ABSTRACT Title of Dissertation: TOXINS AND TOXICITY FROM THE COSMOPOLITAN, BLOOM-FORMIMG DINOFLAGELLATE Karlodinium micrum. Jonathan R. Deeds, Doctor of Philosophy, 2003 Dissertation directed by: Professor Allen R. Place University of Maryland Biotechnology Institute Center of Marine Biotechnology Marine, Estuarine, and Environmental Science Program Karlodinium micrum (Leadbeater and Dodge) Taylor was first described in the United States during an investigation into the cause of repeated fish kills at an estuarine aquaculture facility located on a tributary of the Chesapeake Bay, USA. As part of that investigation we described toxins in this species for the first time. Named karlotoxins (KmTx), these compounds possess hemolytic, cytotoxic, and anti-fungal properties. However, the primary harmful effect associated with blooms of this organism is ichthyotoxicity. Karlotoxins are lethal to fish through damage to gill epithelia. This research focused on two of these toxins, KmTx 1 (1338 Da.) and KmTx 2 (1344 Da.) These two toxins have been the main toxins, in terms of amount and potency, in all US isolates tested to date. Using a range of mammalian cell types, the mode of KmTx 2 cytotoxicity was shown to be non-selective permeabilization of cell membranes to a range of small ions and molecules resulting in cell death through osmotic lysis. Membrane sterol composition appears to play a role in the sensitivity of different species to KmTx’s membrane disrupting effects. This sterol specificity also appears to be responsible for the apparent immunity of K. micrum from its own toxins. We have described various karlotoxins in K. micrum isolates and bloom samples from US east coast estuaries from Maryland to Florida. Among US east coast isolates, a geographic strain variation appears to exist in that KmTx 1 has only been found in Maryland isolates while KmTx 2 has been found in all other isolates tested from North Carolina, South Carolina, and Florida. Recently, a KmTx 2-like compound (1342 Da.) has been isolated from bloom samples from Western Australia, being the first confirmation of karlotoxin production outside of the United States. This work confirms the association between blooms of K. micrum and fish kills that has been observed in temperate estuaries around the world for over half a century. It also lays the foundation for future studies to determine the ecological function of toxin production in this species and the consequences of this production both on K. micrum’s environment and ours. TOXINS AND TOXICITY FROM THE COSMOPOLITAN, BLOOM-FORMING DINOFLAGELLATE Karlodinium micrum By Jonathan R. Deeds Thesis 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 2003 Advisory Committee: Allen R. Place, Ph.D., Chair Renate Reimschuessel, Ph.D., D.V.M. Diane K. Stoecker, Ph.D. Daniel E. Terlizzi, Ph.D. John M. Trant, Ph.D. DEDICATION To my parents, Ron and Marie, who sacrificed so much for my education. You may never have fully understood what I was doing with my life but you never questioned why I was doing it. And to my wife Bethany whose support and understanding made this accomplishment possible. I thank her for agreeing to postpone our lives for a little while. ii ACKNOWLEDGEMENTS First and foremost I must acknowledge my advisor and friend Allen Place. I know he took a chance on me five years ago and I will always be grateful to him for that. We may not have always agreed on our interpretation of the data, but trying to prove my theories to him made me a better scientist. I also wish to acknowledge my research committee members, Renate Reimschuessel, Diane Stoecker, Danial Terlizzi, and John Trant. Each contributed significantly to the quality of the research contained in this text. Lastly, I must acknowledge Tony Mazaccarro, owner and proprietor of HyRock Fish Farm. The observations of Tony and Dan back in 1996 were the inspiration for this entire line of research. Your questioning of the party line did not go in vain. Additional acknowledgements are contained at the end of each chapter. iii TABLE OF CONTENTS List of Tables -------------------------------------------------------------------------------------- vi List of Figures ------------------------------------------------------------------------------------- vii Chapter 1 – Introduction ---------------------------------------------------------------------- 1 Tables/Figures 7 Chapter 2 – Toxic Activity from Cultures of Karlodinium micrum (=Gyrodinium galatheanum) (Dinophyceae) – A Dinoflagellate Associated with Fish Mortalities in an Estuarine Aquaculture Facility. -------------------------- 9 Abstract 10 Introduction 12 Materials and Methods 14 Results 26 Discussion 33 Tables/Figures 42 Chapter 3 - Treatment Options for the Control of the Ichthyotoxic Dinoflagellate Karlodinium micrum in an Estuarine Aquaculture Facility. ------------- 62 Abstract 63 Introduction 64 Materials and Methods 66 Results 69 Discussion 70 Tables/Figures 75 Chapter 4 - Mode of Cytotoxicity of KmTx 2, A New Fish-Killing Toxin from the Dinoflagellate Karlodinium micrum. --------------------------------------- 81 Abstract 82 Introduction 84 Materials and Methods 85 Results 99 Discussion 103 Tables/Figures 112 Chapter 5 - Mode of Ichthyotoxicity of the Toxins from the Dinoflagellate Karlodinium micrum. -------------------------------------------------------- 138 Abstract 139 iv Introduction 140 Materials and Methods 141 Results 144 Discussion 146 Tables/Figures 151 Chapter 6 - Geographic Strain Variation in Toxin Production in Karlodinium micrum (Dinophyceae) from Southeastern Estuaries of the United States. --- 163 Abstract 164 Introduction 165 Materials and Methods 166 Results 168 Discussion 169 Tables/Figures 172 Addendum – Investigation of the 2003 Swan and Canning River Fish Kills (Perth, Western Australia) Associated with Blooms of Karlodinium micrum. ------------------------------------------------------------------------ 182 Abstract 183 Introduction 184 Materials and Methods 185 Results 185 Discussion 186 Tables/Figures 190 Chapter 7 – Summary and Future Research Directions. --------------------------------- 198 Tables/Figures 211 Literature Cited---------------------------------------------------------------------------------- 221 Chapter 1 221 Chapter 2 223 Chapter 3 230 Chapter 4 232 Chapter 5 239 Chapter 6 243 Chapter 7 245 v LIST OF TABLES Table 2.1. Copper chelation experiments measuring free copper in solution after the addition of EDTA (2 mM). p. 43 Table 2.2. Summary of toxic activity from cultures of Karlodinium micrum. p. 45 Table 4.1. Comparison of anti-fungal and hemolytic activities of KmTx 2 and amphotericin B. p. 113 Table 6.1 Karlodinium micrum samples screened for the presence of KmTx 1 and KmTx 2. p. 173 Table 7.1 Summary of current data for toxins from U.S. populations of Karlodinium micrum. p. 212 vi LIST OF FIGURES Figure 1.1. SEM image of Karlodinium micrum. p. 8 Figure 2.1. Map of the Chesapeake Bay showing location of HyRock Fish Farm. p. 47 Figure 2.2. Dose dependence for the lysis of rainbow trout erythrocytes, compared to cells lysed with 10 µg saponin. p. 49 Figure 2.3. Percent hemolysis, measured as release of hemoglobin compared to cells lysed with 10 µg saponin, in aliquots of separated lipid classes from Karlodinium micrum (CCMP 1974). p. 51 Figure 2.4. Hemolytic activity in 0.5 min. HPLC fractions (flow rate 1 ml/min) of 80% MeOH C18 elutions from Karlodinium micrum (CCMP 1974) cells and culture filtrate. p. 53 Figure 2.5. UV absorbance spectra (mAU) of reversed phase HPLC fractions Tox A and Tox B. p. 55 Figure 2.6. In-vitro cytotoxicity assay, based on the release of lactate dehydrogenase, testing 0.5 min. reversed phase HPLC fractions of a 50 µl injection of a concentrated 80% MeOH C18 elution of Karlodinium micrum (CCMP 1974) culture filtrate. p. 57 Figure 2.7. Cell lysis in Karlodinium micrum (CCMP 1974) and Prorocentrum minimum (strain PM-1) cultures exposed to either CuSO4 or KMnO4 . p. 59 Figure 2.8. Time course of hemolytic material release, measured as release of hemoglobin from rainbow trout erythrocytes, compared to cells lysed with 10 µg saponin, in Karlodinium micrum (CCMP 1974) cultures exposed to either CuSO4 or KMnO4. p. 61 Figure 3.1. Schematic diagram of HyRock Fish Farm. p. 76 Figure 3.2. Effect of algicidal treatments on cell lysis of Karlodinium micrum. p. 78 Figure 3.3. Effect of algicidal treatments on hemolytic activity from both cells and isolated toxins from Karlodinium micrum. p. 80 Figure 4.1. Purification and molecular weight determination of KmTx 2. p. 115 Figure 4.2. Purification and identification of gymnodinosterol isolated from Karlodinium micrum (CCMP 2282). p. 117 vii Figure 4.3. Fura 2 pre-loaded rat cardiac myocyte exposed to 0.25 µg/ml KmTx 2. p. 119 Figure 4.4. Measurement of Ca2+ flux into rat embryonic fibroblast (REF 52) cells using the intracellular fluorescent indicator fura-2. p. 121 Figure 4.5. Additional intracellular fluorescent indicator experiments to test the specificity of cation permeability in REF-52 cells upon exposure to KmTx 2. p. 123 Figure 4.6. KmTx 2 induced current in whole cell patch-clamped rabbit vagal sensory nodose ganglion neuron (NGN). p. 125 Figure 4.7. Voltage ramp constructed-current voltage (I-V) relationship for KmTx 2 induced current in whole cell patch clamped rabbit vagal sensory nodose ganglion neuron (NGN). p. 127 Figure 4.8. Inhibition of hemolysis of rainbow trout erythrocytes due to exposure to 0.25, 0.5 or 1 µg/ml KmTx 2 after co-incubation with 30 mM of various molecular weight osmolytes (300-10,000 Da.). p. 129 Figure 4.9. Effect of KmTx 2 on Oxyrrhis marina and Karlodinium micrum (CCMP 2282). p. 131 Figure 4.10. Hemolysis of rainbow trout erythrocytes due to exposure to 0, 0.1, 0.5, or 1 µg/ml KmTx 2 after co-incubation with various membrane sterols. p. 133 Figure 4.11. Hemolysis of rainbow trout erythrocytes due to exposure to 0, 0.1, 0.5, or 1 µg/ml KmTx 2 after co-incubation with membrane lipids. p 135 Figure 4.12. H&E stained sections of whole 60-day old zebrafish (Danio rerio) exposed to an increasing concentration of KmTx 2.