 1 ) 0 20 40 60 80 100 Figure 2-12 The total suspended solids (TSS) of samples with different eutrophic levels of Chl a and harmful algae densities: Prorocentrum minimum, Karlodinium veneficum and cyanobacteria in the Chesapeake Bay. Box plots - line: median; box: 25th to 75th percentiles; whiskers: 10th and 90th percentiles; dots: outliers. Hypereutrophic: > 60 ?g Chl a L-1; high eutrophic: > 20 but ? 60 ?g Chl a L-1; medium eutrophic: > 5 but ? 20 ?g Chl a L-1; low eutrophic: > 0 but ? 5 ?g Chl a L-1. For P. minimum and K. veneficum,Type I: > 5,000,000 cell L-1; Type II: > 1,000,000 but ? 5,000,000 cell L-1; Type III: >1,000 but ? 1,000,000 cell L-1; Type IV: > 0 but ? 1,000 cell L-1. For cyanobacteria, Type I: > 50,000,000 cell L-1; Type II: > 10,000,000 but ? 50,000,000 cell L-1; Type III: >10,000 but ? 10,000,000 cell L-1; Type IV: > 0 but ? 10,000 cell L-1 66 Chl a Hyper High Medium Low D O ( m g L - 1 ) 0 2 4 6 8 10 12 14 16 18 P. minimum I II III IV D O ( m g L - 1 ) 0 2 4 6 8 10 12 14 16 18 K. veneficum I II III IV D O ( m g L - 1 ) 0 2 4 6 8 10 12 14 16 18 Cyanobacteria I II III IV D O ( m g L - 1 ) 0 2 4 6 8 10 12 14 16 18 Figure 2-13 The dissolved oxygen (DO) of samples with different eutrophic levels of Chl a and harmful algae densities: Prorocentrum minimum, Karlodinium veneficum and cyanobacteria in the Chesapeake Bay. Box plots - line: median; box: 25th to 75th percentiles; whiskers: 10th and 90th percentiles; dots: outliers. Hypereutrophic: > 60 ?g Chl a L-1; high eutrophic: > 20 but ? 60 ?g Chl a L-1; medium eutrophic: > 5 but ? 20 ?g Chl a L-1; low eutrophic: > 0 but ? 5 ?g Chl a L-1. For P. minimum and K. veneficum,Type I: > 5,000,000 cell L-1; Type II: > 1,000,000 but ? 5,000,000 cell L-1; Type III: >1,000 but ? 1,000,000 cell L-1; Type IV: > 0 but ? 1,000 cell L-1. For cyanobacteria, Type I: > 50,000,000 cell L-1; Type II: > 10,000,000 but ? 50,000,000 cell L-1; Type III: >10,000 but ? 10,000,000 cell L-1; Type IV: > 0 but ? 10,000 cell L-1 67 Chl a Hyper High Medium Low D O C ( ? M - C ) 0 200 400 600 800 1000 P. minimum I II III IV D O C ( ? M - C ) 0 200 400 600 800 1000 . K. veneficum I II III IV D O C ( ? M - C ) 0 200 400 600 800 1000 Cyanobacteria I II III IV D O C ( ? M - C ) 0 200 400 600 800 1000 Figure 2-14 The dissolved organic carbon (DOC) of samples with different eutrophic levels of Chl a and harmful algae densities: Prorocentrum minimum, Karlodinium veneficum and cyanobacteria in the Chesapeake Bay. Box plots - line: median; box: 25th to 75th percentiles; whiskers: 10th and 90th percentiles; dots: outliers. Hypereutrophic: > 60 ?g Chl a L-1; high eutrophic: > 20 but ? 60 ?g Chl a L-1; medium eutrophic: > 5 but ? 20 ?g Chl a L-1; low eutrophic: > 0 but ? 5 ?g Chl a L-1. For P. minimum and K. veneficum,Type I: > 5,000,000 cell L-1; Type II: > 1,000,000 but ? 5,000,000 cell L-1; Type III: >1,000 but ? 1,000,000 cell L-1; Type IV: > 0 but ? 1,000 cell L-1. For cyanobacteria, Type I: > 50,000,000 cell L-1; Type II: > 10,000,000 but ? 50,000,000 cell L-1; Type III: >10,000 but ? 10,000,000 cell L-1; Type IV: > 0 but ? 10,000 cell L-1 68 Chl a Hyper High Medium Low D I N ( ? M - N ) 0 20 40 60 80 100 P. minimum I II III IV D I N ( ? M - N ) 0 20 40 60 80 100 K. veneficum I II III IV D I N ( ? M - N ) 0 20 40 60 80 100 Cyanobacteria I II III IV D I N ( ? M - N ) 0 20 40 60 80 100 Figure 2-15 The dissolved inorganic nitrogen (DIN) concentration of samples with different eutrophic levels of Chl a and harmful algae densities: Prorocentrum minimum, Karlodinium veneficum and cyanobacteria in the Chesapeake Bay. Box plots - line: median; box: 25th to 75th percentiles; whiskers: 10th and 90th percentiles; dots: outliers. Hypereutrophic: > 60 ?g Chl a L-1; high eutrophic: > 20 but ? 60 ?g Chl a L-1; medium eutrophic: > 5 but ? 20 ?g Chl a L-1; low eutrophic: > 0 but ? 5 ?g Chl a L-1. For P. minimum and K. veneficum,Type I: > 5,000,000 cell L-1; Type II: > 1,000,000 but ? 5,000,000 cell L-1; Type III: >1,000 but ? 1,000,000 cell L-1; Type IV: > 0 but ? 1,000 cell L-1. For cyanobacteria, Type I: > 50,000,000 cell L-1; Type II: > 10,000,000 but ? 50,000,000 cell L-1; Type III: >10,000 but ? 10,000,000 cell L-1; Type IV: > 0 but ? 10,000 cell L-1 69 Chl a Hyper High Medium Low P O 4 3 - ( ? M - P ) 0.0 .5 1.0 1.5 2.0 P. minimum I II III IV P O 4 3 - ( ? M - P ) 0.0 0.5 1.0 1.5 2.0 K. veneficum I II III IV P O 4 3 - ( ? M - P ) 0.0 .5 1.0 1.5 2.0 Cyanobacteria I II III IV P O 4 3 - ( ? M - P ) 0.0 .5 1.0 1.5 2.0 Figure 2-16 The phosphate (PO43-) concentration of samples with different eutrophic levels of Chl a and harmful algae densities: Prorocentrum minimum, Karlodinium veneficum and cyanobacteria in the Chesapeake Bay. Box plots - line: median; box: 25th to 75th percentiles; whiskers: 10th and 90th percentiles; dots: outliers. Hypereutrophic: > 60 ?g Chl a L-1; high eutrophic: > 20 but ? 60 ?g Chl a L-1; medium eutrophic: > 5 but ? 20 ?g Chl a L-1; low eutrophic: > 0 but ? 5 ?g Chl a L-1. For P. minimum and K. veneficum,Type I: > 5,000,000 cell L-1; Type II: > 1,000,000 but ? 5,000,000 cell L-1; Type III: >1,000 but ? 1,000,000 cell L-1; Type IV: > 0 but ? 1,000 cell L-1. For cyanobacteria, Type I: > 50,000,000 cell L-1; Type II: > 10,000,000 but ? 50,000,000 cell L-1; Type III: >10,000 but ? 10,000,000 cell L-1; Type IV: > 0 but ? 10,000 cell L-1 70 Chl a Hyper High Medium Low D I N : D I P 0 100 200 300 400 500 P. minimum I II III IV D I N : D I P 0 100 200 300 400 500 K. veneficum I II III IV D I N : D I P 0 100 200 300 400 500 Cyanobacteria I II III IV D I N : D I P 0 100 200 300 400 500 Figure 2-17 The dissolved inorganic nitrogen (DIN) to dissolved inorganic phosphorus (DIP) ratios of samples with different eutrophic levels of Chl a and harmful algae densities: Prorocentrum minimum, Karlodinium veneficum and cyanobacteria in the Chesapeake Bay. Box plots - line: median; box: 25th to 75th percentiles; whiskers: 10th and 90th percentiles; dots: outliers. Hypereutrophic: > 60 ?g Chl a L-1; high eutrophic: > 20 but ? 60 ?g Chl a L-1; medium eutrophic: > 5 but ? 20 ?g Chl a L-1; low eutrophic: > 0 but ? 5 ?g Chl a L-1. For P. minimum and K. veneficum,Type I: > 5,000,000 cell L-1; Type II: > 1,000,000 but ? 5,000,000 cell L-1; Type III: >1,000 but ? 1,000,000 cell L-1; Type IV: > 0 but ? 1,000 cell L-1. For cyanobacteria, Type I: > 50,000,000 cell L-1; Type II: > 10,000,000 but ? 50,000,000 cell L-1; Type III: >10,000 but ? 10,000,000 cell L-1; Type IV: > 0 but ? 10,000 cell L-1 71 PM KV Cyn D I N : D I P 0 100 200 300 400 500 600 PM KV Cyn P C : P N 0 20 40 60 80 100 120 140 ` PM KV Cyn P C : P P 0 200 400 600 800 1000 PM KV Cyn P N : P P 0 20 40 60 80 100 120 Figure 2-18 The ambient carbon, nitrogen and phosphorus rates of Prorocentrum minimum (PM), Karlodinium veneficum (KV) and cyanobacteria (Cyn) in blooms in the Chesapeake Bay. Box plots - line: median; box: 25th to 75th percentiles; whiskers: 10th and 90th percentiles; dots: outliers. Hypereutrophic: > 60 ?g Chl a L-1; high eutrophic: > 20 but ? 60 ?g Chl a L-1; medium eutrophic: > 5 but ? 20 ?g Chl a L-1; low eutrophic: > 0 but ? 5 ?g Chl a L-1. For P. minimum and K. veneficum,Type I: > 5,000,000 cell L-1; Type II: > 1,000,000 but ? 5,000,000 cell L-1; Type III: >1,000 but ? 1,000,000 cell L-1; Type IV: > 0 but ? 1,000 cell L-1. For cyanobacteria, Type I: > 50,000,000 cell L-1; Type II: > 10,000,000 but ? 50,000,000 cell L-1; Type III: >10,000 but ? 10,000,000 cell L-1; Type IV: > 0 but ? 10,000 cell L-1 72 Chl a Hyper High Medium Low P C ( ? M - C ) 0 200 400 600 800 1000 P. minimum I II III IV P C ( ? M - C ) 0 200 400 600 800 1000 K. veneficum I II III IV P C ( ? M - C ) 0 200 400 600 800 1000 Cyanobacteria I II III IV P C ( ? M - C ) 0 200 400 600 800 1000 Figure 2-19 The particle carbon (PC) concentration of samples with different eutrophic levels of Chl a and harmful algae densities: Prorocentrum minimum, Karlodinium veneficum and cyanobacteria in the Chesapeake Bay. Box plots - line: median; box: 25th to 75th percentiles; whiskers: 10th and 90th percentiles; dots: outliers. Hypereutrophic: > 60 ?g Chl a L-1; high eutrophic: > 20 but ? 60 ?g Chl a L-1; medium eutrophic: > 5 but ? 20 ?g Chl a L-1; low eutrophic: > 0 but ? 5 ?g Chl a L-1. For P. minimum and K. veneficum,Type I: > 5,000,000 cell L-1; Type II: > 1,000,000 but ? 5,000,000 cell L-1; Type III: >1,000 but ? 1,000,000 cell L-1; Type IV: > 0 but ? 1,000 cell L-1. For cyanobacteria, Type I: > 50,000,000 cell L-1; Type II: > 10,000,000 but ? 50,000,000 cell L-1; Type III: >10,000 but ? 10,000,000 cell L-1; Type IV: > 0 but ? 10,000 cell L-1 73 Chl a Hyper High Medium Low P N ( ? M - N ) 0 10 20 30 40 50 P. minimum I II III IV P N ( ? M - N ) 0 10 20 30 40 50 K. veneficum I II III IV P N ( ? M - N ) 0 10 20 30 40 50 Cyanobacteria I II III IV P N ( ? M - N ) 0 10 20 30 40 50 Figure 2-20 The particle nitrogen (PN) concentration of samples with different eutrophic levels of Chl a and harmful algae densities: Prorocentrum minimum, Karlodinium veneficum and cyanobacteria in the Chesapeake Bay. Box plots - line: median; box: 25th to 75th percentiles; whiskers: 10th and 90th percentiles; dots: outliers. Hypereutrophic: > 60 ?g Chl a L-1; high eutrophic: > 20 but ? 60 ?g Chl a L-1; medium eutrophic: > 5 but ? 20 ?g Chl a L-1; low eutrophic: > 0 but ? 5 ?g Chl a L-1. For P. minimum and K. veneficum,Type I: > 5,000,000 cell L-1; Type II: > 1,000,000 but ? 5,000,000 cell L-1; Type III: >1,000 but ? 1,000,000 cell L-1; Type IV: > 0 but ? 1,000 cell L-1. For cyanobacteria, Type I: > 50,000,000 cell L-1; Type II: > 10,000,000 but ? 50,000,000 cell L-1; Type III: >10,000 but ? 10,000,000 cell L-1; Type IV: > 0 but ? 10,000 cell L-1 74 Chl a Hyper High Medium Low P P ( ? M - P ) 0 5 10 15 20 P. minimum I II III IV P P ( ? M - P ) 0 5 10 15 20 P. minimum I II III IV P P ( ? M - P ) 0 2 4 6 8 10 Cyanobacteria I II III IV P P ( ? M - P ) 0 2 4 6 8 10 Figure 2-21 The particle phosphorus (PP) concentration of samples with different eutrophic levels of Chl a and harmful algae densities: Prorocentrum minimum, Karlodinium veneficum and cyanobacteria in the Chesapeake Bay. Box plots - line: median; box: 25th to 75th percentiles; whiskers: 10th and 90th percentiles; dots: outliers. Hypereutrophic: > 60 ?g Chl a L-1; high eutrophic: > 20 but ? 60 ?g Chl a L-1; medium eutrophic: > 5 but ? 20 ?g Chl a L-1; low eutrophic: > 0 but ? 5 ?g Chl a L-1. For P. minimum and K. veneficum,Type I: > 5,000,000 cell L-1; Type II: > 1,000,000 but ? 5,000,000 cell L-1; Type III: >1,000 but ? 1,000,000 cell L-1; Type IV: > 0 but ? 1,000 cell L-1. For cyanobacteria, Type I: > 50,000,000 cell L-1; Type II: > 10,000,000 but ? 50,000,000 cell L-1; Type III: >10,000 but ? 10,000,000 cell L-1; Type IV: > 0 but ? 10,000 cell L-1 75 Chl a Hyper High Medium Low P C : P N 0 20 40 60 80 100 P. minimum I II III IV P C : P N 0 20 40 60 80 100 K. veneficum I II III IV P C : P N 0 20 40 60 80 100 Cyanobacteria I II III IV P C : P N 0 20 40 60 80 100 Figure 2-22 The particle carbon (PC) to particle phosphorus (PP) ratios of samples with different eutrophic levels of Chl a and harmful algae densities: Prorocentrum minimum, Karlodinium veneficum and cyanobacteria in the Chesapeake Bay. Box plots - line: median; box: 25th to 75th percentiles; whiskers: 10th and 90th percentiles; dots: outliers. Hypereutrophic: > 60 ?g Chl a L-1; high eutrophic: > 20 but ? 60 ?g Chl a L-1; medium eutrophic: > 5 but ? 20 ?g Chl a L-1; low eutrophic: > 0 but ? 5 ?g Chl a L-1. For P. minimum and K. veneficum,Type I: > 5,000,000 cell L-1; Type II: > 1,000,000 but ? 5,000,000 cell L-1; Type III: >1,000 but ? 1,000,000 cell L-1; Type IV: > 0 but ? 1,000 cell L-1. For cyanobacteria, Type I: > 50,000,000 cell L-1; Type II: > 10,000,000 but ? 50,000,000 cell L-1; Type III: >10,000 but ? 10,000,000 cell L-1; Type IV: > 0 but ? 10,000 cell L-1 76 Chl a Hyper High Medium Low P C : P P 0 200 400 600 800 1000 P. minimum I II III IV P C : P P 0 200 400 600 800 1000 K. veneficum I II III IV P C : P P 0 200 400 600 800 1000 Cyanobacteria I II III IV P C : P P 0 200 400 600 800 1000 Figure 2-23 The particle carbon (PC) to particle phosphorus (PP) ratios of samples with different eutrophic levels of Chl a and harmful algae densities: Prorocentrum minimum, Karlodinium veneficum and cyanobacteria in the Chesapeake Bay. Dash line: PC:PP = 106. Box plots - line: median; box: 25th to 75th percentiles; whiskers: 10th and 90th percentiles; dots: outliers. Hypereutrophic: > 60 ?g Chl a L-1; high eutrophic: > 20 but ? 60 ?g Chl a L-1; medium eutrophic: > 5 but ? 20 ?g Chl a L-1; low eutrophic: > 0 but ? 5 ?g Chl a L-1. For P. minimum and K. veneficum,Type I: > 5,000,000 cell L-1; Type II: > 1,000,000 but ? 5,000,000 cell L-1; Type III: >1,000 but ? 1,000,000 cell L-1; Type IV: > 0 but ? 1,000 cell L-1. For cyanobacteria, Type I: > 50,000,000 cell L-1; Type II: > 10,000,000 but ? 50,000,000 cell L-1; Type III: >10,000 but ? 10,000,000 cell L-1; Type IV: > 0 but ? 10,000 cell L-1 77 Chl a Hyper High Medium Low P N : P P 0 20 40 60 80 100 P. minimum I II III IV P N : P P 0 20 40 60 80 100 K. veneficum I II III IV P N : P P 0 20 40 60 80 100 Cyanobacteria I II III IV P N : P P 0 20 40 60 80 100 Figure 2-24 The particle nitrogen (PN) to particle phosphorus (PP) ratios of samples with different eutrophic levels of Chl a and harmful algae densities: Prorocentrum minimum, Karlodinium veneficum and cyanobacteria in the Chesapeake Bay. Dash line: the Redfield ratio. Box plots - line: median; box: 25th to 75th percentiles; whiskers: 10th and 90th percentiles; dots: outliers. Hypereutrophic: > 60 ?g Chl a L-1; high eutrophic: > 20 but ? 60 ?g Chl a L-1; medium eutrophic: > 5 but ? 20 ?g Chl a L-1; low eutrophic: > 0 but ? 5 ?g Chl a L-1. For P. minimum and K. veneficum,Type I: > 5,000,000 cell L-1; Type II: > 1,000,000 but ? 5,000,000 cell L-1; Type III: >1,000 but ? 1,000,000 cell L-1; Type IV: > 0 but ? 1,000 cell L-1. For cyanobacteria, Type I: > 50,000,000 cell L-1; Type II: > 10,000,000 but ? 50,000,000 cell L-1; Type III: >10,000 but ? 10,000,000 cell L-1; Type IV: > 0 but ? 10,000 cell L-1 78 N: P r ati o 1 10 100 ECS SFS CB Redfield ratio Figure 2-25 Comparison of the range in ambient N:P ratios during different phase of the blooms in the East China Sea (ECS) during May 2005, in different phytoplankton assemblages along the southwest Florida Shelf (SFS) during May 2003 and different phase of blooms in the Chesapeake Bay from 1991 to 2008. Black: Karenica spp. in ECS and SFS, and Karlodinium veneficum in Chesapeake Bay; Green: Prorocentrum spp.; Red: Diatom. The ECS data were derived from Li et al. (2009). The SFS data were derived from Heil et al. (2007) 79 May Distance from Head of the Bay (km) 0 50 100 150 200 250 300 Su rfa ce Sa lin ity 0 5 10 15 20 25 30 2000-2008 1991-1999 1982-1987 August Distance from Head of the Bay (km) 0 50 100 150 200 250 300 Su rfa ce Sa lin ity 0 5 10 15 20 25 30 Figure 2-26 The spatial distribution of salinities in surface waters along the length of Chesapeake Bay major axis in May and August in the time range of 2000-2008, 1991-1999, and 1982-1987 (data from Fisher et al. 1992). 80 May Distance from Head of the Bay (km) 0 50 100 150 200 250 300 DI N (? M ) 0 20 40 60 80 100 120 140 2000-2008 1991-1999 1982-1987 August Distance from Head of the Bay (km) 0 50 100 150 200 250 300 DI N (? M ) 0 20 40 60 80 100 120 140 Figure 2-27 The spatial distribution of dissolved inorganic nitrogen (DIN) in surface waters along the length of Chesapeake Bay major axis in May and August in the time range of 2000- 2008, 1991-1999, and 1982-1987 (data from Fisher et al. 1992). 81 May Distance from Head of the Bay (km) 0 50 100 150 200 250 300 PO 43 - ( ? M) 0.0 0.2 0.4 0.6 0.8 1.0 2000-2008 1991-1999 1982-1987 August Distance from Head of the Bay (km) 0 50 100 150 200 250 300 PO 43 - (? M) 0.0 0.2 0.4 0.6 0.8 1.0 Figure 2-28 The spatial distribution of phosphate (PO43-) in surface waters along the length of Chesapeake Bay major axis in May and August in the time range of 2000-2008, 1991-1999, and 1982-1987 (data from Fisher et al. 1992). 82 May Distance from Head of the Bay (km) 0 50 100 150 200 250 300 N: P r ati o 0.1 1 10 100 1000 2000-2008 1991-1999 1982-1987 August Distance from Head of the Bay (km) 0 50 100 150 200 250 300 N:P ra tio 0.1 1 10 100 1000 Figure 2-29 The spatial distribution of dissolved inorganic nitrate: dissolved inorganic phosphate (N:P) ratio in surface waters along the length of Chesapeake Bay major axis in May and August in the time range of 2000-2008, 1991-1999, and 1982-1987 (data from Fisher et al. 1992). 83 May Distance from Head of the Bay (km) 0 50 100 150 200 250 300 Ch l a ( ? g m l-1 ) 0 10 20 30 40 50 60 2000-2008 1991-1999 1982-1987 August Distance from Head of the Bay (km) 0 50 100 150 200 250 300 Ch l a (? g m l-1 ) 0 10 20 30 40 50 Figure 2-30 The spatial distribution of chlorophyll a in surface waters along the length of Chesapeake Bay major axis in May and August in the time range of 2000-2008, 1991-1999, and 1982-1987 (data from Fisher et al. 1992). 84 Figure 2-31. The summer surface water salinity in the Chesapeake Bay (modified from http://www.ncnr.nist.gov/programs/CHRNS/pdf/salinity_fall.png) 85 DIN - Zone I Month of year 1 2 3 4 5 6 7 8 9 10 11 12 ? M 0 50 100 150 DIN - Zone II Month of year 1 2 3 4 5 6 7 8 9 10 11 12 ? M 0 50 100 DIN - Zone III Month of year 1 2 3 4 5 6 7 8 9 10 11 12 ? M 0 10 20 30 40 50 Figure 2-32 The seasonal variation of monthly average dissolved inorganic nitrogen (DIN) concentrations in 3 zones of Chesapeake Bay. Box plots - line: median; box: 25th to 75th percentiles; whiskers: 10th and 90th percentiles; dots: outliers. 86 PO43- - Zone I Month of year 1 2 3 4 5 6 7 8 9 10 11 12 ?M 0.0 0.5 1.0 1.5 2.0 PO43- - Zone II Month of year 1 2 3 4 5 6 7 8 9 10 11 12 ?M 0.0 0.5 1.0 1.5 2.0 PO43- - Zone III Month of year 1 2 3 4 5 6 7 8 9 10 11 12 ?M 0.0 0.5 1.0 1.5 2.0 Figure 2-33 The seasonal variation of monthly average phosphate (PO43-) concentrations in 3 zones of Chesapeake Bay. Box plots - line: median; box: 25th to 75th percentiles; whiskers: 10th and 90th percentiles; dots: outliers. 87 N:P ratio - Zone I Month of year 1 2 3 4 5 6 7 8 9 10 11 12 0 100 200 300 400 500 N:P ratio - Zone II Month of year 1 2 3 4 5 6 7 8 9 10 11 12 0 100 200 300 400 500 N:P ratio - Zone III Month of year 1 2 3 4 5 6 7 8 9 10 11 12 0 100 200 300 400 500 Figure 2-34 The seasonal variation of ambient dissolved inorganic nitrogen (N): phosphate (P) ratios in 3 zones of Chesapeake Bay. Dash line: the Redfield ratio. Box plots - line: median; box: 25th to 75th percentiles; whiskers: 10th and 90th percentiles; dots: outliers. 88 Chl a - Zone I Month of year 1 2 3 4 5 6 7 8 9 10 11 12 mg L- 1 0 10 20 30 40 50 Chl a - Zone II Month of year 1 2 3 4 5 6 7 8 9 10 11 12 mg L- 1 0 10 20 30 40 50 Chl a - Zone III Month of year 1 2 3 4 5 6 7 8 9 10 11 12 mg L- 1 0 10 20 30 40 50 Figure 2-35 The seasonal variation of monthly average chlorophyll a concentrations in 3 zones of Chesapeake Bay. Box plots - line: median; box: 25th to 75th percentiles; whiskers: 10th and 90th percentiles; dots: outliers. 89 Cyanobacteria Month of year 1 2 3 4 5 6 7 8 9 10 11 12 ce lls L-1 0 1e+8 2e+8 3e+8 4e+8 5e+8 P. minimum Month of year 1 2 3 4 5 6 7 8 9 10 11 12 ce ll L -1 0 1e+7 2e+7 3e+7 4e+7 5e+7 K. veneficum Month of year 1 2 3 4 5 6 7 8 9 10 11 12 ce ll L -1 0 1e+7 2e+7 3e+7 4e+7 5e+7 Figure 2-36 The seasonal variation of monthly maximum cell densities of cyanobacteria in Zone I and Prorocentrum minimum and Karlodinium veneficum in Zone II of Chesapeake Bay. Box plots - line: median; box: 25th to 75th percentiles; whiskers: 10th and 90th percentiles; dots: outliers. 90 Chapter 3: Effects of Ambient DIN : DIP Ratio on the Nitrogen Uptake of Harmful Dinoflagellate Prorocentrum minimum and Prorocentrum donghaiense in Turbidistat* Abstract The effects of varying nitrogen (N): phosphorus (P) ratios on the growth and N- uptake /assimilation of the harmful dinoflagellate Prorocentrum minimum and Prorocentrum donghaiense were examined in turbidistat culture experiments. Algal cultures were supplied with media containing PO43- in various concentrations to obtain a wide range of N:P ratios. Experiments to determine rates of N uptake and assimilation of different N sources (NO3?, NH4+, urea and glycine by P. minimum and NO3?, NH4+ by P. donghaiense) were conducted using 15N tracer techniques at each N:P ratio. The growth rates suggested nutrient limitation at both high and low N:P ratios relative to the Redfield ratio. On a diel basis, the growth of both species was regulated by the light-dark cycle, which may be a result of regulation of both light- dependent growth and light-independent nutrient uptake. Maximum growth rates of both species always occurred at the beginning of the light phase. In P-rich medium (low N:P ratio), both species had higher N assimilation rates, suggesting N limitation. Low assimilation coefficients at high N:P ratios suggested P limitation of N uptake and assimilation. NO3? and NH4+ contributed more than 90% of the total N uptake of P. minimum. Reduced N sources were more quickly assimilated than NO3?. Highest average daily growth rates were recorded near N:P ratio of 12 for both species. The N uptake rates of cultures at N:P ratio near Redfield ratio were more balanced with the * Li J, Glibert PM, Alexander JA Chinese Journal of Oceanology and Limnology (in press) 91 growth rates. The linkage between growth rates and N uptake/assimilation rates were conceptually described by the variation of cell N quota. The N:P ratios affect the N uptake and growth of Prorocentrum spp., and may regulate their bloom progression in eutrophic ecosystems. Introduction Prorocentrum minimum, a bloom forming dinoflagellate, has been shown to be widely distributed globally (Heil et al. 2005, Glibert et al. 2008b). Its expanding geographical distribution is indicative of a strong relationship between both dissolved inorganic nitrogen (DIN) export and dissolved organic nitrogen (DON) export into coastal waters globally (Heil et al. 2005, Glibert and Burkholder 2006, Glibert et al. 2008a). A related pelagic Prorocentrum species, Prorocentrum donghaiense, has also been suggested to be an eutrophication-related species, becoming one of the dominant bloom species observed along coast of China in recent years (Lu and Goebel 2001, Zhou et al. 2003, Li et al. 2009). The nitrogen nutritional strategies of P. minimum have been well studied, and the understanding of those of P. donghaiense are advancing. Both species can take up a range of nitrogen substrates, including NO3-, NH4+, and urea (Lomas and Glibert 1999a, b, Burns et al. 2000, Fan et al. 2003a, Li et al. 2010). The affinity for different nitrogen substrates varies with growth condition (Fan et al. 2003b) and with the rate of resupply (Sciandra 1991). Organic forms of nitrogen have also been found to be taken up via a range of mechanisms. The protease leucine aminopeptidase (LAP), one of the enzymes that liberates amino acids, has been found to be active in this species 92 in both laboratory and field studies (Stoecker and Gustafson 2003, Salerno and Stoecker 2009). Moreover, P. minimum is mixotrophic and has been shown to graze upon a wide range of organisms, from organic particles (Li et al. 1996) to other microalgae (Stoecker et al. 1997, Heil et al. 2005, Jeong et al. 2005b). The nutrient strategies of P. donghaiense are less well documented, but from what is currently known, they appear to be similar to those of P. minimum. P. donghaiense has also been shown to graze on cyanobacteria (Synechococcus etc.) and cryptophytes (Jeong et al. 2005a, Jeong et al. 2005b). The uptake of NO3- by P. minimum under varying light-dark regimes was contrasted with that of several other dinoflagellates in studies by Paasche et al. (1984). They found that P. minimum continuously took up NO3-, while many other dinoflagellates stopped NO3- uptake at night. P. minimum also has been shown to display considerable plasticity in its photosynthetic parameters and can adapt to low light and be sustained for long periods of darkness (Tyler and Seliger 1978, Harding et al. 1983, Harding and Coats 1988). However, mixotrophy, as an adaptive strategy, is more likely to be a response to nutrient limitation than to carbon limitation (Stoecker et al. 1997). Less is known about P. donghaiense either in terms of photosynthetic adaption or its mixotrophic tendencies. As with many other flagellate species, both P. minimum and P. donghaiense are more commonly observed when inorganic N:P ratios are at the Redfield ratio or below due to their relatively high requirement for P (Fan and Glibert 2005, Heil et al. 2007, Li et al. 2009). A key question that has not been addressed is whether N 93 nutritional strategies vary across a spectrum of N:P ratios. In order to assess this question, a series of laboratory experiments were conducted on both species. The conceptual framework for these experiments is the cell quota equation of Droop (Droop 1968, 1973), who described the growth of phytoplankton growing under a nutrient limited environment using the equation: where the cell quota (Q) is the intracellular content of the limiting chemical element (N, P etc.), ? is the growth rate, and ?max is the maximum growth rate for a given set of environmental conditions. In this model framework, cells can not grow if the cell quota is lower than the minimum quota (Qmin), which defines the minimum requirement of the cells. Nutrients are taken up, then subsequently assimilated into the nitrogenous components of the cell. P. minimum has previously been shown to have a large, but varying cell quota (Sciandra 1991). In this study, cells of P. minimum and P. donghaiense were grown across an N:P spectrum in order to achieve a range of growth rates and a range of physiological states. The rates of uptake of different forms of nitrogen were compared, as were the comparative rates of uptake and assimilation. Materials and Methods Algae Culture Cultures of Prorocentrum minimum were isolated from Choptank River, a tributary of Chesapeake Bay in spring 1995 and were maintained in f/2 medium minmax (1 )Q Q? ?? ? 94 (Guillard and Ryther 1962) in the Horn Point Laboratory culture collection. For the experiments, P. minimum cells were first transferred to f/20 batch cultures for 2 weeks, then grown in turbidistat, an automatic continuous culture system, as described below. Cultures of Prorocentrum donghaiense were obtained from University of Connecticut Department of Marine Sciences, USA. These cultures were originally isolated from the East China Sea. They, too, were transferred to f/2 batch cultures for several weeks, then grown in turbidistat. Continuous Culture Design A continuous turbidistat culturing device (Fig. 3-1) was used to grow the Prorocentrum cultures. A modulated infrared (880 mm) LED beam was directed though the culture vessel (a 2 L glass carboy) to a photosensor, the analog signal output of which was related to optical transmission (i.e., turbidity). Real time turbidity signals were sent to a computer that controlled a peristaltic pump which diluted the culture based on attaining a threshold value. The system was programmed to dilute the culture by 5% in terms of the optical signal. This corresponds to a volume dilution of approx. 10%, based on calibrations using a volumetric dilution. The threshold was set to a turbidity that represented a P. minimum cell density of ~ 16,000 cell ml-1 and a P. donghaiense cell density of ~ 30,000 cell ml-1. The culture was stirred at a speed of ~ 60 rpm and generally bubbled with air to keep the culture well mixed and aerated. In this condition, the cells grew at a maximum rate of growth as set by the environmental conditions of the culture. The specific growth rate over a 20 minute period was calculated from the optical signal and recorded every minute. 95 The use of growth rate, in this study, refers to the rate calculated based on the change in the optical signals. Experimental Design and Algal Growth Unialgal, but not axenic, P. minimum were grown in the turbidistats in filtered Choptank River water, at a salinity of 15, a temperature of 22 ?C and a 14: 10 h light dark cycle at 430 ?mol photons m-2 sec-1. Modified f/2-medium without silicate was used, modified by reducing nitrate (NO3-) to 40 ?M-N, but by adding PO43- in varying concentration from 0.2 to 10 ?M-P in different turbidistats, therefore providing N:P ratios of 5, 16, 40 and 200. The cells were thus grown in a N:P gradient from relatively low P to low N conditions. Unialgal, but not axenic, cultures of P. donghaiense cells were grown in the turbidistats, in filtered Choptank River water, at a salinity of 30, a temperature of 22 ?C and a 12: 12 h light dark cycle at 430 ?mol photons m-2 sec-1. Modified f/2- medium without silicate was used. The turbidistats were supplied with media NO3- at 80 ?M and PO43- at 8 ?M. Then, the media N:P ratios were gradually changed following day 20. In one treatment, media NO3- concentrations were enriched to ~ 220 ?M-N and PO43- concentrations were gradually decreased to 2 ?M-P. In the second treatment, media NO3- concentrations were enriched to 200 ?M-N and PO43- concentrations were gradually enriched to 140 ?M-P by adding additional PO43- every 3 days. The volume of each turbidistat culture was kept about 1.6 L in the 2 L glass carboys. Before each day of experiments, 100 ml water samples were collected from each tubidostat culture carboy and filtered though pre-combusted (2 h at 400 ?C) 96 Whatman GF/F filters for ambient nutrients analysis. The filtrates were stored frozen at -20?C for later analysis as described below. Subsamples (10 ml) were taken each day of experiments and preserved in 4% glutaraldehyde for algae cell counts. Cells were manually counted by optical microscopy. Nitrogen Uptake and Assimilation Experiments N Uptake Kinetics and Rates of P. minimum P. minimum cells were grown in turbidistat at each N:P ratio for at least 3 days to get a steady growth state before the N uptake experiments were initiated. 15N isotopically-labeled dissolved N substrates were used to measure the culture uptake and assimilation rates (Glibert and Capone 1993) at each N:P ratio. Experiments were conducted around 10 am. Culture samples of 40 ml were dispensed into a series of acid-clean 50 ml incubation cuvettes to which 15N substrates (Cambridge Isotope Laboratories, Inc.) were added respectively. Different experiments involved different substrates and/or concentrations of 15N. Due to high background NO3? concentration in the media, 15N labelled NO3? was only added at a concentration of 5 ?M-N, representing ~ 8% of NO3? media concentration. For the kinetic experiments, 15N labelled NH4+, urea and glycine were added in a gradient of 0.2, 0.5, 1, 5, 20 ?M-N. All the cuvettes were incubated for 0.5 h at 430 ?mol photons m-2 sec-1, and then the incubations were terminated by filtration onto pre-combusted GF/F filters. A 20 ml aliquot for each treatment was filtered directly onto one filter, which was treated with 10% ice-cold Tri-Chloroacetic Acid (TCA) for 10 seconds after filtration. For samples with incoming media at N:P ratio 16 and 200, an extra 20 ml aliquot was filtered to a second filter without further treatment. The TCA-treated filter represents 97 the N substrate assimilated into proteinaceous material, while the untreated filter represents the total N substrate which was taken into the cell. The filters were stored frozen until dried at 50 ?C for 48 h, then analyzed as described below. N Uptake Kinetics and Rates of P. donghaiense Similar to the P. minimum experiments, for P. donghaiense at each N:P ratio, culture samples of 40 ml were dispensed into acid-clean 50 ml incubation cuvettes to which 15N substrates were added. These experiments only involved 15N labelled NO3? and NH4+ added at a concentration of 20 ?M. All the cuvettes were incubated for 0.5 h at 430 ?mol photons m-2 sec-1, and then the incubations were terminated as described above, with one filter untreated and one treated with 10% ice-cold TCA for 10 seconds after filtration. The filters were stored frozen until dried at 50 ?C for 48 h, then subsequently analyzed as described below. Analytical Methods Concentrations of ambient inorganic nutrients (NO3?+NO2?-N, NH4+-N, PO43-- P) were determined using a Technicon Auto-Analyzer (Lane et al. 2000). Concentrations of dissolved free amino acid (DFAA) were determined by fluorometric analysis according to Lindroth and Mopper (1979) and urea was analyzed using the method of Revilla et al. (2005). DFAA concentrations were used to approximate the glycine concentrations in the N uptake rate calculations. Glycine is generally considered to be a dominant amino acid (Degens and Mopper 1976), but their rates of uptake should be considered potential rates based on the uncertainty of their contribution to the DFAA pool. All the 15N sample filters were analyzed for 98 isotope enrichment using a Sercon Mass Spectrometer. Rates of 15N uptake and assimilation were calculated according to Glibert and Capone (1993). Data Analysis The nitrogen uptake parameters were plotted and fitted to the Michaelis-Menten formulation: max s SA A K S? ? or SK SVV s ? ? max where A is the N specific assimilation rate (h-1), Amax is the maximal N specific uptake rate, V is the N specific uptake rate (h-1), Vmax is the maximal N specific uptake rate (h-1), S is the substrate concentration (?M-N) and Ks is the half-saturation constant (?M-N) for the substrate. Amax, Vmax and Ks for each of 4 N sources during the blooms were calculated by the regression of uptake dynamic by Sigmaplot software (Systat Software, Inc.). One way ANOVA analyses were conducted to compare the uptake and assimilation data in the different N:P ratio and light intensity treatments. Results Ambient Parameters and Cell Counts In the P. minimum culture, the ambient N:P ratios in the culture vessels at the time of the experiments were 3.5, 12, 21, 300 (Table 3-1), which were slightly different from the ratios of incoming media (5, 16, 40 and 200). NO3- became depleted (< 2 ?M-N) in the 2 relatively high P treatments (N:P ratio 3.5 and 12), but 99 high concentrations (>20 ?M-N) remained in the two relatively low P treatments (N:P ratio 21 and 300). PO43- was depleted in the treatment where N:P was 13. P. minimum cell densities on the day of experiments were in the range of 14,000 to 18,000 cells ml-1. In the P. donghaiense culture, the ambient NO3? concentration varied from 32.8 ?M to 197 ?M. NO3? concentration decreased with time when supplied with relatively P rich media, and NO3? concentration increased with time when supplied with relatively N rich media. Ambient PO43- concentration varied from 0.33 ?M to 127 ?M. PO43- concentration decreased with time when supplied with relatively N rich media, and PO43- concentration increased with time when supplied with relatively P rich media. The imbalance of N and P resulted in ambient N:P ratios from 0.9 to 600. P. donghaiense cell densities on the day of 15N experiments were relatively constant in the range of 28, 000 to 33, 000 cells ml-1. Culture Growth in the Turbidistat When supplied with a constant media supply, the growth rates of both P. minimum and P. donghaiense in turbidistat showed highly repeatable-diel cycles. Figure 2 is an example of the high frequency, optical-signal growth rates of P. donghaiense during 4 continuous light-dark cycles, supplied with a steady ambient media in which the N:P ratio was 12. Both species showed a clear diel cycle (Fig. 3- 3). Growth rapidly increased at the beginning of the light period and the rates increased to the highest value of the day in less than an hour. Growth rates decreased rapidly when the light was off and the calculated rates were negative in the dark. Similar diel growth cycles were observed for all treatments of both species. 100 In the P. minimum culture, at ambient N:P ratios of 3.5, 12, 21 and 300, the average hourly growth rates averaged over the full day (rday) were 0.0085, 0.024, 0.013 and 0.012 h-1, respectively (Fig.4 a), while the average growth rates for only the light phase (rL) were 0.016, 0.047, 0.027 and 0.026 h-1, respectively (Fig. 3-4 b). In the P. donghaiense culture, the rday varied from ~ 0.004 h -1 to 0.021 h -1 (Fig. 3-4 c), while the rL varied from ~ 0.017 h -1 to 0.052 h -1 (Fig. 3-4 d), when the ambient N:P ratios varied from 0.9 to 600. The maximum rday was recorded at an N:P ratio of 12.0 (157 ?M NO3?, 13.1 ?M PO43-). The maximum rL was recorded at an N:P ratio of 5.8 (51 ?M NO3?, 9.0 ?M PO43-). Both the minimum rday and minimum rL were recorded at an N:P ratio of 1.5 (135 ?M NO3?, 94.0 ?M PO43-), followed by the values at N:P ratios of 264.5 (114 ?M NO3?, 0.44 ?M PO43-) and 288.4 (143 ?M NO3?, 0.5 ?M PO43-). For both species, relative high maximum growth rate (0.021 h-1) were recorded near an N:P ratio of 12, and the maximum growth rates decreased to less than 0.01 h-1 at both very low and very high N:P ratios (Fig. 3-4 c, d). Both rday and rL of the high N:P ratio cultures (N:P ratio > 50) were significantly lower than the lower N:P ratio culture (P = 0.008). Nitrogen Uptake Experiments In the P. minimum experiments, both N uptake and assimilation rates of NH4+, urea, and glycine at the N:P ratios of 12.5 and 300 (the only ratios at which full kinetics were tested) displayed saturating-type kinetics in the concentration range of the experiments (Fig. 3-5). Samples from the low P cultures (i.e., N:P ratio = 300) had higher Vmax and higher Ks than samples from the culture where N:P ratio was 12.5 101 (Table 3-2). Samples from P-rich cultures (i.e., N:P ratio = 3.5) had highest rates of Amax for all 3 N nutrients, but have the lowest Ks, except for glycine (Table 3-2). Samples from the low P cultures had the lowest NH4+ Amax. There were no significance differences between the rates of the 2 relatively N:P balanced cultures (N:P ratio 12.5 and 20). To further our understanding of the N uptake and assimilation rates at ambient concentrations, the uptake and assimilation rates at the lowest 15N enrichment concentration (NO3? 5 ?M-N; other 3 N sources 0.2 ?M-N) were compared (Fig. 3-6). The NO3- uptake rate (V) at N:P ratio 300 (0.030 h-1) was higher than that at an N:P ratio of 12.5 (0.021 h-1). Assimilation rates (A) of NO3- (0.021 h-1) in the relatively P- rich cultures (i.e., N:P ratio = 3.5) were 3 - 4 times higher than the rates at the rest of the N:P treatments. The ambient assimilation rates of the reduced N forms (NH4+, urea and glycine) were also highest in the N:P ratio treatments of 3.5 in all 4 treatments. When N uptake rates were measured at approximate ambient concentrations, NO3- contributed more than 60% percent of the total of the 4 N substrates. In contrast, NH4+ contributed more than 30% of total N uptake rate when N:P ratios were 12 and 300. However, when comparing the assimilation rates with uptake rates, relatively less NO3- (50%), but more NH4+ (over 40%), was assimilated of the 4 N sources. The assimilation efficiencies (assimilation rate/ uptake rate) of all N substrates were higher in P-rich cultures (N:P ratio = 12) relative to P-poor cultures (N:P ratio 300). Also, the assimilation efficiencies of the reduced forms of N (NH4+, urea and glycine) were higher than those of NO3-. The assimilation efficiencies of NO3- were less than 102 40%. The assimilation efficiencies also showed evidence of P limitation, which were lower in high N:P ratio cultures. The assimilation efficiencies of NH4+ in P-poor cultures (21%) were less than half of those in culture with N:P ratio near Redfield ratio. All 4 N:P ratio treatments were supplied with the same concentration of NO3- in the media, and were kept at similar biomass and all grew at their maximum growth rates for the condition of the culture, but the ambient nutrient concentrations varied in the 4 treatments at the time of experiments (Table 3-1). NO3- was the primary N source in the media; however, NO3- must be reduced to NH4+ before being assimilated by the algae. This process requires NO3- reductase as well as ATP and NADPH for energy supply (Lomas and Glibert 1999b, 2000). Phosphorus can be a critical limiting nutrient for the assimilation of NO3-. Therefore, the assimilation of NO3- in the high N:P ratio cultures (i.e., N:P ratio = 300) showed more P limitation, and as a result, there was high NO3- concentration remaining in this culture (Table 3-1). However, in the P-rich cultures (i.e., N:P ratio 12.5 and 3.5), P. minimum took up the NO3- rapidly, resulting in the depletion of ambient NO3- to ~ 1 ?M-N, even though there was a supply of new media during the daytime to dilute the culture as the cells grew. Cells in the P-rich cultures (i.e., N:P ratio 3.5) had significantly higher (p < 0.05) N assimilation rates at all concentrations, indicating that N limitation resulted in high P residuals in the media (Fig. 3-6). Cells in the low N:P ratio cultures also had relative higher assimilation efficiencies (A/V), especially for NO3- and NH4+, indicating that the N substrates were more quickly used by the cells in the P-rich media (Fig. 3-7). 103 There were no significant differences in the relative contributions of the 4 N substrates to total N uptake and assimilation at different N:P ratios. In the P. donghaiense experiments, the N-specific uptake rates (V, h-1) and assimilation rates (A, h-1) varied over different ambient N:P ratios (Fig. 3-7). The uptake rates of NO3? not only decreased from an average of 0.021 h-1 to 0.01 h-1 when the ambient N:P ratios decreased from ~ 7 to ~ 1, but they also significantly decreased to 0.006 h-1 (p < 0.05) when ambient N:P ratio increased from ~ 7 to over 100 (Fig. 3-7 a). The assimilation rates of NO3? were close to 0.01 h-1 where ambient N:P ratio was <10, but they decreased to 0.003 h-1 when ambient N:P ratio were between 10 and 50 (Fig. 3-7 b). They further decreased significantly to ~ 0.0007 h-1 at higher N:P ratios (p < 0.05). The uptake rates of NH4+ varied from 0.046 h-1 to 0.080 h-1, and the assimilation rates of NH4+ varied from 0.028 h-1 to 0.058 h-1 (Fig. 3-7 c, d). The assimilation efficiencies (assimilation rate/ uptake rate) of NO3? significantly decreased from 70 % at low N:P ratios to ~ 6% at high N:P ratios (p < 0.05), while the assimilation efficiencies varied from ~ 60 % to 88 % (Fig. 3-8). Similar to the results in the P. minimum experiments, the uptake and assimilation of NO3- in the high N:P ratio cultures were significantly lower than those in lower N:P ratio cultures (p < 0.05), suggesting strong P control on the uptake and assimilation of NO3-. However, the uptake and assimilation of NH4+, which are less energy costly processes, did not show evidence of N:P ratio regulation. Application of Droops Model The nutrient uptake and assimilation rates represent the processes whereby nutrients are taken up from ambient water, which was measured as V in this 104 experiment, and then assimilated to build the cell, which measured as A. The balance of nutrient uptake and assimilation further affects the growth rate. The ratio of these processes to growth rate allows comparison across treatments. In the P. minimum experiments, although growth rates were lower at highest and lowest N:P ratios compared to near Redfield ratios, the patterns of how A/? values for the 4 N substrates varied at different N:P ratios was opposite (Fig. 3-9). A/? values were therefore higher at the highest and lowest N:P ratios. Under low N concentrations, P. minimum quickly responded to the N pulse and assimilated more than their growth needs in the N:P treatment of 3.5, where P. minimum had lowest growth rate, but highest A/? value. The growth of P. minimum at N:P ratio 12.5 were most balanced of all N:P treatments with the highest average growth rates and lowest A/? value. The A/? values increased when P was reduced. In the P. donghaiense experiments, the N-poor treatments (low N:P ratios) also had a significantly enhanced (p < 0.05) N uptake and assimilation relative to growth rates (Fig. 3-10), and this enhancement decreased as the N:P ratios increased to near- Redfield proportion. The V/? values increased again at high N:P ratios (Fig. 3-10 a, b). However, the A/? values did not increase at the high N:P values, since N assimilation was under a greater degree of P regulation (Fig.10 c, d). Discussion Herein we took an approach to examine the effect of different ambient N:P ratios on the growth and N uptake kinetics of P. minimum and P. donghaiense in turbidistat. For P. minimum, two relatively N-P balanced treatments (N:P ratio 12.5 and 21) and 105 two relatively N:P unbalanced treatments (N:P ratio 3.5 and 300) were tested. For P. donghaiense, over 20 N:P ratio treatments were tested. For both species, the highest growth rates were recorded near the Redfield ratio (16), and growth rate decreased when ambient N:P ratios were more unbalanced, which suggested the regulation of growth rate by the ambient N:P ratio. The N uptake and assimilation rates of the 2 species also showed a response to the different N:P ratio treatments. Value of Tubidostat Approach Batch culture and continuous culture are widely used in laboratory experiments for microalgae culture. The growth of algae in a batch culture typically includes 4 phases: lag phase, exponential, stationary phase, and death phase. The physiology in the exponential phase is very valuable to help understand algal growth. However, the growth of batch cultures are regulated by the limiting nutrient, and batch cultures can not provide a relatively long and steady exponential phase of growth without causing significant change in irradiance caused by self-shading and nutrient depletion. In continuous culture, cells can keep growing in exponential phase and the growth rate can be calculated (Monod 1950). There are two common continuous culture devices, chemostat and turbidistat. Both systems include a medium reservoir and a culture incubator. New media can be pumped into the incubator to support the continuous growth of the cells, and extra culture is removed from the incubator to keep a steady culture volume. In chemostat, steady-state can be established at a fixed rate of limiting nutrient inflow (Yoshida et al. 1979, Rhee and Gotham 1981). In another words, the cell growth rate is equal to the culture dilution rate and nutrient uptake rate is equal to the growth rate when steady state is established (Hirsbrunner 1981, 106 Goldman and Glibert 1982). The dilution rate must be set at a value less than the maximum growth rate to avoid washout. The culture can reach steady state, and the nutrient uptake and cell quota of limited nutrient are assumed to be invariant with time (Rhee 1973, Rhee 1978, Gotham and Rhee 1981). Higher biomass can be achieved by increasing the nutrient concentration of the media. Furthermore, chemostat cultures are typically operated in continuous light (Falkowski et al. 1985), which does not exist in natural condition. A cyclostat is a modified chemostat system in which culture grows under light-dark cycles (Gotham and Frisch 1981, Gotham and Rhee 1982). In cyclostats, nutrient uptake rate, growth rate, and cell quota are not invariant, but have been demonstrated to have periodicities relating to light-dark cycles (Eppley and Renger 1974, Chisholm et al. 1975, Malone et al. 1975, Frisch and Gotham 1977). The culture can reach rhythmic steady state with a period of light- dark cycle. Alternatively, turbidistats are run under turbidity control, and new medium inflow only occurs in response to an increase in the culture biomass to a pre- set value. Biomass is set by the optical threshold of dilution. Cell growth is limited by light and temperature; nutrients are usually added in excess of demand (Watson 1972, Skipnes et al. 1980, Fenaux et al. 1985, Hill et al. 1985, Kalyuzhin 1998). Light in turbidistat can run in a light-dark cycle which is more close to real situations (Falkowski et al. 1985). The independent and dependent variables are opposite in chemostat and turbidistat. For example, cell density is dependent and growth rate is independent in chemostat, but cell density is independent and growth rate is dependent in turbidistat (Bennett and Boraas 1989). Turbidistats are better than chemostats for the 107 experiments conducted which study cells growing near their maximum specific growth rate. In the experiments conducted herein, both species achieved continuous growth and repeatable diel cycle in turbidistat. The cells grew at their maximum growth, which was regulated by the ambient nutrient condition (N:P ratios). However, due to the dilution process, the mass balance in the turbidistats changed due to the difference between N and P mass in the culture removed and the new media pumped in. For example, a P. minimum cell in exponential phase has ~ 2.1 ?10-6 ?M-N per cell (Li unpublished data), which represents ~ 34 ?M-N and ~ 2.1 ?M-P mass of cells at a cell density of 16, 000 cell ml-1, with the assumption that the N:P ratio of cells was near the Redfield ratio. The fastest growing culture (ambient N:P ratio 12) took up ~1.6 ?M-N h-1 in the light phase, based on an average growth rate of 0.047 h-1. While the total ambient N (sum of 4 N sources) was only 3.2 ?M- N, which could only support the growth for 2 hrs, there was very limited residual N in the culture, and the growth relied on the new media pumped into the culture. The total N and P mass pumped from new media (enriched with 40 ?M-N and 2.5 ?M-P) was approximately equal to the N removed from the culture in cells and media. The growth of culture near the Redfield ratio was balanced. However, in the low N:P ratio culture (N:P ratio 3.5), there were also very limited residual N and P in the culture. The P in the new media was much higher than that in the culture and in the cells, as a result, the culture became more N limited. In the culture growing at ambient N:P ratio of 300, the residual N was high (over 40 ?M-DIN), but the P was low (0.15 ?M-DIP), which supported the growth for less than 3 hrs. Furthermore, the P in the new media was very low (0.2 ?M-N), which was much less than the P in the cells. When the 108 dilution started, the P pumped in the culture was less than the P removed in the cells, and the culture became more P limited. Value of Droop?s Model Approach Based on the growth rates recorded by the turbidistat, the growth of both species showed strong light-regulated diel cycles. Cell growth, measured as the change of turbidity in the culture vessel, started at the beginning of light cycle. Dinoflagellate species have been reported to have a maximum growth limit of 1 division per day (McDuff and Chisholm 1982), as a result of dark: light regulated diel synchrony. The growth rates in the tubidistat system were consistent with these reports. The maxima growth rates in the light phase were around 1 day-1. Maximum growth rate generally occurred in the first hour at the beginning of the light phase, and growth rate decreased during the day time. The growth rate, in this study, refers to the rate of changing of the optical signals, which attenuated with the turbidity of the culture. The turbidity was caused by the total mass concentration in the culture. Therefore, the growth rate is interpreted as the variation rate of total mass density of the culture, instead of cell density, which was calibrated by diluted the culture by volume. During the dark phase, there were negative growth rates, suggesting decreases in mass concentration in the culture. When the photosynthesis activities stopped in the dark phase, the production rates of these species were less than the respiration rate, causing the negative growth rate. This state lasted until the start of the light cycle, when production rates increased quickly over respiration and the turbidity of the culture increased again. 109 Based on Droop?s model, these Prorocentrum species had their maximum cell quotas when they reached the highest growth rates at the beginning of light phase. The decreasing growth rates during the day are indicative of decreasing cell quotas. However, the low growth rates in the dark cycle were not necessary indicative of a small cell quota, since the cells were light regulated instead of nutrient regulated. P. minimum has been reported to continuously uptake the NO3- in the dark (Paasche et al. 1984). Cells stopped growth at night due to light limitation, but nutrients were likely continuously taken up into and stored in the cell, causing an increasing cell nutrient quota (Fig. 3-11). The cell quota reached its maximum value of the day at the beginning of light phase, as did growth rates. Since the experiments conducted here were not focused on the diel cycle of Prorocentrum growth and nutrient uptake, the conceptual model of diel cell quota variation (Fig. 3-11) needs to be tested by further studies. The N:P Ratios in the Coastal Ecosystems There has been evidence of a global increase of harmful algal blooms (HABs), which have increased not only in frequency, but also in intensity and geographic distribution in the coastal area, and have caused ecological, economical and human health problems (Smayda 1997, Anderson et al. 2002). In many cases, the increase in HABs has been strongly correlated to the global increase of eutrophication (Hallegraeff 1993, Glibert and Terlizzi 1999, Anderson et al. 2002, Smayda 2002, Glibert et al. 2005a, Glibert and Burkholder 2006, Glibert et al. 2006, Anderson et al. 2008, Glibert et al. 2008a, Heisler et al. 2008). Growth of human population, agriculture fertilization and urbanization has caused remarkable increase in N and P 110 loading to the coastal water. The progress toward controlling N loading has been relatively slow, and N load to the coastal environments has been increasing in the past several decades (NRC 2000). In the U.S., N fertilizer consumption has increased over 4 fold since 1960s, however, P fertilizer consumption only increased 80% up to 2006 (USDA 2008). Actually, the consumption of P fertilizer reached its peak in 1979 and has slightly decreased after that year (USDA 2008). There were also downward trends of P concentrations in many riverine sources (Litke 1999). One of reasons is that the principal source of effluent P was from P in laundry detergent, which has been gradually replaced by P-free detergent since the late 1970s (Doemel and Brooks 1975, Sharfstein et al. 1977, Hartig and Horvath 1982, Maki et al. 1984). Therefore, the increases of N and P loading to coastal ecosystems have not been proportionate. As the P-detergent banned expanded globally, export of dissolved inorganic P (DIP, mostly in form of phosphate), the most important form of bio-available P source, did not increase as much as N. This unbalanced increase of N and P nutrients loading has contributed to the change of the N:P ratios in the coastal water. Changes of ambient N:P ratios could regulate the phytoplankton community composition and HAB developments (Hodgkiss and Ho 1997). In freshwater, N- fixing cyanobacteria could dominate in a low N:P ratio water column by fixing its own N source (Vrede et al. 2009). Good N competitors, including diatoms, may dominate the low N:P ratio community, but good P competitors (e.g. Synechococcus) may dominate the high N:P ratio community (Suttle and Harrison 1988). In seawater, a shift of dominant species in the phytoplankton community caused by the alternation of ambient N:P ratios could happen in time ranges of seasons to decades (Phillips and 111 Tanabe 1989, Bulgakov and Levich 1999, Lagus et al. 2004, Paerl et al. 2004). For example, the phytoplankton community of downstream Neuse River, USA, shifted from an N-limited cyanobacteria dominant freshwater phytoplankton community to a P-limited community after the P-detergent ban in 1988 in this area (Paerl et al. 2004). As a result of P loading reduction, N substrates taken up by phytoplankton decreased and more N was delivered to the lower estuary which stimulated HABs comprised of dinoflagellates, cryptomonads, and diatoms (Paerl et al. 2004). Spatially, high N:P ratio runoff mixed with relatively low N:P ratio seawater can develop a gradient of N:P ratio, which may regulate not only the HAB distribution and biomass, but also dominant species and its nutrient uptake rates and preference (Fisher et al. 1992, Li et al. 2009, Li et al. 2010). In summary, the study of the growth and N uptake of P. minimum (a global HAB species) and P. donghaiense (a regional HAB species) on different N:P ratios showed evidence that N:P ratios could not only affect the growth, but also N uptake and assimilation. Relatively N:P balanced cultures reached the highest growth rates. N- poor cultures showed an enhanced N uptake in N enrichment experiments. Highest average daily growth rates of both species were recorded near N:P ratios of 12. Highest N assimilation rates for 4 N sources by P. minimum were recorded at N:P ratio of 3.5. P. donghaiense also has highest N assimilation coefficient at low N:P ratio. 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Journal of Fermentation Technology 57:546-553 Zhou M, Yan T, Zou J (2003) Preliminary analysis of the characteristics of red tide areas in Changjiang River estuary and its adjacent sea. Chinese Journal of Applied Ecology 14:1031-1038 120 Tables Table 3-1 Ambient nutrient concentrations in 4 different turbidistat systems supplied by media at different DIN:DIP ratios. Medium Enriched N:P Ratio NO3-?M-N NH4+?M-N Urea ?M-N DFAA ?M-N DIP ?M-P Culture DIN:DIP Ratio 5 1.95 0.05 1.18 0.063 0.57 3.5 16 0.37 1.05 3.14 0.011 0.11 12.5 40 21.98 3.59 2.09 0.038 1.17 21 200 42.4 2.52 2.05 1.123 0.15 300 121 Table 3-2 Calculated parameters of uptake kinetics of nitrogen sources (NH4+, urea, and glycine) by Prorocentrum minimum at different ambient DIN:DIP ratio in turbidistat based on the Michaelis-Menten equation. Vmax and Amax are the specific nitrogen uptake and assimilation rates. Ks is the Michaelis constant where uptake rates equal to ? of maximum uptake rates. Culture DIN:DIP NH4+ Urea Glycine NH4+ Urea Glycine Ratio Vmax Ks Vmax Ks Vmax Ks Amax Ks Amax Ks Amax Ks 3.5 0.031 0.20 5E-04 0.43 0.004 1.27 12.5 0.046 1.248 3E-04 0.052 0.004 0.48 0.024 2.32 7E-04 16.50 0.001 0.04 21 0.02 1.43 4E-04 2.63 0.001 0.10 300 0.065 4.646 8E-04 5.43 0.007 3.54 0.016 2.70 4E-04 1.56 0.003 3.39 122 Figures Figure 3-1 The setup of a turbidistat system 123 00:00 12:00 00:00 12:00 00:00 12:00 00:00 12:00 00:00 Gr ow th Ra te pe r H ou r -0.10 -0.05 0.00 0.05 0.10 0.15 Figure 3-2 Diurnal growth rates of Prorocentrum donghaiense in turbidistat system for 4 constant 12 h: 12 h light dark cycles (96 hrs). Each point stands for the growth rate calculated based on the change in the optical signals over a 20-minute time period. 124 a. P. minimum 0 2 4 6 8 10 12 14 16 18 20 22 24 Gr ow th Ra te pe r H ou r -0.10 -0.05 0.00 0.05 0.10 0.15 b. P. donghaiense 0 2 4 6 8 10 12 14 16 18 20 22 24 Gr ow th Ra te pe r h ou r -0.10 -0.05 0.00 0.05 0.10 0.15 Figure 3-3 a. Diurnal growth rates of Prorocentrum minimum in turbidistat system at 14: 10 h light dark cycle. b. Diurnal growth rates of Prorocentrum donghaiense in turbidistat system at 12: 12 h light dark cycle. Box plots ? line: median; box: 25th and 75th percentiles; whiskers: 10th and 90th percentiles; dots: outliers. 125 N:P ratio 3.5 12 21 300 G r o w t h R a t e p e r H o u r 0.00 0.01 0.02 0.03 0.04 0.05 0.06 a. P. minimum b. P. minimum N:P ratio 3.5 12 21 300 G r o w t h R a t e p e r H o u r 0.00 0.01 0.02 0.03 0.04 0.05 0.06 c. P. donghaiense Lg (N:P ratio) 0.0 .5 1.0 1.5 2.0 2.5 3.0 G r o w t h R a t e p e r h o u r 0.00 .01 .02 .03 .04 .05 .06 d. P. donghaiense Lg (N:P ratio) 0.0 .5 1.0 1.5 2.0 2.5 3.0 G r o w t h R a t e p e r h o u r 0.00 .01 .02 .03 .04 .05 .06 Figure 3-4 a. Average growth rates of Prorocentrum minimum in a 14h: 10h light dark cycle at different ambient N:P ratios. b. Average growth rates of P. minimum in the light phase of 14h: 10h light dark cycle at different ambient N:P ratios. c. Average growth rates of P. donghaiense in a 12h: 12h light dark cycle at different ambient N:P ratios. d. Average growth rates of P. donghaiense in the light phase of 12h: 12h light dark cycle at different ambient N:P ratios 126 NH4+ ?M-N 0 5 10 15 20 25 V h o u r - 1 0.00 0.02 0.04 0.06 0.08 Urea ?M-N 0 5 10 15 20 25 V h o u r - 1 0.0000 0.0002 0.0004 0.0006 0.0008 Glycine ?M-N 0 5 10 15 20 25 V h o u r - 1 0.000 0.002 0.004 0.006 0.008 NH4+ ?M-N 0 5 10 15 20 25 A h o u r - 1 0.00 0.02 0.04 0.06 0.08 Urea ?M-N 0 5 10 15 20 25 A h o u r - 1 0.0000 0.0002 0.0004 0.0006 0.0008 Glycine ?M-N 0 5 10 15 20 25 A h o u r - 1 0.000 0.002 0.004 0.006 0.008 Figure 3-5 Kinetic relationships between nitrogen specific uptake rates V (h-1, upper panels) and assimilation rates A (h-1, lower panels) of NH4+, urea, and glycine with 15N substrate concentration (?M-N) by Prorocentrum minimum at different DIN:DIP ratio cultures. All curves R2 > 0.6. 127 NO3- DIN:DIP Ratio 3.5 12.5 21 300 p e r H o u r 0.00 0.01 0.02 0.03 0.04 Uptake Rate Assimilation Rate NH4+ DIN:DIP Ratio 3.5 12.5 21 300 p e r H o u r 0.00 0.01 0.02 0.03 0.04 Uptake Rate Assimilation Rate Urea DIN:DIP Ratio 3.5 12.5 21 300 p e r H o u r 0.0000 0.0001 0.0002 0.0003 0.0004 0.0005 Uptake Rate Assimilation Rate Glycine DIN:DIP Ratio 3.5 12.5 21 300 p e r H o u r 0.000 0.001 0.002 0.003 0.004 0.005 Uptake Rate Assimilation Rate Figure 3-6 Nitrate specific uptake rates V (h-1) and assimilation rates A (h-1) of NH4+, urea, and glycine with 15N substrate concentration (?M-N) by Prorocentrum minimum grown at different DIN:DIP ratios. 128 a. NO3- Lg (N:P ratio) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 U p t a k e R a t e p e r h o u r 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 b. NO3- Lg (N:P ratio) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 A s s i m i l a t i o n R a t e p e r h o u r 0.000 0.005 0.010 0.015 0.020 0.025 c. NH4+ Lg (N:P ratio) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 U p t a k e R a t e p e r h o u r 0.00 0.02 0.04 0.06 0.08 0.10 Lg (N:P ratio) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 A s s i m i l a t i o n R a t e p e r h o u r 0.00 0.02 0.04 0.06 0.08 0.10 d. NH4+ Figure 3-7 a. The NO3? specific uptake rates (V, h-1) of Prorocentrum donghaiense grown at different ambient N:P ratios; b. The NO3? specific assimilation rates (A, h-1) of P. donghaiense grown at different ambient N:P ratios. c. The NH4+ uptake rates of P. donghaiense grown at different ambient N:P ratios; d. The NH4+ assimilation rates of P. donghaiense grown at different ambient N:P ratios. 129 a.NO3- Lg (N:P ratio) 0.0 .5 1.0 1.5 2.0 2.5 3.0 A / V % 0 20 40 60 80 100 b. NH4+ Lg (N:P ratio) 0.0 .5 1.0 1.5 2.0 2.5 3.0 A / V % 0 20 40 60 80 100 Figure 3-8 The assimilation efficiencies % (assimilation rate/ uptake rate) of NO3? by Prorocentrum donghaiense grown at different ambient N:P ratios; b. The assimilation efficiencies % (assimilation rate/ uptake rate) of NH4+ by P. donghaiense grown at different ambient N:P ratios 130 N:P Ratio A / ? 0 1 2 3 4 NO3- NH4+ Urea Glycine 3.5 12.5 20 300 Figure 3-9 The ratio of specific N assimilation rate/ growth rate (A/?) of Prorocentrum minimum for major N sources at different DIN:DIP ratios 131 a.V/ ????NO3- Log10(N:P ratio) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0 1 2 3 4 5 b. V/ ????NH4+ Log10(N:P) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.0 0.5 1.0 1.5 2.0 c. A/ ????NO3- Lg(N:P ratio) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.0 0.5 1.0 1.5 2.0 d. A/ ????NH4+ Lg(N:P ratio) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Figure 3-10 The ratio of specific N uptake rate/ growth rate (V/?) of Prorocentrum donghaiense for NO3- (a) and NH4+ (b) at different ambient N:P ratios; the ratio of specific N assimilation rate/ growth rate (A/?) of P. donghaiense for NO3- (c) and NH4+ (d) at different ambient N:P ratios 132 Figure 3-11 The conceptual model of the diel cell nutrient quota variation in a light-dark cycle. Q is the cell nutrient quota; the shading area stands for dark phase in a diel cycle; the filled arrows stand for the nutrients uptake from ambient water into the cell quota; the dash arrows stand for the process that nutrient in the cell quota is assimilated to build the cell. 133 Chapter 4: Effects of Light on Growth and Nitrogen Uptake of Prorocentrum minimum and Prorocentrum donghaiense Abstract The effects of varying light irradiance on the growth, nitrogen (N) uptake and assimilation of the harmful dinoflagellate Prorocentrum minimum and Prorocentrum donghaiense were examined in turbidistat culture experiments. Algal cultures were grown in a wide range of ambient light intensities from high light to dark, when their growth rates and N acquisition were studied. Experiments to determine rates of N uptake and assimilation of different N sources (NO3?, NH4+, urea and glycine by P. minimum and NO3?, NH4+ by P. donghaiense) were conducted using 15N tracer technique at each light irradiance. The growth rates suggested strong light regulation on the growth of P. donghaiense. On a diel basis, the growth of both species was regulated by the light-dark cycle, which may be a result of regulation of both light- dependent growth and light-independent N nutrient uptake. Maximum growth rates of both species always occurred at the beginning of light phase. N uptake by P. minimum for all tested substrates showed no obvious light limitation on a short-term basis. However, NH4+ uptake of P. donghaiense showed significant light limitation on long-term basis. The light independent N uptake by P. minimum and P. donghaiense allow them to be competitive in the relative low light available near-shore water. However, the capability of light independent N uptake between the two Prorocentrum species might be different. 134 Introduction An increasing frequency and intensity of harmful algae blooms (HABs) has been observed in coastal waters all over the world and these events threaten the structure and function of coastal ecosystems (Smayda 1990, Hallegraeff 1993, Anderson 1997, Moncheva et al. 2001, Heil et al. 2005, Wang and Wu 2009). It has been suggested that many HABs are correlated with the acceleration of eutrophication in coastal ecosystems (Hallegraeff 1993, Anderson et al. 2002, Glibert et al. 2005, Glibert and Burkholder 2006, Glibert et al. 2006, Glibert et al. 2008a, Heisler et al. 2008). Smayda (2001) suggested that dinoflagellates, a major group of bloom- forming algae, have diverse habitat preferences and adaptive strategies and characterized dinoflagellate bloom species in coastal areas in a nutrient-turbulence matrix (Smayda and Reynolds 2001, Smayda 2002). Each dinoflagellate species has its specific niche for bloom expansion, and the niches are described by available nutrients and turbulence/mixing intensity. In this matrix, Prorocentrum spp. (length 15~22 ?m width 9~14 ?m) are Type II species which are small, rapidly growing competitors blooming in nutrient-rich near-shore regions. Prorocentrum minimum has caused HAB problems in both tropical and temperate coastal waters (Heil et al. 2005, Glibert et al. 2008b). It is a common HAB species in coastal and estuarine systems in USA, and frequently develops blooms in the Chesapeake Bay and southwest Florida shelf (Glibert et al. 2001, Tango et al. 2005, Heil et al. 2007). Prorocentrum donghaiense, is a major bloom species near the Changjiang Estuary and in the coastal area of East China Sea. Large scale P. donghaiense blooms have been observed in the past two decades (Zhou et al. 2006, Li 135 et al. 2009, Li et al. 2010). Prorocentrum minimum and P. donghaiense are very similar species and can be classified in the same category in the Smayda?s model, and their expanding geographical distribution is indicative of a strong relationship to terrestrial nutrients discharge into coastal waters (Heil et al. 2005, Glibert and Burkholder 2006, Glibert et al. 2008a). However, there are still differences between the habitats of these two species. Prorocentrum donghaiense can only grow well in high salinity (25 - 35) water (Xu et al. 2010). The high frequent bloom areas of P. donghaiense have been documented mostly in the adjacent area of the Changjiang River estuary, which is the largest river of the China (Zhou et al. 2003, Zhou et al. 2006). The Changjiang River water develops huge plumes from the river mouth, with characteristically high nutrients, high turbidity, but low salinity water (Lie et al. 2003). The distribution of P. donghaiense is usually observed relatively off-shore near the edge of the river plume, where these cells could access the riverine nutrients, but still remain in the water column with high clarity (Li et al. 2009). On the other hand, P. minimum can grow over a broad salinity range, and develops blooms in the coastal water with salinity as low as 5 (Heil et al. 2005, Tango et al. 2005). In the Chesapeake Bay, high frequent P. minimum were documented in the muddy tributaries, where the secchi depth was only ~ 1m (Tango et al. 2005). Considering these field observation, it is reasonable to suggest these two species can be exposed to different light in these habitats, when they develop blooms. Studying the effects of light on the growth and nutrient uptake of these two species will be helpful to understand the mechanisms of bloom development. Previous studies suggested that P. minimum grew well under low light conditions (Grzebyk and 136 Berland 1996), and show light-independent N uptake ability (Fan and Glibert 2005). However, according to recent literature review, there is no information on the influence of light on the N uptake of P. donghaiense. Herein, the utilization of N nutrients by the phytoplankton are described as two steps. Nitrogen uptake is the step in which N nutrients are transported across the cell membrane into the nutrient pool inside the cell. When taken up into the cell, NO3? and NO2? are reduced to NH4+with the aid of NO3?reductase or NO2?reductase, or both (Lomas and Glibert 2000). Reduced forms of N nutrients can be utilized in the synthesis of organic compounds, which is the step of assimilation (Conway 1977, Glibert and McCarthy 1984). In this study, cultures of P. minimum and P. donghaiense were grown across a light irradiance spectrum in order to achieve a range of growth rates and a range of physiological states. Ambient N:P ratio, which has been reported to be related to the regulating growth and physiology of these two species (Li et al. 2009, Li et al. in press), was also considered as a variable in the experiments of P. minimum. The effect of light on these two species was tested on growth rate and the rates of uptake of different forms of N, as were the comparative rates of uptake and assimilation. Materials and Methods Algal Culture Cultures of Prorocentrum minimum were isolated from Choptank River, a tributary of Chesapeake Bay in spring 1995 and were maintained in f/2 medium in the 137 Horn Point Laboratory culture collection. For the present experiments, P. minimum cells were first transferred to f/2 batch cultures for 2 weeks, then grown in turbidistat, an automatic continuous culture system, as described in Li et al. (in press), at a cell density ~ 16,000 cell ml-1. Cultures of Prorocentrum donghaiense were obtained from University of Connecticut Department of Marine Sciences, USA. These cultures were originally isolated from the East China Sea. They, too, were transferred to f/2 batch cultures for several weeks, then grown in turbidistat at a cell density ~ 30,000 cell ml-1. Continuous Culture Design A continuous turbidistat culturing device was used to grow the Prorocentrum cultures. A modulated infrared (880 mm) LED beam was directed though the culture vessel (a 2 L glass carboy) to a photosensor, the analog signal output of which was related to optical transmission (i.e., turbidity). Real time turbidity signals were sent to a computer that controlled a peristaltic pump which diluted the culture based on attaining a threshold value. The system was programmed to dilute the culture by 5% in terms of the optical signal. This corresponds to a volume dilution of approx. 10%, based on calibrations using a volumetric dilution. The threshold was set to a turbidity that represented a P. minimum cell density of ~ 16,000 cell ml-1 and a P. donghaiense cell density of ~ 30,000 cell ml-1. The culture was stirred at a speed of ~ 60 rpm and generally bubbled with air to keep the culture well mixed and aerated. In this condition, the cells grew at a maximum rate of growth as set by the environmental conditions of the culture. The specific growth rate over each 20 minute period was 138 calculated from the optical signal and recorded every minute. The use of growth rate, in this study, refers to the rate calculated based on the change in the optical signals. Experimental Design and Algal Growth Unialgal, but not axenic, P. minimum were grown in the turbidistats in filtered Choptank River water, at a salinity of 15, a temperature of 22 ?C and a 14 h: 10 h light dark cycle at 430 ?mol photons m-2 sec-1. Modified f/2-medium without silicate was used, modified by reducing nitrate (NO3-) to 40 ?M-N, but by adding PO43- in varying concentrations from 0.2 to 10 ?M-P in different turbidistats, therefore providing N:P ratios of 5, 16, 40 and 200. The cells were thus grown in a N:P gradient from relatively low P to low N conditions. Unialgal, but not axenic, cultures of P. donghaiense cells were grown in the turbidistats, in filtered Choptank River water, at a salinity of 30, a temperature of 22 ?C and a 12 h: 12 h light dark cycle at 500 ?mol photons m-2 sec-1. Modified f/2- medium without silicate was used. The turbidistat were supplied with media NO3- at 80 ?M and PO43- at 5 ?M. The volume of each turbidistat culture was kept about 1.6 L in the 2 L glass carboys. Before the initiation of experiments, 100 ml water samples were collected from each turbidistat culture and filtered though pre-combusted (2 h at 400 ?C) Whatman GF/F filters for ambient nutrient analysis as described below. 139 Nitrogen Uptake and Assimilation Experiments N Uptake of P. minimum Prorocentrum minimum cells were grown in turbidistat at each N:P ratio for at least 3 days to obtain a steady growth state before the N uptake experiments were initiated. 15N isotopically labeled dissolved N substrates were used to measure the culture uptake rates (Glibert and Capone 1993) across a light irradiance spectrum at each N:P ratio. Experiments were conducted around 12 pm. Culture samples of 40 ml were dispensed into a series of acid-clean 50 ml incubation cuvettes to which 15N substrates (Cambridge Isotope Laboratories, Inc.) were added respectively. Considering NO3? was the primary N nutrient in the media, 15N labelled NO3? was added at a concentration of 20 ?M-N. 15N labelled NH4+, urea and glycine were added at 2 ?M-N. Each group of cuvettes, after adding the 4 substrates, were incubated for 0.5 h in a light irradiance gradient of 9, 35, 200, 430 ?mol photons m-2 sec-1. Irradiance was obtained by covering the cuvettes with different number layer of black mesh screens. The incubations were terminated by filtration onto pre-combusted GF/F filters. A 20 ml aliquot for each treatment was filtered directly onto one filter. The filters were stored frozen until dried at 50 ?C for 48 h, then analyzed as described below. N Uptake and Assimilation of P. donghaiense Prorocentrum donghaiense cells were grown in turbidistats at a light irradiance gradient of 500, 200, 66, 16, 5 ?mol photons m-2 sec-1. Cells were grown at each light irradiance for at least 3 days. Experiments were conducted around 10 am. Culture samples of 40 ml were dispensed into acid-clean 50 ml incubation cuvettes to 140 which 15N substrates were added. These experiments only involved 15N labelled NO3? and NH4+. NO3? was added in a gradient of 1, 2, 5, 10, 25, 37.5, 50 ?M-N. NH4+ was added in a gradient of 0.1, 0.5, 2, 5, 10, 20 ?M-N. All the cuvettes were incubated for 0.5 h at the same light irradiance which the cultures were grown at the time of experiments. The incubations were terminated by filtration onto pre-combusted GF/F filters. A 20 ml aliquot for each treatment was filtered directly onto one filter, which was treated with 10% ice-cold Tri-Chloroacetic Acid (TCA) for 10 seconds after filtration. An extra 20 ml aliquot was filtered to a second filter without further treatment. The TCA-treated filter represents the N substrate assimilated into proteinaceous material, while the untreated filter represents the total N substrate which was taken into the cell. The filters were stored frozen until dried at 50 ?C for 48 h, then analyzed as described below. Analytical Methods Concentrations of ambient inorganic nutrients (NO3?+NO2?-N, NH4+-N, PO43- -P) were determined using a Technicon Auto-Analyzer (Lane et al. 2000). Concentrations of dissolved free amino acid (DFAA) were determined by fluorometric analysis according to Lindroth and Mopper (1979) and urea was analyzed using the method of Revilla et al. (2005). DFAA concentrations were used to approximate the glycine concentrations in the N uptake rate calculations. Glycine is generally considered to be a dominant amino acid (Degens and Mopper 1976), but their rates of uptake should be considered potential rates based on the uncertainty of their contribution to the DFAA pool. All the 15N sample filters were analyzed for 141 isotope enrichment using a Sercon Mass Spectrometer. Rates of 15N uptake and assimilation were calculated according to Glibert and Capone (1993). Data Analysis The nitrogen uptake parameters relating to ambient N concentrations were plotted and fitted to the Michaelis-Menten formulation: SK SVV s ? ? max where V is the N specific uptake rate (h-1), Vmax is the maximal N specific uptake rate (h-1), S is the substrate concentration (?M-N) and Ks is the half-saturation constant (?M-N) for the substrate. Vmax and Ks for each N source were calculated by using Sigmaplot software (Systat Software, Inc.). The growth rate parameters relating to ambient light irradiances were also plotted and fitted to a modified formulation (Baly 1935, Aalderink and Jovin 1997): max I Ir r DK I? ?? where r is the specific growth rate (h-1), rmax is the maximal growth rate, I is the ambient light irradiance (?mol photons m-2 sec-1), KI is the half-saturation light irradiance (?mol photons m-2 sec-1), and D is the specific dark respiration rate (h-1), which is generally considered opposite to the growth process. rmax, KI and D for P. donghaiense were also calculated by the regression of dynamic by Sigmaplot software (Systat Software, Inc.). Two way ANOVA analyses were conducted to compare the growth and uptake and assimilation data in the different N:P ratio and light intensity treatments. 142 Results Ambient Parameters In the P. minimum culture, the ambient N:P ratios in the culture vessels at the time of the experiments were 3.5, 12, 21, 300 (Table 4-1), which were slightly different from the ratios of incoming media (5, 16, 40 and 200). NO3- became depleted (< 2 ?M-N) in the two relatively high P treatments (N:P ratios 3.5 and 12), but high concentrations (>20 ?M-N) remained in the two relatively low P treatments (N:P ratios 21 and 300). PO43- was depleted in the treatment where N:P ratio was 13. In the P. donghaiense culture, the ambient NO3- concentration varied from 1.9 ?M-N to 17.8 ?M-N, and ambient NH4+ concentration varied from 0.72 ?M-N to 2.57 ?M- N. Growth Rate in the Turbidistat When supplied with a constant media supply, the growth rates of both P. minimum and P. donghaiense in turbidistats showed highly repeatable diel cycles, and cells reached high frequency optical-signal continuous growth in light-dark cycles (e.g. Fig. 4-1). Both species showed a clear diel cycle (Fig. 4-2). Growth rapidly increased at the beginning of the light period and the rates increased to the highest value of the day in less than an hour. Growth rates decreased rapidly when the light was off and the calculated rates were negative in the dark, which indicated the decrease of turbidity in the dark. Similar diel growth cycles were observed for all treatments of both species. 143 In the P. minimum cultures, at ambient N:P ratios of 3.5, 12, 21 and 300, the hourly growth rates averaged over the full day (rday) were 0.0085, 0.024, 0.013 and 0.012 h-1, respectively (Fig. 4-3 a), while the average growth rates for only the light phase (rL) were 0.016, 0.047, 0.027 and 0.026 h-1, respectively (Fig. 4-3 b). The maximum rday and rL was recorded at a N:P ratio of 12.0 (157 ?M NO3?, 13.1 ?M PO43-). In the P. donghaiense cultures, the growth rates decreased as the ambient irradiance decreased (Fig. 4-4). The rday decreased from ~ 0.022 h -1 at 500 ?mol photons m-2 sec-1 to ~ 0.011 h -1 at 5 ?mol photons m-2 sec-1 (Fig. 4-5), while the rL decreased from ~ 0.071 h -1 at 500 ?mol photons m-2 sec-1 to -0.014 h -1 at 5 ?mol photons m-2 sec-1. There was no evidence of high light growth inhibition in the light range of experiments. For P. donghaiense, the average rmax over one light-dark cycle was 0.042 h -1, with half saturation coefficient (KI) of 34 ?mol photons m-2 sec-1 and respiration rate (D) of 0.017 h -1. The rmax averaged in the light phase was 0.097 h -1, with KI of 56 ?mol photons m-2 sec-1 and D of 0.021 h -1. Nitrogen Uptake Experiments In the P. minimum experiments, the N uptake rates did not show significant light limitation in the irradiance range of experiments (Fig. 4-6). The uptake rates of NO3- , NH4+, urea and glycine in the relatively high-P culture (N:P ratio 3.5) were significantly higher than in the low-P cultures (p< 0.05). There were no significant differences among the NO3- , NH4+ uptake rates in these higher N:P ratio cultures. A significant light inhibition of N uptake rates was only observed when the average uptake rates at relatively low light (9 and 35?mol photons m-2 sec-1) and relatively high light (200 at 430 ?mol photons m-2 sec-1) were compared for the culture growing 144 at ambient N:P ratio of 3.5 (Fig. 4-7). Although NO3- was the major N nutrient in the total of the 4 N substrates in the culture, NH4+ contributed about 70% of total N uptake rate (sum of uptake rate of 4 N nutrients), while NO3- contributed about 18% of total N uptake rate. Glycine contributed about 11% of total N uptake rate. The uptake rates of urea were very low. In the P. donghaiense experiments, the VNO3- was about 0.002 h-1 near ambient concentration, and increased to 0.005 - 0.01 h-1, when enriched with 50 ?M-NO3-. Although V NO3- did not saturate over the concentration range tested, Vmax values can be estimated mathematically (Fig. 4-8). The highest calculated V maxNO3- was 0.032 h- 1, measured in lowest light. However, the second highest calculated V maxNO3- was 0.022 h-1, measured in highest light. The differences among NO3- uptake rates under different light treatments were not significant. V NH4+ reached saturation over the concentration range tested. The VNH4+ in the 2 highest light treatments were significantly higher than the 2 lowest light treatments. V max NH4+ varied from 0.02 to 0.07 h-1. The half-saturation constant Ks can be considered an index of competitive ability of bloom species at low nutrient concentration. Since the measured rates of NO3- uptake at some irradiance levels did not saturate over the concentration range tested, the Ks values were also estimated mathematically to be very high. Calculated Ks values of NO3- uptake were in range of 13.5 to 156.6 ?M-N. The uptake rate curve was almost linear with a high slope with high Ks (Table 4-2). The KsNH4+ values were negatively correlated to the ambient light, and increased from ~1 to over 20 ?M-N as the ambient light irradiance decreased. 145 Not all the N substrate taken up into the cells was assimilated into proteinaceous material during the incubation time. Here, the two highest light irradiance, 500 and 200 ?mol photons m-2 sec-1, were averaged, and the two lowest light irradiance, 5 and 16 ?mol photons m-2 sec-1, were also averaged to compare high and low light rates. Near the ambient concentration level, P. donghaiense had higher VNO3- (~ 0.003 h-1) and ANO3- (~ 0.0007 h-1) in the low light than high light, however, it had higher VNH4+ (~ 0.017 h-1) and ANH4+ (~ 0.07 h-1) in the high light than low light (Fig. 4-9). The VNH4+ and ANH4+ is an order of magnitude higher than VNO3- and ANO3- respectively. The assimilation coefficients (assimilation rate : uptake rate) of NH4+ were also much higher than those of NO3-. Prorocentrum donghaiense growing in high light could assimilate average 35% of the NH4+ taken up during the incubation, but those in low light only assimilate less than 20%. Prorocentrum donghaiense could only assimilate on average less than 10% of the NO3- taken up. Discussion Growth Regulation in Turbidistat Turbidistat is run under turbidity control, and new medium inflow only occurs in response to an increase in the culture biomass to a pre-set value. Biomass is set by the optical threshold of dilution. Cell growth is limited by light and temperature; nutrients are usually added in excess of demand (Watson 1972, Skipnes et al. 1980, Fenaux et al. 1985, Hill et al. 1985, Kalyuzhin 1998). Light in turbidistat can run on a light-dark cycle which is more close to real situations (Falkowski et al. 1985). Herein, turbidistats provided a relatively long and steady exponential phase of growth without 146 causing significant change in irradiance caused by self-shading and nutrient depletion, and also provided detailed records of real-time growth rates. The growth rate, in this study, refers to the rate of changing of the optical signals, which attenuate with the turbidity of the culture. The turbidity is caused by the total mass concentration in the culture. Therefore, the growth rate is interpreted as the variation rate of total mass density of the culture, instead of cell density, which is calibrated by diluted the culture by volume. Based on the growth rates recorded by the turbidistat, the growth of both species showed strong light-regulated diel cycles. Cell growth, measured as the change of turbidity in the culture vessel, started at the beginning of light cycle. Dinoflagellate species have been reported to have a maximum growth limit of 1 division per day (McDuff and Chisholm 1982), as a result of dark-light regulated diel synchrony. Here, the growth rates recorded in the high light treatments were around 0.7 division per day. Maximum growth rate generally occurred in the first hour at the beginning of the light phase, and growth rate decreased during the day time. During the dark phase, the cultures were still mixed and gently bubbled and to keep them well mixed. Therefore, the negative growth rates suggested decreases in mass concentration in the culture. Negative growth rate, therefore, were not to due to setting of cells. When the photosynthesis stopped in the dark phase, the production rates of these species were less than the respiration rate, causing the negative growth rate. This state lasted until the start of the light cycle, when production rates increased quickly over respiration and the turbidity of the culture increased again. 147 Diel phasing of the cell cycle is common among photoautotrophic dinoflagellates, although the precise temporal phasing varies in a species-specific or environment-specific manner (Van Dolah et al. 1995, Van Dolah and Leighfield 1999, Leighfield and Van Dolah 2001). In one cell cycle Prorocentrum micans and P. minimum divide (the M phase) during a narrow window of time in the early hours of the light phase (Pan and Cembella 1998). The dark phase of the diel cycle of Prorocentrum spp. may include the S and G2 phase of the cycle which is light independent in dinoflagellates (Olson and Chisholm 1986). The entry of M phase is suggested to be dictated by the time of S phase entry instead of light. However, the G1 phase is suggested to be light dependent (Olson and Chisholm 1986). Incubation in low light for multiple diel cycle could arrest cells in the G1 phase, instead of entering the S phase and finishing a division cycle. As the result, the cell growth was inhibited. Light limitation impacts ATP and reductant production, which is derived from photosynthesis, and therefore affects N uptake, reduction and assimilation. In a previous study, in contrast to tested a diatom (Thalassiosira weissflogii), in which the N uptake and cell division were regulated by the new N pulses, the cell division of a dinoflagellate (Amphidinium carteri) was strictly regulated by the light-dark cycle, regardless the time of new N pulses (Wheeler et al. 1983). However, light-dark cycle had a relatively weak effect on N uptake and assimilation. N was metabolized rapidly, under conditions of N limitation. This light regulated dinoflagellate cell cycle led to a large degree of uncoupling between N assimilation and cell division (Wheeler et al. 1983). In the present study, the culture was diluted by new media from 0 to 6 times a day, depending on the growth rates. Each dilution could be considered as a N pulse. 148 However, there were no significant variations on the growth rate pattern after each pulse, and the growth rates were still strictly regulated by the dark-light cycle, which was measured as the change of turbidity. Wheeler et al. (1989) also reported maximal N uptake during the daytime and release (regeneration) of NH4+ exclusively at night, suggesting the capacity for large internal N pools for the N. Cells were able to take up and assimilate N, but could not proceed through the cell cycle until the correct portion of the dark-light cycle. At the beginning of the photoperiod, this pool of intracellular N may allow the assimilation process to proceed at an enhanced rate to support the fast growth, which was recorded by the turbidistats. This uncoupled N uptake and cell growth can also be explained by Droop?s model (Droop 1968, 1973), which describes the growth rate regulated by the cell quota (intracellular content of the limiting chemical element). These Prorocentrum species had their maximum cell quotas (internal N pool), and reached the highest growth rates at the beginning of light phase. Decreasing growth rates during the day are indicative of decreasing cell quotas. However, low growth rates in the dark cycle were not necessary indicative of a small cell quota, since the cells were light regulated instead of nutrient regulated. Prorocentrum minimum has been reported to continuously uptake the NO3- in the dark (Paasche et al. 1984). Cell stopped growth at night due to light limitation, but nutrients were likely continuously taken up into and stored in the cell, causing an increasing cell nutrient quota. The cell quota reached its maximum value of the day at the beginning of light phase, as did growth rates. 149 The regulation of light on the growth of P. donghaiense was significant. Based on the kinetics simulation, the half saturation coefficient (KI) was low (30 ~ 50 ?mol photons m-2 sec-1) in the range of light tested growth rates. The growth rates dropped rapidly when the ambient light was lower than the KI. The negative growth rate in the dark phased indicated very low production, which could not compensate the respiration lost, as a result, negative growth rates were recorded. In the low light treatments, the growth activities of P. donghaiense were light limited. N Uptake versus Irradiance Nitrogen uptake by phytoplankton is an energy consuming process. Light dependent nutrient uptake of phytoplankton has been studied in both laboratory culture and field communities, and the responses to light are varied among different species (Anderson and Roels 1981, Megard et al. 1985, Lomas et al. 1996, Kudela et al. 1997, Sinclair et al. 2009). High light may enhance the rate of N uptake, as the energy for N uptake and NO3- reduction is derived primarily from photosynthesis in the light (Kanda et al. 1989). The uptake rates might be cyclic over diel cycles (Anderson and Roels 1981). Numerous studies have shown that uptake of nutrient responding to light were different among taxonomic groups (Eppley et al. 1971, Wheeler et al. 1983). For example, the diatom Skeletonema costatum had lower dark uptake rates of NO3- and NH4+, and coccolithophore Emiliania huxleyi was more consistent in both day and night (Eppley et al. 1971). Light dependent N uptake rates have also been shown to be different for different nutrients. NO3- needs to be reduced before it can be assimilated by the algae, and this process needs light, as an energy consuming process. NO3- uptake rates have been shown to decrease more than uptake 150 of NH4+ in diatom dominant assemblages at night, suggesting higher light dependence of NO3- uptake than reduced N sources (Cochlan et al. 1991). For dinoflagellates, the light dependence of N uptake also varied with species. Prorocentrum minimum could continuous uptake the NO3- in the dark; while Karenia mikimotoi showed almost no NO3- uptake in the dark (Paasche et al. 1984). The light dependence might be weaken with increasing nutrient limitation (Healey 1977). In this experiment, the N uptake rates rate of P. minimum showed similar results to those reported by as Fan and Glibert (2005), which suggested little or no effect on N uptake during short term (~ half hour) incubations. Compared to the growth rates measured in the turbidistat, which had quick response to light and which decreased to near zero in 0.5 hour after the lights turned offs, the light independence of N uptake of P. minimum suggested that N uptake was not affected by low light. In this case, the growth stopped and but the cell N quota still increased. However, after days of incubation in different irradiance levels, the regulation of light on the N uptake of P. donghaiense was significant. Although the uptake and assimilation rates of NO3- in the low light were even slightly higher than those in high light treatments, the uptake and assimilation rates of NO3- were an order of magnitude lower than those of NH4+. The contribution of NO3- to the total N uptake and assimilation was very limited. Both the uptake and uptake assimilation rates of NH4+ suggested significant light limitation when comparing the low and high light treatments. This light limitation of N uptake was consistent with light limitation on the growth rate (Fig. 4-9). The NH4+ uptake rates were less than the growth rate, 151 suggesting the contribution of other N nutrients to support growth (i.e., organic N, Li et al. 2009). Light Condition and Prorocentrum Blooms The annual recirculation of cell transportation in the Chesapeake Bay has been suggested as an important mechanism for the periodic outbreak of P. minimum blooms (Tyler and Seliger 1978). P. minimum cells are transported to upper Chesapeake Bay and its tributaries by the northward current from the bottom layer from the mouth of the Chesapeake Bay in the winter and early spring. It could take over a month before the P. minimum is transported to the tributaries, and more time to reach the surface water. During this period, the cells are in near or total dark environment. When P. minimum rises to the surface, the cells grow and develop blooms. Dinoflagellates have multiple strategies to survive extended periods of darkness. For example, both P. minimum and P. donghaiense have been reported to be mixotrophic dinoflagellates, and may directly feed on a wide range of food sources, including other algae, bacteria, and organic particles (Li et al. 1999, Stoecker 1999, Jeong et al. 2004, Burkholder et al. 2008, Jeong et al. 2010). Light independent N uptake is another strategy for the cells to keep taking up N nutrients supply during the dark phase in the bottom layer, which would allow them to be competitive in turbid coastal waters where light is low. When cells are transported to the deeper layer, although the growth may be light limited, cells can still take up N and keep a large cell N quota. When they reach surface water, cells with large cell nutrient quota can quickly reach high growth rate and develop blooms. When N is depleted in the surface layer, cells can also obtain N sources in the dark deep layer by taking the 152 advantage of vertical migration (Holmes et al. 1967, Eppley et al. 1968, Haraguchi et al. 2010, Rines et al. 2010). Dinoflagellates can access both the deep nutrient pool and near-surface light required for photosynthesis, at different times of the day. Bloom-forming dinoflagellates are capable of maintaining a relatively high nutrient uptake rate in a low-light or even completely dark environment (Heaney and Eppley 1981, Paasche et al. 1984, Cullen et al. 1985, Olsson and Gran?li 1991, Cloern 2001, Fan and Glibert 2005), thus allowing them to store extra nutrients (especially regenerated nutrient PO43- and NH4+) by luxury uptake in the deep layer before ascending to the surface for photosynthesis. However, the regulation of light on N uptake between the two Prorocentrum species might be different. Although the growth rate of P. donghaiense only decreased about 40%, when ambient light decreased from 500 to 66 ?mol photons m- 2 sec-1, there was significantly greater light limitation on NH4+ uptake rate. Prorocentrum donghaiense also had significantly lower Ks for NH4+ in high light treatments. These results suggested P. donghaiense might not adapt to low light as well as P. minimum, which is consistent with the field observation that P. minimum can bloom in very turbid near-shore estuarine waters, while P. donghaiense blooms are relatively off-shore. 153 References Aalderink RH, Jovin R (1997) Estimation of the photosynthesis/irradiance (P/I) curve parameters from light and dark bottle experiments. Journal of Plankton Research 19:1713-1742 Anderson DM (1997) Turning back the harmful red tide - Commentary. Nature 388:513-514 Anderson DM, Glibert PM, Burkholder JM (2002) Harmful algal blooms and eutrophication: Nutrient sources, composition, and consequences. 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Chinese Journal of Applied Ecology 14:1031-1038 Zhou W, Yin K, Zhu D (2006) Phytoplankton biomass and high frequency of Prorocentrum donghaiense harmful algal bloom in Zhoushan sea area in spring. Chinese Journal of Applied Ecology (In Chinese) 17:887-893 161 Table 4-1 Ambient nutrient concentrations in 4 different turbidistat systems supplied by medium at different DIN:DIP ratios. Tables Medium Enriched N:P Ratio NO3-?M-N NH4+?M-N Urea ?M-N DFAA ?M-N DIP ?M-P Culture DIN:DIP Ratio 5 1.95 0.05 1.18 0.063 0.57 3.5 16 0.37 1.05 3.14 0.011 0.11 12.5 40 21.98 3.59 2.09 0.038 1.17 21 200 42.4 2.52 2.05 1.123 0.15 300 162 Table 4-2 Calculated parameters of uptake kinetics of nitrogen sources (NH4+, urea, and glycine) by Prorocentrum minimum at different ambient DIN:DIP ratio in turbidistat based on the Michaelis-Menten equation. Vmax and Amax are the specific nitrogen uptake and assimilation rates. Ks is the Michaelis constant where uptake rates equal to ? of maximum uptake rates. Culture DIN:DIP NH4+ Urea Glycine NH4+ Urea Glycine Ratio Vmax Ks Vmax Ks Vmax Ks Amax Ks Amax Ks Amax Ks 3.5 0.031 0.20 5E-04 0.43 0.004 1.27 12.5 0.046 1.248 3E-04 0.052 0.004 0.48 0.024 2.32 7E-04 16.50 0.001 0.04 21 0.02 1.43 4E-04 2.63 0.001 0.10 300 0.065 4.646 8E-04 5.43 0.007 3.54 0.016 2.70 4E-04 1.56 0.003 3.39 163 Figures 00:00 12:00 00:00 12:00 00:00 12:00 00:00 12:00 00:00 Gr ow th Ra te pe r H ou r -0.10 -0.05 0.00 0.05 0.10 0.15 Figure 4-1 Diurnal growth rates of Prorocentrum donghaiense in turbidistat system for 4 constant 12 h: 12 h light dark cycles (96 hrs). Each point stands for the growth rate calculated based on the change in the optical signals over a 20-minute time period. 164 P. minimum 0 2 4 6 8 10 12 14 16 18 20 22 24 Gr ow th Ra te pe r H ou r -0.10 -0.05 0.00 0.05 0.10 0.15 P. donghaiense 0 2 4 6 8 10 12 14 16 18 20 22 24 Gr ow th Ra te pe r h ou r -0.10 -0.05 0.00 0.05 0.10 0.15 Figure 4-2 a. Diurnal growth rates based on changes in optical signals of Prorocentrum minimum in turbidistat system at 14 h: 10 h light dark cycle. b. Diurnal growth rates of Prorocentrum donghaiense in turbidistat system at 14 h: 10 h light dark cycle. Box plots - line: median; box: 25th to 75th percentiles; whiskers: 10th and 90th percentiles; dots: outliers. 165 N:P ratio 3.5 12 21 300 Gr ow th Ra te pe r H ou r 0.00 0.01 0.02 0.03 0.04 0.05 0.06 a. P. minimum b. P. minimum N:P ratio 3.5 12 21 300 Gr ow th Ra te pe r H ou r 0.00 0.01 0.02 0.03 0.04 0.05 0.06 Figure 4-3 a. Average growth rates of Prorocentrum minimum in a 14 h: 10 h light dark cycle at different ambient N:P ratios. b. Average growth rates of P. minimum in the light phase of 14h: 10h light dark cycle at different ambient N:P ratios. 166 00 06 12 18 00 Gr ow th Ra te pe r H ou r -0.1 0.0 0.1 0.2 0.3 a. 00 06 12 18 00 Gr ow th Ra te pe r H ou r -0.1 0.0 0.1 0.2 0.3 b. 00 06 12 18 00 Gr ow th Ra te pe r H ou r -0.1 0.0 0.1 0.2 0.3 c. 00 06 12 18 00 Gr ow th Ra te pe r H ou r -0.1 0.0 0.1 0.2 0.3 d. 00 06 12 18 00 Gr ow th Ra te pe r H ou r -0.1 0.0 0.1 0.2 0.3 e. Figure 4-4 Diurnal (12 h: 12 h light dark cycle) growth rates based on changes in optical signals of Prorocentrum donghaiense in turbidistat systems at different light intensities. From 00 h to 00 h the next day, lights were on at 08 h and off at 20 h (X-axis). a. 500; b. 200; c. 66; d. 16; e. 5 (?mol photons m-2 sec-1). Each point stands for the growth rate calculated based on the change in the optical signals over a 20-minute time period. 167 Irradiance (?mol photons m-2 sec-1) 0 100 200 300 400 500 600 Gr ow th Ra te pe r H ou r -0.02 0.00 0.02 0.04 0.06 0.08 Figure 4-5 Average diurnal growth rates (?) and average growth rates based on changes in optical signals of Prorocentrum donghaiense in the light phase (?) in a 12 h: 12 h light dark cycle at different ambient light irradiance. 168 NO3- Irradiance (?mol photons m-2 s-1) 0 100 200 300 400 500 V pe r h ou r 0.000 0.005 0.010 0.015 0.020 0.025 DIN:DIP 3.5 DIN:DIP 12.5 DIN:DIP 21 DIN:DIP 300 NH4+ Irradiance (?mol photons m-2 s-1) 0 100 200 300 400 500 V pe r h ou r 0.00 0.02 0.04 0.06 0.08 0.10 Urea Irradiance (?mol photons m-2 s-1) 0 100 200 300 400 500 V pe r h ou r 0.0000 0.0002 0.0004 0.0006 0.0008 0.0010 Glycine Irradiance (?mol photons m-2 s-1) 0 100 200 300 400 500 V pe r h ou r 0.000 0.002 0.004 0.006 0.008 0.010 Figure 4-6 The relationship of N (Nitrate (NO3-), ammonium (NH4+), urea and glycine) uptake rates V (h-1) of Prorocentrum minimum at different irradiance levels (?mol photons m-2 sec-1) with different ambient DIN:DIP ratio treatments. 169 NO3- N:P ratio V pe r h ou r 0.000 .005 .010 .015 .020 .025 High Light Low Light 3.5 12.5 20 300 NH4+ N:P ratio V pe r h ou r 0.00 .02 .04 .06 .08 3.5 12.5 20 300 Urea N:P ratio V pe r h ou r 0.0000 .0002 .0004 .0006 .0008 .0010 3.5 12.5 20 300 Glycine N:P ratio V pe r h ou r 0.000 .002 .004 .006 .008 .010 3.5 12.5 20 300 Figure 4-7 Average specific uptake rates V (h-1) of 4 nitrogen substrates (Nitrate (NO3-), ammonium (NH4+), urea and glycine) near ambient concentration by Prorocentrum minimum in high light (average of treatments 9 and 35 ?mol photons m-2 sec-1) and low light (average of treatments 200 and 430 ?mol photons m-2 sec-1) incubation at different ambient N:P ratios. 170 ?M-N 0 20 40 60 80 V pe r h ou r 0.000 .002 .004 .006 .008 .010 NO3- NH4+ ?M-N 0 5 10 15 20 25 V pe r h ou r 0.00 .02 .04 .06 .08 Figure 4-8 Kinetic relationships between nitrogen specific uptake rates V (h-1) of NO3- and NH4+ with 15N substrate concentration (?M-N) by Prorocentrum donghaiense in different irradiance levels (?mol photons m-2 sec-1). 171 NO3- pe r h ou r 0.000 0.001 0.002 0.003 0.004 0.005 Uptake Assimilation High Light Low Light NH4+ pe r h ou r 0.00 0.01 0.02 0.03 0.04 0.05 Growth Uptake Assimilation High Light Low Light Figure 4-9 Average specific uptake rates V (h-1) and assimilation rates A (h-1) of nitrogen substrates nitrate (NO3-) and ammonium (NH4+) near ambient concentration by Prorocentrum donghaiense growing in high light (average of treatments 9 and 35 ?mol photons m-2 sec-1) and low light (average of treatments 200 and 430 ?mol photons m-2 sec-1) treatments. The average growth rate r (h-1) of that light-dark cycle was also compared with V and A of NH4+. 172 NO3- As sim ilat ion : U pta ke Ra tio 0.0 0.2 0.4 0.6 0.8 1.0 NH4+ High Light Low Light High Light Low Light Figure 4-10 Ratio of assimilation rates A : uptake rates V of nitrogen substrates nitrate (NO3-) and ammonium (NH4+) by Prorocentrum donghaiense growing in high light and low light treatments. Box plots - line: median; box: 25th to 75th percentiles; whiskers: 10th and 90th percentiles; dots: outliers. 173 Chapter 5: Growth and Competition of Several Harmful Dinoflagellates under Different Nutrient and Light Condition Abstract Three near-shore type harmful dinoflagellates, Prorocentrum minimum, P. donghaiense and Karlodinium veneficum, and one off-shore dinoflagellate, Karenia brevis, were grown in laboratory monoculture and mixed batch cultures. The dinoflagellate cultures were grown on treatments of two ambient nitrogen (N) : phosphorus (P) ratios; two N substrates (nitrate and urea) and two light intensities. The microalgae Rhodomonas and Synechococcus were also added in separate treatments to the mixed culture treatments as potential food sources. All tested species grew well on both N substrates. In mixed culture, P. minimum outgrew K. veneficum, and P. donghaiense outgrew K. brevis in most treatments reaching higher growth rates and higher biomass. However, when a third algal, Rhodomonas, was added, the growth of P. minimum was inhibited relative to that of K. veneficum. In contrast, when grown with K. brevis, the growth rate of P. donghaiense was not significantly affected. K. brevis had a longer growth phase, and kept growing after P. donghaiense reached stationary phase, suggesting better adaptation of K. brevis to low inorganic nutrient conditions. The growth of K. brevis was also significantly limited in the low light treatment. Karlodinium veneficum overgrew P. minimum with the presence of Rhodomonas, a potential nutrient source. The growth of both K. 174 brevis and P. donghaiense were reduced with the presence of Synechococcus. Other mechanisms (e.g. allelopathy), in addition to nutrient competition, should be considered and may also play a role in determining the dominant species. Introduction Prorocentrum spp. are one of the major groups of bloom forming harmful dinoflagellates that have been reported to develop harmful algal blooms (HABs) in coastal waters worldwide, and to cause ecological damage by high biomass and to have related effects (Heil et al. 2005, Glibert et al. 2008b). Prorocentrum minimum is a common HAB species in coastal and estuarine systems in USA, and it is well documented in the Chesapeake Bay and southwest Florida shelf among many other parts of the world (Glibert et al. 2001, Tango et al. 2005, Heil et al. 2007, Glibert et al. 2008b). In the past two decades, over 800 events of P. minimum with cell density over 100, 000 cell L-1 were recorded in the Chesapeake Bay by the State of Maryland Department of Natural Resources (MD DNR) monitoring program. Another Prorocentrum species, P. donghaiense, is a major bloom species near the Changjiang River estuary and in the coastal area of East China Sea. Large scale P. donghaiense blooms have been observed in those waters in the past two decades (Zhou et al. 2006, Li et al. 2009, Li et al. 2010). Another HAB species, which is increasing in both frequency and abundance in Chesapeake Bay, is Karlodinium veneficum. Karlodinium veneficum is a potentially toxic dinoflagellate and has been implicated to cause fish kill events (Heil et al. 2001, Deeds et al. 2002, Adolf et al. 2006a, Deeds et al. 2006). Both P. minimum and K. veneficum are recognized as dominant late spring and summer 175 bloom species in the Chesapeake Bay area, and often co-occur (Glibert et al. 2001, Tango et al. 2005). In the southwest Florida shelf, P. minimum has also been recorded to bloom with Karenia brevis, a prevalent, potentially toxic dinoflagellate (Heil et al. 2007, Vargo et al. 2008, Vargo 2009). In the East China Sea, the dinoflagellate Karenia mikimotoi was reported to bloom just before or contemporaneously with P. donghaiense blooms (Li et al. 2009). Thus, there are numerous questions with regard to relative success of Prorocentrum species in the presence of the competing dinoflagellates, Karlodinium or Karenia. In all these cases, bloom areas were enriched by large terrestrial nutrient inputs. The relationships between nutrient loading and bloom development are very complex (Glibert et al. 2005a, Glibert and Burkholder 2006, Glibert et al. 2006, Anderson et al. 2008, Glibert et al. 2008a). While dissolved inorganic nitrogen (DIN) still is the major N sources enriched to the coastal systems, urea and other dissolved organic N (DON) have became important N sources for many HAB species (Glibert et al. 2004, Glibert et al. 2006, Heil et al. 2007, Li et al. 2009). The nutrient composition might regulate the community composition of the blooms. In addition to nutrient composition, the relative ambient N:P ratio may also regulate the phytoplankton community composition and HAB development (Phillips and Tanabe 1989, Hodgkiss and Ho 1997, Bulgakov and Levich 1999, Lagus et al. 2004). For P. minimum, P. donghaiense and K. brevis, the N:P ratio may not only relate to the bloom distribution and biomass, but also may regulate dominant species due to their differential nutrient uptake rates and preference (Fisher et al. 1992, Heil et al. 2007, Li et al. 2009, Li et al. 2010). In both East China Sea and southwest 176 Florida shelf, the Prorocentrum spp. communities were observed to be associated with DIN:DIP ratios that were slightly higher than the Redfield ratio (16), while the Karenia spp. dominated communities were found in association with DIN:DIP ratios that were below the Redfield ratio, but high DON:DOP ratio (>20), suggesting that Karenia spp. in both environments may have been consuming the organic N source (Heil et al. 2007, Li et al. 2009). As an alternative nutrient strategy, mixotrophy, a combination of phototrophic and phagotrophic nutrition, has been reported for P. minimum, P. donghaiense, K. veneficum and K. brevis (Li et al. 1999, Stoecker 1999, Jeong et al. 2004, Jeong et al. 2005a, Adolf et al. 2007, Burkholder et al. 2008, Glibert et al. 2009, Jeong et al. 2010). Phagotrophy may provide mixotrophic dinoflagellates a source of nutrients and/or carbon from other organisms that supplement that which they assimilate from the water. Some dinoflagellates (e. g. K. veneficum and K. brevis) approach a higher maximum growth rate when growing mixotrophically (Adolf et al. 2006b, Glibert et al. 2009). Therefore, phagotrophy of dinoflagellates could serve as a competition strategy during nutrient limitation and could be a means of sustaining the growth of mixotrophic dinoflagellates. Mixotrophic activity may play a substantial role in sustaining blooms (Glibert et al. 2009). Dinoflagellates feed on a wide range of food sources, including other algae, bacteria, and organic particles (Burkholder et al. 2008). In estuaries with high terrestrial input, organic particles and their primary decomposers, heterotrophic bacteria, could be important food sources for mixotrophic dinoflagellates. 177 The effects of various nutrient sources and growth conditions on the growth competition of P. minimum, P. donghaiense, K. veneficum and K. brevis singly and in combination were tested in the present study. The factors tested included different type of N sources (nitrate and urea), different ambient N:P ratios (12 and 145), different light availability (50 and 300 ?mol photons m-2 s-2) and the potential of mixotrophic food sources. The euryhaline cryptophyte, Rhodomonas, and cyanobacteria, Synechococcus, were grown with the dinoflagellates as potential food sources to test their contribution as a nutrient source in sustaining the growth of the dinoflagellates. Materials and Methods Algae Cultures and Methods of Growth Cultures of Prorocentrum minimum were isolated from Choptank River, a tributary of Chesapeake Bay in spring 1995 and were maintained in f/2 medium in the Horn Point Laboratory culture collection. Cultures of Prorocentrum donghaiense were obtained from University of Connecticut Department of Marine Sciences, USA. These cultures were originally isolated from the East China Sea. Cultures of Karlodinium veneficum, Karenia brevis (CCMP 2229), Rhodomonas and Synechococcus (CCMP 1768) were obtained from the Provasoli-Guillard National Culture Collection, Bigelow Laboratory for Ocean Sciences, Maine, USA. The K. brevis strain was originally isolated from Manasota Key, Florida. Synechococcus strain CCMP1768 was originally isolated from the Gulf of Mexico. All the cultures in the present study were unialgal, but not axenic. 178 The P. minimum and K. veneficum cultures were grown in modified f/2 media at a salinity of 12; P. donghaiense and K. brevis cultures were grown in modified f/2 media at a salinity of 30. The N and P source concentration varied with the experiments, as described below. Maintenance cultures were grown at 22 ?C and on a 12 h light: 12 h dark cycle in a walk-in temperature-controlled incubator. The seawater used in media preparation was artificial seawater. Fluorescent lights were set to provide a range of light intensities, which varied by experiments. Cultures were not axenic, but aseptic techniques were used to minimize additional bacterial contamination during the growth periods. Experimental Design Experiment 1: Phototrophic growth of P. minimum and K. veneficum monoculture with NO3- or urea at high or low light levels and 2 N:P ratios. Prorocentrum minimum and K. veneficum were diluted by fresh media at salinity of 12 to a final concentration of ~1.0 ? 104 cells ml-1 for each species. Monocultures were grown in triplicate 50 ml glass cuvettes with the N substrate as NO3- or urea and P substrate as PO43- at two N:P ratios (12 and 145) and two light intensities (50 and 300 ?mol photons m-2 s-2). One set of cultures was enriched to a final nutrient concentrations of 42.8 ?M-N and 3.56 ?M-P to reach an N:P ratio of 12, and the other set was enriched to 224.9 ?M-N and 1.55 ?M-P to reach the N:P ratio of 145. Experiment 2: Phototrophic growth competition between P. minimum and K. veneficum with NO3- or urea at high or low light levels and 2 N:P ratios. 179 Prorocentrum minimum and K. veneficum were mixed and diluted by fresh media at a salinity of 12 to a final concentration of ~1.0 ? 104 cells ml-1 for each species. Cells were grown in duplicate 1.5 L glass bottles with two N substrates (NO3- and urea) at two N:P ratios (12 and 145) and two light intensities (50 and 300 ?mol photons m-2 s-2). Nutrients were added as in experiment 1. All bottles were gently bubbled with air to keep the culture well mixed and aerated. Experiment 3: Phototrophic/ mixotrophic growth competition between P. minimum and K. veneficum with Rhodomonas as potential prey, and with NO3- or urea at high or low light levels and 2 N:P ratios. Prorocentrum minimum and K. veneficum were mixed and diluted by fresh media at a salinity of 12 to a final concentration of ~2.5 ? 104 cells ml-1 for each species. Rhodomonas were also added at a final concentration of ~7.5 ? 104 cells ml- 1. Cells were grown in duplicate 1.5 L glass bottles with NO3- at two N:P ratios (12 and 145) and two light intensities (50 and 300 ?mol photons m-2 s-2). Nutrients were added as in experiment 1. All bottles were gently bubbled with air to keep the culture well mixed and aerated. Experiment 4: Phototrophic growth of P. donghaiense and K. brevis with NO3- or urea at high or low light levels and 2 N:P ratios. Prorocentrum donghaiense and K. brevis were diluted by fresh media at salinity of 30 to a final concentration of ~1.0 ? 103 cells ml-1 for each species. Monocultures were grown in triplicate 50 ml glass cuvettes with two substrates (NO3- and urea) at two N:P ratios (12 and 145) and two light intensities (50 and 300 ?mol photons m-2 s-2 ). Nutrients were added as in experiment 1. 180 Experiment 5: Phototrophic growth competition between P. donghaiense and K. brevis with NO3- or urea at high or low light levels and 2 N:P ratios. Prorocentrum donghaiense and K. brevis were mixed and diluted by fresh media at salinity of 30 to a final concentration of ~1.0 ? 103 cells ml-1. Cells were grown in duplicate 1.5 L glass bottles with 2 N substrates (NO3- and urea), at two N:P ratios (15.5 and 145) and two light intensities (50 and 300 ?mol photons m-2 s-2). Nutrients were added as in experiment 1. All bottles were gently bubbled with air to keep the culture well mixed and aerated. Experiment 6: Phototrophic growth competition between P. donghaiense and K. brevis with Synechococcus as potential prey, and with NO3- or urea at high or low light levels and 2 N:P ratios. Prorocentrum donghaiense and K. brevis were mixed and diluted by fresh media at salinity of 30 to a final concentration of ~1.0 ? 103 cells ml-1. Synechococcus were also added at a final concentration of ~7.5 ? 104 cells L-1. Cells were grown in duplicate 1.5 L glass bottles with two forms of N sources (NO3- and urea), at two N:P ratios (15.5 and 145) and two light intensities (50 and 300 ?mol photons m-2 s-2). Nutrients were added as in experiment 1. All bottles were gently bubbled with air to keep the culture well mixed and aerated. Analytical Protocols The growth of each dinoflagellate monoculture was tracked by the change of its fluorescence using a TD-700 Fluorometer. The growth of mixed dinoflagellates and Rhodomonas in the mixed culture was tracked by optical microscopy. On an approximate daily basis, 7 ml of culture were sampled from each bottle and fixed 181 with Lugol's solution for cell counting. The cell densities of Synechococcus were also measured on a daily basis. To do so, fresh culture was added to a quartzose cuvette and the fluorescence emission of 560 nm was recorded on a Jobin Yvon Fluoro Max- 3 spectrofluorometer according to Glibert et al. (2009). The results were calibrated by microscopy cell counting. Data Analysis and Simulation Statistical comparisons between groups and treatments were compared and analyzed using ANOVA and t-tests. All comparisons were made at a significance level of 0.05. The exponential growth rates of each culture in the early stage of the growth phase were determined with Sigmaplot software (Systat Software, Inc.) by the equation: (1) where ? is the specific growth rate (d-1); N0 is the initial cell density (cells ml-1); N(t) is the cell density at time t. Herein, only the cell densities of the early exponential phase were used for the growth rate regression to estimate the exponential growth rate. Results Experiment 1 Prorocentrum minimum monocultures in NO3- media grew for about 3 weeks before reaching the stationary phase, except for the treatment with low light and high 0( ) tN t N e?? 182 N:P ratio, which started to decrease in 2 weeks (Fig. 5-1 a). The growth rates were in the range of 0.44 day-1 to 0.49 day-1 (Table 5-1) in high light, which were significantly higher than the rates (~0.25 day-1) in low light (p<0.001). There was no significant difference between growth rates with low and high N:P ratio treatments. The P. minimum monocultures in urea media had a long lag phase, and rapid growth started after one week of incubation and only lasted for a week, with a growth rate about 0.39 day-1 in the high light treatment. In low light, exponential growth lasted about 2 weeks; however, the growth rates (~0.24 day-1) were significantly lower than those at high light (Fig. 5-1 b). There was no significant difference between growth rates with low and high N:P ratio treatments. Karlodinium veneficum monocultures in NO3- media also grew exponentially for more than 3 weeks, except for the treatments with low light and high N:P ratio, which started to decrease at day 18 (Fig. 5-1 c.). The growth rates were about 0.32 day-1 in high light, significantly higher than those in low light (0.19 day-1, p=0.004). There was no significant difference in growth rates between the low and high N:P ratio treatments. In NO3- media, the growth rates of P. minimum were significantly higher than K. veneficum for all treatments (p<0.05). The K. veneficum monoculture in urea media also had a long lag phase, and growth started after over one week of incubation and only lasted for a week at a growth rate of ~ 0.24 day-1 in high light treatment, and 0.16 day-1 in low light (Fig. 5-1 d.). There was no significant difference between growth rates with low and high N:P ratio treatments. 183 Experiment 2 Prorocentrum minimum biomass in the mixed culture increased in the low N:P ratio treatment for about 10 days (Fig. 5-2), while exponential growth lasted more than 20 days in the high N:P ratio in low light. The growth rates in the low N:P ratio treatment were in the range of 0.17 day-1 to 0.27 day-1 (Table 5-2). The growth rates in the high N:P ratio treatment were in the range of 0.23 day-1 to 0.33 day-1, which were significantly higher than those of the low N:P ratio treatment (p<0.001). There was no significant difference between the P. minimum growth rates at high light and low light treatments. Prorocentrum minimum grown in urea media in low light had significantly higher growth rates than the comparable cultures growing on NO3- (p<0.05). The exponential growth phase of K. veneficum in the mixed culture lasted from a week to 10 days. The growth rates of K. veneficum were in the range of 0.06 day-1 to 0.28 day-1. However, the difference among growth rates of K. veneficum was not significant among all treatments. The growth rates of K. veneficum were significantly lower than those of P. minimum (p<0.001). As a result of a longer period of exponential growth and faster growth rates, P. minimum reached significantly higher cell densities than K. veneficum in most treatments, except for the culture in urea at a N:P ratio of 12 in high light. Neither species grew well in the urea treatment in high light. Experiment 3 With the addition of Rhodomonas, the growth rate of P. minimum was very low, only ~ 0.04 day-1, in high light (Table 5-3), and no significant growth was observed in low light. The growth rate of K. veneficum was only ~ 0.085 day-1, and no 184 significant difference in growth rates was observed between light or N:P ratio treatments. The increase in biomass for both species only lasted 4-5 days. However, K. veneficum outgrew P. minimum. The cell densities of Rhodomonas decreased over 50% during first 2-3 days and were subsequently held at < 5% of the initial densities (Fig. 5-3). There were no significant differences of growth rates among different treatments for each species. Experiment 4 Prorocentrum donghaiense in monoculture grew exponentially for about 20 days before reaching the stationary phase, except for the treatments in high N:P ratio, which started to decrease in 10 days (Fig. 5-4). The highest growth rates were 0.37 day-1 in NO3- media at low N:P ratio and high light (Table 5-4). The lowest growth rates were 0.16 day-1 in NO3- media at low N:P ratio and low light, but the high and low light growth rates were not significantly different. The growth rates in the low N:P ratio media were significantly higher than those in high N:P ratio media in high light (p<0.001). The growth rates on urea were significantly higher than those in NO3- media in low light (p<0.001). Karenia brevis biomass in monoculture increased for about 10 days in high light, then started to decrease. However, K. brevis in low light kept increasing during the entire study (Fig. 5-4). The growth rates in high light were in the range of 0.22 day-1 to 0.28 day-1. The growth rates in low light were in the range of 0.08 day-1 to 0.14 day-1, and were significantly lower than those in high light (p<0.001). There were no significant differences in growth rates between the low and high N:P ratio treatments. In monoculture, the growth rates of P. donghaiense were significantly higher than K. brevis for all treatments (p=0.015). 185 Experiment 5 In high light, Prorocentrum donghaiense growth rates were in the range of 0.30 day-1 to 0.38 day-1 (Table 5-5). P. donghaiense biomass in the mixed culture kept increasing at high light for about 7-10 days, and reached cell densities of ~ 40, 000 cell ml-1 (Fig. 5-5). Karenia brevis growth rates were in the range of 0.17 day-1 to 0.25 day-1in high light, and reached cell densities of ~ 20, 000 cell ml-1. However, at low light, P. donghaiense grew in the range of 0.12 day-1 to 0.23 day-1, and the growth lasted longer and reached maximal cell densities of ~ 80, 000 cell ml-1. Karenia brevis only grew at 0.025 day-1 or lower, and only reached cell densities of ~ 4, 000 cell ml-1. Both species grew significantly faster in high light than low light (p<0.01). There were no significant differences between NO3- and urea treatments. The growth rates of P. donghaiense were significantly higher than K. brevis (p=0.004). Experiment 6 In experiments 6 with addition of Synechococcus, the growth rates of P. donghaiense in high light were in the range of 0.36 day-1 to 0.39 day-1, which were significant higher than rates in low light (0.14 day-1, p=0.001). The growth rates of K. brevis at low light were in the range of 0.019 day-1 to 0.055 day-1. The growth rates in high light were about 0.17 day-1, which were significantly higher than those at low light (p<0.001). The growth rates of P. donghaiense were significantly higher than K. brevis (p<0.05). The Synechococcus growth rates were about 0.32 day-1 in high light and 0.06 day-1 in low light. Synechococcus grew exponentially for a week before it decreased at high light, but it continued growing at low light (Fig. 5-6). 186 Discussion Growth Rate Numerous studies have been conducted on the four dinoflagellate species studied herein. The growth rates in the present study were compared with previous work (Table 5-7).The growth rates of P. minimum monocultures (0.23-0.46 day-1) in this study were within the range of the previous work, which mostly were in the range of 0.13 to 1 day-1 except for one report of rates reaching 3.54 day-1 (Smayda 1996). The growth rates of P. minimum in mixed culture with K. veneficum (0.17-0.32 day-1) fell in the lower range of previous work, and the growth rates of P. minimum in cultures mixed with K. veneficum and Rhodomonas (<0.05 day-1) were lower than previous results. The growth rates of K. veneficum monoculture (0.19-0.33 day-1) in this study were within the range of the previous work (< 0.75 day-1, Table 5-7). The growth rates of K. veneficum in mixed culture with P. minimum (0.06-0.28 day-1) were also in the in the range of previous work, and the growth rates of K. veneficum with P. minimum and Rhodomonas (<0.09 day-1) were in the lower range of previous results. The growth rates of P. donghaiense were in the range of 0.12-0.39 day-1, which were within the range of the previous work (0.1-0.78 day-1). There was only one report of growth rates reaching 1.40 day-1 (Wang and Tang 2008). The growth rates of K. brevis monoculture (0.08-0.28 day-1) in this study were within the range of the previous work (0-0.36 day-1, Table 5-7). The growth rates of K. brevis in mixed culture with P. donghaiense (0.0-0.26 day-1) were in also in the range of previous work, and the growth rates of K. brevis with P. donghaiense and Synechococcus (0.02-0.15day-1) were in the lower range of previous results. 187 Dinoflagellates are one of the major groups of algae causing HABs in coastal waters. Smayda suggested that dinoflagellates have diverse habitat preferences and adaptive strategies. He characterized the dinoflagellate bloom species in coastal areas as 5 types, which fit to a nutrient-turbulence matrix (Smayda and Reynolds 2001, Smayda 2002). Each dinoflagellate species had its specific niche for bloom expansion, and the niches were described by available nutrients and turbulence/mixing intensities. In Smayda?s model, small, rapidly growing species (e.g. Prorocentrum spp.) are better competitors in near-shore regions. Conversely, relative slower growing, larger, motile species (e.g. Karenia spp.) are better suited to off-shore regions. The near-shore regions are generally considered nutrient rich but with high turbidity, while light might be the limiting factor for the phytoplankton. The off-shore regions are generally considered to have relatively low nutrients but with higher light availability. These two groups of algae have different adaptive strategies to the environment. Therefore, not only are the nutrient preferences but also light adaptation strategies of these two general genera of algae potentially differ. In this study, the growth and competition between the near-shore dinoflagellates (P. minimum and K. veneficum) and between near-shore and off-shore species (P. donghaiense and K. brevis) were tested under different ambient conditions, including variable nutrient conditions and light intensities. The two near- shore dinoflagellate species, P. minimum and P. donghaiense, both had higher growth rates in monoculture than their competitors in this study. When grown with K. veneficum, the average growth rates of P. minimum were inhibited to about 40% of the rates observed in monocultures. After adding Rhodomonas, K. veneficum outgrew 188 P. minimum, and reached higher cell densities than P. minimum. However, when grown with a different type (off-shore) of dinoflagellate, K. brevis, the growth rate of P. donghaiense was not significantly affected. Although K. brevis grew slower than P. donghaiense, they still reached similar cell densities in mixed culture in high light treatment. K. brevis had a longer growth phase, and kept growing after P. donghaiense reached stationary phase, suggesting better adaptation of K. brevis to low inorganic nutrient conditions. The growth of K. brevis was mostly limited by the low light treatment of the four species. This off-shore species only had an advantage at low ambient inorganic nutrient condition, and appeared to be more limited by light. Nutrient Strategy ? Autotrophy and Mixotrophy All four tested dinoflagellates have developed HABs in eutrophic estuarine and marine coastal waters. The increase of many HABs has been suggested to correspond with high nutrient loading (Glibert et al. 2001, Anderson et al. 2002, Glibert et al. 2005a). Dissolved inorganic nitrogen forms (DIN, i.e., NO3-, NH4+) are generally considered the principal N sources for phytoplankton. Diatoms are often found to use NO3- at low temperatures and to develop blooms in the early spring (Lomas and Glibert 1999a, b). Spring blooms also deplete the NO3- in the ambient water in coastal systems, as often seen in the Changjiang River estuary (Li et al. 2009, Li et al. 2010), and the Chesapeake Bay (Jordan et al. 1991, Fisher et al. 1992, Glibert et al. 1995). Although the NO3-concentration decreases after the spring blooms, it is still an important N source, and it has been widely used as N source in the media in the culture experiments (Guillard and Ryther 1962). 189 The succession of dominant phytoplankton assemblages in eutrophic waters has been associated with shifts in nutrient supply ratios, especially decreased DIN:DIP ratios that reflect regional disproportionate P loading relative to N, which relates to anthropogenic and regenerated inorganic sources (Suttle and Harrison 1988, Jordan et al. 1991, Koerselman and Meuleman 1996, Bulgakov and Levich 1999, Stelzer and Lamberti 2001, Heil et al. 2007, Vrede et al. 2009). All four tested dinoflagellates have been documented to bloom in relatively lower DIN:DIP ratio waters (close to the Redfield ratio) than diatom blooms (Jordan et al. 1991, Heil et al. 2007, Li et al. 2009). For dinoflagellates, another important N source is dissolved organic nitrogen (DON), which may represent 14 - 90% of the total N in lower river estuaries from natural and anthropogenic sources (Glibert et al. 1991, Seitzinger et al. 2002, Glibert et al. 2004). DON may also represent an important N source for phytoplankton in eutrophic coastal waters (Bronk et al. 2004, Glibert et al. 2004, Wiegner et al. 2006, Heil et al. 2007, Li et al. 2010). Urea has become a very important DON source because of its accelerated global use, which has increased more than 100-fold in the past 4 decades and contributes > 50% of global nitrogenous fertilizer usage (Glibert et al. 2006). Urea is increasingly found to be an important N nutrient source for HAB species and is positively correlated to some blooms (Glibert et al. 2004, Glibert et al. 2005b, Glibert et al. 2006). In Chesapeake Bay tributaries, high concentrations of urea (> 10 ?M-N) have been observed frequently, generally in late spring when urea or poultry manure is applied to winter wheat and corn; this is also the time when high frequent P. minimum blooms have been recorded (Glibert et al. 2005b). P. minimum 190 have been observed to follow heavy rainfall and fertilizer application which result in short term increases of urea in the water column (Glibert et al. 2001). Urea can be directly taken up by the dinoflagellates. A high affinity of P. minimum for urea has previously been found (Fan et al. 2003). In the Western Florida Shelf, urea concentrations were also significantly greater over the entire region adjacent to the mouths of both the Shark and Caloosahatchee Rivers after hurricane flooding (Heil et al. 2007). In the East China Sea, urea contributed to over 20% of reduced N (NH4+, urea and glycine) uptake during the K. brevis and P. donghaiense bloom progression. Urea has also been reported to increased toxicity in some harmful algae. Toxigenic Pseudo-nitzschia australis assemblages have also been shown to double toxin production when grown on urea compared to growth on NO3- and NH4+ (Howard et al. 2007). K. brevis in culture was observed to increase in toxin content up to 6-fold after enrichment with urea compared to treatments without urea enrichment (Shimizu et al. 1995). All four tested dinoflagellates have been reported to have mixotrophic abilities as a mechanism to supplement nutrient supplies (Jeong et al. 2005a, Stoecker et al. 2006, Burkholder et al. 2008, Jeong et al. 2010). This mechanism is suggested to be important in nutrient acquisition and growth of these species for the development and maintenance of their blooms in eutrophic waters (Burkholder et al. 2008). Although the high frequent bloom areas of these tested dinoflagellates have variable nutrient loads and concentrations, dissolved forms of nutrients still can be limiting factors in bloom progression, due to high biomass of the algae and the disproportionate nature of nutrient supply (e.g. very high DIN:DIP ratio). Light deficiency is also considered 191 common in eutrophic systems, especially in shallow water and estuarine systems, where algal blooms, sediment from river input, precipitation, wind/river mixing process and other disturbance can cause limiting light availability for the phytoplankton (Hecky and Kilham 1988, Cloern and Jassby 2010). These low light and/or nutrient deficiency habitats may promote the mixotrophy of algae. Mixotrophy might be triggered by nutrient limitation (Gran?li et al. 1999), light limitation (Caron et al. 1993, Jones et al. 1995), or high prey densities (Sanders et al. 1990). Stoecker (1998) proposed three physiological types of protistan mixotrophs: Type I - ??ideal?? mixotrophs that can use phototrophy and phagotrophy equally well; Type II - predominantly phototrophic algae, which included many of the harmful algae in eutrophic estuarine and coastal waters; and Type III - predominantly heterotrophic algae. The four tested dinoflagellates are generally considered type II algae. Many Type II species become mixotrophic when DIN and/or DIP become limiting, and their feeding rates can be directly related to available light (Stoecker 1998, 1999, Li et al. 2000). Prorocentrum minimum can not only consume DON sources via osmotrophy (Fan et al. 2003, Heil et al. 2005, Solomon and Glibert 2008), but also has been reported to ingest cyanobacteria and cryptophytes, including Rhodomonas salina (Stoecker et al. 1997, Jeong et al. 2005a, Jeong et al. 2005b, Jeong et al. 2010). Mixotrophy by P. minimum was found to be higher in spring than the summer in the Chesapeake Bay, when ingestion of cryptophytes was observed in over 50% of the cells in natural assemblages (Stoecker et al. 1997). But, ingestion was low when the cryptophyte densities were high. Ingestion was also inhibited after nutrient 192 enrichment (DIN or DIP), suggesting it a mechanism for obtaining organic nutrients rather than organic carbon (Stoecker et al. 1997). Karlodinium veneficum also takes up urea to support growth, and can ingest eubacteria and cryptophytes Rhodomonas salina (Nygaard and Tobiesen 1993, Li et al. 1996, Li et al. 1999, Li et al. 2000, Adolf et al. 2003, Adolf et al. 2006a, Adolf et al. 2007, Adolf et al. 2008). Growth rates of mixotrophic K. veneficum (0.52 - 0.75 day-1) have been found to be comparable to or greater than maximum growth rate in phototrophic mode (0.55 day-1) (Adolf et al. 2006b). Phagotrophy of K. veneficum has been intensively studied in Chesapeake Bay (Li et al. 1996, Li et al. 1999, Li et al. 2000, Adolf et al. 2003, Adolf et al. 2006a, Adolf et al. 2007, Adolf et al. 2008). From field and laboratory data, phagotrophy of K. veneficum was positively correlated with prey density, and negatively correlated with depth, salinity, and PO43- concentration (Li et al. 2000). N and/or P deficiency, or N:P ratios that were substantially higher or lower than the optimum N:P ratio (~10), increased phagotrophic activity, suggesting feeding as a supplement for major nutrients (N, P) (Li et al., 2000). Phagotrophic activity might also supplement organic carbon sources or assist in acquiring trace growth factors (Li et al., 2000). Prorocentrum donghaiense also has been shown to ingest cyanobacteria, cryptophytes and other dinoflagellates (P. minimum) (Jeong et al. 2005a, Jeong et al. 2010). In low ambient light (20 ?mol photons m-2 sec-1), mixotrophic P. donghaiense reached higher maximum growth rate (0.051 day-1) by feeding on cryptophytes than in autotrophic mode (0.038 day-1) (Jeong et al. 2004, Jeong et al. 2005b). 193 Karenia brevis has been shown to take up DON from Trichodesmium blooms (Mulholland et al. 2006), and also urea to support growth (Glibert et al. 2009). K. brevis can ingest cyanobacteria Synechococcus (Jeong et al. 2005b, Glibert et al. 2009). K. brevis was observed grazing on Synechococcus at higher rate in low light (43 ?mol photons m-2 sec-1) when N-depleted (Glibert et al. 2009). In the present study, the four tested dinoflagellates were grown on treatments which promoted different growth strategies, from autotrophic growth on NO3- at high light, to osmotrophic growth on urea and mixotrophic growth on eukaryote prey, the co-cultured dinoflagellate, or phagotrophic growth on the prey, Rhodomonas or Synechococcus. The initial ambient nutrients were replete. There were no significant differences of the growth rate among the monocultures growing on NO3- and urea. A significant light limitation on the growth rate was observed. All four species could use these dissolved N sources well. When two dinoflagellates were grown together, the growth of P. minimum and K. veneficum were both inhibited in high light, while only the growth of K. brevis was inhibited when P. donghaiense was co-cultured. After adding the Rhodomonas or Synechococcus, the cell densities of these potential food sources either decreased at the beginning of the incubation or decreased when the culture became more nutrient limited. The tested dinoflagellates could feed on the prey, however, the competition between two dinoflagellates is more complicated and needs further experimental study. Both P. minimum and K. veneficum have been documented to bloom in the Chesapeake Bay (Deeds et al. 2002, Glibert and Magnien 2004, Goshorn et al. 2004). Prorocentrum minimum generally blooms in the late spring and early summer, when 194 ambient water N:P ratios are higher than the Redfield ratio, suggesting P limitation (Fisher et al. 1992, Tango et al. 2005, Ptacnik et al. 2010). Prorocentrum minimum outgrew K. veneficum in relative N-rich natural water, which was consistent with the present study. In contrast, K. veneficum generally develops more blooms in the later summer and autumn (Li et al. 2000, Glibert and Magnien 2004), when ambient inorganic N is depleted, and ambient N:P ratios are lower than the Redfield ratio, suggesting N limitation (Fisher et al. 1992, Ptacnik et al. 2010). High cryptophyte abundance has been suggested to trigger K. veneficum blooms (Adolf et al. 2008). Mixotrophic K. veneficum may feed on the cryptophyte as an N source. Herein, in the experiments simulating this condition, K. veneficum outgrew P. minimum after enrichment with Rhodomonas. In the East China Sea, P. donghaiense has been the dominant bloom forming dinoflagellate in recent years, even when the off-shore type species Karenia mikimotoi and Alexandrium tamarense have been documented to mix with the P. donghaiense (Zhou et al. 2006, Li et al. 2009). The only recent exception to this pattern was in 2005, when K. mikimotoi developed blooms before the P. donghaiense in the East China Sea. However, P. donghaiense still outgrew K. mikimotoi in approximately two weeks (Li et al. 2009). Herein, Prorocentrum donghaiense also outgrew the off-shore type K. brevis in present study. In summary, dinoflagellates have multiple nutrient strategies to grow in coastal waters, which depend on the nutrients and light availabilities. The dinoflagellate species in present study have slightly different nutrient strategies, which may drive the results of the growth competition. The effectiveness of utilizing 195 dissolved organic nutrients and prey appears to play important roles in determining the competition between P. minimum and K. veneficum. It may be advantageous for K. brevis to feed on Rhodomonas, but this needs further direct study. The growth of off-shore type species K. brevis was mostly limited by the low light treatments. However, K. brevis had longer growth phase, and kept growing after P. donghaiense reached stationary phase, suggesting better adaptation of K. brevis to low nutrient (or nutrient depleted) condition, because of its greater mixotrophic tendencies. The laboratory experiment results were consistent with the field observation in waters where the blooms of these species were documented. 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Aquatic Microbial Ecology 43:277-287 204 Zhou W, Yin K, Zhu D (2006) Phytoplankton biomass and high frequency of Prorocentrum donghaiense harmful algal bloom in Zhoushan sea area in spring. Chinese Journal of Applied Ecology (In Chinese) 17:887-893 205 Table 5-1 Monoculture exponential growth rates (? day-1) of Prorocentrum minimum and Karlodinium veneficum. R2 is the coefficient of determination of growth rate from the regression of daily cell counts. N source Light N:P ratio Prorocentrum minimum Karlodinium veneficum ? day-1 p R2 ? day-1 p R2 NO3- high 12 0.485 0.0012 0.949 0.321 <0.0001 0.986 NO3- high 145 0.443 0.0003 0.969 0.312 <0.0001 0.990 Urea high 12 0.377 <0.0001 0.829 0.249 <0.0001 0.940 Urea high 145 0.405 <0.0001 0.898 0.237 <0.0001 0.960 NO3- low 12 0.277 <0.0001 0.999 0.187 <0.0001 0.984 NO3- low 145 0.232 <0.0001 0.993 0.185 <0.0001 0.982 Urea low 12 0.257 <0.0001 0.989 0.175 <0.0001 0.918 Urea low 145 0.235 <0.0001 0.958 0.150 <0.0001 0.844 Tables 206 Table 5-2 Exponential growth rates (? day-1) of Prorocentrum minimum and Karlodinium veneficum in mixed culture experiments. R2 is the coefficient of determination of growth rate from the regression of daily cell counts. N source Light N:P ratio Prorocentrum minimum Karlodinium veneficum ? day-1 p R2 ? day-1 p R2 NO3- high 12 0.173 0.0189 0.675 0.06 0.0399 0.507 NO3- high 145 0.328 0.0029 0.862 0.145 0.0369 0.568 Urea high 12 0.182 0.0287 0.593 0.135 0.0391 0.492 Urea high 145 0.245 0.0056 0.729 0.283 0.0033 0.796 NO3- low 12 0.187 0.0006 0.897 0.079 0.0122 0.646 NO3- low 145 0.225 <0.0001 0.939 0.102 0.0057 0.701 Urea low 12 0.276 0.0022 0.861 0.124 0.002 0.799 Urea low 145 0.276 <0.0001 0.935 0.104 0.0024 0.728 207 Table 5-3 Exponential growth rates (? day-1) of Prorocentrum minimum and Karlodinium veneficum with Rhodomonas in mixed culture experiments. R2 is the coefficient of determination of growth rate from the regression of daily cell counts. N source Light N:P ratio Prorocentrum minimum Karlodinium veneficum ? day-1 p R2 ? day-1 p R2 NO3- high 12 0.048 0.3521 0.204 0.086 0.0041 0.843 NO3- high 145 0.038 0.1054 0.434 0.09 0.1181 0.461 NO3- low 12 ~ 0 0.081 0.0712 0.532 NO3- low 145 ~ 0 ~0 208 Table 5-4 Monoculture exponential growth rates (? day-1) of Prorocentrum donghaiense and Karlodinium brevis. R2 is the coefficient of determination of growth rate from the regression of daily cell counts. N source Light N:P ratio Prorocentrum donghaiense Karlodinium brevis ? day-1 p R2 ? day-1 p R2 NO3- high 12 0.374 <0.0001 0.999 0.267 <0.0001 0.948 NO3- high 145 0.206 0.0002 0.911 0.219 <0.0001 0.942 Urea high 12 0.292 0.0001 0.939 0.278 <0.0001 0.971 Urea high 145 0.18 0.0016 0.913 0.278 <0.0001 0.989 NO3- low 12 0.158 <0.0001 0.992 0.136 <0.0001 0.979 NO3- low 145 0.216 <0.0001 0.988 0.124 <0.0001 0.971 Urea low 12 0.269 <0.0001 0.996 0.116 <0.0001 0.968 Urea low 145 0.282 <0.0001 0.986 0.078 <0.0001 0.943 209 Table 5-5 Exponential growth rates (? day-1) of Prorocentrum donghaiense and Karlodinium brevis in mixed culture experiments. R2 is the coefficient of determination of growth rate from the regression of daily cell counts. N source Light N:P ratio Prorocentrum donghaiense Karlodinium brevis ? day-1 p R2 ? day-1 p R2 NO3- high 12 0.380 0.0004 0.871 0.173 <0.0001 0.974 NO3- high 145 0.301 0.002 0.79 0.249 <0.0001 0.987 Urea high 12 0.334 0.0014 0.818 0.235 <0.0001 0.97 Urea high 145 0.344 0.0069 0.709 0.224 <0.0001 0.987 NO3- low 12 0.127 0.0164 0.566 ~0 NO3- low 145 0.153 0.0001 0.88 0.024 0.0131 0.552 Urea low 12 0.116 <0.0001 0.92 ~0 Urea low 145 0.233 0.166 0.926 0.026 0.1391 0.266 210 Table 5-6. Exponential growth rates (? day-1) of Prorocentrum donghaiense and Karlodinium brevis with Synechococcus in mixed culture experiments. R2 is the coefficient of determination of growth rate from the regression of daily cell counts. N source Light N:P ratio Prorocentrum donghaiense Karlodinium brevis ? day-1 p R2 ? day-1 p R2 NO3- high 12 0.385 <0.0001 0.951 0.172 0.0563 0.587 NO3- high 145 0.355 0.0061 0.866 0.16 0.0086 0.802 NO3- low 12 0.137 0.0003 0.894 0.019 0.2738 0.166 NO3- low 145 0.141 <0.0001 0.922 0.055 0.0011 0.762 211 Table 5-7 Summary of the growth rates of 4 dinoflagellates reported in laboratory experiments. 212 Day 0 2 4 6 8 10 12 14 16 18 20 22 24 26 f l u o r e s c e n c e u n i t 0 200 400 600 800 1000 1200 1400 1600 LNP HL HNP HL LNP LL HNP LL Day 0 2 4 6 8 10 12 14 16 18 20 22 24 26 f l u o r e s c e n c e u n i t 0 200 400 600 800 1000 1200 1400 1600 LNP HL HNP HL LNP LL HNP LL Day 0 2 4 6 8 10 12 14 16 18 20 22 24 26 f l u o r e s c e n c e u n i t 0 100 200 300 400 500 LNP HL HNP HL LNP LL HNP LL Day 0 2 4 6 8 10 12 14 16 18 20 22 24 26 f l u o r e s c e n c e u n i t 0 100 200 300 400 500 LNP HL HNP HL LNP LL HNP LL Figure 5-1 Time course of growth of Prorocentrum minimum (a) and Karlodinium veneficum (b) in monoculture with 2 nitrogen sources NO3- (a, c) and urea (b, d) at high and low N:P ratios (HNP, LNP) and high and low light intensities (HL, LL). The error bar is the standard deviation of triplicate treatments. a. P. minimum b. P. minimum c. K. veneficum d. K. veneficum 213 a. NO3- N:P 12 high light Day 0 10 20 30 40 C e l l s p e r m l 0.0 5.0e+4 1.0e+5 1.5e+5 2.0e+5 2.5e+5 K. veneficum P. minimum b. NO3- N:P 145 high light Day 0 10 20 30 40 C e l l s p e r m l 0.0 5.0e+4 1.0e+5 1.5e+5 2.0e+5 2.5e+5 K. veneficum P. minimum c. urea N:P 12 high light Day 0 10 20 30 40 C e l l s p e r m l 0.0 5.0e+4 1.0e+5 1.5e+5 2.0e+5 2.5e+5 K. veneficum P. minimum d. urea N:P 145 high light Day 0 10 20 30 40 C e l l s p e r m l 0.0 5.0e+4 1.0e+5 1.5e+5 2.0e+5 2.5e+5 K. veneficum P. minimum 214 e. NO3- N:P 12 low light Day 0 10 20 30 40 C e l l s p e r m l 0.0 5.0e+4 1.0e+5 1.5e+5 2.0e+5 2.5e+5 K. veneficum P. minimum f. NO3- N:P 145 low light Day 0 10 20 30 40 C e l l s p e r m l 0.0 5.0e+4 1.0e+5 1.5e+5 2.0e+5 2.5e+5 K. veneficum P. minimum g. urea N:P 12 low light Day 0 10 20 30 40 C e l l s p e r m l 0.0 5.0e+4 1.0e+5 1.5e+5 2.0e+5 2.5e+5 K. veneficum P. minimum h. urea N:P 145 low light Day 0 10 20 30 40 C e l l s p e r m l 0.0 5.0e+4 1.0e+5 1.5e+5 2.0e+5 2.5e+5 K. veneficum P. minimum Figure 5-2 Time course of growth of Prorocentrum minimum and Karlodinium veneficum in mixed culture with 2 nitrogen sources at 2 N:P ratios and 2 light intensities. The error bar is the standard deviation of duplicate treatments. 215 a. N:P 12 high light Day 0 5 10 15 20 25 C e l l s p e r m l 0.0 5.0e+4 1.0e+5 1.5e+5 K. veneficum P. minimum Rhodomonas b. N:P 145 high light Day 0 5 10 15 20 25 C e l l s p e r m l 0.0 5.0e+4 1.0e+5 1.5e+5 K. veneficum P. minimum Rhodomonas c. N:P 12 low light Day 0 5 10 15 20 25 C e l l s p e r m l 0.0 5.0e+4 1.0e+5 1.5e+5 K. veneficum P. minimum Rhodomonas d. N:P 145 low light Day 0 5 10 15 20 25 C e l l s p e r m l 0.0 5.0e+4 1.0e+5 1.5e+5 K. veneficum P. minimum Rhodomonas Figure 5-3 Time course of growth of Prorocentrum minimum and Karlodinium veneficum with Rhodomonas in mixed culture with NO3- at 2 N:P ratios and 2 light intensities. The error bar is the standard deviation of duplicate treatments. 216 Day 0 2 4 6 8 10 12 14 16 18 20 f l u o r e s c e n c e u n i t 0 20 40 60 80 100 LNP HL HNP HL LNP LL HNP LL Day 0 2 4 6 8 10 12 14 16 18 20 f l u o r e s c e n c e u n i t 0 20 40 60 80 100 LNP HL HNP HL LNP LL HNP LL Day 0 2 4 6 8 10 12 14 16 18 20 f l u o r e s c e n c e u n i t 0 10 20 30 40 50 LNP HL HNP HL LNP LL HNP LL Day 0 2 4 6 8 10 12 14 16 18 20 f l u o r e s c e n c e u n i t 0 10 20 30 40 50 LNP HL HNP HL LNP LL HNP LL Figure 5-4 Time course of growth of Prorocentrum donghaiense (a., b.) and Karenia brevis (c., d.) in monoculture with 2 nitrogen sources NO3- (a., c.) and urea (b., d.) at high and low N:P ratios (HNP, LNP) and high and low light intensities (HL, LL). The error bar is the standard deviation of triplicate treatments. a. b. c. d. 217 a. NO3- N:P 12 high light Day 0 5 10 15 20 25 C e l l s p e r m l 0 2e+4 4e+4 6e+4 8e+4 1e+5 K. brevis P. donghaiense b. NO3- N:P 145 high light Day 0 5 10 15 20 25 C e l l s p e r m l 0 2e+4 4e+4 6e+4 8e+4 1e+5 K. brevis P. donghaiense c. urea N:P 12 high light Day 0 5 10 15 20 25 C e l l s p e r m l 0 2e+4 4e+4 6e+4 8e+4 1e+5 K. brevis P. donghaiense d. urea N:P 145 high light Day 0 5 10 15 20 25 C e l l s p e r m l 0 2e+4 4e+4 6e+4 8e+4 1e+5 K. brevis P. donghaiense 218 e. NO3- N:P 12 low light Day 0 5 10 15 20 25 C e l l s p e r m l 0 2e+4 4e+4 6e+4 8e+4 1e+5 K. brevis P. donghaiense f. NO3- N:P 145 low light Day 0 5 10 15 20 25 C e l l s p e r m l 0 2e+4 4e+4 6e+4 8e+4 1e+5 K. brevis P. donghaiense g. urea N:P 12 low light Day 0 5 10 15 20 25 C e l l s p e r m l 0 2e+4 4e+4 6e+4 8e+4 1e+5 K. brevis P. donghaiense h. urea N:P 145 low light Day 0 5 10 15 20 25 C e l l s p e r m l 0 2e+4 4e+4 6e+4 8e+4 1e+5 K. brevis P. donghaiense Figure 5-5 Time course of growth of Prorocentrum donghaiense and Karenia brevis in mixed culture with 2 nitrogen sources at 2 N:P ratios and 2 light intensities. The error bar is the standard deviation of duplicate treatments. 219 a. N:P 12 high light Day 0 5 10 15 20 C e l l s p e r m l 0.0 5.0e+4 1.0e+5 1.5e+5 K. brevis P. donghaiense synechococcus b. N:P 145 high light Day 0 2 4 6 8 10 12 14 16 18 20 C e l l s p e r m l 0.0 5.0e+4 1.0e+5 1.5e+5 K. brevis P. donghaiense synechococcus c. N:P 12 low light Day 0 2 4 6 8 10 12 14 16 18 20 C e l l s p e r m l 0.0 5.0e+4 1.0e+5 1.5e+5 K. brevis P. donghaiense synechococcus d. N:P 145 low light Day 0 2 4 6 8 10 12 14 16 18 20 C e l l s p e r m l 0.0 5.0e+4 1.0e+5 1.5e+5 K. brevis P. donghaiense synechococcus Figure 5-6 Time course of growth of Prorocentrum donghaiense, Karenia brevis and Synechococcus in mixed culture at 2 N:P ratios and 2 light intensities. The error bar is the standard deviation of duplicate treatments. 220 a. Hight Light Low N:P Ratio Gr ow th Ra te pe r D ay 0.0 0.1 0.2 0.3 0.4 0.5 0.6 I II III PM PD b.Hight Light High N:P Ratio Gr ow th Ra te pe r D ay 0.0 0.1 0.2 0.3 0.4 0.5 0.6 I II III PM PD c. Hight Light Low N:P Ratio Gr ow th Ra te pe r D ay 0.0 0.1 0.2 0.3 0.4 0.5 0.6 I II III PM PD n.a. n.a. d. Hight Light High N:P Ratio Gr ow th Ra te pe r D ay 0.0 0.1 0.2 0.3 0.4 0.5 0.6 I II III PM PD n.a. n.a. e. Low Light Low N:P Ratio Gr ow th Ra te pe r D ay 0.0 0.1 0.2 0.3 0.4 0.5 0.6 I II III PM PD f. Low Light High N:P Ratio Gr ow th Ra te pe r D ay 0.0 0.1 0.2 0.3 0.4 0.5 0.6 I II III PM PD g. Low Light Low N:P Ratio Gr ow th Ra te pe r D ay 0.0 0.1 0.2 0.3 0.4 0.5 0.6 I II III PM PD n.a. n.a. h. Low Light High N:P Ratio Gr ow th Ra te pe r D ay 0.0 0.1 0.2 0.3 0.4 0.5 0.6 I II III PM PD n.a. n.a. NO3- NO3- Urea Urea 221 i. Hight Light Low N:P Ratio Gr ow th Ra te pe r D ay 0.0 0.1 0.2 0.3 0.4 0.5 0.6 I II III KV KB j. Hight Light High N:P Ratio Gr ow th Ra te pe r D ay 0.0 0.1 0.2 0.3 0.4 0.5 0.6 I II III KV KB k. Hight Light Low N:P Ratio Gr ow th Ra te pe r D ay 0.0 0.1 0.2 0.3 0.4 0.5 0.6 I II III KV KB n.a. n.a. l. Hight Light High N:P Ratio Gr ow th Ra te pe r D ay 0.0 0.1 0.2 0.3 0.4 0.5 0.6 I II III KV KB n.a. n.a. m. Low Light Low N:P Ratio Gr ow th Ra te pe r D ay 0.0 0.1 0.2 0.3 0.4 0.5 0.6 I II III KV KB n. Low Light High N:P Ratio Gr ow th Ra te pe r D ay 0.0 0.1 0.2 0.3 0.4 0.5 0.6 I II III KV KB o. Low Light Low N:P Ratio Gr ow th Ra te pe r D ay 0.0 0.1 0.2 0.3 0.4 0.5 0.6 I II III KV KB n.a. n.a. p. Low Light High N:P Ratio Gr ow th Ra te pe r D ay 0.0 0.1 0.2 0.3 0.4 0.5 0.6 I II III KV KB n.a. n.a. Figure 5-7 The exponential grow rates of 4 harmful dinoflagellates Prorocentrum minimum (PM), P. donghaiense (PD), Karlodinium veneficum (KV) and Karenia brevis (KB) in 3 sets of culture setting with treatments of high light (a, b, c, d, i, j, k, l) and low light (e, f, g, h, m, n, o, p); low N:P ratio (a, c, e, j, i, k, m, o) and high N:P ratio (b, d, f, h, j, l, n, p). Culture setting I: Monoculture; II: mixed culture of P. minimum and K. veneficum; mixing culture of P. donghaiense and K. brevis; III: mixed culture of Rhodomonas, P. minimum and K. veneficum; mixed culture of Synechococcus, P. donghaiense and K. brevis NO3- NO3- Urea Urea 222 Chapter 6: Research Conclusions Dinoflagellate Blooms and the Matrix of Nutrients and Light Smayda (Smayda and Reynolds 2001, Smayda 2002) has suggested that the increase in dinoflagellate blooms can be categorized as responses to a changing environment (e.g. eutrophication). Each dinoflagellate requires a common set of ecological characteristics (niche) that support it to develop blooms (Smayda 1990, Smayda 1997, 2002). Smayda (2002) refined this environmental adaptation of dinoflagellates by classifying them into different functional groups in a matrix, based on ecophysiological preferences organized by a near-shore / off-shore gradient in decreasing nutrients, reduced mixing, and increasing light. In this matrix, estuarine species are defined as the dinoflagellates which are better adapted to low-light high- nutrient waters, but oceanic species are better adapted to high-light low-nutrient waters. Estuarine dinoflagellate Prorocentrum minimum and P. donghaiense have been reported to coexist with Karenia brevis and K. mikimotoi in the southwest Florida shelf and the East China Sea respectively (Heil et al. 2007, Li et al. 2009). Each pair of algae developed blooms in either spatial or temporal succession in these coastal waters, and a remarkable similarity was found between the dominant species and their ambient N:P ratios (Li et al. 2009). This dissertation study was conducted to answer the research question whether this relationship between the ambient N:P ratio and the dominance species can be expanded to other regions, where estuarine species 223 P. minimum and Karlodinium veneficum develop blooms, and to address the physiological basis for such a relationship. The research conducted for this dissertation set out to study the growth and physiological mechanisms of nitrogen acquisition with respect to nutrient and light, which are two factors used to characterize the niches of dinoflagellates by Smayda?s matrix. The following hypotheses were tested: 1) In Chesapeake Bay, P. minimum develops blooms in relative high DIN:DIP ratio water; while K. veneficum blooms near or lower than the Redfield ratio. 2) These species will grow faster in the N:P ratio in which they develop blooms, even when these nutrients are not at limiting levels. 3) Prorocentrum spp. preferentially take up more DIN in high DIN:DIP ratio water, while Karlodinium can better use other source of N in the low DIN water (e.g. DON, particulate N). 4) Low-light-adapted nitrogen acquisition by Prorocentrum spp. serves as an adaptive advantage to grow in low light waters. From this dissertation study, the following results were found, corresponding to the hypotheses: In the Chesapeake Bay, the estuarine species P. minimum developed blooms in high DIN, high N:P ratio, turbid water; while K. veneficum bloomed after DIN was depleted (Chapter 2) and ambient N:P ratio was near the Redfield ratio. These trends are consistent with East China Sea and west Florida shelf for similar species (Chapter 224 2). K. veneficum needs additional N sources to bloom, which may be the relatively high concentrations of DON in the water column. However, in culture, the estuarine species P. minimum and P. donghaiense did not reach the highest maximum growth rates in N-rich high N:P ratio water, instead, they reached the highest maximum growth rates at more N:P balanced ratio (near the Redfield ratio) in turbidistat (Chapter 3). When ambient N:P ratio varied, but nutrients were sufficient, there was also no significant variation (p > 0.05) of the growth rates for both P. minimum and K. veneficum in batch culture (Chapter 5). Instead, P. minimum grew faster on NO3-, a DIN source, while, K. veneficum grew faster on urea, a DON source. Light is another important factor in the Smayda?s matrix. All four tested species showed light limitation in the low light treatments in batch culture (Chapter 5) and turbidistat (Chapter 4). However, light affected the growth rate differently: low- light adapted estuarine species Prorocentrum spp. reached about 60% of their high- light growth rate; while, oceanic species K. brevis only reached about 30% of its high-light growth rate. Similar results were recorded in both batch culture and turbidistat. The diel growth of P. minimum and P. donghaiense is strictly light regulated. Positive growth was only recorded during the light phase by the turbidistat (Chapter 4). The highest growth rates were recorded at the beginning of the light phase, and the growth rates decreased during the light phase. This diel cycle of growth rate can be interpreted by Droop?s model as the light-dark regulation of the N acquisition by Prorocentrum species. The growth rate appears to be more driven by the variation of 225 cell quota, than by ambient nutrient concentrations. Prorocentrum spp. continuously take up nitrogen at night to supplement the cell quota, when the growth stopped. The cells have the maximum cell N quota and reach highest growth rate at the beginning of light phase (Chapter 3). In contrast, oceanic species K. brevis and K. mikimotoi take up N in the light phase only, even when nutrient is saturated (Paasche et al. 1984, Sinclair et al. 2009). Therefore, the physiological mechanisms of nitrogen acquisition are regulated by light differentially between these two types of algae. In turbid estuarine water, Prorocentrum spp. can take up N at high rates and may be better competitors than Karenia and Karlodinium spp., which have a greater light requirement for dissolved N uptake. When two types of algae were grown in mixed culture, the estuarine species Prorocentrum spp. overgrew the opponents in most of the treatments (Chapter 5). However, Karlodinium veneficum overgrew P. minimum with the presence of Rhodomonas, a potential nutrient source. The growth of both K. brevis and P. donghaiense were reduced with the presence of Synechococcus. Other mechanisms (e.g. allelopathy), in addition to nutrient competition, should be considered and may also play a role in determining the dominant species. Therefore, in the mixed culture, growth rates do not need to be at the physiological maximum for the given set of conditions for species to become dominant. There might be other competition mechanisms (e.g. allelopathy), other than nutrient competition, to determine the dominant species. In summary, there are high DIN (mostly NO3-) sources accumulated in the upper Chesapeake Bay and its tributaries during the winter and spring (Chapter 2). 226 When the water temperature increased over 10-15 ?C, estuarine species P. minimum can take the advantage of high DIN uptake rate in turbid water, and overgrow other dinoflagellates or diatoms (Fig. 6-1). However, DIN is depleted in the summer, but there are still relatively high organic forms of nutrients available in the water column. Karlodinium veneficum may be mixotrophic and feed on the organic sources, or directly feed on other micro-algae in the water column to develop blooms. In eutrophic coastal systems (e.g. Chesapeake Bay), there are high residual nutrient sources or/and nutrient inputs. The exponential growth phase of batch culture and continuous culture are used to simulate the growth of dinoflagellates in such environment. From this study, the availability of different nutrient sources (not limited to inorganic nutrients), but not the ratio of those nutrients, are critical for the nutrient acquisition and bloom development. P-limitation may not develop in high N:P ratio eutrophic water, due to the development of light-limitation (Flynn 2010b). Light, instead of ambient N:P ratio, significantly affected the growth of all species in this study. In Chesapeake Bay, the eutrophic areas are very turbid, and the two types of bloom-forming dinoflagellates have different nutrient acquisition strategies: Prorocentrum minimum keeps taking N in the dark, while K. veneficum can be mixotrophic and may directly use organic sources. Availability of nutrient sources and light are two key environmental factors necessary to understand the bloom mechanisms in eutrophic waters. The Application of Droop Model Nutrient limitation of phytoplankton grow can either be described by the Monod model (Monod 1950) or by the Droop model (Droop 1968, 1973). Although 227 the Monod model originated as a description of steady-state bacterial growth, it has been used as the default description of nutrient acquisition in dynamic systems in many ecosystem models (Flynn 2010a). It is widely used, because it is the simplest mathematical description of nutrient limited growth, and only considers external nutrients. The Redfield ratio (Redfield 1958), originated as an empirical observation, has been used as a key index to identify the limited nutrient in the application of Monod model, in which the model simulation will follow the fate of the limiting nutrient and the consumption of other nutrients follow in proportion of the Redfield ratio (Flynn 2010a). However, the ambient N:P ratio is the sum of both abiotic and biotic processes. Not only nutrient consumption, but also nutrient availability affects the ratios. For example, in Chesapeake Bay (Chapter 2), when the water column is dominated by the abiotic processes (i.e., terrestrial input) in the winter, the ambient N:P ratio converge to the ratios of input nutrients, which is much higher than the Redfield ratio. However, when the water column is dominated by the biotic processes (i.e., algal blooms), the ambient N:P ratio converge to Redfield ratio, the stoichiometric ratio of phytoplankton cells. In this dissertation study, the effects of ambient N:P ratio on the growth and N acquisition Prorocentrum species were studied. The N uptake rates of Prorocentrum spp. were more balanced with the growth rates at N:P ratios near Redfield ratio. However, when nutrients were sufficient, there was no significant variation of the growth rates at different N:P ratios. There was no direct evidence that growth was regulated by the ambient N:P ratio, instead, the growth is regulated by the nutrient acquisition ability of the cells. 228 When studying N acquisition, both the N uptake and assimilation process can be measured and linked to the growth rates by the Droop cell quota model (Droop 1968, 1973). An intracellular nutrient pool (cell quota) is regulated by nutrient taken into the cell and assimilated to support the growth. The growth rate is regulated by the size of cell quota, instead of the external nutrient pool (Droop 1968, 1973). The quota concept is considered more realistic than the Monod model, because growth can now continue at the expense of previously accumulated nutrient (Flynn 2008). It is easier to configure a relationship between growth rate and the internal nutrient concentration (quota) than between growth and the external nutrient concentration (Monod) (Goldman 1977, Flynn 2010a). Thus, growth rate is solely a function of the quota equation with no reference to external nutrient concentrations in the application of Droop model. In this dissertation study, the physiological mechanisms of cell quota regulated by nutrient uptake are supported by the turbidistat study, and can be described using individual cell quota based model. Prorocentrum spp. can take up N at night, and supplement the cell quota, which is consistent with the observation of maximum growth rate of Prorocentrum spp. at the beginning of the light phase in a diel cycle. Therefore, the Droop model may better describe the physiological activities and growth of phytoplankton cells, and can be further applied to ecosystem modeling work. The Monod model can not biologically capture this fundamental dynamic. The growth and physiology of phytoplankton should be directly linked to the cell quota, which is regulated by their ability to acquire nutrients (Flynn 2010b), instead of the ambient nutrient concentrations or ratios. 229 The Improvement of Droop Model The cell quota model initially described in term of cell (e.g. N per cell), but cell size varies not only with cell cycle, but also with various environmental and physiological factors, including nutrient stress (Lehman 1976, Gotham and Frisch 1981). Therefore, the changes of cell quota may not directly respond to the nutrient content on a cell, but due to the change of cell size. To overcome these issues, the quota relationship has been strongly suggested to be normalized to biomass and described in terms of carbon (Flynn 2008). However, in this dissertation study, both the uptake and assimilation rates were measured as specific rates (h-1). The cell N quota can be described as a dynamic system of N acquisition and consumption, and the growth rate can be mathematically described as variation rate of cell quota. Compared to the Monod model, the application of Droop model needs more parameters. In the future research, experiments should be designed to get complete data sets for the Droop model. For example, in a growth experiment, not only the nutrients and cell number variation during the growth phase should be measured, the variation of particulate C, N, and P in the culture also should be measured, if possible. Also, when using C or N isotope tracer techniques to study the C-fixation or N- uptake, the variation of C and N in the cell should be linked with the growth rate during the incubation. 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