Characterizing the Link between Biological Membranes and Thermal Physiology in Antarctic Notothenioid Fishes A dissertation presented to the faculty of the College of Arts and Sciences of Ohio University In partial fulfillment of the requirements for the degree Doctor of Philosophy Amanda M. Biederman August 2019 © 2019 Amanda M. Biederman. All Rights Reserved. 2 This dissertation titled Characterizing the Link between Biological Membranes and Thermal Physiology in Antarctic Notothenioid Fishes by AMANDA M. BIEDERMAN has been approved for the Department of Biological Sciences and the College of Arts and Sciences by Elizabeth L. Crockett Professor of Biological Sciences Joseph Shields Interim Dean, College of Arts and Sciences 3 ABSTRACT BIEDERMAN, AMANDA M., Ph.D., August 2019, Biological Sciences Characterizing the Link between Biological Membranes and Thermal Physiology in Antarctic Notothenioid Fishes Director of Dissertation: Elizabeth L. Crockett The Antarctic notothenioid fishes are among the most stenothermal animals on the planet and are likely to be vulnerable to the effects of global climate change. The physiological mechanisms that govern the thermal tolerance of Antarctic notothenioids are not fully understood. Membrane integrity and structure are highly sensitive to temperature and are critical to maintenance of cellular function. The two central hypotheses of this work are: (1) Variation in physical and biochemical membrane properties exists among notothenioids that display differences in thermal tolerance and thermal sensitivity of physiological processes; and (2) Membranes of Notothenia coriiceps undergo lipid remodeling in response to long-term thermal change in order to conserve membrane properties. Physical and biochemical properties of biological membranes from several tissues (cardiac ventricles and brain) were analyzed in several species of notothenioids in order to characterize variation in properties of biological membranes within this suborder of fishes. I also sought to determine whether notothenioids possess the capacity for acclimation to elevated temperature by determining the extent of compensation of membrane properties in several tissues (cardiac ventricles, brain, gill). Findings from this work provide novel insight into how notothenioids are likely to fare within a warmer climate. 4 An interspecific comparative analysis was performed between notothenioids that exhibit variation in thermal tolerance (Chapters 2, 3). Membrane fluidity and composition were measured in several brain (synaptic, myelin, mitochondria) and cardiac (mitochondria) membranes from the red-blooded (more thermotolerant) Notothenia coriiceps and the white-blooded Chaenocephalus aceratus. Synaptic membranes and cardiac mitochondria were more fluid in the icefish, compared to the red-blooded species. Hyperfluidization of membranes, particularly in the less thermotolerant species, C. aceratus, is consistent with the failure of the nervous and cardiovascular systems upon acute warming. Additionally, properties of membranes from N. coriiceps were analyzed following several weeks of acclimation to 0°C or 5ºC (Chapters 4, 5). In Chapter 4, fluidity was compared between thermal treatment groups in brain (synaptic membranes, myelin, mitochondria) and cardiac (mitochondria, microsomes) membranes. Biochemical analyses of membrane composition were performed on select membranes. Results suggest evidence of homeoviscous adaptation in the cardiac, but not brain, membranes. Both cardiac mitochondria and microsomes displayed reduced fluidity following acclimation to 5°C, indicating full thermal compensation when the membrane fluidity is compared at the animal’s respective acclimation temperature. In Chapter 5, fluidity, composition, and osmotic permeability were compared between thermal treatment groups in plasma membranes from gill epithelia. Results provide evidence for membrane remodeling, consistent with the observed preservation of membrane fluidity upon acclimation. 5 Further, measurements of osmotic uptake in gill epithelia suggest membrane permeability is reduced during acclimation to 5°C, possibly to compensate for the effects of higher temperatures that would otherwise render the membrane more permeable. For cardiac and branchial membranes, differences in fluidity were achieved by modulation of membrane cholesterol contents and/or fatty acyl chain length. Taken together, these results provide evidence for thermal plasticity of membrane properties in the cardiac and branchial systems of this species. The lack of a homeoviscous response and membrane restructuring in the brain would appear to limit the capacity for thermal acclimation in N. coriiceps. In total, these data indicate that the nervous system is likely to be the most susceptible to failure with increased warming in the Southern Ocean. 6 DEDICATION This work is dedicated to my fiancé, Steven, for always supporting me and for waiting patiently for me to finish my dissertation while we lived 335 miles apart. I love you so much. 7 ACKNOWLEDGMENTS First and foremost, I would like to express my sincere gratitude to my advisor, Dr. Lisa Crockett, for her mentorship and constant moral support over the past five years. I would also like to thank my committee members Dr. Janet Duerr, Dr. Daewoo Lee, Dr. Sarah Wyatt and Dr. Theresa Grove for their help and guidance. Thanks to members of the Crockett lab, especially Dr. Donald Kuhn and Elizabeth Evans, for help and support. I would like to acknowledge my advisor’s collaborators, especially Dr. Kristin O’Brien, as well as my fellow student field researchers, Anna Rix, William Joyce, and Jordan Scharping. Thanks to Mary Roth, Dr. Ruth Welti, Dr. John Robertson, Dr. Bruce Carlson, Dr. Luisa Diele Viegas, Juan Pablo Aguilar Cabezas, and Marilyn Seyfi for advice and analytical assistance. I am grateful to Dr. Chris Griffin, Dr. Ahmed Faik and Tasleem Javaid for generously lending equipment. This project would not have been possible without the logistic support of the staff at Palmer Station and the masters and crew of the ARSV Laurence M. Gould. Special thanks to Dan Nielsen, Tom Adams, Matt Boyer, Adina Scott, Emily Olson and Emily Longano. Financial support for this work was provided by the Ohio University Student Enhancement Award and the Ohio University Department of Biological Sciences. I was also supported through an NSF award granted to my advisor [PLR 1341602]. I would like to thank my friends and family, especially my fiancé, Steven, for their love and encouragement. I would also like to thank my undergraduate advisor, Dr. Gene Williams. Lastly, thank you to my parents, Angela and Michael, for encouraging my love of learning and for always challenging me to pursue my dreams. 8 TABLE OF CONTENTS Page Abstract ............................................................................................................................... 3 Dedication ........................................................................................................................... 6 Acknowledgments............................................................................................................... 7 List of Tables .................................................................................................................... 12 List of Figures ................................................................................................................... 13 Chapter 1: Introduction ..................................................................................................... 15 The Evolution of Antarctic Fishes .............................................................................. 15 Icefishes: Emergence of a Novel Trait ........................................................................ 18 Life in a Dynamic Thermal Climate ........................................................................... 20 What Physiological System(s) Set(s) Thermal Limits? .............................................. 22 Are Antarctic Notothenioids Thermally Plastic? ........................................................ 24 Membranes are Critical to Cell Structure and Function ............................................. 26 Membranes Are Highly Thermosensitive ................................................................... 28 Biological Membranes are Involved in Thermal Acclimation ................................... 29 Dissertation Overview and Specific Aims .................................................................. 31 Chapter 2: Variation in Properties of Brain Membranes in Notothenioids Helps Explain Differences in Acute Warming Behavior and Thermal Tolerance ................................... 36 Introduction ................................................................................................................. 36 Materials and Methods ................................................................................................ 38 Animal and Tissue Collection ............................................................................... 38 Membrane Preparations and Marker Enzyme Analyses ....................................... 39 Membrane Physical and Chemical Properties ...................................................... 39 Statistical Analyses ............................................................................................... 40 Results ......................................................................................................................... 40 Cell Fractionation