HEMOLYTIC ACTIVITY OF SIX CARIBBEAN GAMBIERDISCUS SPECIES William Christopher Holland A Thesis Submitted to the University of North Carolina Wilmington in Partial Fulfillment of the Requirements for the Degree of Master of Science Center for Marine Science University of North Carolina Wilmington 2012 Approved by Advisory Committee Wayne Litaker John Morrison Carmelo Tomas Chair Accepted by Dean, Graduate School This thesis was submitted in the format for the journal TOXICON ii TABLE OF CONTENTS ABSTRACT .....................................................................................................................................v ACKNOWLEDGEMENTS .......................................................................................................... vii LIST OF TABLES ....................................................................................................................... viii LIST OF FIGURES .........................................................................................................................x APPENDIX .................................................................................................................................... xi INTRODUCTION ...........................................................................................................................1 MATERIALS AND METHODS .....................................................................................................4 Algal cultures .......................................................................................................................4 Cultures tested for hemolytic activity ..................................................................................6 Optimizing the extraction protocol ......................................................................................6 Hemolytic assays .................................................................................................................8 Hemolytic activity versus growth temperature ....................................................................9 Statistical analysis ..............................................................................................................11 RESULTS ......................................................................................................................................11 Species-specific hemolytic activity....................................................................................11 Regional differences in toxicity .........................................................................................13 Variations in toxicity with growth phase ...........................................................................14 The effect of growth temperature on hemolytic activity ...................................................15 Membrane associated hemolytic activity ...........................................................................16 DISCUSSION ................................................................................................................................16 Species-specific differences in hemolytic activity .............................................................16 Regional differences in hemolytic activity ........................................................................17 Variations in hemolytic activity with growth phase ..........................................................20 The effect of growth temperature on hemolytic activity ...................................................22 Stability of hemolytic activity through time ......................................................................23 iii Hemolytic activity associated with the membrane fraction ...............................................24 CONCLUSIONS............................................................................................................................24 LITERATURE CITED ..................................................................................................................36 FIGURES .......................................................................................................................................35 TABLES ........................................................................................................................................40 APPENDIX ....................................................................................................................................53 iv ABSTRACT This study examined the variation in hemolytic activity (HA) among six of the dominant Gambierdiscus species found in the Caribbean. In all, 56 isolates were tested. The data showed that certain species had significantly greater HA on average than others. Geographically dispersed isolates of G. caribaeus and G. carolinianus were examined to determine if regional difference in toxicity exist. Isolates from the western Caribbean and Gulf of Mexico had ten times more HA than isolates from the eastern Caribbean as far north as the Florida Keys. Isolates of G. carolininaus from the continental shelf off North Carolina, had HAs 10-fold lower than those from the eastern Caribbean and 100-fold less than those from the western Caribbean. Intra specific variation in HA of isolates the same region was low, indicating regionally differentiated populations selected for varying levels of HA. Depending on the species, HA consistently increased by 40% from log phase to late log phase and then declined in stationary phase to levels below those observed in log phase growth. This same trend of maximal HA in late log phase was observed whether the toxicity data were normalized on either a per cell or per biomass basis indicating a definite increase in HA per unit biomass. Increasing growth temperatures from 20 to 31 °C for clones of G. caribaeus from different regions showed only a slightly significant increase in HA between 20 and 27 °C. Gambierdiscus carolinianus isolates from different regions grown at the same range of temperatures remained constant. These data suggest that growth temperature is not a significant factor in modulating the inter isolate and inter specific differences in HA. The HA of various isolates measured repeatedly over a 2 year period remained constant, indicating that toxins were constitutively produced and under strong genetic control. Depending on species, 60 to 90% of the total HA was associated with the cell membranes. The HA of a limited number of Gambierdiscus isolates examined from outside the v Caribbean were similar to those from the Caribbean. This suggests that variations in HA among Gambierdiscus species is similar worldwide. vi ACKNOWLEDGEMENTS I would like to thank Dr. Carmelo Tomas for allowing me the opportunity to learn about hemolytic activity and the processing of data. Additional thanks go out to the former members of the Tomas lab; Avery Tatters and Joanne Kelly who helped me run samples and process data. Also this work would have not been able without the full support of Dr. Patricia Tester who allowed me to pursue my graduate degree and for opening my eyes to ecology and the physiology of phytoplankton. A special thanks to Dr. Wayne Litaker for helping me throughout the project and especially in drafting my thesis. Thanks to Erik Davenport for the statistical analyses. I would also like to thank the follow individuals from the NOAA Beaufort lab and colleagues assisting on diving operations for the lab for their willingness to collect samples from various locations in the Gulf of Mexico and the Caribbean: Paula Whitfield, Christine A. Buckel, Brian Degan, Don Field, Jenny VanderPlyum, Roldan Muñoz, John Burke, Dave Cerino, Michael Dowgiallo, Wilson Freshwater, David Johnson, Brett Harrison, Doug Kesling, William Lee, Roger Mays, James Morris, Brandon Puckett, and Sherry Reed. Reference to tradenames does not imply product endorsement by the National Ocean Service, NOAA. vii LIST OF TABLES Table Page 1. Species, strain identification and relevant location collection information .......................40 2. Homogeneity of variance test (Levene) estimated for the data obtained from analyzing hemolytic activity in six Gambierdiscus species found in the Caribbean. The analysis specifically examined the differences among species, isolates, growth phase, and species vs. growth phase interaction ........................42 3. Non-parametric Kruskall-Wallis ANOVA tested whether hemolytic activity was significantly different among all six species (G. belizeanus, G. caribaeus, G. carolininaus, G. carpenteri, G. ribotype 2, and G. ruetzleri) when the log, late log and stationary phase data were considered together or separately .......................................................................................43 4A. Multiple comparison test to determine which species (G. belizeanus, G. caribaeus, G. carolininaus, G. carpenteri, G. ribotype 2, and G. ruetzleri) had statistically different hemolytic activity ...................................................................44 4B. The same non-parametric multiple comparisons test based on the EC50 data normalized to biovolume for (G. belizeanus, G. caribaeus, G. carolininaus, G. carpenteri, G. ribotype 2, and G. ruetzleri) ........................................... 45 5. Growth rates of G. caribaeus, G. carolinianus, and G. ruetzleri versus EC50 cells ...........................................................................................................................46 6. The data for G. caribaeus and G. carolinianus were further