View and Specific Aims

Home , Bald notothen, Membrane lipid

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

This dissertation titled

Characterizing the Link between Biological Membranes and Thermal Physiology in

Antarctic Notothenioid Fishes

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.

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.

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 Revealed Three Distinct Membrane Types ...... 40 Fluidity of Synaptic Membranes was Greatest in the Less Thermotolerant Species ...... 42 Lipid Profiles of Myelin Showed Variation between Species ...... 44 Discussion ...... 46 Species Differences in Synaptic Membrane Fluidity Are Associated with Variation in Thermal Tolerance ...... 46 9

Greater Sensitivity to Thermal Change of Myelin from N. coriiceps May Account for Spasmodic Behavior with Acute Warming ...... 47 Chapter 3: Properties of Cardiac Mitochondrial Membranes Vary Among Notothenioids with Differing Cardiac Responses to Acute Warming ...... 50 Introduction ...... 50 Materials and Methods ...... 51 Animal and Tissue Collection ...... 51 Membrane Fluidity Measurements ...... 52 Lipid Extraction and Quantification ...... 52 Statistical Analyses ...... 53 Results ...... 53 Fluidity of Mitochondrial Membranes was Greatest in the Less Thermotolerant Species ...... 53 Membrane Compositions Varied Among Notothenioid Species ...... 54 Discussion ...... 56 Differences in Proportions of Polar Lipid Classes are Consistent with Variation in Fluidity between Species ...... 57 Greater Fluidity in Icefish Mitochondria is Consistent with Functional Consequences Associated with Warming ...... 59 Conclusions ...... 61 Chapter 4: Membranes from Brain and Cardiac Tissues Display Variable Homeoviscous Responses to Thermal Acclimation in Notothenia coriiceps ...... 62 Introduction ...... 62 Materials and Methods ...... 64 Animal Collection and Thermal Acclimation ...... 64 Membrane Preparations and Marker Enzyme Analyses ...... 65 Membrane Physical and Biochemical Properties ...... 65 Statistical Analyses ...... 66 Results ...... 67 Membrane Fluidity Differed Between Tissues and With Thermal Treatment ..... 67 Polar Lipid Profiles Differed Between Tissues and With Thermal Treatment ..... 70 Discussion ...... 72 Measurements of Membrane Fluidity Demonstrate a Homeoviscous Response in Cardiac, but Not Brain Membranes ...... 72 10

An Absence of Change in Membrane Unsaturation is Consistent with Previous Literature in Notothenioids ...... 73 Alterations in Membrane Cholesterol and Fatty Acyl Chain Length are Consistent with Changes in Fluidity ...... 76 Decreases in Proportion of Hydrolyzed Phospholipids May Reflect Functional Changes with Environmental Warming ...... 77 Compromises in Brain Function May Limit Organismal Performance at Elevated Temperatures...... 79 Chapter 5: Thermal Acclimation Alters Membrane Fluidity and Osmotic Permeability of Branchial Epithelia in Notothenia coriiceps ...... 82 Introduction ...... 82 Materials and Methods ...... 85 Animal Collection and Thermal Acclimation ...... 85 Membrane Preparations and Marker Enzyme Analyses ...... 86 Membrane Composition...... 87 Membrane Fluidity Assays ...... 87 Osmotic Permeability Assays ...... 87 Oxygen Partition Coefficient Measurements ...... 88 Statistical Analyses ...... 88 Results ...... 89 Membranes Displayed Enrichment and Altered Enzyme Activities upon Acclimation ...... 89 Cholesterol and Acyl Chain Length Increased with Acclimation ...... 91 Osmotic Permeability was Reduced with Acclimation, but Oxygen Solubility was Unchanged...... 93 Discussion ...... 95 Lipid Remodeling Is Consistent with a Homeoviscous Response ...... 95 Reduced Membrane Fluidity Accounts for Decreased Osmotic Permeability ..... 97 Changes in Enzymatic Activity May Reflect Altered Membrane Fluidity ...... 99 Conclusions ...... 100 Chapter 6: Dissemination of Notothenioid Research to the Public Sphere through Wikipedia ...... 102 Introduction ...... 102 Materials and Methods ...... 105 Importance ...... 105 11

Quality...... 106 Relevance ...... 107 Article Selection...... 107 Results and Discussion ...... 109 Overview ...... 109 Article Revisions: Notothenioidei...... 110 Article Revisions: Channichthyidae ...... 111 Article Revisions: Dissostichus ...... 112 Article Creation: Notothenia coriiceps ...... 112 Article Revisions: Chaenocephalus aceratus ...... 113 Mass Revisions: Icefish versus Cod Icefish...... 114 Summary ...... 115 Chapter 7: Perspectives ...... 117 Interspecies Comparisons Reveal Differences in Potential Vulnerability of Membranes to Acute Warming ...... 117 Investigation of Thermal Acclimation Reveals Potential Weak Link to Physiological Function During Long-Term Warming ...... 118 How Do Antarctic Notothenioids Modulate Membrane Fluidity? ...... 118 Future Directions ...... 119 References ...... 122 Appendix A: Animal Collection and Maintenance ...... 156 Appendix B: Tissue Collection And Membrane Preparation Techniques ...... 158 Appendix C: Membrane Marker Enzymes ...... 162 Appendix D: Membrane Fluidity Assays ...... 165 Appendix E: Lipid Extraction and Compositional Analyses ...... 166 Appendix F: Thermal Acclimation Treatments ...... 167 Appendix G: Measurement of Osmotic Permeability in Gill Tissue ...... 168 Appendix H: Measurement of Oxygen Solubility in Gill Plasma Membranes ...... 169

LIST OF TABLES

Table 2.1. Distribution of Membrane Fractions as Indicated by Marker Enzyme Enrichment...... 41 Table 2.2. Distribution of Polar Lipids in Myelin...... 44 Table 2.3. Distribution of Fatty Acid Unsaturation in Myelin Samples by Number of Double Bonds...... 45 Table 3.1. Polar Lipid Class Distribution and Unsaturation Index in Mitochondrial Membranes from Cardiac Ventricles in White- and Red-Blooded Notothenioids...... 55 Table 4.1. Relative Distribution of Polar Lipid Classes in Mitochondria from Cardiac and Brain Tissue Following Acclimation to 0°C or 5°C in Notothenia coriiceps...... 71 Table 4.2. Relative Abundance of Fatty Acids with at Least 20 Carbon Atoms Per Chain in Mitochondria from Cardiac and Brain Tissue Following Acclimation to 0°C or 5°C In Notothenia coriiceps...... 71 Table 4.3. Cholesterol Contents of Cardiac Microsomes, Synaptic Membranes, and Myelin Following Acclimation to 0°C or 5°C In Notothenia coriiceps...... 72 Table 5.1. Enrichment Factors for Plasma Membrane Preparations for Gill Epithelial Tissues from Notothenia coriiceps...... 89 Table 5.2. Polar Lipid Class Distribution and Unsaturation Index in Plasma Membranes from Gill Epithelia in 0°C and 5°C Acclimation Groups...... 92 Table 6.1. Summary of Articles Chosen for Modification...... 107

LIST OF FIGURES

Figure 1.1. The Antarctic Circumpolar Current. (Wikimedia Commons) ...... 16 Figure 1.2. Notothenioid families (a) Bovichtidae (b) Nototheniidae (c) Harpagiferidae (d) Artedidraconidae (e) Bathydraconidae (f) Channichthyidae (Wikimedia Commons, Kock, 1992)...... 17 Figure 1.3. Representation of (a) solid-ordered, (b) liquid-ordered, and (c) liquid- disordered membrane phases...... 29 Figure 2.1. Steady state polarization values (i.e., inverse of membrane fluidity) for the fluorescent probe DPH in Chaenocephalus aceratus (ACE, open circles) and Notothenia coriiceps (COR, closed circles) (N=8) in (a) synaptic membranes, (b) myelin and (c) mitochondria. Error bars represent means + s.e.m...... 43 Figure 2.2. Cholesterol-to-phospholipid ratios for Chaenocephalus aceratus (ACE, white bars) and Notothenia coriiceps (COR, black bars) in synaptic membranes and myelin (N=8). One asterisk indicates P<0.05 between species. Error bars represent + s.e.m...... 46 Figure 3.1. Steady state polarization values (i.e., inverse of membrane fluidity) for the fluorescent probe DPH in mitochondrial membranes from cardiac ventricles from C. aceratus (open circles) and N. coriiceps (closed circles) (N=6). Error bars represent + s.e.m...... 54 Figure 4.1. Steady state polarization values (i.e., inverse of membrane fluidity) for the fluorescent probe DPH in (a) cardiac mitochondria and (b) cardiac microsomes from hearts of animals subjected to 0°C and 5°C acclimation treatments. Error bars represent means + s.e.m. (N=5)...... 68 Figure 4.2. Steady state polarization values (i.e., inverse of membrane fluidity) for the fluorescent probe DPH in (a) synaptic membranes, (b) myelin, and (c) mitochondria from brains of animals subjected to 0°C and 5°C acclimation treatments. Error bars represent means ± s.e.m. (N=10)...... 69 Figure 5.1. Protein-specific activities of Na+/K+-ATPase (NKA) and gamma glutamyltransferase (GGT) from gill epithelia in 0°C and 5°C acclimation groups. Error bars represent means ± s.e.m. (N=7) One asterisk indicates P<0.05...... 90 Figure 5.2. Steady state polarization values (i.e., inverse of membrane fluidity) for the fluorescent probe DPH in plasma membranes from gill epithelia in 0°C and 5°C acclimation groups. Error bars represent means ± s.e.m. (N=10 for 0°C and N=9 for 5°C)...... 91 Figure 5.3. Cholesterol contents of plasma membranes from gill epithelia in 0°C and 5°C acclimation groups. Error bars represent means ± s.e.m. (N=8). Two asterisks indicate P<0.01...... 92 Figure 5.4. Relative abundance of fatty acids with >20 carbon atoms per chain in plasma membranes from gill epithelia in 0°C and 5°C acclimation groups. Error bars represent means ± s.e.m. (N=8). One asterisk indicates P<0.05...... 93 14

Figure 5.5. Osmotic permeability of plasma membranes from gill epithelia in 0°C and 5°C acclimation groups. Assays were performed at 0°C and 4°C. Error bars represent means ± s.e.m. (N=10 for 0°C and N=8 for 5°C). One asterisk indicates P<0.05 between acclimation groups...... 94 Figure 5.6. Oxygen partition coefficient (i.e., the ratio of oxygen solubility to membrane lipids to that of water) of plasma membranes from gill epithelia in 0°C and 5°C acclimation groups. (N=10 for 0°C and N=8 for 5°C). Error bars represent means ± s.e.m...... 94 Figure 6.1. Relative distribution of importance and quality rankings within Wikipedia notothenioid literature...... 106

CHAPTER 1: INTRODUCTION

The Evolution of Antarctic Fishes

The Southern Ocean, which forms at the southern sector of the Atlantic, Indian, and Pacific Oceans, has supported a finfish fauna for 400 million years (1). The Antarctic ice sheet likely formed during the Cenozoic Period (2), and expansion of the sheet likely prompted an extended cooling period, which gave rise to the stable, extreme cold temperatures that have characterized the Southern Ocean habitat for 10-14 MYBP (3).

Waters encompassing and extending somewhat beyond the continental shelf of

Antarctica typically remain between -1.9ºC and +1.5ºC year-round (4) and are characterized by productive, ice-filled habitats that support a variety of marine organisms

(5). In addition, the Southern Ocean and its marine fauna became isolated approximately

25 million years ago through the formation of the Antarctic Circumpolar Current and

Antarctic Polar Front as well as deep ocean basins, all of which preclude migration to and from the region (6, 7) (Fig. 1.1). 16

Figure 1.1. The Antarctic Circumpolar Current. (Wikimedia Commons)

The perciform suborder Notothenioidei, which accounts for 90 percent of the fish biomass in the shelf waters surrounding Antarctica, evolved under these unique geological conditions (Fig. 1.2) (1). The notothenioids evolved from a benthic, nearshore ancestor and underwent rapid radiation within the suborder (8, 9). Eight notothenioid families have been classified, constituting 43 genera and at least 122 confirmed species

(10). Approximately 80% of notothenioids are found south of the Antarctic Polar Front; the least derived family, Bovichtidae, is the only notothenioid family with a primarily non-Antarctic distribution (10). The notothenioids evolved within a low-diversity habitat 17 and in the absence of high competition; thus, notothenioid evolution was characterized by rapid speciation to fill a broad range of ecological niches (11). Consistent with this, polar- adapted marine fishes generally exhibit greater rates of speciation, relative to temperate species (12).

Figure 1.2. Notothenioid families (a) Bovichtidae (b) Nototheniidae (c) Harpagiferidae (d) Artedidraconidae (e) Bathydraconidae (f) Channichthyidae (Wikimedia Commons, Kock, 1992)

Notothenioid morphology is largely typical of other coastal perciform fishes, yet members of the suborder display a unique combination of phenotypes (1). These include the presence of three pectoral radials, a nostril on either side of the head, the lack of a 18 swim bladder, and the presence of multiple lateral lines (1). Because notothenioids lack a swim bladder, most species lack neutral buoyancy and thus exhibit demersal swimming behavior in order to maintain their position near the seafloor (8, 13). Some notothenioids have undergone morphological modifications, including the accumulation of lipid deposits and reduced skeletal ossification, to attain neutral buoyancy and inhabit the water column (i.e., the pelagic zone) (13).

Antarctic notothenioids exhibit several biochemical characteristics that reflect their adaptation to an extreme cold (i.e., freezing) environment. These adaptive traits include cold-stable proteins that contain amino acid modifications to increase hydrophobic interactions (14), highly concentrated enzymes with enhanced catalytic efficiency (i.e., kcat) (15), and biological membranes that can maintain fluidity even at freezing temperatures (16). In addition, most Antarctic notothenioids produce antifreeze glycoproteins (17), which serve to preclude accumulation of ice crystals in the blood and body tissues. Lipids tend to serve as a preferred fuel source, rather than carbohydrates

(18). Most active notothenioids tend to possess a greater capacity for fatty acid oxidation, relative to temperate fishes (18, 19).

Icefishes: Emergence of a Novel Trait

Members of the notothenioid family Channichthyidae, referred to commonly as the “icefishes,” are the only known vertebrate animals to lack hemoglobin (Hb), the oxygen-carrying protein in blood, as adults (20). This trait, which includes full and partial deletions of the two genes encoding the beta and alpha units of hemoglobin, respectively, has resulted in blood that lacks functional erythrocytes (21). Icefish blood lacks 19 hemoglobin and possesses an oxygen carrying capacity reduced to 10% of that of their red-blooded relatives (22).

The loss of hemoglobin likely occurred in response to the unique physical and ecological conditions within the Southern Ocean (20). First, competition within Southern

Ocean habitats is relatively low, allowing for the emergence of traits that would likely otherwise be lethal (21). Additionally, cold water is highly enriched in oxygen due to the inverse relationship between oxygen solubility and water temperature. Further, notothenioids maintain relatively low metabolic rates, compared to temperate species at physiological temperatures (23). Thus, oxygen availability is maximized, and oxygen demands are reduced.

Icefishes exhibit a variety of adaptive traits, largely within the cardiovascular system, to counter the absence of hemoglobin and to maximize oxygen delivery to tissues. These include the development of an enlarged, slow-beating heart, increased blood volume and distribution of the capillary network, enlarged blood vessels, and increased mitochondrial density (21, 24–26). While all notothenioids appear to lack skeletal muscle myoglobin (Moylan and Sidell, 2000), six species of icefishes also lack cardiac myoglobin (Mb) (21, 27). This trait appears to have emerged several times during the evolution of icefishes due to changes in gene expression (28).

It is unclear whether the losses of hemoglobin and myoglobin served of some adaptive value to the icefishes. “Back-of-the envelope” calculations suggest the increased blood volume in icefishes, relative to red-blooded notothenioids, results in increased cardiac work (21). Additionally, myoglobin has been shown to enhance cardiac 20 performance in icefishes (29). Although the physiological mechanisms that allow icefishes to survive without hemoglobin and myoglobin are not yet fully understood, icefishes represent perhaps the most striking example of notothenioid evolution within a unique environment.

Life in a Dynamic Thermal Climate

All ectothermic organisms must work to maintain physiological constancy in the face of an ever-changing abiotic environment. Temperature affects organisms at every level of biological organization (30). Consequently, for ectotherms, any shift in temperature poses a threat to the maintenance of physiological function. To cope with a dynamic climate, these organisms must harness physiological mechanisms to counter the effect of thermal change. These adjustments to biological function often involve alterations in kinetic parameters of enzymes (15, 31, 32), gene and protein expression

(33–35), and membrane composition (36–38).

When discussing thermal physiology, it is essential to consider physiological responses to temperature from three perspectives. The first, thermal adaptation, refers to the evolutionary mechanisms by which a species adjusts to its environment. These mechanisms most often involve changes at the genomic level (39). Typically, evolutionary adaptations to the environment are relatively fixed within individuals (40).

The second mechanism, thermal acclimation or acclimatization, refers to an individual’s capacity to optimize organismal performance within a shifting environment

(41). These optimizations may involve alterations in gene or protein expression, often resulting in changes to compositions of biological membranes (42), metabolic function 21

(43), and organ morphology (44). An organism’s capacity to acclimatize in response to environmental fluctuations is referred to as its “plasticity” (45, 46). Capacity for plasticity represents a type of evolutionary adaptation and may serve to facilitate adaptive radiation in response to environmental change (46).

The third mechanism, acute response, refers to an organism’s reaction to abrupt changes in temperature. Responses often involve rapid changes in behavior and/or physiological function (47–49). At thermal extremes, virtually all ectotherms will ultimately reach a point of loss of performance. Beyond these temperatures, these organisms are unable to cope with further deviations from their optimal temperature (47).

In situations where the temperature is elevated relatively rapidly (e.g., over hours), the point of loss of function is known as the critical thermal maximum (CTMAX) (50). In fishes, CTMAX is typically defined as the temperature at which righting ability is lost (51), although some researchers have defined upper thermal limits in fish as the point of onset of cardiac arrhythmia (52) or body spasms (53).

Ectothermic organisms are likely to be particularly vulnerable to the effects of climate change, and aquatic animals are likely to be among the most critically threatened, as these species often exhibit narrow survival ranges, relative to non-aquatic animals (39,

54). Possible biochemical consequences of climate change include misfolded and/or malfunctioning proteins (39), exacerbated oxidative damage in cells (55), and compromised membrane integrity (42). Many species have recently altered their geographic distributions, shifting towards more polar climates (56–58). The species most 22 vulnerable to climate change are likely to be those currently living close to their thermal limits (41).

What Physiological System(s) Set(s) Thermal Limits?

Researchers have traditionally posited that thermal tolerance is likely limited by the failure of a single organ system (39). Previous work suggests that for many species, the cardiovascular system may serve as this limiting factor, possibly resulting from the loss of aerobic scope (the difference between minimum and maximum rates of oxygen consumption) during warming (59, 60).

Fish hearts are sensitive to acute thermal change; acute cooling slows the rate of impulse conduction in cardiomyocytes and thus, the speed of contraction (61).

Eventually, a point is reached beyond which the fish can no longer keep pace with increased cardiac demand (62). Fish display elevated heart rates with warming to a particular temperature, beyond which cardiac function collapses (reported as the onset of cardiac arrhythmia) (63). This loss of function appears to occur prior to the onset of molecular damage (14). However, fish hearts also display high plasticity and can undergo functional adjustments to preserve function upon chronic thermal change (45, 64). This may be carried out through a variety of mechanisms including changes in heart size, contraction duration, and/or swimming activity. Adjustments to cardiac function will likely be critical to the survival of fishes in a warmer climate.

While a large body of literature points to cardiac function as a limiting factor in the thermal tolerance of ectotherms (59, 60), other reports provide data to support the hypothesis that brain function plays a role in loss of performance during acute 23 temperature change (65). Neurons of the central nervous system display high thermosensitivity in their function (66, 67). For example, with acute cooling, the amplitude of action potentials decreases, and synaptic delay increases. In addition, receptor affinity is reduced during cooling (67).

Additional evidence suggests brain function is impeded, and possibly terminated, during acute warming. Neuron firing rates increase during acute warming until reaching a critical temperature, beyond which point the firing becomes more random. This phenomenon is particularly pronounced at some inhibitory synapses (68). It is critical that the brain preserve metabolic function during acute warming, and some evidence suggests that brain anoxia occurs shortly before CTMAX, possibly resulting from cardiac failure and compromised circulation (67). Further, the blood-brain barrier may be compromised at elevated temperatures, possibly resulting in exposure to neurotoxic agents (69).

Adjustments in brain function, as is also the case for the heart, are essential to long-term acclimatization to altered temperatures. These adjustments include modulations of the composition of brain membrane lipids (70, 71), enzymatic activities (72), and channel regulation (68).

The cardiovascular and nervous systems, and thus their responses to acute and thermal change, are closely intertwined. Brain function is likely to impact cardiac function. For example, brain anoxia causes sharp depressions in blood flow and heart rate in trout (73). Additionally, changes in blood flow are known to impact brain oxygenation and function (74, 75). Further, cardiac failure compromises cerebral blood flow and can lead to brain damage (75, 76). Thus, it is of particular value to consider both the 24 cardiovascular and the nervous systems when evaluating the physiological parameters that set limits to organismal performance during thermal change.

Are Antarctic Notothenioids Thermally Plastic?

Antarctic notothenioids are among the most stenothermal animals on the planet

(77). Many notothenioids lack a heat shock response (33), and their physiological processes have been optimized to function in a cold, yet thermally stable environment.

Global warming is currently occurring most rapidly in the polar regions, and the Western

Antarctic Peninsula region has been shown to be among the most critically impacted (78).

Consequently, many researchers are interested in the capacity of Antarctic notothenioids to adjust their physiological function in response to long-term thermal change.

In notothenioids, thermal tolerance appears related to hematocrit (Hct), as icefishes with a Hct value of zero, and red-blooded notothenioids with a low Hct exhibit a reduced CTMAX, relative to red-blooded notothenioids possessing a relatively high Hct

(79). Notothenia coriiceps (Hct 35%) has a CTMAX of 17°C, while Gobionotothen gibberifrons (Hct 27%) and Lepidonotothen squamifrons (Hct 20%) have CTMAX values of 15°C and 14°C, respectively (79). Thermal limits tend to be further reduced in icefishes; CTMAX values of 14°C, 13°C and 12°C have been reported for Chaenocephalus aceratus, Chionodraco rastrospinosus, and Pseudochaenichthys georgianus, respectively

(Beers & Sidell, 2011; Egginton et al., unpublished observations). However, thermal tolerance does not appear to be governed by oxygen delivery directly, as CTMAX is unchanged in both red-blooded N. coriiceps and in the icefish C. aceratus upon hyperoxia treatment (80). 25

Some notothenioids have exhibited the ability to extend their thermal limits upon acclimation to warmer temperatures (81, 82). For example, with acclimation to 4ºC for a minimum of one week, eight species of red-blooded notothenioids from the Western

Antarctic Peninsula and McMurdo Sound displayed an extended upper thermal limit, typically increased by approximately 2-3ºC (82). Further, six of these species also exhibited an extension of upper thermal limits of approximately 1ºC following acute exposure to CTMAX (83). Thus, despite the evolution of notothenioids in an extreme, yet stable environment, some Antarctic notothenioids possess the capacity to adjust their physiological function in response to thermal change.

The physiological mechanisms by which thermal limits may be extended in these species are not yet fully understood. However, using experimental acclimation to elevated temperatures, some research is beginning to shed light on what physiological systems may contribute to preservation of performance. In N. coriiceps, cardiac output and oxygen consumption increase following acclimation to 5ºC for a minimum of six weeks (84). In addition, enhanced defenses against oxidative damage have been observed in gill and liver tissue from Trematomus bernacchii, Pagothenia borchgrevinki, and

Trematomus newnesi upon acclimation to 4ºC for a minimum of four weeks (85). Further,

T. bernacchii exhibits a shift towards anaerobic metabolism following acclimation to

4.5ºC for two weeks (86). Further, P. borchgrevinki is capable of adjusting plasma glucose, hematocrit, and oxygen consumption, but not plasma osmolality, in response to acclimation to 4ºC for a minimum of four weeks (87, 88). In summary, while many notothenioids appear to exhibit some degree of thermal plasticity, the mechanisms by 26 which these organisms respond to long-term thermal change have yet to be characterized fully.

Membranes are Critical to Cell Structure and Function

Biological membranes are essential to cellular function. They surround the perimeter of the cell, governing cell shape and serving as a semipermeable barrier to regulate the movement of solutes and water between intra- and extracellular compartments. Further, the organelles of eukaryotic cells are also encased by biological membranes (89).

Biological membranes are arranged in a bilayer structure comprised largely of polar lipids including phospholipids, which contain a polar phosphate head group and a pair of nonpolar fatty acid tails, or glycosphingolipids, which contain a sphingosine backbone bound in an amide with a fatty acid and a carbohydrate (polar) head group. The nonpolar tails of both types of membrane lipids are oriented inward and comprise the hydrophobic center of the bilayer, while the polar head groups are oriented towards the inner and outer membrane spaces.

Membranes are involved in various physiological processes, including coordination of proteins and protein complexes (e.g., those participating in oxidative phosphorylation), exo- and endocytosis, protein and lipid synthesis, vesicular trafficking, reception of extracellular signals, and coordination of intracellular signaling pathways

(90). Membranes are also involved in ion balance and regulation of cell permeability through the flexibility of lipids and by providing a lipid matrix for insertion of ion channels, protein pumps, and membrane transporters (89, 91). 27

The membrane environment is highly dynamic and heterogeneous. Membranes display lateral and transverse asymmetry and maintain different lipid and protein constituents between the inner and outer leaflets (i.e., hemilayers) of the membrane (92).

Various proteins, which help regulate solute transfer and cellular signaling, are bound to the membrane and stabilized through lipid-protein and protein-protein interactions.

Membranes are known to be organized into microdomains, including lipid rafts, which are comprised of lipids that contribute to high degrees of membrane ordering (93). These microdomains also typically contain proteins associated with signaling cascades (94, 95).

Membrane heterogeneity is governed by variation in the composition and organization of polar lipid classes as well as by the length and unsaturation of acyl chain tails. There are seven major classes of polar lipids that are classified by their head group structure: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidic acid (PA), and sphingomyelin (SM) (89). PC and PE, generally the two most abundant polar lipids in biological membranes, play a critical role in governing membrane fluidity and order (96). PE is particularly important for inducing membrane curvature stress, which is important for the formation of non-lamellar structures that are involved in membrane budding and fusion (97). Additionally, membrane polar lipids, particularly PI, are important in anchoring receptor molecules, thus helping to regulate cell signaling (89, 90). Further, the diphosphatidylglycerol lipid cardiolipin (CL) is critical to the coordination of oxidative phosphorylation in the inner mitochondrial membrane of eukaryotic cells (90, 98). 28

Membrane structure and function are also highly influenced by the neutral lipid cholesterol, which associates with SM to form lipid microdomains, including lipid rafts and caveolae (99). Cholesterol tends to order biological membranes, thus contributing to the stabilization of membrane structure, although it can impart a disordering effect if in excess (100). Cholesterol and SM are also critical to the stability of myelin, a sheath of electrical insulation surrounding the axons of neurons in some animals (101, 102).

Plasmalogens, a type of phospholipid with an ether bond at the glycerol backbone, are also important in the formation and stabilization of membrane rafts (103).

Membranes Are Highly Thermosensitive

Membranes can exist in various physical phases that reflect the degree of phospholipid movement (Fig. 1.3) (97). Membrane phases are governed by both membrane composition and temperature. Because the membrane is heterogeneous, various portions of the membrane can exist in different phases simultaneously (i.e., phase separation) (92). At low temperatures, portions of the membrane are likely to form a solid-ordered state in which lipid movement is relatively limited. At elevated temperatures, portions of the membrane are likely to form a liquid-disordered state and may be vulnerable to hyperfluidity and even possibly the formation of non-lamellar phases (97). Further, membrane permeability, which is influenced by the degree of molecular movement within the membrane and is governed by lipid composition, is compromised at extreme high and low temperatures (104). 29

Figure 1.3. Representation of (a) solid-ordered, (b) liquid-ordered, and (c) liquid-disordered membrane phases.

In order to maintain its structure and function, the membrane must remain largely in the liquid-ordered state. This state allows for sufficient lipid movement and protein flexibility, while membrane integrity remains intact (97). Membrane phases are maintained via a steady-state dynamic in which distinct regions of the membrane exist in separate physical states (89). Even moderate heat stressors can impart critical effects on this dynamic. Membrane thermosensors detect acute changes in fluidity and mount various physiological responses (105). These responses include post-translational modification of membrane proteins, activation of channels and signaling domains, and accumulation of ceramides (105–107). Consequently, the membrane can serve as one of the first responders within the cell during acute thermal change.

Biological Membranes are Involved in Thermal Acclimation

Remodeling of membrane lipids has been shown to play a critical role in thermal acclimation of ectothermic organisms and in the evolutionary adaptation of animals to disparate thermal environments (e.g., Behan-Martin, Jones, Bowler, & Cossins, 1993;

Logue, DeVries, et al., 2000). Because the body temperature (Tb) of an ectothermic organism will parallel the ambient temperature (Ta) of its physical environment (air or 30 water), biological membranes must protect their integrity and function over the organism’s physiological temperature range (109). This process, known as homeoviscous adaptation (HVA), has been documented in prokaryotes (110), insects (111), plants (112), and both poikilothermic and homeothermic animals (71, 108, 113).

Several lipid classes are known to play a role in HVA. For instance, the ratio of

PE to PC tends to decrease with warm acclimation (42). This trend may appear counterintuitive, as PC tends to enhance membrane fluidity while PE tends to decrease fluidity (96). These effects are attributed to differences in the shapes of PC and PE; PC has a large, cylindrical head group that tends to increase membrane hydration, while PE has a small, conical head group that tends to decrease hydration (96). However, when concentrated, the conical shape of PE can induce membrane curvature stress, resulting in the formation of non-lamellar phases (114). Thus, PE is thought to be reduced during warm acclimation to maintain a constant temperature differential between membrane phase transition temperatures (i.e., homeophasic adaptation) (Hazel, 1995).

The fatty acids associated with polar lipids (phospholipids and glycolipids) are also known to play an important role in HVA. Highly unsaturated lipids are known to be more fluid, and membrane lipid unsaturation typically decreases with warm acclimation

(115, 116). Further, fatty acyl chain length of phospholipids plays a role in membrane fluidity; short and asymmetric tails tend to increase fluidity, while long and symmetrical tails tend to be less fluid (89).

The capacities of Antarctic notothenioids to undergo HVA as a result of changes in temperature are not well-studied and are not fully understood. Previous reports have 31 indicated that membrane unsaturation is unchanged following thermal acclimation in several notothenioids (117–119). However, polar lipid classes and other aspects of membrane composition were not reported. Yet in another study, evidence of reduced liver membrane unsaturation was reported in one notothenioid, T. bernacchii, upon acclimation to 6ºC (119).

Dissertation Overview and Specific Aims

In this dissertation, I describe my efforts to characterize the relationships between membrane composition, membrane structure, and thermal physiology of Antarctic notothenioid fishes from the Western Antarctic Peninsula region. Analyses were performed on animals collected at fishing sites near the United States Antarctic Program research base, Palmer Station, during the austral autumns of 2015 and 2017. Antarctic notothenioids are known to display variation in their thermal tolerances, but neither the mechanisms that govern their thermal limits, nor the capacities of Antarctic notothenioids to adjust their physiological function in response to thermal change, have been characterized fully. Membrane fluidity plays a role in loss of organismal performance at elevated temperatures and, thus, is likely to be important in determining the thermal limits of these species.

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. To test these hypotheses, I completed 32 the following specific aims: (1) Assess variation in membrane fluidity and composition in cardiac and brain tissues from notothenioids differing in thermal tolerance (Chapters 2,

3); (2) Characterize differences in membrane fluidity and composition in cardiac, brain, and gill tissues from N. coriiceps following long-term acclimation to 0°C or 5°C

(Chapters 4, 5); and (3) Characterize differences in osmotic permeability and oxygen solubility of gill membranes upon thermal acclimation (Chapter 5).

A technique central to this work is fluorescence depolarization. I utilized the fluorescent molecular probe 1,6-diphenyl-1,3,5-hexatriene (DPH) to measure the degree of movement within biological membranes, and, thus, the degree of membrane fluidity.

Another technique important to this work is quadrupole mass spectrometry. This technique was employed to generate polar lipid profiles of membrane samples. Extracted lipid samples were sent to the Kansas Lipidomics Research Center for these analyses, as this institution possesses equipment to quantify contents of polar lipids at greater sensitivity than is possible with our own equipment. In several cases, certain analyses were not performed on all available membrane types. This reflects limited tissue availability, as opportunities for field seasons were limited and tissues were shared among members of our research team.

In Section 1 (Chapters 2-3), comparative analyses were performed among notothenioids that display variation in thermal tolerance. In Chapter 2, the fluidity of three brain membranes (synaptic membranes, myelin, mitochondria) from C. aceratus

(Hb-/Mb-) and N. coriiceps (Hb+/Mb+) were compared over a range of temperatures.

Select compositional analyses are were described. In Chapter 3, cardiac mitochondrial 33 membrane compositions were compared among three notothenioids: C. aceratus, P. georgianus (Hb-/Mb+), and N. coriiceps. Additionally, membrane fluidity was compared between C. aceratus and N. coriiceps. Results indicate differences in composition and fluidity of some, but not all of these membranes and suggest variation in membrane properties may help account for failure of the nervous and cardiovascular systems upon acute warming in these species. Cardiac mitochondria and synaptic membranes were more fluid in C. aceratus, compared to N. coriiceps. In cardiac mitochondria, the difference in fluidity may be related to variation in the ratio of total PE to total PC.

Additionally, the in vitro thermosensitivity of myelin was comparatively greater in N. coriiceps, a finding that appears related to the contents of cholesterol in myelin and may help to explain behavioral differences between species during acute, sub-critical warming.

In Section 2 (Chapters 4-5), membrane properties of N. coriiceps were characterized following acclimation to 0°C or 5ºC. In Chapter 4, fluidity was compared between acclimation groups in brain (synaptic membranes, myelin, mitochondria) and cardiac (microsomes, mitochondria) membranes. Select compositional analyses were also performed. Results suggest evidence of HVA in cardiac, but not brain, membranes.

Cardiac mitochondria and microsomes displayed greater ordering following acclimation to 5°C and exhibited 100% homeoviscous efficacy. In contrast, there was no significant effect of acclimation on the fluidity of the brain membranes. Differences in cardiac mitochondrial fluidity appear related to reduced levels of lipid hydrolysis as well as 34 increased fatty acyl chain length, while differences in microsomal fluidity appear related to modulation of membrane cholesterol.

In Chapter 5, membrane fluidity, composition, and osmotic permeability were analyzed in plasma membranes from gill epithelia. Results provide evidence for lipid remodeling to maintain membrane integrity and permeability upon warm acclimation.

Membranes became more ordered following warm acclimation, displaying 100% homeoviscous efficacy. Differences in fluidity appear related to changes in membrane cholesterol and in fatty acyl chain length. Further, measurements of osmotic uptake suggest membrane permeability is reduced during acclimation, possibly to compensate for the effect of temperature on permeability.

In Section 3 (Chapter 6), I describe my work to disseminate known research on notothenioid biology to the public. I assessed the existing notothenioid Wikipedia literature and selected five articles for revision. Improvements to content included the addition of references, images, links, and taxonomic information. Further, content was added and revised where necessary. Together, this work represents an effort to increase the accessibility of scientific information to the public.

Work contained in Chapter 2 has been published and has been reprinted with permission from the publisher (license number 4615741180507) – Biederman et al. (2019). Physical, chemical, and functional properties of neuronal membranes vary between species of antarctic notothenioids differing in thermal tolerance. Journal of Comparative Physiology

B. 189: 213-222

CHAPTER 2: VARIATION IN PROPERTIES OF BRAIN MEMBRANES IN

NOTOTHENIOIDS HELPS EXPLAIN DIFFERENCES IN ACUTE WARMING

BEHAVIOR AND THERMAL TOLERANCE

Introduction

Sensitivity of brain function to warming is likely to be critical to the thermal tolerances of ectothermic organisms. Several species of fishes exhibit behaviors that indicate brain failure with warming (44, 65, 120). In vitro experiments with notothenioids indicate brain metabolism fails at temperatures approaching the organisms’ upper thermal limits, possibly due to fluidization of biological membranes (77). In addition, it has been demonstrated that synaptic transmission is impaired with warming in several Antarctic notothenioids (121). Because several processes of the nervous system are influenced by physical properties of biological membranes, membrane stability and integrity are likely to play important roles in setting thermal limits. In addition, the activities of synaptic membrane-associated enzymes, such as acetylcholinesterase (AChE) and Na+/K+-ATPase

(NKA), are highly sensitive to membrane properties, including fluidity and lipid composition. For example, increased bilayer movement enhances AChE activity, but activity declines beyond a critical point of fluidity (122). Further, NKA activity, which is central to the maintenance of resting membrane potential, can be influenced by cholesterol and fatty acid composition of polar lipids (123, 124).

The central nervous system of notothenioids is largely similar to that of other coastal perciform fishes, with a segmented brain that connects to a rostral spinal cord and ten cranial nerves (1). The brains of Antarctic notothenioids must maintain sufficient 37 membrane fluidity at sub-zero temperatures in order to preserve brain function. The synaptic membranes of Antarctic fishes have been shown to display greater absolute fluidity relative to temperate ectotherms and mammals, an adaptation involving an increase in unsaturation of acyl chain tails of membrane phospholipids (16). This trend has also been shown to differentiate species of notothenioids that reside in non-Antarctic and Antarctic regions (16).

While extreme membrane unsaturation allows for optimal function at subzero temperatures, this phospholipid composition is likely to be unsuitable at elevated temperatures. Critical processes that may be impacted by warming include ion channel activities, neurotransmitter release, and maintenance of conduction velocity (16, 70, 108,

125). Membranes are involved in numerous neural processes including axon insulation by the myelin sheath, membrane fusion of synaptic membranes, and energy production by mitochondria (102, 126, 127). In the brains of temperate fishes exposed to thermal variation over an acclimation or acclimatization time course, homeoviscous adaptation

(i.e., preservation of membrane fluidity at physiological temperatures) has been demonstrated in synaptic membranes (71, 128–130), indicating that lipid restructuring is likely to be necessary for adjustment to relatively long-term thermal changes.

Consequently, loss of membrane integrity in brain membranes may limit thermal tolerance during acute warming.

How the physical and chemical properties of brain membranes vary in different

Antarctic notothenioid species is not yet fully understood. In particular, these questions have not been examined for membranes beyond the synaptic membranes, nor within the 38 context of known differences in thermal tolerance limits. I hypothesized that species differences in thermal tolerance might be explained by physical (i.e., fluidity) or chemical

(i.e., lipid composition) attributes of biological membranes in the nervous system. To this end, I quantified fluidity in three types of membranes (synaptic membranes, myelin, and brain mitochondria) in two species of Antarctic notothenioids that differ in their thermal tolerance: the red-blooded Notothenia coriiceps (CTMAX=17°C) and the white-blooded

(i.e., hemoglobinless) Chaenocephalus aceratus (CTMAX=14°C). Differences in polar lipid composition were characterized in myelin, and cholesterol was measured in myelin and synaptic membranes.

I hypothesized that in at least one of the brain membranes analyzed, C. aceratus would exhibit greater (excessive) fluidity than the more thermotolerant N. coriiceps. This would suggest membrane integrity is compromised beyond a lower critical temperature in the icefish species. Results reveal species differences in synaptic membrane fluidity, as well as dissimilarities between the responses to temperature variation in myelin fluidity.

Together, these findings help shed light on the underpinnings of thermal tolerance and neural failure associated with acute warming in Antarctic notothenioids.

Materials and Methods

Animal and Tissue Collection

Adult specimens (800 to 2000 g) of N. coriiceps and C. aceratus were collected in the Western Antarctic Peninsula region during the austral autumn of 2015 (Appendix A).

Animals were held in circulating seawater tanks on the vessel before being transferred to

Palmer Station, Antarctica, where they were held at ambient temperatures (0+1°C) for a 39 maximum of three weeks (for N. coriiceps) or 1.5 weeks (for the icefishes). Animals were euthanized by a single blunt blow to the head followed by severing the spinal cord.

Membrane preparations were carried out on freshly extracted brain tissue.

Membrane Preparations and Marker Enzyme Analyses

Myelin, synaptic membranes, and mitochondria were fractionated from brain tissue as described (131), with modifications (Appendix B). Membrane pellets were collected and resuspended in 250 μl storage buffer (25 mM Tris, pH 7.4 at 25°C) and stored at -80°C.

Enrichments of each membrane fraction were determined by measuring the protein-specific activities of marker enzymes: acetylcholinesterase (AChE) for synaptic membranes, cyclic nucleotide phosphodiesterase (CNPase) for myelin, and succinate dehydrogenase (SDH) for mitochondria (Appendix C). All marker assays were conducted at ~23°C and adapted to a microplate reader.

Membrane Physical and Chemical Properties

Membrane fluidity was quantified by fluorescence depolarization as described

(132) (Appendix D). Change in polarization (excitation=356 nm, emission=430 nm) was measured between 2°C and 40°C. Temperatures were elevated at 2°C intervals (for myelin) and 5°C intervals (for synaptic membranes and mitochondria).

Lipids were extracted from myelin as described (133) (Appendix E). Extracts were sent to the Kansas Lipidomics Research Center for polar lipid analysis, and a diacyl polar lipid profile dataset was generated by quadrupole mass spectrometry using an

Applied Biosystems 4000 QTRAP mass spectrometer as described (134). Relative 40 abundances of the major phospholipid classes were compared between species. The unsaturation index (UI) was calculated as described (135). Polar lipid compositions were not analyzed in synaptic membranes due to lack of sufficient material.

Cholesterol was quantified in myelin and synaptic membranes using a Cayman fluorometric assay kit and normalized to total phospholipid content (Appendix E).

Statistical Analyses

All statistical analyses were performed in SPSS Statistics. Membrane fluidity was analyzed using an analysis of covariance (ANCOVA). Polar lipid class abundances, unsaturation indices, unsaturation distributions, and cholesterol-to-phospholipid ratios were analyzed using unpaired two-tailed t-tests to compare lipid profiles between species.

Analyses of polar lipids and unsaturation distributions were adjusted for the Bonferroni correction to account for multiple t-tests, with a minimum P-value of 0.0042. All assumptions were tested before performing statistical analyses. Although the fluidity measurements for the synaptic membranes displayed heterogeneity of variance, all other assumptions were met and sample sizes were equivalent between these groups.

Results

Cell Fractionation Revealed Three Distinct Membrane Types

Fraction 2 (between 3% and 10% Percoll layers) was enriched approximately 3.5- fold with myelin in C. aceratus and N. coriiceps. Fraction 4 (between 15% and 23%

Percoll layers) was enriched 2.9- and 3.9-fold with synaptic membranes in C. aceratus and N. coriiceps, respectively. Fraction 5 (below the 23% Percoll layer) was enriched 41

5.4- and 4.3-fold with mitochondria for C. aceratus and N. coriiceps, respectively. (Table

2.1)

Table 2.1. Distribution of Membrane Fractions as Indicated by Marker Enzyme Enrichment.

CNPase ACE COR Fraction Enrichment (s.e.m.) Enrichment (s.e.m.) 1 1.0 (0.048) 1.2 (0.17) 2 3.4 (0.17) 3.5 (0.34) 3 0.90 (0.023) 0.88 (0.010) 4 0.75 (0.63) 0.75 (0.033) 5 0.68 (0.27) 0.73 (0.039) AChE ACE COR Fraction Enrichment (s.e.m.) Enrichment (s.e.m.) 1 0.96 (0.13) 1.1 (0.18) 2 1.0 (0.18) 1.7 (0.18) 3 0.74 (0.11) 0.64 (0.18) 4 2.9 (0.17) 3.9 (0.44) 5 0.29 (0.073) 0.41 (0.083) SDH ACE COR Fraction Enrichment (s.e.m.) Enrichment (s.e.m.) 1 0.97 (0.20) 0.34 (0.093) 2 0.59 (0.073) 0.29 (0.069) 3 0.88 (0.11) 1.1 (0.21) 4 1.7 (0.049) 1.4 (0.096) 5 5.4 (0.46) 4.3 (0.26)

All enzyme activities were normalized to the activity of the crude homogenate to calculate enrichment in C. aceratus (ACE) and N. coriiceps (COR) (N=8). Acetylcholine esterase (AChE) was used to assess the localization of synaptic membranes, cyclic nucleotide phosphodiesterase (CNPase) was used to assess the localization of myelin, and succinate dehydrogenase (SDH) to assess the localization of mitochondria. All animals in this group were held at ambient temperatures (~0°C) before membrane preparation.

Fluidity of Synaptic Membranes was Greatest in the Less Thermotolerant Species

No significant discontinuities in slope were present in membrane fluidity measurements of synaptic membranes, myelin, or mitochondria (Fig. 2.1). Polarization values of synaptic membranes from C. aceratus were consistently lower than those of N. coriiceps (P<0.0001), indicating a greater degree of fluidity in synaptic membranes from the icefish compared with membranes from the red-blooded species (Fig. 2.1.a). In contrast, the absolute fluidity of both myelin and mitochondria did not differ significantly between species (Fig. 2.1.b-c). However, myelin fluidity of N. coriiceps was significantly more influenced by thermal variation in vitro (i.e., greater thermal sensitivity) than in C. aceratus. Specifically, the change in polarization with temperature was approximately

1.3-fold (30%) greater in myelin from N. coriiceps than in myelin from C. aceratus

(P<0.001). 43

Figure 2.1. Steady state polarization values (i.e., inverse of membrane fluidity) for the fluorescent probe DPH in Chaenocephalus aceratus (ACE, open circles) and Notothenia coriiceps (COR, closed circles) (N=8) in (a) synaptic membranes, (b) myelin and (c) mitochondria. Error bars represent means + s.e.m.

Lipid Profiles of Myelin Showed Variation between Species

The most abundant polar lipid classes in myelin from both notothenioid species were phosphatidylcholine (PC), representing more than 50% of total polar lipids, and phosphatidylethanolamine (PE), representing approximately 25% (Table 2.2).

Plasmalogen PC (ePC) and phosphatidylserine (PS) each represented 5-7.5% of the total phospholipid content. The relative abundance of PC in myelin was significantly greater in

N. coriiceps than in C. aceratus (P<0.001). Additionally, the ratio of PC to PE was significantly greater in N. coriiceps (P<0.0042).

Table 2.2. Distribution of Polar Lipids in Myelin.

Myelin from C. aceratus contained a significantly greater unsaturation index (UI) than myelin from N. coriiceps (P<0.0042) (Table 2.3). Specifically, myelin from C. aceratus contained a greater proportion of highly unsaturated fatty acids (11-12 double bonds per pair of acyl chains), while myelin from N. coriiceps contained a greater proportion of mono- and di-unsaturated fatty acids (Table 2.3). Cholesterol-to- phospholipid ratios in myelin were approximately two-fold greater in C. aceratus than in

N. coriiceps (P<0.05), whereas cholesterol contents in synaptic membranes did not differ significantly between species (Fig. 2.2).

Table 2.3. Distribution of Fatty Acid Unsaturation in Myelin Samples by Number of Double Bonds. 46

Figure 2.2. Cholesterol-to-phospholipid ratios for Chaenocephalus aceratus (ACE, white bars) and Notothenia coriiceps (COR, black bars) in synaptic membranes and myelin (N=8). One asterisk indicates P<0.05 between species. Error bars represent + s.e.m.

Discussion

Species Differences in Synaptic Membrane Fluidity Are Associated with Variation in

Thermal Tolerance

In this chapter, properties of three types of brain membranes from two species of notothenioids that vary in thermal tolerance were investigated. Because the absolute fluidity varied between species only in synaptic membranes, disruption of physical properties in the synaptic junctions is likely to be of greater importance in governing limits to acute thermal stress rather than in either myelin or mitochondrial membranes.

This interpretation is consistent with the observation that synaptic transmission is inhibited at elevated temperatures in several Antarctic notothenioids (121). I posit that in the icefish, C. aceratus, synaptic transmission is adversely affected at a lower temperature during thermal ramping than in N. coriiceps. 47

Greater Sensitivity to Thermal Change of Myelin from N. coriiceps May Account for

Spasmodic Behavior with Acute Warming

The absolute fluidity of myelin did not vary significantly between species, and this similarity in myelin fluidity can be explained, at least in part, by the polar lipid profiles of each species. While myelin in C. aceratus exhibited a higher UI, which should enhance fluidity (109), myelin in C. aceratus also had a lower PC-to-PE ratio, which should reduce fluidity (96). The fluidizing effect of a greater UI is likely to be countered by the lower PC-to-PE ratio, as well as the greater cholesterol contents, in myelin from C. aceratus compared with that of N. coriiceps.

In contrast with the similarity in myelin fluidity between species, the perturbation in fluidity with in vitro changes in temperature was greater in myelin from N. coriiceps than in C. aceratus. This finding can be explained by the observation that cholesterol-to- phospholipid ratios were significantly lower in myelin of N. coriiceps. Because cholesterol is known to stabilize the structure of the membrane (89), the higher cholesterol contents in myelin of the less thermotolerant C. aceratus do, at first glance, seem counterintuitive. However, the greater thermal dependence of myelin fluidity in the red-blooded species may play a role in some of the irregular animal behaviors observed by our group during warming (Crockett and O’Brien, unpublished observations).

For example, N. coriiceps displays spasms that suggest some disruption to brain function, even at temperatures well below the species’ CTMAX of 17ºC. Such irregular behaviors, however, were not observed in C. aceratus. Frequency of impulse conduction 48 has been shown to increase with acute warming in both mammals and poikilotherms, but often becomes irregular beyond a critical temperature (67, 136).

This spasmodic behavior in N. coriiceps may reflect ineffective myelin sheath insulation that is brought on with warming, perhaps as a result of perturbations in the fluidity of myelin. I propose that the spasms exhibited by N. coriiceps at subcritical temperatures reflect the beginning of breakdown of signal propagation due to loss of myelin integrity. Consistent with this, knockdown of ceramide galactosyltransferase, an enzyme essential for myelin structural stability, results in loss of locomotive control and induces whole-body tremors in mice (137). It seems possible that a breakdown in synaptic transmission may precede the destabilization of myelin in the icefish species, while in N. coriiceps, a disruption of myelin integrity could contribute to the irregular behaviors observed at temperatures below CTMAX.

Work contained in Chapter 3 is in press and has been reprinted with permission from the publisher – Biederman et al. (2019). Mitochondrial membranes in cardiac muscle from antarctic notothenioid fishes vary in phospholipid composition and membrane fluidity.

Comparative Biochemistry and Physiology B. September 2019 issue.

CHAPTER 3: PROPERTIES OF CARDIAC MITOCHONDRIAL MEMBRANES

VARY AMONG NOTOTHENIOIDS WITH DIFFERING CARDIAC RESPONSES TO

ACUTE WARMING

Introduction

Loss of aerobic scope and cardiac function are thought to limit the capacities of ectothermic organisms to endure elevated temperatures (45, 47, 138, 139) and at some point in the future, Antarctic notothenioid fishes are likely to encounter this physiological challenge as a consequence of anthropogenic drivers of climate warming (140). For example, in Notothenia coriiceps, cardiac arrhythmia occurs at temperatures below

CTMAX, with persistent ventricular asystole at 16.7°C, a temperature comparable to

CTMAX (26).

At the subcellular level, it has been shown in various ectothermic and endothermic taxa that several major catabolic pathways are particularly vulnerable to alterations in membrane fluidity, more specifically the kinetics and/or activities of enzymes and protein complexes associated with mitochondrial membranes, such as succinate dehydrogenase (141, 142), cytochrome c oxidase (55, 143), and carnitine palmitoyltransferase (144).

Recently, it has been demonstrated that cardiac mitochondrial metabolism is impaired with warming in two Antarctic notothenioids, the red-blooded N. coriiceps and in the white-blooded (i.e., hemoglobinless) Chaenocephalus aceratus. For example, energy charge is greater in the more thermotolerant N. coriiceps, relative to C. aceratus, at ambient temperatures and following exposure to CTMAX (145). In addition, rates of 51 proton leak (reflected in rates of state 4 respiration) in cardiac mitochondria increase following exposure to CTMAX in C. aceratus, but not in N. coriiceps (145).

At a critical point with rising temperature, hyperfluidization of the membrane can occur, thus potentially limiting ATP supply, cardiac performance, and ultimately thermal tolerance. Elevated temperatures are likely to compromise membrane structure and function in particular in Antarctic notothenioids due to the preponderance of polyunsaturated fatty acids (PUFAs). While acyl chains of polar lipids containing PUFAs permit maintenance of membrane fluidity at sub-zero temperatures (146–148), this composition will likely render the membrane more susceptible to hyperfluidization and possibly more permeable to flux of ions and uncharged solutes.

In this chapter, biochemical compositions and membrane fluidity were characterized in cardiac mitochondria from white- and red-blooded notothenioids in order to explore potential relationships between membrane properties and known cardiac responses to elevated temperature. I hypothesized that membrane fluidity differs among species, possibly helping to account for differences in mitochondrial and cardiac function. Results suggest that membrane fluidity indeed differs between notothenioids varying in thermal tolerance, and lipid composition appears to help explain variation in membrane fluidity.

Materials and Methods

Animal and Tissue Collection

Adult specimens of the white-blooded (i.e., icefishes) C. aceratus and

Pseudochaenichthys georgianus, and the red-blooded N. coriiceps, were collected in the 52 region of the Western Antarctic Peninsula during the austral autumn of 2015 (Appendix

A). Specimens were held in circulating seawater tanks on the ARSV Laurence M. Gould before being transferred to tanks at Palmer Station, Antarctica, where they were held at ambient temperatures (0+1°C) for up to three weeks (for N. coriiceps) or 1.5 weeks (for the icefishes).

Animals were euthanized by a single blunt blow to the head followed by severing the spinal cord. Hearts were extracted immediately and allowed to contract several times in ice-cold notothenioid Ringer’s solution (240 mM NaCl, 2.5 mM MgCl2, 5 mM KCl,

2.5 mM NaHCO3, 5 mM NaH2PO4, pH 8.0 at 4°C). Membrane preparations were carried out immediately (Appendix B).

Membrane Fluidity Measurements

Membrane fluidity was quantified by fluorescence depolarization as described

(132) in C. aceratus and N. coriiceps (Appendix D). Fluidity was not measured in P. georgianus due to lack of sufficient material. Change in polarization (excitation=356 nm, emission=430 nm) was measured between 2°C and 30°C. Temperatures were elevated at

2°C intervals at a rate of ~0.3°C min-1. Polarization measurements were performed in triplicate after the temperature stabilized at each temperature interval.

Lipid Extraction and Quantification

Lipids were extracted from membranes as described (133) (Appendix E). Extracts were sent to the Kansas Lipidomics Research Center for analysis, and a diacyl polar lipid profile dataset was generated by quadrupole mass spectrometry using an Applied

Biosystems 4000 QTRAP mass spectrometer as described (134). The relative abundances 53 of the major polar lipid classes were compared among species. The unsaturation index

(UI) was calculated as described previously (135).

Statistical Analyses

Membrane fluidity was compared between C. aceratus and N. coriiceps using an analysis of covariance (ANCOVA) in SPSS Statistics. Potential breaks in fluidity were analyzed by a two-phase linear regression test in SPSS Statistics. Differences in polar lipid class abundances and unsaturation indices were analyzed by a one-way analysis of variance in R Studio followed by Tukey’s post-hoc tests to compare lipid profiles among species. All necessary assumptions were tested before performing analyses.

Results

Fluidity of Mitochondrial Membranes was Greatest in the Less Thermotolerant Species

Mitochondrial membranes from the heart ventricle of C. aceratus displayed consistently lower polarization values than those of N. coriiceps (P<0.0001) (Fig. 3.1), indicating significantly greater fluidity in membranes from the icefish. No significant discontinuities in slope for either C. aceratus or N. coriiceps were observed over the measured temperature range of 2 to 30ºC.

Figure 3.1. Steady state polarization values (i.e., inverse of membrane fluidity) for the fluorescent probe DPH in mitochondrial membranes from cardiac ventricles from C. aceratus (open circles) and N. coriiceps (closed circles) (N=6). Error bars represent + s.e.m.

Membrane Compositions Varied Among Notothenioid Species

Consistent with the data presented in Chapter 2, phosphatidylcholine (PC) was the most abundant polar lipid class, constituting ~40% of the polar lipids. It was followed by phosphatidylethanolamine (PE), and cardiolipin (CL), which accounted for ~25 and

~13% of polar lipids, respectively (Table 3.1). 55

Table 3.1. Polar Lipid Class Distribution and Unsaturation Index in Mitochondrial Membranes from Cardiac Ventricles in White- and Red- Blooded Notothenioids.

The polar lipid compositions of mitochondrial membranes of the icefishes C. aceratus and P. georgianus were similar to one another, but markedly dissimilar to the t of N. coriiceps (Table 3.1). For the eight lipid classes that differed among species, seven were similar between the two icefishes and dissimilar to N. coriiceps. The PC contents of membranes from the two icefishes were approximately 1.1-fold (i.e., 10%) greater than 56 those of N. coriiceps (P<0.001). In contrast, levels of PE and CL were both similar among all species of notothenioids.

Hydrolyzed phospholipids (LPC and LPE) were lower in the icefishes, relative to

N. coriiceps (P<0.001). The abundance of the plasmalogen ePC (i.e., ether phospholipid of PC) was almost two-fold greater in the membranes of the icefishes compared to membranes of N. coriiceps (P<0.001). Similarly, the abundance of the anionic phospholipid phosphatidylinositol (PI) (P<0.01) was higher in membranes from both icefishes than in the membranes of N. coriiceps. In addition, the proportion of total PE

(PE, LPE, ePE) to total PC (PC, LPC, ePC) was 1.1-fold (10%) greater in N. coriiceps, relative to the icefishes (P<0.05). The unsaturation index (UI), a measure of double bonds present in the polar lipids, differed significantly among species, with a greater UI in N. coriiceps than in the icefishes (Table 3.1) (P<0.0001).

Discussion

Significant differences were reported in membrane fluidity and in polar lipid compositions of cardiac mitochondria from white- and red-blooded notothenioid fishes, with mitochondrial membranes from the icefish C. aceratus displaying significantly greater fluidity than those from the red-blooded species, N. coriiceps. Polar lipid profiles were distinct among species and exhibited similarity between the two species of icefishes, relative to the red-blooded species. Although I was unable to quantify fluidity in mitochondrial membranes from P. georgianus due to lack of sufficient material during sampling, given the observation that the polar lipid compositions between both C. aceratus and P. georgianus were similar, it appears likely that the fluidity of 57 mitochondrial membranes from P. georgianus is comparable to that of its confamilial relative, C. aceratus. Consistent with this, preliminary data by our group comparing biophysical and biochemical properties of cardiac plasma membranes among six species of icefishes and red-blooded notothenioids indicate grouping among familial affiliations

(Evans et al., unpublished observations).

Differences in Proportions of Polar Lipid Classes are Consistent with Variation in

Fluidity between Species

The differences in membrane fluidity between the icefish C. aceratus and the red- blooded N. coriiceps can be accounted for by differences in membrane lipid composition.

Because PC and PE are the dominant polar lipids in biological membranes (89), their influences on the membrane are likely to be particularly impactful. PC tends to impart a fluidizing effect within membranes, while PE can impart a rigidifying effect (96, 149).

Thus, the lower total PE-to-total PC ratio in C. aceratus is consistent with the greater membrane fluidity in this species.

While (unhydrolyzed) PC contents were greatest in membranes from icefishes, amounts of lyso-PC (LPC) were greater in membranes from N. coriiceps. Because hydrolyzed phospholipids fluidize membranes, relative to their unhydrolyzed counterparts (150), variation in (unhydrolyzed) PC alone is unlikely to account for the variation in fluidity we observed between N. coriiceps and C. aceratus. However, because the proportion of total PE (PE, LPE, ePE) to total PC (PC, LPC, ePC) was greater in N. coriiceps, this difference in the ratio of total PE-to-total PC may explain the variation in membrane fluidity. 58

In contrast to hydrolyzed lipids, plasmalogens generally impart an ordering effect on membranes (103, 151, 152). Yet the relatively large choline head group in both ePC and LPC can promote fluidization (relative to ePE and LPE, respectively) (150, 153), which is likely to contribute to the observed greater fluidity in membranes of the icefish.

Furthermore, as plasmalogens generally limit membrane permeability (152, 154), greater contents of ePC in the membranes of the icefishes may ensure that membrane permeability remains uncompromised, as more fluid membranes may otherwise exhibit greater permeability (155).

Levels of PI, a polar lipid class involved in lipid signaling, were found to be significantly greater in the membranes of the two icefish species than in membranes from

N. coriiceps. However, because this phospholipid class constitutes only 3% of membrane lipids measured, it is unlikely that PI accounts directly for the significant variation in fluidity between species.

The greater fluidity in membranes from C. aceratus cannot be explained by species differences in fatty acyl unsaturation, as membranes from the icefishes exhibited a significantly lower UI than the red-blooded species. Thus, it appears most likely that distribution of polar lipid classes contributed more significantly to variation in fluidity among the species, even countering the effects of unsaturation on membrane fluidity.

In total, these findings suggest a link between variation in polar lipid classes (i.e., polar lipid head group) distribution and membrane fluidity among notothenioids. It is important to note, however, that some of the variation within some of the less prominent 59 polar lipid classes may reflect restructuring within the major classes, as adjustments within one class can, and likely will, impact overall composition.

Further, these results may be somewhat complicated by differences in mitochondrial architecture among species, specifically the proportion of inner and outer mitochondrial membranes. The relative proportion of outer-to-inner membrane is greater in C. aceratus than in N. coriiceps; the surface density of outer membrane (per gram tissue) is enhanced in icefishes while the surface density of inner membrane is largely similar among species (25, 156).

Consistent with this, I found that the abundance of PC (the most abundant polar lipid in this study) was greatest in the icefishes. PC has been shown to be more abundant in the outer, relative to the inner, membrane (157). Levels of CL, which is localized almost exclusively to the inner membrane (158), were greater in N. coriiceps when normalized to total phospholipid by mass (P<0.05) (data not shown), although CL contents are equivalent between the two species when normalized to total protein by mass

(156). Also consistent with the present data, the outer mitochondrial membrane is typically more fluid than the inner membrane (157). Thus, it is possible that the observed differences in membrane composition and physical properties are present, at least in part, due to the proliferation of outer mitochondrial membrane lipids in icefishes.

Greater Fluidity in Icefish Mitochondria is Consistent with Functional Consequences

Associated with Warming

Because the cardiac mitochondrial membranes of C. aceratus were inherently more fluid than those of N. coriiceps, mitochondria in this icefish (and likely too the 60 icefish P. georgianus) may be more prone, at a given elevated temperature, to hyperfluidization than those of the red-blooded species, thus possibly contributing to the lower CTMAX in the icefishes. Consistent with this, it is also worth noting that membrane fluidity at both species’ approximate CTMAX (14ºC for C. aceratus and 18ºC for N. coriiceps) were comparable. These results are consistent with the interpretation that as the animal’s body temperature approaches its CTMAX, the membranes of the cardiac mitochondria may reach a critical threshold beyond which membrane integrity is significantly compromised.

It has been postulated that membrane fluidization associated with warming is likely to lead to loss of mitochondrial integrity at a critical temperature, thus incurring loss of some cellular function (105, 159, 160), specifically oxidative phosphorylation

(161). Because work by our group has shown that polar lipid contents are unchanged upon CTMAX exposure in cardiac mitochondria of C. aceratus, P. georgianus, and N. coriiceps (Biederman et al., in press at Comparative Biochemistry and Physiology B), it is likely that, without membrane remodeling, the integrity of the membrane will be compromised during acute warming in these species. Fluidization of the mitochondrial membrane is likely to incur major consequences for mitochondrial function. For example, greater fluidity in C. aceratus may promote the formation of the mitochondrial permeability transition pore, which is involved in the onset of apoptosis (162). It is worth noting, however, that neither the greater fluidity in cardiac mitochondria of C. aceratus, nor the greater unsaturation in those of N. coriiceps, appeared to significantly impact 61 susceptibility to lipid peroxidation, as levels of lipid peroxidation did not differ among species (Biederman et al., in press at Comparative Biochemistry and Physiology B).

Conclusions

Together, these data demonstrate differences in properties of cardiac mitochondrial membranes and, potentially, differences in maintenance of membrane structure during acute warming. Compromised membrane integrity may help account for differences in cardiac function among notothenioids. It is also possible, however, that the greater fluidity serves to enhance cardiac function at ambient (i.e., cold) temperatures.

Work in mammals has demonstrated that cardiac mitochondrial membranes become less fluid with aging; this has been postulated to contribute to compromised cardiac function in these organisms (163, 164). Further, greater fluidity has been shown to elevate the amplitude and frequency of calcium transient in cardiac mitochondria, enhancing energy transduction (164). The greater fluidity observed in C. aceratus may serve to enhance the cardiovascular function of icefishes at physiological temperatures but may limit function at elevated temperatures as a result of compromised membrane integrity and, consequently, impediment of mitochondrial function. The particular vulnerability of

Antarctic icefishes to hyperfluidization of mitochondrial membranes at elevated temperatures may contribute to significant perturbation of oxidative phosphorylation, leaving the cardiac ventricle with an inadequate energy charge necessary for regular contractility.

CHAPTER 4: MEMBRANES FROM BRAIN AND CARDIAC TISSUES DISPLAY

VARIABLE HOMEOVISCOUS RESPONSES TO THERMAL ACCLIMATION IN

NOTOTHENIA CORIICEPS

Introduction

The Antarctic notothenioid fishes have adapted to the ice-cold Southern Ocean and, consequently, are among the most stenothermal animals on the planet (77). Yet despite their stenothermy, several species of notothenioids have demonstrated the ability to extend their thermal tolerance limits (critical thermal maxima, CTMAX) upon acclimation to warmer temperatures (82). The physiological mechanisms that govern thermal tolerance in Antarctic notothenioid fishes are not yet fully understood.

Many researchers have proposed that thermal tolerance is limited by the failure of a single organ system (39). Cardiac and brain function have both been identified as potential limiting factors in the thermal tolerances of ectotherms. Cardiac arrhythmia typically occurs just prior to the point of CTMAX, suggesting that cardiac failure may help account for loss of organismal performance during acute warming (63). However, additional evidence suggests brain function is also compromised upon acute warming

(65, 77, 165). During warming, neuron firing rates increase until a critical temperature is reached, beyond which point the firing becomes more random (68). The interplay of the cardiovascular and nervous systems, in relation to the thermal tolerances of the Antarctic notothenioids, is not fully understood.

The fluidity of synaptic membranes (Chapter 2) and cardiac mitochondria

(Chapter 3) may explain variation in thermal physiology between different species of 63 notothenioids. Homeoviscous adaptation (HVA) is critical to the survival of ectothermic organisms in fluctuating thermal environments (114). Membrane integrity is typically maintained via modulation of the membrane lipid environment. Highly unsaturated lipids serve to decrease membrane order, and membrane unsaturation is often reduced upon warm acclimation (71, 89, 166). Further, properties of membrane fatty acyl chains are known to play a role in fluidity; typically, short and asymmetric (i.e., heterogeneous in length) tails contribute to a more fluid membrane (89).

The capacities of Antarctic notothenioids to undergo membrane remodeling during acclimatization will likely be critical to their survival in a warmer climate.

Previous reports have demonstrated a lack of change in membrane composition following thermal acclimation in several Antarctic notothenioids (117–119). While these previous reports have provided valuable information about certain aspects of membrane remodeling with thermal acclimation in notothenioids, the distributions of polar lipid classes, as well as other aspects of membrane composition, were not reported in these studies. In three reports, evidence of decreased unsaturation in liver membranes as well as in crude gill and white muscle, was demonstrated in an Antarctic notothenioid,

Trematomus bernacchii, upon exposure to warmer temperatures (119, 167, 168).

Evidence of HVA in notothenioids has not been reported in either cardiac or brain tissue.

In this chapter, membrane fluidity was measured in several membranes from cardiac ventricles (microsomes, mitochondria) and brains (synaptic membranes, myelin, mitochondria) of the Antarctic notothenioid Notothenia coriiceps following acclimation to 0°C or 5°C for a minimum of six weeks. Biochemical analyses (polar lipid profiling 64 and cholesterol measurements) were performed in select membranes. I hypothesized that membrane remodeling would occur to preserve membrane fluidity following thermal acclimation. My results provide evidence of HVA in the cardiac membranes, yet an absence of HVA in brain membranes. These data are indicative of thermal plasticity in membranes of the cardiac system, but not of the nervous system, in N. coriiceps.

Materials and Methods

Animal Collection and Thermal Acclimation

Adult specimens of N. coriiceps were collected in the Western Antarctic

Peninsula region during the austral autumn of 2017 (Appendix A). Animals (Group A) were collected by trawl nets and baited pots and transferred to circulating seawater tanks at Palmer Station, Antarctica. A second set of animals (Group B) was caught using baited lines at Arthur Harbor. These individuals were transferred immediately to 5-gallon buckets containing seawater and transported to seawater tanks on station. Animals were held for three days before initiation of acclimation experiments.

After a three-day recovery period, the animals were subjected to a thermal acclimation experiment (Appendix F). In brief, animals were assigned randomly to designated warm (5+1°C) or control (0+1°C) groups. The tanks used for the warm acclimation were increased at a rate of 1°C per day until the acclimation temperature of

5°C was reached. Animals were then held at their respective acclimation temperature

(0°C or 5°C) for a period of either 10 (Group A) or six (Group B) weeks. 65

Membrane Preparations and Marker Enzyme Analyses

Animals were euthanized by a single blunt blow to the head followed by severing the spinal cord. Membrane preparations were carried out on freshly extracted tissues.

Hearts were extracted immediately and allowed to contract several times in ice-cold notothenioid Ringer’s solution (240 mM NaCl, 2.5 mM MgCl2, 5 mM KCl, 2.5 mM

NaHCO3, 5 mM NaH2PO4, pH 8.0 at 4°C). Mitochondrial and microsomal preparations were carried out immediately in cardiac ventricles (Appendix B). Next, brains were extracted and synaptic membranes, myelin and mitochondria were fractionated from tissue as described (131), with modifications (Appendix B). Membrane marker assays were performed as described previously (Chapter 2, Appendix C).

Membrane Physical and Biochemical Properties

Membrane fluidity was quantified in all preparations by fluorescence depolarization as described (132) (Appendix D). Change in polarization (excitation=356 nm, emission=430 nm) was measured between 2°C and 40°C. Temperatures were elevated at 2°C intervals at a rate of ~0.3°C min-1. Homeoviscous efficacy was calculated as described previously (129).

Lipids were extracted from cardiac and brain mitochondria as described (133)

(Appendix E). Extracts were sent to the Kansas Lipidomics Research Center, and a diacyl polar lipid profile dataset was generated by quadrupole mass spectrometry using an

Applied Biosystems 4000 QTRAP mass spectrometer as described (134). Relative abundances of the major polar lipid classes were compared between acclimation groups and tissues. The unsaturation index (UI) was calculated as described (135). 66

Cholesterol was quantified in membranes likely to display cholesterol enrichment

(myelin, synaptic membranes, and cardiac microsomes) (169–171) using a Cayman fluorometric assay kit. Cholesterol contents were normalized to total phospholipid

(Appendix E).

Statistical Analyses

Fluidity of cardiac microsomes, synaptic membranes, myelin, and brain mitochondria was compared between treatment groups by an analysis of covariance

(ANCOVA) using SPSS Statistics. Due to differences in slope (i.e., thermal dependence of polarization), the data for cardiac mitochondrial fluidity did not meet the requirements for an ANCOVA and were therefore analyzed by generating a mixed effects model in R

Studio, with temperature designated as a random factor. Polar lipid compositions were compared among treatment groups and membrane types by a two-way analysis of variance (ANOVA). Cholesterol contents and fatty acyl chain lengths were compared between thermal treatment groups by a two-tailed t-test.

For the brain membranes, samples from the two acclimation cohorts (Groups A and B) were found to be statistically equivalent in all reported analyses (P>0.50). For this reason, data from both groups were pooled to account for logistical issues during fieldwork that otherwise limited sample size. Data from cardiac membranes represent samples from the six-week acclimation (Group B) only, as cardiac membranes were not prepared from the 10-week acclimation group (Group A).

Results

Membrane Fluidity Differed Between Tissues and With Thermal Treatment

Both microsomal and mitochondrial membranes in cardiac tissues from the 5°C group displayed significantly greater polarization values, indicating reduced membrane fluidity compared to the 0°C group (P<0.0001) (Fig. 4.1.a-b). Homeoviscous efficacy

(calculated as the ratio of polarization values for both treatment groups, at their respective physiological temperatures) was determined to be 100% for both membrane types, indicating full compensation for the difference in membrane order that would otherwise be expected at the respective acclimation temperatures. For the three brain membranes, polarization values did not differ significantly between thermal treatment groups (Fig.

4.2.a-c); thus, no evidence of a homeoviscous response was observed.

Figure 4.1. Steady state polarization values (i.e., inverse of membrane fluidity) for the fluorescent probe DPH in (a) cardiac mitochondria and (b) cardiac microsomes from hearts of animals subjected to 0°C and 5°C acclimation treatments. Error bars represent means + s.e.m. (N=5). 69

Figure 4.2. Steady state polarization values (i.e., inverse of membrane fluidity) for the fluorescent probe DPH in (a) synaptic membranes, (b) myelin, and (c) mitochondria from brains of animals subjected to 0°C and 5°C acclimation treatments. Error bars represent means ± s.e.m. (N=10). 70

Polar Lipid Profiles Differed Between Tissues and With Thermal Treatment

Polar lipid profiles were generated for mitochondrial membranes prepared from cardiac ventricle and brain tissue. In cardiac mitochondria, the relative abundances of major polar lipids (e.g., phosphatidylcholine, PC; and phosphatidylethanolamine, PE) were unchanged with thermal acclimation. However, the proportion of hydrolyzed phospholipids (i.e., lyso-PC and lyso-PE, relative to PC and PE) was reduced 1.3-fold

(30%) in the 5°C acclimation group (P<0.01) (Table 4.1). Additionally, the relative abundance of the specific class lyso-PC (LPC) was reduced 1.4-fold (40%) in cardiac mitochondria from the 5°C acclimation group (P<0.01). Neither the UI, nor the relative abundance of plasmalogens, were altered upon thermal acclimation (Table 4.1).

However, the proportion of long-chain fatty acids (>20 carbon atoms per acyl chain) was increased 1.2-fold (20%) in the 5°C acclimation group (P<0.01) (Table 4.2).

Similar to the cardiac mitochondria, the relative abundance of major polar lipid classes and the UI were both unchanged following thermal acclimation in brain mitochondria (Table 4.1). However, in contrast to the cardiac mitochondria, phosphatidylinositol (PI) and phosphatidylserine (PS) were increased 1.3- (30%) and 1.6- fold, (60%) respectively, with 5°C acclimation in this membrane type (P<0.05). Neither the degree of hydrolysis, nor the acyl chain length, differed between treatment groups

(Table 4.1).

Table 4.1. Relative Distribution of Polar Lipid Classes in Mitochondria from Cardiac and Brain Tissue Following Acclimation to 0°C or 5°C in Notothenia coriiceps.

Mitochondria (cardiac) Mitochondria (neuronal) 0°C 5°C 0°C 5°C PC 56.1 + 2.8 54.3 + 2.6 51.8+ 4.2 48.4 + 1.6 PE 15.9 + 3.6 21.1 + 4.0 29.6 + 6.8 35.9 + 1.8 ePC 9.7+ 0.7 8.3 + 0.8 N 6.9 + 1.0 5.6 + 0.2 PI 4.2 + 0.2 N 4.5 + 0.4 N 1.8 + 0.25 2.4 + 0.07 C LPC 5.9 + 0.6 N,C 4.3 + 0.6 N 1.7 + 0.7 0.76 + 0.2 LPE 2.8 + 0.3 2.3 + 0.3 N 1.7 + 0.6 0.82 + 0.1 PG 1.3 + 0.5 0.97 + 0.3 2.3 + 0.42 1.5 + 0.19 ePE 1.1 + 0.2 N 1.1 + 0.1 N 2.3 + 0.14 2.4 + 0.14 PS 0.6 + 0.09 N 0.8 + 0.1 N 1.2 + 0.14 1.9 + 0.17 C [LPE+LPC] / [PE+PC] 0.12 + 0.01 N,C 0.090 + 0.01 0.019 + 0.003 0.044 + 0.02 UI 537 + 41 572 + 44 548 + 31 513 + 13 Abbreviations: phosphatidylcholine (PC), phosphatidylethanolamine (PE), plasmalogen PC (ePC), phosphatidylinositol (PI), lyso-PC (LPC), lyso-PE (LPE), phosphatidylglycerol (PG), plasmalogen PE (ePE), phosphatidylserine (PS), unsaturation index (UI) Data are expressed as mol%. Error bars represent mean + s.e.m. (N=5) N denotes a significant difference from neuronal membranes of the same treatment group. C denotes a significant difference from the control group of the same membrane type.

Table 4.2. Relative Abundance of Fatty Acids with at Least 20 Carbon Atoms Per Chain in Mitochondria from Cardiac and Brain Tissue Following Acclimation to 0°C or 5°C In Notothenia coriiceps.

Of the nine polar lipid classes analyzed, six differed between brain and cardiac

mitochondria (Table 4.1). The hydrolyzed polar lipids (LPE, LPC) were greater in the

cardiac, relative to brain, mitochondria (P<0.05). The plasmalogen form of PC (ePC) was

greater in the cardiac mitochondria, while the plasmalogen form of PE (ePE) was greater 72 in the brain mitochondria. The two major polar lipid classes, PC and PE, did not differ significantly between membrane types.

Cholesterol contents (relative to total phospholipid) were measured in membrane types known to display cholesterol enrichment (synaptic membranes, myelin, and cardiac microsomes). In the cardiac microsomes, cholesterol contents were increased 1.6-fold

(60%) in the 5°C acclimation group (Table 4.3). In marked contrast, cholesterol contents did not differ between thermal treatment groups for either myelin or synaptic membranes

(Table 4.3).

Table 4.3. Cholesterol Contents of Cardiac Microsomes, Synaptic Membranes, and Myelin Following Acclimation to 0°C or 5°C In Notothenia coriiceps.

Discussion

Measurements of Membrane Fluidity Demonstrate a Homeoviscous Response in Cardiac,

but Not Brain Membranes

Cardiac mitochondria and microsomes displayed 100% efficacy, demonstrating full compensation for acclimation to warmer temperatures. In contrast, no significant effect of acclimation on membrane fluidity was observed in any of the three brain 73 membranes measured. These results indicate thermal plasticity of membrane structure in the heart ventricle, yet a lack of plasticity in membranes from the brain in this species.

An Absence of Change in Membrane Unsaturation is Consistent with Previous Literature

in Notothenioids

In this chapter, no differences in the extent of membrane unsaturation in polar lipids were observed even though modulation of membrane unsaturation and polar lipid class distribution are common mechanisms to ensure membrane constancy with thermal acclimation in temperate fishes (37, 71, 129, 132, 166). However, work in plants suggests alterations in membrane unsaturation may be more energetically costly than those of polar lipid classes (172). While the distributions of major polar lipid classes (i.e., PE and

PC) were also unchanged during this study, it is possible that the indicators of remodeling reported in this chapter represent the most energetically efficient responses to thermal variation in this species.

Previous membrane-focused studies of Antarctic notothenioids suggest some species, and perhaps some biological tissues, may be unable to mount a response to remodel their biological membranes with thermal acclimation (117–119). These studies, however, have examined the effects of acclimation on aspects of biochemical composition (i.e., unsaturation and cholesterol) of membranes. The effect of thermal acclimation on membrane fluidity in Antarctic notothenioids has not yet been investigated directly

These previous studies sought to determine the capacity of notothenioids for membrane remodeling upon acclimation to a warmer temperature. Results suggest the 74 capacity for remodeling varies among tissue, membrane type, and species. However, most of these previous studies assessed biochemical properties of membranes not characterized with marker enzymes. Because the structural and biochemical properties of membranes differ among cellular and intracellular membranes, it is important to characterize their properties independently. In this study, I determined whether, in fact, there exists a homeoviscous response in three brain membranes (synaptic membranes, myelin, mitochondria), as well as cardiac mitochondrial membranes and microsomes, the latter of which are likely to be enriched in plasma membranes (169, 171). The physical and biochemical characteristics of these particular membrane types have not been assessed in previous studies aimed at elucidating the capacities of notothenioids to acclimate to an elevated body temperature.

Results of earlier studies, combined with data herein, show that responses to long- term changes (i.e., weeks) in temperature can vary among notothenioid species as well as among tissues. For example, Malekar et al. (2018) reported a lack of lipid remodeling in non-specific liver membranes of two Antarctic notothenioids, Trematomus bernacchii and Pagothenia borchgrevinki, upon acclimation to 4°C for two and four weeks, respectively. When acclimated to 6°C for only one week, an increase in saturates and a decrease in monounsaturates was reported in T. bernacchii, but not in P. borchgrevinki.

In this same study, membrane cholesterol was unchanged with acclimation (119).

Similarly, a decrease in saturates, and an increase in mono- and polyunsaturated, was observed in crude gill and white muscle of T. bernacchii following acclimation to 2°C for

10 days (167, 168). 75

To date, no other studies have reported changes in membrane composition following thermal acclimation in an Antarctic notothenioid. Strobel et al. (2013) reported no change in membrane unsaturation of liver mitochondria in Notothenia rossii (a congener to the species used in the present chapter), nor in the confamilial notothenioid

Lepidonotothen squamifrons, following acclimation to 7°C and 9°C, respectively, for 4-6 weeks. Similarly, Gonzalez et al. (1995) reported no change in the level of unsaturation in the phospholipids from membranes associated with the gill, trunk kidney, liver, and white muscle tissues of Trematomus newnesi or T. bernacchii following acclimation to

4°C for four weeks.

I posit that the high degree of membrane unsaturation in notothenioids, known to serve as an adaptation to their extreme cold environment, may provide some advantage to physiological function that counters the need for remodeling in response to thermal change. Polyunsaturated fatty acids (PUFAs) have been shown to stabilize and enhance the activities of membrane-bound enzymes, including UDP-glucuronosyltransferase and cytochrome-b5 reductase (173). It seems possible that the preponderance of PUFAs has become relatively fixed in at least some Antarctic notothenioids. For this reason, other forms of membrane remodeling may be necessary for the generation of a homeoviscous response in notothenioids.

It is also critical to note that the analytical method employed in this work (i.e., the generation of a diacyl lipid dataset that reflects the sum of the acyl chains, rather than the abundance of individual fatty acids) may incur limitations in the lipid analysis, as the previous studies reported significant changes in the preponderance of individual fatty 76 acids (119, 167, 168). Although I was able to calculate the total membrane unsaturation index, neither the abundance of individual fatty acids, nor their specific degree of unsaturation, could not be determined using this analytical method. Thus, it is plausible that changes to membrane unsaturation, not detected in this analysis, indeed occurred during the course of this study.

Alterations in Membrane Cholesterol and Fatty Acyl Chain Length are Consistent with

Changes in Fluidity

Membrane cholesterol was elevated in cardiac microsomes (a preparation likely enriched in plasma membranes) following warm acclimation. Cholesterol stabilizes the membrane by restraining movement of fatty acyl tails (159) and typically imparts an ordering effect (100). Other studies have reported a positive association between membrane cholesterol and acclimation temperature (174–176), although this trend is not always observed (132, 174). Because membrane cholesterol was unchanged in myelin and synaptic membranes, it appears likely that modulation of membrane cholesterol may help explain in part the homeoviscous response observed in the cardiac microsomes.

Because mitochondria typically do not contain significant amounts of cholesterol

(169, 171), it is unlikely that modulation of cholesterol would occur to conserve membrane fluidity in this membrane. Analyses of polar lipids (which were not characterized in cardiac microsomes due to lack of available tissue), suggest modulation of acyl chain length, and in the extent of lipid hydrolysis, following acclimation. In the data herein, the proportion of long-chain fatty acids (defined as >20 carbons) increased

1.2-fold (20%) with warm acclimation in cardiac mitochondria. Extended acyl chains 77 typically serve to increase membrane order (177). Further, alterations in chain length

(178) have been reported in membranes from temperate fishes following thermal acclimation, consistent with the data reported in this chapter.

Decreases in Proportion of Hydrolyzed Phospholipids May Reflect Functional Changes

with Environmental Warming

The proportion of hydrolyzed phospholipids (relative to their unhydrolyzed forms) were reduced 1.2-fold (20%) in cardiac mitochondria from the 5°C acclimation group. Because hydrolysis of phospholipids is associated with enhanced membrane fluidity (159), the reduced hydrolysis is consistent with the observed homeoviscous response in mitochondria from cardiac ventricles.

Previous work suggests lipid hydrolysis may be decreased during environmental warming. Because phospholipase A is enhanced at cold growth temperatures in

Escherichia coli and is modulated by alterations in membrane order (179, 180), it appears plausible that the observed reduction in hydrolysis at 5°C was modulated by membrane- induced changes in a phospholipase enzyme. Consistent with this, enzymatic activity of phospholipase A2, which cleaves fatty acids from phospholipids, was increased with cold acclimation in Oncorhynchus mykiss (181). Nevertheless, this compositional change is consistent with the observed physical alterations of membrane order with thermal acclimation in cardiac mitochondria.

The change in hydrolyzed phospholipids may reflect an adaptation of notothenioids to subzero temperatures. Similar responses to thermal acclimation have been recorded in LPC and LPE in membranes from Arabidopsis thaliana (182). In plants, 78 changes in lipid hydrolysis are thought to be an important component of freeze tolerance

(182) and also serve to provide intermediates for membrane restructuring (183).

It seems possible that lipid hydrolysis may play a role in preservation of membrane structure at freezing temperatures for Antarctic notothenioids. As observed in this chapter, this dynamic would likely be shifted upon acclimation to elevated temperatures. Additionally, LPC and LPE are modulated following exposure to immune stressors in plants, likely due to the tendency of hydrolyzed phospholipids to induce protein phosphorylation (183). The alteration in lipid hydrolysis may thus serve a dual function, serving to preserve membrane fluidity upon thermal acclimation and to influence various cellular pathways (e.g., DNA repair, immune response, metabolic processes) that occur in response to environmental change (184).

The relative abundances of two relatively minor phospholipids, PI and PS, decreased following acclimation to 5°C in brain mitochondria. Changes in PI and PS are not commonly associated with thermal acclimation, although decreases in PS with warm acclimation have been recorded in brain membranes of Carassius auratus (130, 185), and the opposite trend was observed in liver tissue of O. mykiss upon warm acclimation (42).

Increases in PI have been recorded previously with warm acclimation in sperm plasma membranes of O. mykiss (186).

I posit that the changes in these (relatively minor) polar lipid classes may have occurred to preserve cellular function upon acclimation. Because PS and PI each represented less than 2% of polar lipids in this membrane type, changes in their relative abundance are unlikely to impart a significant effect on membrane fluidity. Yet both play 79 critical roles in coordination of signaling and membrane-associated processes such as apoptosis, cell proliferation, and blood coagulation (187, 188). Thus, it seems plausible that the changes in PS and PI with acclimation reflect changes in cellular processes, rather than a direct effort to modulate membrane fluidity.

Compromises in Brain Function May Limit Organismal Performance at Elevated

Temperatures

In summary, the data presented in this chapter demonstrate a homeoviscous response in cardiac membranes (microsomes, mitochondria), but not brain membranes

(synaptic membranes, myelin, mitochondria) in the Antarctic notothenioid fish N. coriiceps following acclimation to ambient conditions (0°C) and an elevated temperature

(5°C). Because preservation of membrane fluidity is critical to the maintenance of physiological function, these results suggest membrane restructuring may serve to preserve some aspect(s) of cardiac function at elevated temperatures. In cardiac plasma membranes, changes in membrane composition and fluidity are likely to impact calcium signaling (189), endo- and exocytosis (190), and the activities of membrane-bound transporters such as Na+/K+-ATPase (191). Further, the mitochondrial membrane is critical to synthesis of ATP in cardiac tissue and thus is likely to be important for maintenance of cardiac function at elevated temperatures (164). Consistent with these findings, work by our collaborators provides evidence of thermal plasticity in cardiovascular function of N. coriiceps following acclimation to 5°C (84).

Neither a homeoviscous response (i.e., maintenance of membrane fluidity) nor evidence of major membrane remodeling (i.e., cholesterol, major polar lipid classes, 80 membrane unsaturation) were detected in the three types of brain membranes considered.

These findings suggest a lack of thermal plasticity in the brain of this species, at least via modulation of membrane structure. Because fluidity did not differ between cardiac and brain mitochondria (i.e., comparable membrane types) between 0°C and 5°C, and because fluidity also did not differ significantly by tissue type (at a common acclimation temperature), it appears possible that disruption within the structure of biological membranes associated with the brain may serve as a limiting factor to performance within warmer environments in this species.

It is also possible that acclimation to 5°C was insufficient to prompt a homeoviscous response in the brain. Work in Cyprinus carpio demonstrates a capacity for modulation of blood flow to minimize changes in brain temperature, following fluctuations in environmental temperature (192). Consequently, membrane remodeling in the brain may be less critical than in other tissues during periods of (relatively) minor shifts in temperature. Additionally, the changes in PI and PS in brain mitochondria suggest that some degree of lipid modulation is possible within the brain in response to thermal variation.

It seems possible that, for notothenioids, a threshold exists beyond which lipid remodeling is necessary for the maintenance of membrane integrity. Consistent with this idea, Malekar et al. (2018) reported evidence of membrane remodeling following acclimation to 6°C, but not 4°C, in Trematomus bernacchii. Thus, it appears that this threshold is likely to vary among species, as well as among different membranes and tissue types. Taken together, these findings suggest variation in the homeoviscous 81 responses of different tissues of N. coriiceps. These differences in membrane remodeling with long-term warming may incur consequences for organismal performance at elevated temperatures.

CHAPTER 5: THERMAL ACCLIMATION ALTERS MEMBRANE FLUIDITY AND

OSMOTIC PERMEABILITY OF BRANCHIAL EPITHELIA IN NOTOTHENIA

CORIICEPS

Introduction

The Antarctic notothenioid fishes arguably represent one of the most striking cases of an organism’s potential vulnerability to the effects of global climate change. The extreme stenothermy under acute deviations in temperature of Antarctic notothenioids has been documented for more than 50 years (77). Less well-understood is their capacity to maintain physiological function at elevated temperatures with more gradual warming.

Homeoviscous adaptation (HVA), the preservation of membrane fluidity in response to thermal variation (110), will likely be critical to the survival of notothenioids in the future.

The physical state of the membrane is highly dependent on temperature (37). In the absence of any compensatory changes, an increase in temperature will render biological membranes more fluid, resulting in an increased vulnerability to excess passive movement of solutes across the membrane (155, 193, 194). Furthermore, the permeabilities of both oxygen and water are highly sensitive to membrane fluidity (155,

195). For ectothermic organisms, whose body temperatures match that of their environment, HVA serves to preserve membrane integrity upon shifts in environmental temperature (114).

The membranes of Antarctic notothenioid fishes contain high proportions of polyunsaturated fatty acids (PUFAs) within phospholipids, which serve to enhance lipid 83 movement at subzero temperatures (16). Researchers have previously shown that the proportion of saturated fatty acids in phospholipids increases with long-term warming

(e.g., weeks) in temperate species, serving to increase membrane rigidity (71, 130, 159,

196), a trend that has been observed in membranes prepared from liver of an Antarctic notothenioid, Trematomus bernacchii, as well as in crude gill and white muscle of this species (119, 167, 168). Yet other studies have demonstrated limited, or lack of membrane restructuring (i.e., phospholipid remodeling) in notothenioids (117–119). The capacity of notothenioids to adjust membrane compositions in response to thermal variation is not yet fully understood.

The gill constitutes a highly vascularized network of epithelial and connective tissues and serves as the gatekeeper between the environment and the organism (197). In teleost fishes, the gill is a structurally complex, multifunctional organ that is responsible for gas transfer, osmoregulation, excretion of nitrogenous waste, and pH balance. In teleosts, the gills are located near the pharynx and consist of several paired arches (197,

198). A set of bony gill rays radiate laterally from the base of each arch, forming a structural support for the gill filaments. The filaments consist of soft tissue and comprise the functional unit of the gill. The surface area of a filament is characterized by the presence of lamellae, which serve to facilitate gas exchange across the gill filament (197).

Pavement cells, the most abundant cell type in the gill (~90%), are responsible for the exchange of gases at the surface of the gill filament and contain apical projections, such as microvilli, which serve to increase the surface area of the membrane (198, 199). 84

Mitochondria-rich cells comprise a smaller proportion of the gill (~10%) but serve as the primary site for osmoregulatory processes (200).

Because the teleost gill exists at the interface between the organism and its environment, it is likely to be one of the organs most affected by a shifting dynamic of temperature and oxygen availability associated with climate change. Fishes must optimize gill function, as oxygen uptake incurs a significant cost in ion and water movement across the epithelium (201). For marine fishes, oxygen uptake imparts a high metabolic cost, due in part to the relatively low solubility of oxygen, as well as the relatively high density and viscosity of seawater (202). Secondary lamellae serve to enhance available surface area for gas exchange but incur a cost of increased osmoregulation (203).

For notothenioids, optimization of gill function during thermal change is likely to be critical to maintenance of various aspects of physiological function. Currently, for ectotherms residing in the Southern Ocean, oxygen demand is low and oxygen availability is high (204). However, this balance will likely shift with climate change, and thus optimization of oxygen uptake will be altered. Oxygen demands will likely increase as a consequence of temperature-dependent increases in metabolic rates, while oxygen availability will likely decrease as a result of the inverse relationship between oxygen solubility in seawater and temperature (60).

Many fishes can modify their gill structure, via the development of interlamellar cell masses that modulate the functional surface area of the gill, in response to fluctuations in oxygen availability (201). This suggests a capacity for plasticity in oxygen 85 uptake and gill function in teleost fishes. Although relatively little work has focused attention on structural remodeling in the gills of Antarctic notothenioid fishes, previous work in Trematomus bernacchii and T. newnesi demonstrated a decrease in serum osmolality following acclimation to 4°C (118), suggesting at least some degree of plasticity in gill function of notothenioids.

In this chapter, indicators of thermal compensation were measured in plasma membranes from gill epithelial tissue of the Antarctic notothenioid Notothenia coriiceps in response to acclimation to 0°C or 5°C. More specifically, I examined how acclimation to an elevated temperature affected membrane physical properties (e.g., fluidity and osmotic permeability) as well as biochemical membrane compositions. I hypothesized that the plasma membranes would exhibit a homeostatic response upon acclimation, becoming less fluid (and less permeable) in order to compensate for the fluidizing effects of elevated temperature. This response would allow for modulation of membrane permeability to solutes as well as the functional conservation of ion-regulating pumps, such as Na+/K+-potassium ATPase (NKA). The data reported in this chapter demonstrate that N. coriiceps is capable of membrane remodeling, as well as the maintenance of membrane fluidity and permeability, in gill tissue following long-term thermal change.

Materials and Methods

Animal Collection and Thermal Acclimation

Adult specimens of N. coriiceps were collected in the Western Antarctic

Peninsula region during the austral autumn of 2017 (Appendix A). Animals (Group A) were collected by trawl nets and baited pots and transferred to circulating seawater tanks 86 at Palmer Station, Antarctica. Additionally, a second set of animals (Group B) were caught using baited lines at Arthur Harbor. These samples were transferred immediately to 5-gallon buckets containing seawater and transported to circulating seawater tanks on station. Animals were allowed to recover for 3 days before initiation of the acclimation experiments.

5°C was reached. Animals were then held at their respective acclimation temperature

(0°C or 5°C) for a period of either 10 (Group A) or six (Group B) weeks.

Animals were euthanized by a single blunt blow to the head followed by severing the spinal cord. Whole gill arches were excised and flash frozen in liquid nitrogen for subsequent experiments. All animal experiments were approved by the Ohio University

Animal Care and Use Committee (14-L-004).

Membrane Preparations and Marker Enzyme Analyses

Plasma membranes were prepared from gill tissue as described previously (174), with modifications for preparations in previously flash-frozen tissue (Appendix B). Gill arches were selected randomly for preparation and were pooled as necessary. A maximum of two individuals were pooled per preparation.

Enrichments of membrane fractions were determined by measuring the protein- specific activities of marker enzymes: NKA for basolateral membranes, gamma- 87 glutamyltransferase (GGT) for apical membranes, and succinate dehydrogenase (SDH) for mitochondrial contamination (Appendix C). Enrichment was calculated as the enzymatic activity detected in membrane preparations, relative to crude homogenate.

Membrane Composition

Lipids were extracted as described (133) (Appendix E). Extracts were sent to the

Kansas Lipidomics Research Center for analysis, and a diacyl polar lipid profile dataset was generated by quadrupole mass spectrometry using an Applied Biosystems 4000

QTRAP mass spectrometer as described (134). Relative abundances of the major phospholipid classes were compared between species. The unsaturation index (UI) was calculated as described (135).

Membrane Fluidity Assays

Membrane fluidity was quantified by fluorescence depolarization as described

(132) (Appendix D). Change in polarization (excitation=356 nm, emission=430 nm) was measured between 0°C and 30°C using a Perkin-Elmer LS-50B spectrophotometer.

Temperatures were elevated at 2°C intervals at a rate of ~0.3°C min-1. Homeoviscous efficacy was calculated as described previously (129).

Osmotic Permeability Assays

A subset of freshly extracted whole gill arches were bathed immediately in notothenioid Ringer’s solution (240 mM NaCl, 2.5 mM MgCl2, 5 mM KCl, 2.5 mM 88

NaHCO3, 5 mM NaH2PO4, pH 8.0 at 4°C) for osmotic permeability assays, which were performed on the day of collection (Appendix G). Measurements were performed in the anterior arch of the right and left sides for all selected individuals. All samples were kept on ice during collection and tissue preparation.

Oxygen Partition Coefficient Measurements

Oxygen solubility was quantified in plasma membranes from gill epithelia as described previously (206), with modifications (Appendix H). Oxygen concentrations were measured using a dissolved oxygen probe, which was fitted to a transbridge and an analog-to-digital converter. Temperature was maintained at 4°C using a circulating water bath, which was connected directly to the sample chamber. Oxygen concentrations were recorded using WinDAQ data acquisition software.

Statistical Analyses

Osmotic permeability measurements were calculated for each gill arch as described above. Rates of linear osmotic gain were compared between treatment groups by an analysis of covariance (ANCOVA). Protein-specific enzymatic activities were calculated as described above and compared between groups by a two-tailed t-test.

Phospholipid compositions and cholesterol contents were compared between treatment groups by two-tailed t-tests. Analyses of polar lipids and unsaturation distributions were adjusted for the Bonferroni correction to account for multiple t-tests, with a minimum P- value of 0.00625. Membrane fluidity was compared between treatment groups by an

ANCOVA. Oxygen partition coefficients were calculated as described above and compared between groups by a two-tailed t-test. 89

Individuals from the two acclimation cohorts (Groups A and B) were found to be statistically equivalent in all reported analyses (P>0.50). For this reason, data from both groups were pooled to account for logistical issues during fieldwork that otherwise limited sample size.

The effect of gill arch position (anterior-to-posterior) was assessed for all relevant assays (fluidity, oxygen partitioning, biochemical composition, enzymatic assays) and was found to be insignificant.

Results

Membranes Displayed Enrichment and Altered Enzyme Activities upon Acclimation

Membrane preparations were enriched in enzymes associated with the plasma membrane, demonstrating 4.8- and 5.2-fold enrichment in NKA and GGT, respectively

(Table 5.1). SDH activity was found to be a relatively minor component of membrane fractions (Table 5.1). The enrichments of NKA, SDH and GGT were found to be comparable between thermal treatment groups.

Table 5.1. Enrichment Factors for Plasma Membrane Preparations for Gill Epithelial Tissues from Notothenia coriiceps. Enzyme Location Enrichment NKA Basolateral membrane 4.8 (0.4) GGT Apical membrane 5.2 (0.5) SDH Mitochondria 0.54 (0.04) Notes: Values in parentheses represent means ± s.e.m. (N=7).

Abbreviations: Na+/K+-potassium ATPase (NKA), gamma glutamyltransferase (GGT), succinate dehydrogenase (SDH)

In whole-tissue homogenates, NKA activity increased 1.4-fold (40%) with 5°C acclimation, compared with tissues from animals held under ambient conditions (P<0.01)

(Fig. 5.1). In contrast, GGT activity was reduced 1.5-fold (50%) in 5°C-acclimation group (P<0.05) (Fig. 5.1). Although data were normalized to protein, the trends were consistent when normalized to wet tissue weight.

Figure 5.1. Protein-specific activities of Na+/K+-ATPase (NKA) and gamma glutamyltransferase (GGT) from gill epithelia in 0°C and 5°C acclimation groups. Error bars represent means ± s.e.m. (N=7) One asterisk indicates P<0.05.

Polarization values were elevated in the 5°C group, relative to the 0°C group

(P<0.0001), indicating a lesser degree of membrane fluidity in the animals acclimated to the warmer temperature (Fig. 5.2). Homeoviscous efficacy (calculated as the ratio of polarization values for both treatment groups, at their respective physiological temperatures) was determined to be 100%. No significant discontinuities in membrane fluidity were observed, indicating the lack of a detectable phase transition over the temperature range measured. 91

Figure 5.2. Steady state polarization values (i.e., inverse of membrane fluidity) for the fluorescent probe DPH in plasma membranes from gill epithelia in 0°C and 5°C acclimation groups. Error bars represent means ± s.e.m. (N=10 for 0°C and N=9 for 5°C).

Cholesterol and Acyl Chain Length Increased with Acclimation

Cholesterol contents increased 1.2-fold (20%) (P<0.01) in membranes from the

5°C group, relative to membranes from the animals acclimated to 0°C (Fig. 5.3). Neither distribution of polar lipid classes, nor acyl chain unsaturation, were changed with temperature acclimation. Phosphatidylcholine (PC), phosphatidylethanolamine (PE) and plasmalogen PC (ePC) represented the most abundant classes in the gill plasma membranes (Table 5.2). PC, PE, and ePC accounted for approximately 54, 15, and 12% of phospholipids, respectively (Table 5.2). In contrast to the lack of change in acyl chain unsaturation or composition of polar lipid class, the relative abundance of long-chain fatty acids (i.e., fatty acids with >20 carbon molecules per chain) was increased by 1.1- fold (10%) (P<0.05) with 5°C acclimation (Fig. 5.4). 92

Figure 5.3. Cholesterol contents of plasma membranes from gill epithelia in 0°C and 5°C acclimation groups. Error bars represent means ± s.e.m. (N=8). Two asterisks indicate P<0.01.

Table 5.2. Polar Lipid Class Distribution and Unsaturation Index in Plasma Membranes from Gill Epithelia in 0°C and 5°C Acclimation Groups.

Polar lipid class 0°C 5°C PC 53.9 (1.5) 54.4 (1.2) PE 14.5 (1.4) 15.3 (0.75) ePC 12.1 (0.51) 12.3 (0.22) PS 6.38 (0.50) 7.38 (0.48) PI 5.17 (0.56) 5.42 (0.56) LPC 1.10 (0.19) 0.852 (0.075) ePS 0.622 (0.31) 0.45 (0.082) UI 276 (9.1) 286 (6.9) Notes: Polar lipid class values are expressed as mol%. Values in parentheses represent means ± s.e.m. (N=8).

Abbreviations: phosphatidylcholine (PC), phosphatidylethanolamine (PE), plasmalogen PC (ePC), phosphatidylserine (PS), phosphatidylinositol (PI), lysoPC (LPC), plasmalogen PS (ePS), unsaturation index (UI).

Figure 5.4. Relative abundance of fatty acids with >20 carbon atoms per chain in plasma membranes from gill epithelia in 0°C and 5°C acclimation groups. Error bars represent means ± s.e.m. (N=8). One asterisk indicates P<0.05.

Osmotic Permeability was Reduced with Acclimation, but Oxygen Solubility was

Unchanged.

Rates of osmotic gain were 1.5-fold (50%) lower in intact gills from the 5°C group, compared with gills from animals held under ambient temperatures (P<0.05), indicating greater permeability to water in the 0°C group (Fig. 5.5). The oxygen partition coefficient between membrane samples and water was approximately 3.5 and did not differ significantly between acclimation groups (Fig. 5.6).

* *

Figure 5.6. Oxygen partition coefficient (i.e., the ratio of oxygen solubility to membrane lipids to that of water) of plasma membranes from gill epithelia in 0°C and 5°C acclimation groups. (N=10 for 0°C and N=8 for 5°C). Error bars represent means ± s.e.m. 95

Discussion

In this chapter, I report alterations to properties of gill plasma membranes, including lipid composition, physical properties, and activities of enzymes, from the

Antarctic notothenioid N. coriiceps after a six or 10-week acclimation to an elevated temperature of 5°C. My results indicate that, when faced with a significant elevation in body temperature, this species possesses the capacity to remodel plasma membranes of the gill epithelium. Furthermore, my results indicate that several physiological properties of the gill are conserved upon acclimation to 5°C. Given the projections for warming, and the resulting loss of sea ice coverage (by 10-50%), in the Southern Ocean over the next century (207), Antarctic notothenioids will face challenges not only to whole-animal, but also organ-specific function. I suggest at least some of these challenges may be offset via remodeling within the plasma membranes of the gill.

Lipid Remodeling Is Consistent with a Homeoviscous Response

For a given assay temperature, gill membranes from the 5°C-acclimated fish were less fluid than those of the 0°C group, suggesting thermal plasticity in this key membrane property. Homeoviscous efficacy was 100%, indicating complete conservation of membrane fluidity at each group’s respective acclimation temperature.

The specific patterns of lipid remodeling observed in plasma membranes of the gill epithelium are consistent with the observed homeoviscous response in N. coriiceps.

For example, changes in cholesterol contents and alterations to fatty acyl chain length in polar lipids are compatible with the modulation of membrane fluidity. Cholesterol stabilizes the membrane by restraining movement of fatty acyl tails, thus reducing 96 membrane fluidity (100). Increases in cholesterol with acclimation to elevated temperatures have been reported previously in temperate ectotherms (174–176), although the trend is not universal (132, 174).

Long-chain fatty acids (in this study, defined as >20 carbons) can serve to decrease membrane fluidity by enhancing acyl chain packing and, consequently, decreasing membrane permeability (177). Further, alterations in acyl chain length (178) have been reported in membranes from temperate fishes following thermal acclimation, consistent with the data reported in this chapter. Thus, a cholesterol-enriched membrane, combined with longer fatty acyl chains, is likely to exhibit reduced fluidity, consistent with the homeoviscous response observed in the gills of animals acclimated to the elevated (5°C) temperature.

Alterations in the extent of unsaturation of membrane polar lipids represent a common mechanism to preserve membrane fluidity (71). However, in this work, unsaturation was unchanged with acclimation in the gill plasma membranes of N. coriiceps. Similarly, a lack of change in unsaturation was reported in cardiac mitochondria in this species with acclimation, despite the observed homeoviscous response in this membrane type (Chapter 4). Although the methodology employed may account for the lack of an observable change in membrane unsaturation (Chapter 4), it also appears plausible that other factors may help account for the homeoviscous response observed in gill plasma membranes of N. coriiceps.

No changes in the relative abundances of PC or PE with acclimation were observed in this study, consistent with findings in brain and cardiac mitochondria from N. 97 coriiceps (Chapter 4). The PE/PC ratio is often elevated upon warm acclimation in temperate organisms (42, 208). This adjustment is likely related to the destabilizing nature of PE, as the PE head group contributes to the curvature necessary for the formation of non-lamellar vesicles, which are required for membrane fusion and fission

(97). In my study, a 5°C increase in environmental temperature may have been insufficient to stimulate excessive formation of non-lamellar vesicles, perhaps rendering restructuring of polar lipid classes unnecessary during the course of this acclimation treatment.

I posit that the observed changes in membrane lipids, particularly cholesterol, are ideal for membrane restructuring. Membrane cholesterol is critical to the conservation of various factors related to membrane function, including permeability and stability of membrane-associated proteins, as well as preserving membrane fluidity; further, cholesterol can increase membrane resistance to acute thermal fluctuations (209). In this membrane type and species, alterations in cholesterol content appear to serve as an important mechanism in the response to thermal change.

Reduced Membrane Fluidity Accounts for Decreased Osmotic Permeability

The decrease in membrane fluidity in the 5°C group (relative to the 0°C group) was matched by a reduction in osmotic permeability. The alteration in osmotic permeability is likely attributable, at least in part, to the change in cholesterol content, as cholesterol is well-known to reduce membrane permeability (210, 211). In a numerical simulation, acyl chain length was also shown to alter membrane permeability, although the effects were relatively small compared to those brought about by cholesterol (212). 98

Alternatively, changes to the distributions of other membrane components, such as aquaporins, may serve to modulate osmotic permeability (213). Aquaporin-1 levels were enhanced in gill tissue of the giant tiger shrimp Penaeus monodon upon warm acclimation (214). Aquaporins serve to increase membrane permeability to water and oxygen without altering membrane fluidity (213, 215). However, because osmotic permeability was reduced (at a common assay temperature) in warm-acclimated N. coriiceps, consistent with the decrease in membrane fluidity, it appears unlikely that aquaporin levels were increased with warm acclimation in gills of this species.

Similarly, it is also possible that alterations of claudins may account for modulation of osmotic permeability with acclimation. Claudins are transmembrane proteins that provide the basic structural support for tight junctions, which form functional barriers between cells and are important for cell coordination and function

(216). In teleosts, tight junctions are particularly important for control of permeability to ions and other compounds of low molecular weight (217). The expression of claudins in gills of fishes is highly sensitive to environmental parameters, particularly salinity (218).

Further, in hybrid catfish, several genes encoding claudins were upregulated following heat stress in gill tissue (219). Because they are both likely to influence membrane permeability, the expression of aquaporins and claudins in gill epithelia following thermal acclimation in this species warrants study in future work.

While the degree of water permeability was reduced in the 5°C group, the partition coefficient of oxygen, a measure of solubility to oxygen (in this case, measured at a common temperature of 5°C), did not differ among treatment groups. This suggests 99 that, in this species, oxygen diffusion is not likely to be impeded by altered membrane composition. Although oxygen diffusion across the membrane is highly sensitive to membrane fluidity and lipid composition, particularly cholesterol (195, 220), oxygen flux across the gill is largely perfusion-limited due to the high solubility of oxygen in lipids, relative to water (221).

Changes in Enzymatic Activity May Reflect Altered Membrane Fluidity

Consistent with the present data, elevated NKA activity in gill has been reported following warm acclimation in the Antarctic notothenioids T. bernacchii and T. newnesi and was accompanied by a reduction in serum osmolality (118, 222). Similarly, NKA activity was positively correlated with acclimation temperature in the gills of a non-

Antarctic notothenioid, Eleginops maclovinus (223).

In this study, alterations in NKA activity may reflect changes in the lipid environment with thermal acclimation. Although NKA activity tends to correlate positively with fluidity, NKA activity is enhanced by several membrane lipids including cholesterol, which stabilizes the enzyme (100, 123). Alternatively, alterations in NKA activity following acclimation may reflect differences in gene expression (224) or post- translational modifications, such as phosphorylation (225).

In this study, GGT activity was reduced upon warm acclimation. GGT, an important regulatory component of the gamma-glutamyl cycle, is involved in the degradation of glutathione (226). Elevated levels of glutathione with warm acclimation have been reported previously in tissues of N. coriiceps and N. rossii (227), as well as the

Atlantic killifish, Fundulus heteroclitus (228). It is worth noting that levels of glutathione 100 were unchanged in liver tissue of an Antarctic notothenioid, Pagothenia borchgrevinki, following acclimation to 4°C (229). The reduced GGT activity may reflect alterations in membrane properties, as GGT activity is enhanced by bilayer movement (230). GGT is sensitive to the lipid microenvironment, particularly by its interactions with PC and PE

(231). However, the direct effects of cholesterol and fluidity on GGT activity have not been assessed previously.

I posit that alterations in GGT activity may reflect alterations in the gamma- glutamyl cycle with warm acclimation, possibly suggesting a physiological response to oxidative stress (232). Membrane lipids are particularly susceptible to peroxidation at elevated temperatures (229, 233), and the high proportion of PUFAs in the membranes of

Antarctic fishes may render these species more vulnerable to oxidative damage (148), as

PUFAs are primary targets of lipid peroxidation (234, 235). Because GGT is thought to play a protective role in response to oxidative stress, it is possible that alterations in GGT reflect potential oxidative damage associated with elevated temperatures (232).

Additional research might focus on the question of whether antioxidant capacities are elevated with warm acclimation in Antarctic notothenioids.

Conclusions

In this chapter, I report the capacity for membrane remodeling and modulation of membrane fluidity and permeability in gill epithelia of an Antarctic notothenioid in response to warming. This chapter (and Chapter 4) represent the first reports providing direct evidence of HVA with thermal acclimation in an Antarctic notothenioid. These data suggest thermal plasticity in membrane structure and composition, indicating that, 101 despite millions of years of adaptation to an extreme cold and stable thermal environment of the Southern Ocean, N. coriiceps possesses the capacity for conservation of gill properties and functional attributes in response to elevated temperatures. As the environment continues to warm, all teleost fishes, including Antarctic notothenioids, will require maintenance of the physical properties of the gill plasma membranes. These constituents will be especially critical to conserving gill function in the future.

102

CHAPTER 6: DISSEMINATION OF NOTOTHENIOID RESEARCH TO THE

PUBLIC SPHERE THROUGH WIKIPEDIA

Introduction

Public engagement is a critical, yet often overlooked aspect of the scientific research process. Research suggests that traditional science, technology, engineering, and mathematics (i.e., STEM) curriculum in the United States often lacks engagement and integration, potentially causing students to lose interest in science at a young age (236–

238). Perhaps in part as a result, numerous misconceptions exist regarding science within

American culture. Recent reports indicate that American adults remain markedly polarized on scientific issues including climate change (239), evolutionary theory (240), genetic modification technology in the food industry (241), and public vaccination (242).

While most Americans are unlikely to visit the Antarctic, their opinions on climate policy and biology are likely to influence the fate of the Southern Ocean.

Antarctic marine fauna are currently at high risk of extinction due to the unprecedented warming in polar regions (14, 41, 78). While a warming climate may be inevitable, some research suggests that changes in climate policy may help mitigate the global effects of climate change (243). Further, some species of Antarctic fishes are currently vulnerable to overfishing due to lack of fishing regulation in the Southern Ocean (5). Consequently, science policy measures will be critical to the preservation of the Southern Ocean.

Research suggests that most Americans learn about science primarily through popular mass media (244). Thus, creators and journalists have the responsibility to produce accurate and engaging scientific content. Yet creators often fall short of this 103 objective. Research in this field has demonstrated that media coverage often lacks sufficient depth and accuracy in scientific topics (245, 246). Inaccurate science reporting often furthers misconceptions about science, causing the public to lose interest (246).

Many Antarctic scientists work to dispel myths about their research (247). However, a recent search of media coverage related to Antarctic fishes revealed several inaccuracies; the most common issue being misuse of the term icefish (248–255), as well as other misleading descriptions of taxonomic organization (248, 256) and cold adaptation (252,

253, 257) in notothenioid fishes.

Wikipedia is one of the most universal sources of information, both for the general public as well as journalists and other content creators. Wikipedia is arguably the largest and most commonly used database of open information in the world, with a current total of more than 40 million articles (258) and an average of 16 billion monthly views (259). Wikipedia maintains a high Google PageRank status (260) and thus often serves as the public’s first introduction to an unfamiliar topic. A large majority of students self-report utilizing Wikipedia as an introduction to academic topics (261, 262).

Wikipedia is generally regarded as unreliable, because any user can edit a Wikipedia article (262). However, since its creation in 2001, Wikipedia has employed critical efforts to increase the reliability of its content. One of these efforts involves the creation of

WikiProjects (263).

WikiProjects serve as collaborations among designated Wikipedia editors who have committed to improving the body of articles within a specific discipline. The roles of the editors include “develop(ing) criteria, maintain(ing) various collaborative 104 processes and keep(ing) track of work that needs to be done” (263). WikiProjects involve the assessment of relevant content through a self-created “peer review” system and place priority on articles that are the most likely to reach a broad readership (263).

WikiProject Fishes represents an effort to create, categorize, and improve content relating to various fish taxa (264). The existing Wikipedia articles related to notothenioid fishes are encompassed by WikiProject Fishes. These articles are organized taxonomically; all families and genera are designated by their own page. The majority

(~85%) of notothenioid species do not have their own Wikipedia page, although confirmed species are designated as such on corresponding genus pages.

Any internet user can edit a Wikipedia page. The edit history of any Wikipedia page is publicly viewable, and users can revert edits at any time as needed. Wikipedia users are required to abide by the five pillars of Wikipedia: (1) adherence to encyclopedic format; (2) maintenance of neutrality; (3) use and creation of free, publicly-owned content; (4) respect and civility; and (5) adherence to flexible rules that are designed to evolve as needed over time (265). The process of Wikipedia editing is intended to function as an open conversation, where users create and discuss content, making changes and corrections as needed.

In this chapter, I describe my efforts to improve the quality of Wikipedia articles relevant to my field of research. The objectives of this work are as follows: (1) to identify specific content issues within Wikipedia notothenioid articles; (2) to resolve those issues as a Wikipedia user; and (3) to submit the revised articles for quality reassessment to

WikiProject Fishes. 105

Materials and Methods

The work described in this section was performed on Wikipedia pages related to notothenioid fishes. All Wikipedia edits were carried out between the fall of 2015 and the spring of 2019. Wikipedia training was completed in fall 2015 as a part of a Wiki Edu assignment for a graduate college course (PBIO 5150: Writing in the Life Sciences,

Department of Environmental and Plant Biology, Ohio University) (266). All work was carried out on my personal Wikipedia account (username: Ambiederman). All edits were accompanied by a brief summary of work performed, which is publicly viewable in the

“edit history” section of each article. In addition, general comments were made on the article’s talk page as appropriate. Upon completion of substantial edits, selected articles were resubmitted to WikiProject: Fishes for reevaluation. Articles were selected based on the following criteria: (1) importance; (2) quality; and (3) relevance.

Importance

Articles included in any WikiProject are ranked on an importance scale (267). In this context, “importance” is not a general evaluation of a topic’s importance to society or to the scientific community, but rather an assessment of an article’s potential to reach a large audience. Importance is rated on a four-component scale: Top, High, Mid, and Low.

Articles that have not yet been evaluated are classified as None. Within the Wikipedia notothenioid literature, 10 percent of articles were categorized as Mid Importance, 28 percent were categorized as Low Importance, and 62 percent were uncategorized (264)

(Fig. 5.1). For this project, preference was given to articles classified as Mid Importance. 106

Quality

WikiProject articles are also rated on a quality scale. Articles are reviewed by a team of designated editors, who read the articles and post their assessments on the article’s talk page. Quality is assessed on a six-component scale: Featured, Class A, Class

B, Class C, Start, and Stub. Articles that have been revised substantially may be submitted for reassessment. The majority (83%) of Wikipedia notothenioid literature were designated as Stub Class by WikiProject Fishes. Approximately 13 percent were designated as Start Class, and 4.3 percent were designated as Class C (264) (Fig. 6.1).

Many of the low-quality ratings have likely been assigned due to the limited information available on some species and genera of Antarctic fishes, which may be insufficient to constitute full articles. For this reason, I elected to place greater weight on the importance and relevance parameters when selecting articles for substantial revision.

Figure 6.1. Relative distribution of importance and quality rankings within Wikipedia notothenioid literature.

107

Relevance

In this chapter, “relevance” refers to the relevance of a Wikipedia article to my dissertation. Preference was granted to pages that featured organisms collected and analyzed for my dissertation research, as presented in Chapters 2-5. Ten relevant articles were identified: Antarctic fishes (general page), Notothenioidei (suborder), Nototheniidae

(family), Channichthyidae (family), Notothenia (genus), Chaenocephalus (genus),

Pseudochaenichthys (genus); Notothenia coriiceps (species); Chaenocephalus aceratus

(species); and Pseudochaenichthys georgianus (species) (Table 6.1).

Table 6.1. Summary of Articles Chosen for Modification.

Article title Classification Importance Quality Relevance Notothenioidei Sub-order Mid Class C Direct Channichthyidae Family Mid Start Direct Dissostichus Genus Mid Stub Indirect Notothenia coriiceps Species N/A N/A Direct Chaenocephalus Species Uncategorized Stub Direct aceratus

Article Selection

Notothenioidei

This page describes characteristics of the suborder Notothenioidei, the dominant fish taxa in the shelf waters surrounding the Antarctic continent (268). This page was rated as Mid on the importance scale, and as Class C on the quality scale (269). The page has averaged 1,848 views/month since 2015 (270). This page was chosen because, 108 although it is one of the higher-ranked pages in quality for this topic, it is also one of the most influential.

Channichthyidae

This page describes the family Channichthyidae, which encompasses the hemoglobinless Antarctic icefishes (271). Antarctic icefishes possess a novel and interesting mutation in response to an extreme environment, and they are often featured in popular media coverage of Antarctic marine fauna. This page was rated as Mid on the importance scale and as Start on the quality scale (272). The page has averaged 2,259 views/month since 2015 (270). This page was selected due to public interest in the topic, as well as its relevance to my dissertation; two of the species utilized in Chapters 1 and 2

(Chaenocephalus aceratus and Pseudochaenichthys georgianus) are icefishes. Revisions of this article were carried out as a class assignment for PBIO 5150 (266).

Dissostichus

This page describes the genus Dissostichus, which comprises two species of notothenioids commonly known as the toothfishes (273). These species have been studied extensively by biologists. Further, toothfishes been targeted by commercial fisheries and are commonly sold in the United States as Chilean sea bass. Research suggests that overfishing of both species will likely damage the entire ecological structure of the

Southern Ocean (5). This article was ranked as Mid on the importance scale, and as a

Stub on the quality scale (274). The page has averaged 1,162 views/month since 2015, although monthly views have increased nearly five-fold since October 2017 (499 views/month versus 2,356 views/month) (270). Although toothfishes do not relate 109 directly to my work, this page was chosen due to the direct importance of this information to consumers in the United States.

Notothenia coriiceps

This page was selected because it relates directly to my field of study; investigations of N. coriiceps physiology are central to Chapters 2-5. I have worked with this species throughout my graduate career. This page did not previously exist on

Wikipedia. N. coriiceps is one of the more well-studied notothenioids, and it was the first notothenioid to have a fully sequenced genome (275). Because a large body of literature exists on this species, a designated page for N. coriiceps would add value to WikiProject

Fishes.

Chaenocephalus aceratus

This page describes Chaenocephalus aceratus, a species of icefish that is central to Chapters 2 and 3 (276). This page was uncategorized on the importance scale and was categorized as a Stub on the quality scale (277). The page has averaged 205 views/month since 2015, although monthly views have increased more than two-fold since February

2018 (140 views/month to 385 views/month) (270). Although this page garners fewer views than the other articles chosen, it was selected due to the large body of research on this species, as well its relevance to my work.

Results and Discussion

Overview

An estimated 64,000 characters of original content (roughly equivalent to 40 pages of double-spaced, 12-point text). were added to the existing body of articles related 110 to notothenioids in Wikipedia during this project (278). A total of 275 edits were made and 92 pages were edited (278). Minor edits included addition of scholarly references, inclusion of images through Wikimedia Commons, addition and correction of links to existing content, corrections of grammar and spelling, and minor corrections regarding content.

Article Revisions: Notothenioidei

Link to modified version: https://en.wikipedia.org/w/index.php?title=Notothenioidei&oldid=880328424

This revision involved the addition and reorganization of content, as well as quality improvements as needed (268). Approximately 700 words of original content were added. Fifteen scholarly references were added, as only one source was cited previously. Prior to revision, the article contained only one subsection, “Anatomy.” In this project, two new subsections, titled “Evolution and geographic distribution” and

“Physiology,” were added. In the “Evolution and geographic distribution” subsection, content was added describing the evolution of notothenioid species under the unique conditions created by the cooling and isolation of the Southern Ocean. In the

“Physiology” subsection, content was added to describe the general characteristics of notothenioids and to explain how these traits directly reflect these species’ environments.

Portions of this content had been previously included in the “Anatomy” subsection. In addition, minor corrections were performed in the list of notothenioid families and genera. This list was created based on a well-known paper describing notothenioid taxa

(10), but minor changes had been made previously to reflect newly classified species. 111

References and clarifications were included as needed in this subsection. While still not complete (as some of these species classifications remain under debate among taxonomists), the information now more accurately reflects the scientific community’s current consensus on notothenioid taxonomy. Further, an appropriate featured image was added that represented multiple notothenioid families; the previous image had featured only one species of icefish. The new image was obtained through Wikimedia Commons.

Article Revisions: Channichthyidae

Link to modified version: https://en.wikipedia.org/w/index.php?title=Channichthyidae&oldid=880884399

Initial edits of this article involved restructuring of subsections and revision of existing information to improve its scholarly quality (271). A subsection titled “Diet and body size,” which described the species’ maximum body sizes as well as their predatory strategy, was added. In addition, content was added to clarify the conditions under which hemoglobin and myoglobin were lost in the icefishes. Three references were added. The current image was replaced with an original photo, as I contended that the previously used photo, despite being commonly used in media coverage of Antarctic fishes, did not represent the icefishes well. Over the next two years (2016-2018), the article was edited substantially by several Wikipedia users, and the content was further improved, with an additional 1,000 words and six scholarly references. In 2019, I made additional clarifications to some of the new content. These recent changes encompassed aspects of geographic distribution and cardiovascular function. Further, substantial changes by me 112 were made to the evolution section, both in content and organization. Further, the introductory paragraph was restructured and a new featured image was added.

Article Revisions: Dissostichus

Link to modified version: https://en.wikipedia.org/w/index.php?title=Dissostichus&oldid=880911372

This article initially contained minimal content; it included a subsection listing the two species of toothfish and a short paragraph describing the genus’ importance to the commercial fishing industry (273). Approximately 2200 words of content, 38 references, and 57 links were added. Eight new subsections were added as follows: “Distribution,”

“Morphology and body size,” “History,” “Commercial fisheries,” “Illegal, unreported and unregulated fishing,” “Diet and ecological importance,” “Migration and reproductive cycle,” and “Conservation efforts.” Species were discussed separately and compared as appropriate. The “Marketing” subsection, which had been included in previous versions, was deleted. Content from this section was moved to the introductory paragraph.

Article Creation: Notothenia coriiceps

Link to modified version: https://en.wikipedia.org/w/index.php?title=Notothenia_coriiceps&oldid=880863135

Approximately 880 words of original content were added in the creation of this article (279). Fifteen scholarly references were added. A representative image of N. coriiceps was also added from Wikimedia Commons. The revised article was divided into four subsections: “Distribution and diet,” “Morphology,” “Physiology,” and 113

“Genome.” An introductory paragraph was also added, which included an overview of the species’ general characteristics.

In the “Distribution and diet” subsection, the species’ known geographic distribution, as well as physiological and physical explanations for its wide distribution around the continent, were described. Information on the species’ food sources was also included. In the “Morphology” subsection, literature was cited describing the outward appearance and internal structure of the species, with an emphasis on ecological and physiological relevance. In the “Physiology” subsection, work was cited demonstrating this species’ physiological adaptations to an extreme cold environment.

In addition, a section was dedicated to a report of the N. coriiceps draft genome.

While the paper cited focused primarily on discussion of genes related to physiological function, I resolved that this study merited its own section due to its scientific relevance, as the authors reported the first notothenioid genome sequence. Further, this section, while relatively small, has the potential to grow as more literature is contributed to this field. The article was ranked as Start Class and Low Importance (280), although additional content was added after this evaluation. Since its initial creation in January

2019, the article has garnered an average of 85 views/month (270).

Article Revisions: Chaenocephalus aceratus

Link to modified version: https://en.wikipedia.org/w/index.php?title=Chaenocephalus_aceratus&oldid=880888375

This article initially contained a large amount of text but had major issues in content, grammar and referencing (276). Approximately 300 words of original content 114 were added to this article, but more substantial changes included modification of existing content. Approximately 45 links to existing content were added in order to increase accessibility for general readers. Seven scholarly references were added.

Minor content changes were made in the “Morphology” and “Habitat” subsections. Several changes were made to the “Evolution” subsection in order to maintain consistency with other Wikipedia articles that address the topic. In addition, major revisions were made to the “Adaptation” section, as the previous version did not differentiate sufficiently between temperature- and hemoglobin loss-specific adaptations in the species. Previously, the weakest aspect of this article was the writing quality.

While the content was substantial and content issues were relatively minor, the article suffered from spelling, grammatical and formatting errors. In addition, consistency issues existed throughout the article; references to the species often switched between

“Chaenocephalus aceratus” and “blackfin icefish,” and the species name was occasionally not italicized. These issues are now resolved.

Mass Revisions: Icefish versus Cod Icefish

Link to modified version: https://en.wikipedia.org/w/index.php?title=Icefish&oldid=880037116

In addition to substantial edits of chosen articles, minor edits were made as needed across the Wikipedia notothenioid literature. One of the most widespread changes was clarification of the term “icefish. Previously, nearly all articles related to the family

Nototheniidae referred to its members as “cod icefishes.” Although this term is used in a few select peer-reviewed articles, it is not used commonly within the discipline. The term 115 is potentially confusing, because members of Nototheniidae produce hemoglobin and thus not true icefishes. This discrepancy has, arguably, contributed to misreporting by the media. During this project, all references to cod icefishes were removed from articles featuring lower taxa within Nototheniidae; the term “notothen” was written as a replacement. The term “cod icefish” was retained in the main Nototheniidae article, but a note was added to the introduction explaining the distinction between the two terms.

In addition, the disambiguation page for icefish was modified (281). Previously, the page incorrectly stated that “Icefish may mean … Notothenioidei, a suborder of mostly bottom-dwelling fish of the Southern Ocean.” In this project, the page was revised to differentiate between “Icefish (Antarctic)” and “Cod icefish,” with short descriptions explaining the differences between the terms as well as a note that the term “cod icefish” is not used commonly in scientific literature. Lastly, the Wiktionary entry for icefish had incorrectly identified icefishes as “Any of many perciform fish, of the order

Notothenioidei, that live in the cold, continental-shelf waters of the Antarctic.” (281).

This entry was similarly corrected.

Summary

In summary, this report has demonstrated how Wikipedia can be used as a tool to allow for public accessibility to scientific information. By improving the quality, reliability and quantity of science-related public content, readers are granted access to summaries of scientific literature on various topics. Media coverage of notothenioid science has often contained misinformation on these species, due in part to lack of accurate information in open-source websites. The revised articles have been submitted to 116

WikiProject: Fishes and remain under review (as of May 2019). By improving the quality of Wikipedia articles related to my dissertation research, I have worked to disseminate my knowledge from my graduate work into the public sphere. I am hopeful that these efforts, both by myself and by other Wikipedia users, will help to bridge the gap between scientists and the public and, perhaps, encourage a new generation of scientists to pursue research in the Antarctic.

117

CHAPTER 7: PERSPECTIVES

The work described herein adds to the growing literature, by our group and others, aimed at elucidating the physiological mechanisms that govern the thermal tolerances of the Antarctic notothenioid fishes. With the waters surrounding the Western

Antarctic Peninsula continuing to warm rapidly (78), the capacities of notothenioids to ensure membrane constancy will be key to their survival.

Interspecies Comparisons Reveal Differences in Potential Vulnerability of Membranes to

Acute Warming

Two experimental approaches were employed to answer the question of how membrane fluidity and composition relate to physiological function in notothenioids. In the first approach (Section 1), I performed a comparative analysis between notothenioids known to differ in their thermal tolerance.

My findings suggest that different types of biological membranes vary in their vulnerability to (i.e., hyper-) fluidization with environmental warming. Synaptic membranes and cardiac mitochondria displayed significant differences in fluidity between species; for both membrane types, membranes from the more thermotolerant

Notothenia coriiceps displayed lower fluidity, compared to those of Chaenocephalus aceratus. Because compromises to membrane integrity are likely to impede organismal performance, it is likely that these membranes will reach some critical point beyond which nervous and/or cardiovascular functions are compromised. Indeed, previous work suggests signs of impairment to physiological function that are consistent with the present findings (121, 145). Taken together, these data suggest that variation in select membrane 118 properties is likely to contribute to loss of function in both the nervous and cardiovascular systems that have been observed previously (Kristin M. O’Brien et al., 2018, Crockett and O'Brien, unpublished observations) at elevated temperatures in Antarctic notothenioids.

Investigation of Thermal Acclimation Reveals Potential Weak Link to Physiological

Function During Long-Term Warming

In the second approach (Section 2), the capacity for homeoviscous adaptation

(HVA) was assessed in several biological membranes (cardiac, brain, branchial) of N. coriiceps following acclimation to 0°C or 5°C. My findings from this section suggest the presence of a homeoviscous response in the gill (plasma membranes) and cardiac

(microsomes, mitochondria) membranes, but not the brain (synaptic membranes, myelin, mitochondria) membranes. These results suggest membrane remodeling may serve to preserve some aspect(s) of cardiac and branchial function at elevated temperatures.

Neither a homeoviscous response (i.e., maintenance of membrane fluidity) nor evidence of major membrane remodeling (i.e., cholesterol, major polar lipid classes, membrane unsaturation index) were detected in the brain membranes considered. These results suggest a possible lack of thermal plasticity in the brain of this species, at least in modulation of membrane structure.

How Do Antarctic Notothenioids Modulate Membrane Fluidity?

In this work, I also sought to characterize alterations in lipid compositions that help to account for variation in membrane fluidity. In section 1, variation in cardiac mitochondrial fluidity appeared related to differences in the ratios of major polar lipids. 119

Other work by our group in cardiac microsomes suggests interspecies variation in membrane fluidity may be related to membrane unsaturation (Evans et al., unpublished observations).

Alterations of membrane cholesterol and fatty acyl chain length were observed in several membranes exhibiting a homeoviscous response. Both changes were consistent with the observed alterations in membrane fluidity following acclimation, and neither change (as applicable) was reported in the brain membranes. Consequently, cholesterol and chain length may play a role in responses of notothenioids to environmental warming.

Interestingly, neither a change membrane unsaturation, not in the distribution of major polar lipids, were detected in this study. Thus, it appears other mechanisms of remodeling serve to preserve membrane fluidity in this species. Alternatively, because

Malekar et al. (2018) reported evidence of remodeling (i.e., an increase in saturates and a decrease in monounsaturates) in an Antarctic notothenioid following acclimation to 6°C but not to 4°C, is seems possible that, for notothenioids, a thermal threshold exists beyond which lipid remodeling is necessary for the maintenance of membrane integrity.

This threshold may vary among species, as well as among different membranes and tissue types.

Future Directions

1. A future direction of this work would be to examine membrane properties of other

notothenioids in addition to N. coriiceps and C. aceratus, as previous studies (by our

group and others) demonstrate variation in thermal physiology and thermal tolerance 120

among species. Species diversity was limited by tank space at the field station;

however, this approach would allow me to better distinguish between phylogenetic

and physiological effects and to consider the suborder more broadly.

2. Polar lipid analyses were performed in only select membranes due to lack of available

tissue. In the future, it would be beneficial to perform compositional analyses on

membranes (e.g., synaptic membranes and cardiac microsomes) that could not be

measured for this work. For example, synaptic membrane fluidity differed between

species, but because cholesterol contents did not differ, I was unable to fully explain

the compositional basis for the variation in fluidity. Similarly, I was unable to

characterize the polar lipid composition of cardiac microsomes from the acclimation

experiment due to lack of available material. Although cholesterol changes were

consistent with the observed modulation of fluidity in this membrane, it would be

valuable to examine the composition of this membrane more fully.

3. Investigations of other aspects of membrane structure and organization would serve

to better elucidate the relationships between composition, fluidity, and physiological

function. This might include characterization of membrane microdomains, membrane

leakage, the abundance of individual fatty acids (rather than diacyl polar lipids), lipid

peroxidation, and the expression of proteins known to influence membrane function,

such as aquaporins and/or claudins.

4. To test the hypothesis that some aspects of membrane modulation in notothenioids

are bound by a thermal threshold, it would be beneficial to conduct acclimation

experiments to multiple temperatures (0°C, 4°C, 5°C, and 6°C) in N. coriiceps to 121

determine whether a 1°C or 2°C difference in temperature is likely to impact

homeoviscous responses of various tissues and membrane types. Similarly, it would

be of value to perform an experiment examining changes to the membrane at various

time points during the acclimation process, rather than a single endpoint.

5. Because previous work indicates that oxygen sensing may serve as a trigger for

remodeling (282), it would be of value to design a hypoxia acclimation experiment to

help piece apart the effects of temperature and oxygen content on membrane

composition and fluidity in notothenioids, as their warming environment may incur

costs of oxygen limitation in the future.

122

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APPENDIX A: ANIMAL COLLECTION AND MAINTENANCE

Adult specimens of Chaenocephalus aceratus (1000-2800 g),

Pseudochaenichthys georgianus (1000-2600 g), and Notothenia coriiceps (500-2300 g) were collected in the Western Antarctic Peninsula region during the austral autumn of

2015 (all three species) and 2017 (N. coriiceps only), using otter trawls deployed from the ARSV Laurence M. Gould in Dallmann Bay (64°10’S, 62°35’W) and off the southwestern shore of Low Island (63°24’S, 62°10’W). Additionally, N. coriiceps were captured at these sites using baited pots. Animals were held in circulating seawater tanks on the vessel before being transferred to circulating seawater tanks at Palmer Station,

Antarctica, where they were maintained at ambient temperatures (0+1°C) for a maximum of three weeks (for N. coriiceps) or 1.5 weeks (the icefishes) before tissue collection. N. coriiceps were fed to satiation with ~10 g fish muscle every other day. The icefish, which maintain a lower metabolic rate than other notothenioids (283), did not feed in captivity during this study. Previous studies have shown that notothenioids have a high specific dynamic action (i.e., dietary induced thermogenesis) and likely feed at relatively broad, seasonally-dependent time intervals (284, 285). Consistent with this, otolith and scale analyses suggest that notothenioids likely grow, and thus feed, sporadically (286).

2.5 mM NaHCO3, 5 mM NaH2PO4, pH 8.0 at 4°C). Next, brain tissue was excised and placed immediately in ice-cold extraction buffer (0.35 M sucrose, 5 mM EGTA, 10 mM 157

HEPES, pH 7.8 at 1°C). All remaining tissues were collected after brain excision. Gills were flash frozen immediately in liquid nitrogen for subsequent experiments. A subset of fresh gills were selected randomly and were bathed immediately in notothenioid Ringer’s solution for osmotic uptake assays, which were performed on the day of collection. All samples were kept on ice during collection and tissue preparation. Animal work for both field seasons was approved by Ohio University’s Institutional Animal Care and Use

Committee (14-L-004).

158

APPENDIX B: TISSUE COLLECTION AND MEMBRANE PREPARATION

TECHNIQUES

Cardiac Mitochondria and Microsomes

Mitochondrial membranes were prepared from cardiac ventricles as described

(287), with modifications (148). Up to three (for N. coriiceps) or two (for C. aceratus and

P. georgianus) hearts were pooled per preparation. In brief, ventricles were excised as described previously (Appendix A) and sliced on an ice-cold metal block. The tissue was homogenized in 8 volumes of isolation buffer (0.1 M sucrose, 140 mM KCl, 10 mM

EDTA, 10 mM MgCl2, 20 mM HEPES, pH 7.3 at 4°C) using an ice-cold 40 ml

Tenbroeck ground glass homogenizer.

The homogenate was centrifuged at 1400 g for five minutes at 4°C in a Beckman-

Avanti-JE with a JA-17 rotor (spin 1). The supernatant was collected and centrifuged at

9000 g for 10 minutes at 4°C (spin 2). The pellet was resuspended in 11 ml isolation medium and centrifuged at 1400 g for five minutes at 4°C (spin 3). Finally, the supernatant was centrifuged at 11,000 g for 10 minutes at 4°C (spin 4). The mitochondrial pellet was resuspended gently in ~0.5 ml resuspension buffer (10 mM

HEPES, pH 7.4 at 4°C), rapidly frozen in liquid nitrogen, and stored at -70ºC.

During the mitochondrial preparation process, the supernatant from spin 2 was collected and centrifuged at 302,000 g for 90 minutes at 4°C in a Beckman Coulter

Optima XPN centrifuge with a 50.2 Ti rotor. Microsomal pellets were collected and resuspended in ~0.5 ml storage buffer (10 mM HEPES, pH 7.4 at 4°C), rapidly frozen in liquid nitrogen, and stored at -70ºC. 159

Synaptic Membranes, Myelin, and Brain Mitochondria

Synaptic membranes, myelin and mitochondria were fractionated from brain tissue as described (131), with modifications. Brains were excised as described previously (Appendix A), sliced, and homogenized in six volumes of extraction buffer

(0.35 M sucrose, 5 mM EGTA, 10 mM HEPES, pH 7.8 at 1°C) using a motor-driven

Potter-Elvehjem grinder with eight even strokes (approximately five seconds per stroke).

Samples were kept on ice during preparations. Tissues were pooled as needed; generally, two animals were required per membrane preparation. Interspecies variation in tissue consistency necessitated slight differences in the initial centrifugation step to maintain consistent pellet and supernatant generation. The homogenate was centrifuged at 4°C for eight minutes at either 600 g (for N. coriiceps) or 800 g (for C. aceratus) in a Beckman

Coulter Avanti J-E centrifuge with a JA-17 rotor.

The supernatant was pipetted onto discontinuous Percoll gradients. Four gradients per preparation were prepared up to four hours in advance. Percoll was filtered using

Whatman Nuclepore track-etched membranes and made up to appropriate concentrations

(3, 10, 15, and 23%) in gradient buffer (0.32 M sucrose, 1 mM EDTA, 0.25 mM DTT, 5 mM Tris, pH 7.4 at 25°C). Four discrete 2-ml layers were generated using a P1000 micropipettor. After the addition of 2 ml supernatant, gradients were centrifuged at

13,000 g for 31 minutes at 4°C in a Beckman Coulter Avanti J-E centrifuge with a JA-17 rotor. Five bands formed at the gradient interfaces. Fractions were collected using a

Pasteur pipette, resuspended in 20 ml extraction buffer, and centrifuged at 302,000 g for

90 minutes at 4°C in a Beckman Coulter Optima XPN centrifuge with a 50.2 Ti rotor. 160

Membranes were concentrated in a loose pellet above the hard Percoll pellet. Membrane pellets were collected and resuspended in 250 μl storage buffer (25 mM Tris, pH 7.4 at

25°C), separated into three aliquots, and frozen at -70°C.

Gill Epithelial Plasma Membranes

Plasma membranes were prepared from gill tissue as described previously (174), with modifications for preparations in previously flash-frozen tissue. First, samples were thawed in beakers of ice-cold notothenioid Ringer’s solution for approximately 20 minutes. The samples were irrigated with fresh Ringer’s using a 10-ml syringe, fitted with a 32-gauge needle, until the gill tissue was visibly clear. Next, tissue was scraped from the gill using a razor blade and placed on an ice-cold glass tray. Gills were pooled as needed; typically, 2-3 gills were combined per preparation. Tissues were pooled from the same individuals whenever possible.

Next, the sample was homogenized in 15 ml extraction buffer (0.35 M sucrose, 5 mM EGTA, 10 mM HEPES, pH 7.8 at 1°C) using a motor-driven Potter-Elvehjem grinder with six even strokes (approximately 5 seconds per stroke). The homogenate was filtered through cheesecloth then made up to 25 ml with extraction buffer. Next, 15 ml of

41% sucrose were pipetted below the diluted homogenate. The sample was then centrifuged at 23,000 g for 30 minutes at 4°C in a Sorvall RC 6+ high-speed centrifuge with a Fiberlite F21-8x50y rotor. A band formed at the homogenate-sucrose interface.

The band was removed using a glass Pasteur pipette and diluted in 25 ml extraction buffer.

161

The dilute suspension was then centrifuged at 7000 g for 15 minutes. The pellet was collected and resuspended in 1 ml extraction buffer. The sample was then pipetted onto a self-generating Percoll gradient, which consisted of 18% Percoll and 10% 2.5 M sucrose in 20 mM Tris, pH 7.4 at 4°C. The gradient was centrifuged at 33,600 g for 25 minutes. A band formed towards the upper third of the gradient and was collected. The band was then resuspended in 30 ml extraction buffer and centrifuged at 82,000 g for two hours at 4°C in a 50.2 Ti rotor, using a either a Beckman Coulter L5-50 ultracentrifuge or a Beckman Coulter Optima L-80 XP ultracentrifuge. The pellet was reconstituted in four

50-µl aliquots of resuspension buffer (20 mM Tris, pH 7.4 at 4°C) and stored at

-70°C.

162

APPENDIX C: MEMBRANE MARKER ENZYMES

Enrichments of membrane fractions were determined by measuring the protein- specific activities of marker enzymes: acetylcholinesterase (AChE) for synaptic membranes (288), cyclic nucleotide phosphodiesterase (CNPase) for myelin (289), succinate dehydrogenase (SDH) for mitochondria (290), Na+/K+-ATPase (NKA) for gill basolateral membranes (291), and gamma-glutamyltransferase (GGT) for gill apical membranes (292). Brain marker assays were conducted at ~23°C, and gill marker assays were conducted at 4°C. Brain assays were adapted to a microplate reader. CNPase activity was measured using a SpectraMax M2 microplate reader. AChE and SDH activities, as well as protein content, were measured using a Tecan Infinite 200 Pro microplate reader. NKA and GGT activities were measured using a Beckman DU640 spectrophotometer. Protein-specific activities, relative to crude homogenates, were calculated in order to determine enrichment factors for each membrane fraction. Two controls (one in the absence of substrate and one in the absence of sample) were conducted for each marker assay. Total protein content was determined using a Sigma-

Aldrich bicinchoninic acid assay kit.

AChE (EC number 3.1.1.7) activity was determined as described (288), with modifications. The reaction was initiated by adding 20 µl acetylthiocholine iodide (5.6 mM) to 160 µl reaction mixture (2.9 mM DTNB, 88 mM potassium phosphate, pH 8.0 at

25°C) and 20 µl diluted sample. The change in absorbance at 415 nm was measured at

20-second intervals for 10 minutes. 163

CNPase (EC number 3.1.4.37) activity was determined as described (289), with modifications. The reaction was initiated by adding 10 µl sample (diluted as necessary to produce a measurable linear increase in absorbance) to 200 µl reaction mixture (27 mM

MgCl2, 180 mM MES, 1 mM 3’5’-cAMP, 5 mM glucose-6-phosphate, 0.001% glucose-

6-phosphate dehydrogenase, pH 7.4 at 20°C). The change in absorbance at 254 nm was measured at 20-second intervals for 10 minutes.

SDH (EC number 1.3.5.1) activity was determined as described (290), with modifications. The reaction was initiated by adding 20 µl succinic acid (200 mM) to the final reaction mixture, which consisted of 180 µl reaction buffer (311 mM NaN3, 4 mM

MgCl2, 233 mM mannitol, 5 mM KH2PO4, 11 mM K2HPO4, 39 mM DCPIP, pH 7.2 at

25°C) and 20 µl diluted sample. The change in absorbance at 600 nm was measured at

20-second intervals for 10 minutes.

NKA (EC number 3.6.3.9) activity was determined as described (291), with modifications. The reaction was initiated by adding 100 µl diluted sample to 1.5 ml reaction mixture (4 U/ml lactate dehydrogenase, 5 U/ml pyruvate kinase, 2.8 mM phosphoenolpyruvate, 0.7 mM adenosine triphosphate, 0.22 mM NADH, 50 mM imidazole, pH 7.5 at 4°C) and 500 µl salt solution (189 mM NaCl, 10.5 mM MgCl2, 42 mM KCl). Controls containing 0.5 mM ouabain were performed in tandem. The change in absorbance at 340 nm was measured at 15-second intervals for 10 minutes.

GGT (EC number 2.3.2.2) activity was determined as described (292), with modifications. The reaction was initiated by adding 300 µl diluted sample to 2.5 ul reaction buffer (120 mM Tris, 80 mM glycylglycine, 6 mM gamma-glutamyl-p- 164 nitroanilide, pH 8.0 at 4ºC). The change in absorbance at 405 nm was measured at 15- second intervals for 10 minutes.

165

APPENDIX D: MEMBRANE FLUIDITY ASSAYS

Membrane fluidity was quantified by fluorescence depolarization as described

(132). In brief, samples were added to a solution of 1,6-diphenyl-1,3,5-hexatriene (DPH) for a final phosphate-to-probe molar ratio of 500:1 and added to 2.5 ml assay buffer (20 mM Tris, pH 7.4 at 20°C) with constant stirring in a quartz cuvette. Probe incorporation was conducted in a darkened room in a foil-covered amber vial to prevent quenching of the fluorescent signal.

Change in polarization (excitation=356 nm, emission=430 nm) was measured between 0°C and 40°C using a Perkin-Elmer LS-50B spectrophotometer. Measurements were initiated at cold temperatures, and temperatures were elevated at 2°C or 5°C intervals (as denoted in main text) at a rate of ~0.3°C min-1. Temperature was controlled using a circulating water bath containing 50% ethylene glycol. Polarization measurements were performed in triplicate after the temperature stabilized at each interval.

166

APPENDIX E: LIPID EXTRACTION AND COMPOSITIONAL ANALYSES

Lipids were extracted as described (133). Samples were diluted to a volume of

600 µl, added to a 2:1 ratio of methanol and chloroform, and vortexed vigorously for 30 seconds. The mixture was centrifuged at 600 g for 10 minutes and distinct layers formed.

The lower layer was collected using a glass Pasteur pipette. The extraction procedure was repeated three times on each sample. Samples were washed with deionized water and evaporated under dry nitrogen gas in 2-ml borosilicate glass vials with Teflon-lined caps.

Extracts were sent to the Kansas Lipidomics Research Center for analysis, and a diacyl polar lipid profile dataset was generated by quadrupole mass spectrometry using an

Applied Biosystems 4000 QTRAP mass spectrometer as described (134). Relative abundances of the major phospholipid classes were compared between species. The unsaturation index (UI) was calculated as described (135).

Cholesterol was quantified using a Cayman fluorometric assay kit and normalized to total phospholipid content, which was measured as hydrolyzed inorganic phosphate in membranes as described (205). Diluted samples were hydrolyzed in covered glass culture tubes with full-strength perchloric acid at 180°C for approximately two hours until the samples clarified. Samples were cooled to room temperature before quantifying phosphate content by adding 40 µl sample (diluted as necessary to produce absorbance values within standard curve range) to 160 µl reaction medium (1.2 N H2SO4, 2% ascorbate, and 0.5% [NH4]2MoO4). Samples were incubated for seven minutes at 60°C, and absorbance was measured at 820 nm using a SpectraMax M2 microplate reader.

167

APPENDIX F: THERMAL ACCLIMATION TREATMENTS

Notothenia coriiceps (Group A) were collected as described (Appendix A) before being transferred to Palmer Station, where fish were held at ambient seawater temperature (0+1ºC) for at least three days prior to thermal treatment. Additionally, a second group of animals (Group B) were caught using baited lines at Arthur Harbor during the same season. These samples were transferred immediately to seawater buckets and transported to seawater tanks on station. After a three-day recovery, these animals were used for a second, shorter acclimation.

After recovery, the animals were assigned randomly to designated warm (5+1°C) or control (0+1°C) groups. The tanks used for the warm acclimation were increased at a rate of 1°C per day. Animals were then held at 5°C for a period of either 10 (group A) or six (group B) weeks. The 0°C tank was kept constant during this period. All animals were fed to satiation with ~10g muscle fillets every other day. All animal experiments were approved by the Ohio University Animal Care and Use Committee (14-L-004).

Individuals from the two acclimation groups (A and B) were analyzed separately and were found to be equivalent in all reported analyses. Thus, data from both groups were pooled to account for logistical issues during fieldwork that limited sample size

(thus necessitating the second, shorter acclimation treatment).

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APPENDIX G: MEASUREMENT OF OSMOTIC PERMEABILITY IN GILL TISSUE

Gills were excised and bathed immediately in notothenioid Ringer’s solution (240 mM NaCl, 2.5 mM MgCl2, 5 mM KCl, 2.5 mM NaHCO3, 5 mM NaH2PO4, pH 8.0 at

4°C). After incubation, the gills were washed with clean Ringer’s, blotted dry for 3 seconds using four layers of Kimwipe tissues, and weighed. Gills were then transferred to beakers containing 100 ml 20 mM CaCl2. Air stones were fixed to the bottom of the beaker to maintain consistent aeration. Beakers were held in either an ice bath (0+0.5°C) or the countertop of a fixed-temperature cold room (4+0.7°C). Gill masses were measured in 10-minute intervals after using the blotting procedure described above. Gills were then dried at 110°C for 24 hours to obtain dry weights. The osmotic water gain

(푅(%)) for each time point (t) was calculated using the following formula: 푅(%) =

(푊푡 – 푊𝑖)/(푊𝑖 − 푊푑) 푥 100, where 푊푡 = the arch mass at time t, 푊𝑖 = the initial arch mass, and 푊푑 = the dry weight. The rate of osmotic water gain was calculated over the linear range of data (i.e., before the rate of 푅(%) began to stabilize).

169

APPENDIX H: MEASUREMENT OF OXYGEN SOLUBILITY IN GILL PLASMA

MEMBRANES

Oxygen solubility was quantified in plasma membranes from gill epithelia as described previously (206), with modifications. Membrane samples were thawed on ice and transferred to 2-ml glass vials. Samples were flushed with oxygen in 10-minute intervals for three hours. Oxygen was delivered to samples for 30 seconds at 3 psi, using a 20-guage needle attached to a plastic tubing apparatus. Samples were kept on ice during all preparations.

Oxygen concentrations were measured using a Microelectrodes, Inc. MI-730 dissolved oxygen probe, which was fitted to a transbridge and an analog-to-digital converter. Temperature was maintained at 4°C using a circulating water bath, which was connected directly to the sample chamber. The chamber was kept sealed using molding clay.

Oxygen concentrations were recorded using WinDAQ data acquisition software.

A gain of 8 and a sample rate of 30 readings per second were used. Low and high calibrations were performed in the sample chamber. For the low calibration, 2 ml deionized water was deoxygenated using ~0.5 g dithionite. For the high calibration, 2 ml oxygenated water was used in an unsealed chamber.

The corrected relative increase in oxygen (CRI) was calculated using the following formula: 퐶푅퐼 = ([푂2]푆 – [푂2]퐵 푥 푏/([푂2]퐵 푥 푏 – [푂2]푆), where [푂2]푆 = the oxygen content of the injected sample, [푂2]퐵 = the oxygen content of the injected buffer, and 푏 = the assay dilution factor. CRI was plotted against the hydrophobic fractional 170 volume 훼ℎ, which was calculated by correcting phosphate concentration to assay volume.

The oxygen partition coefficient was calculated as the linear slope (CRI versus 훼ℎ).

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