I Nutritional and Photophysiological Approaches to Identifying the Niche
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i Nutritional and Photophysiological Approaches to Identifying the Niche of Gambierdiscus: Insight into the Ecology of Ciguatera Fish Poisoning A Thesis Presented to The Faculty of the College of Arts and Sciences Florida Gulf Coast University In Partial Fulfillment Of the Requirements for the Degree of Master of Science By Alexander Kenneth Leynse 2016 ii APPROVAL SHEET This thesis is submitted in partial fulfillment of the requirements for the degree of Master of Science _______________________________________ Alexander Kenneth Leynse Approved: ________________________ __________________________________ Michael Parsons, Ph.D. Committee Chair / Advisor __________________________________ Serge Thomas, Ph.D. __________________________________ Ai Ning Loh, Ph.D. The final copy of this thesis has been examined by the signatories, and we find that both the content and the form meet acceptable presentation standards of scholarly work in the above mentioned discipline. iii To Mom and Dad without whose love, support and encouragement this would not have been possible. iv Acknowledgments I extend my sincere gratitude first and foremost to the members of my thesis committee: Dr. Mike Parsons for providing his guidance, patience, and ongoing commitment to my research, as well as his expertise on ciguatera, rock and roll and pretty much everything else. I couldn’t have asked for a better mentor/advisor; Dr. Serge Thomas, for providing his expertise on physiology and photosynthesis; and to Dr. Ai Ning Loh, for providing her expertise on analytical procedures and chemical transfer. Without the support of this committee the true potential of this work as well as that of myself as a scientist would not have been possible. I would also like to thank my friends and family for providing their support both in and out of the lab: The original team FiReCResT: Lacey Rains and Ashley Brandt; My fellow graduate students: Rheannon Ketover, Amanda Ellsworth, Mark Barton, Chris Lienhardt, Kevin Tyre, Meghan Hian, Adam Catasus and Jeff Zingre; and my loving and incredibly supportive family: Dad, for providing support in every way, from technical help with any custom electronics or devices, to encouraging me to pursue what seemed impossible at times; Mom, for always being there to provide love and support in any way possible, and last but not least, Marina whose competitiveness and drive will always motivate me. I would also like to thank the Coastal Watershed Institute and the Vester Field Station, namely Meghan Hian and Tim Bryant for help with ordering supplies, reserving vehicles, and providing moral support. Thank you also to the NOAA EcoHAB program (Grant # NA11NO54780028) for their funding and support of this project; to the Blair Foundation Environmental Sciences Scholarship for partially supporting me as I carried out my research, and to the FGCU Office of Research and Graduate Studies for providing funding for travel to relevant meetings and conferences. v TABLE OF CONTENTS Acknowledgments ............................................................................................................ iv LIST OF FIGURES ........................................................................................................ vii LIST OF TABLES ........................................................................................................... ix LIST OF ACRONYMS .....................................................................................................x ABSTRACT ...................................................................................................................... xi Chapter I: Ciguatera Fish Poisoning…………………………………………………... 1 Chapter II: Nutrient Uptake Kinetics Experiments (N.U.K.E) .................................. 14 INTRODUCTION............................................................................................................14 METHODS .......................................................................................................................19 RESULTS .........................................................................................................................28 DISCUSSION ...................................................................................................................48 CONCLUSION ................................................................................................................62 Chapter III: Photoacclimation and Photoprotection of Gambierdiscus .................... 63 INTRODUCTION............................................................................................................63 METHODS .......................................................................................................................76 RESULTS .........................................................................................................................81 DISCUSSION ...................................................................................................................89 CONCLUSION ................................................................................................................96 vi Chapter IV: The Niche of Gambierdiscus……………………………………………..97 References……………………………………………………………………………...100 vii LIST OF FIGURES Chapter II: Nutrient Uptake Kinetics Experiments (N.U.K.E) .................................. 14 Figure 2.1: Example of Michaelis-Menten kinetics .......................................................... 16 Figure 2.2: Flow chart describing the process for arriving at ρassim, p, and alpha (α) .... 24 Figure 2.3: Example of the reverse reciprocal plots ......................................................... 31 - Figure 2.4: RRP for G. carolinianus NO3 assimilation kinetics ...................................... 32 Figure 2.5: Kinetic uptake and assimilation rates of nitrate and ammonium ................... 34 Figure 2.6: Comparison of competitive nitrate and ammonium kinetic uptake rates ....... 38 Figure 2.7: Comparison of competitive kinetic quota satisfaction times.......................... 39 Figure 2.8: Assimilation efficiencies for nitrate at various ambient concentrations ........ 43 Figure 2.9: Assimilation efficiencies for ammonium at various ambient concentrations 43 Figure 2.10: G. silvae intracellular DIN concentrations ................................................... 44 Figure 2.11: G. carolinianus intracellular DIN concentrations ........................................ 44 Figure 2.12: Assimilation efficiency vs. intracellular DIN concentrations ...................... 45 Figure 2.13: Absolute assimilation rate vs. intracellular DIN concentrations .................. 46 Figure 2.14: Conceptual model describing the nutritional strategies of Gambierdiscus .. 47 Chapter III: Photoacclimation and Photoprotection of Gambierdiscus .................... 63 Figure 3.1: Depiction of PSII and PSI .............................................................................. 66 Figure3.2: Example of the PSII Light Harvesting Complex............................................. 67 Figure 3.3: Different possible fates of singlet state chlorophyll ....................................... 69 viii Figure 3.4: Theoretical trace of fluorescence output ........................................................ 73 Figure 3.5: Stepwise exposure process during P-I curves ................................................ 80 Figure 3.6: Kinetic light driven growth rates .................................................................... 81 Figure 3.7 Cell sizes following acclimation to situational irradiance treatments ............. 82 Figure 3.8: Comparison of the RF per cell exhibited across acclimation to SITs ............ 83 Figure 3.9: Comparison of the kinetic photosynthetic yields across SITs ........................ 85 Figure 3.10: Comparison of the grouped mean kinetic photosynthetic yield ................... 86 Figure 3.11: Recovery of photosynthetic yield ................................................................. 86 Figure 3.12: Calculated NPQ exhibited by G. carolinianus ............................................. 87 Figure 3.13: Calculated NPQ exhibited by G. silvae ........................................................ 87 Figure 3.14: G. carolinianus cellular and photophysiological response to SITs .............. 88 Figure 3.15: G. silvae cellular and photophysiological response to SITs ......................... 89 ix LIST OF TABLES Chapter I: Ciguatera Fish Poisoning…………………………………………………... 1 Table 1.1: Gambierdiscus species. Including 11 described and three undescribed. ........... 4 Chapter II: Nutrient Uptake Kinetics Experiments (N.U.K.E) .................................. 14 Table. 2.1. Analytical steps involved in nutrient uptake kinetic experiments .................. 20 Table 2.1: Cellular mass and N composition for G. silvae and G. carolinianus ............... 29 - + Table.2.2: Kinetic parameters for uptake and assimilation of NO3 and NH4 ................. 33 Table 2.3: Comparison of kinetic parameters ................................................................... 37 Table 2.4: Competative comparison of kinetic parameter α ............................................. 40 Table.2.5: Comparison of kinetic parameters exhibited by toxic dinoflagellate species .. 41 Chapter III: Photoacclimation and Photoprotection of Gambierdiscus .................... 63 Table 3.1: Fluorescence terminology ................................................................................ 70 Table 3.2: Summary of chlorophyll