The Impacts of Acute Hypoxic Exposure and Other Concomitant Stressors on the Cardiorespiratory Physiology of Coastal Elasmobranch Fishes

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The Impacts Of Acute Hypoxic Exposure And Other Concomitant Stressors On The Cardiorespiratory Physiology Of Coastal Elasmobranch Fishes

Gail Danielle Schwieterman William & Mary - Virginia Institute of Marine Science, [email protected]

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Recommended Citation Schwieterman, Gail Danielle, "The Impacts Of Acute Hypoxic Exposure And Other Concomitant Stressors On The Cardiorespiratory Physiology Of Coastal Elasmobranch Fishes" (2020). Dissertations, Theses, and Masters Projects. Paper 1593091930. http://dx.doi.org/10.25773/v5-6hq9-zy38

This Dissertation is brought to you for free and open access by the Theses, Dissertations, & Master Projects at W&M ScholarWorks. It has been accepted for inclusion in Dissertations, Theses, and Masters Projects by an authorized administrator of W&M ScholarWorks. For more information, please contact [email protected]. The impacts of acute hypoxic exposure and other concomitant stressors on the cardiorespiratory physiology of coastal elasmobranch fishes

A Dissertation

Presented to

The Faculty of the School of Marine Science

The College of William & Mary

In Partial Fulfillment

of the Requirements for the Degree of

Doctor of Philosophy

Gail Danielle Schwieterman

May 2020

APPROVAL PAGE

This dissertation is submitted in partial fulfillment of

the requirements for the degree of

Doctor of Philosophy

Gail Danielle Schwieterman

Approved by the Committee, April 2020

Richard W. Brill, Ph.D. Committee Chair / Advisor

Mary C. Fabrizio, Ph.D.

Kevin C. Weng, Ph.D.

Emily B. Rivest, Ph.D.

Holly A. Shiels, Ph.D. University of Manchester Manchester, UK

This Ph.D. is dedicated to the makers of escitalopram, without which I never would have completed this document, and to the friends and family who encouraged me to get the medical help I needed

iii TABLE OF CONTENTS

ACKNOWLEDGEMENTS ...... ix

LIST OF TABLES ...... x

LIST OF FIGURES ...... xi

AUTHOR’S NOTE ...... xiv

ABSTRACT ...... xv

CHAPTER I — Introduction: Elasmobranch cardiorespiratory physiology and responses to stress ...... 2

Elasmobranch Fishes ...... 3

Anthropogenic Stress ...... 5

Climate Change ...... 5

Fishing Pressure ...... 7

Cardiorespiratory Physiology of Fishes ...... 10

The Elasmobranch Cardiorespiratory System ...... 14

The Cardiorespiratory Stress Response of Fishes ...... 17

Dissertation Aims and Structure ...... 18

References ...... 22

Figures...... 35

CHAPTER II — Combined effects of acute temperature change and elevated pCO2 on the metabolic rates and hypoxia tolerances of clearnose skate (Rostaraja elganteria), summer flounder (Paralichthys dentatus), and thorny skate (Amblyraja radiata) ...... 38

Abstract ...... 39

Introduction ...... 40

iv Materials and Methods ...... 44

Results ...... 49

Standard Metabolic Rate and Q10 ...... 49

Maximum Metabolic Rate and Aerobic Scope ...... 50

Hypoxia Tolerance ...... 50

Discussion ...... 51

Conclusions ...... 60

Acknowledgements ...... 61

References ...... 62

Tables ...... 73

Figures...... 75

Supplementary Material ...... 80

CHAPTER III — Drivers of hypoxia tolerance in elasmobranch fishes: comparing blood oxygen affinity between clearnose skate and summer flounder ...... 81

Abstract ...... 82

Introduction ...... 83

Materials and Methods ...... 90

Animal Collection and Maintenance ...... 90

Tonometry and Hematology ...... 91

Data Processing and Statistical Analysis ...... 93

Results ...... 95

Discussion ...... 96

Conclusions ...... 104

v Acknowledgements ...... 105

References ...... 106

Tables ...... 116

Figures...... 117

Supplementary Material ...... 128

CHAPTER IV — A search for red blood cell swelling in five elasmobranch fishes ...... 134

Abstract ...... 135

Introduction ...... 136

Materials and Methods ...... 140

Results ...... 143

Discussion ...... 145

Possible Functions of RBC Swelling ...... 149

Possible Mechanisms of Swelling in Elasmobranch Fishes ...... 152

Preservation of CO2 Excretion ...... 153

Future Work ...... 155

Conclusions ...... 155

Acknowledgements ...... 156

References ...... 157

Figures...... 165

CHAPTER V — The effects of elevated potassium, acidosis, reduced oxygen levels, and temperature on the functional properties of isolated myocardium from three elasmobranch fishes: clearnose skate (Rostraja eglanteria), smooth dogfish (Mustelus canis), and sandbar shark (Carcharhinus plumbeus) ...... 177

Abstract ...... 178

vi Introduction ...... 179

Materials and Methods ...... 182

Animal Collection and Maintenance ...... 182

Isolated Heart Strip Procedure ...... 183

Treatments...... 185

Data Processing and Statistical Procedures ...... 186

Results ...... 189

Effects of Hyperkalemia, Acidosis + Low O2, and

Combined Stressors at 0.2 Hz Stimulation Frequency ...... 189

Effects of Hyperkalemia, Acidosis + Low O2, and

Combined Stressors During Force Frequency Trials ...... 190

Effects of Isoproterenol at 0.2 Hz Stimulation Frequency ...... 191

Effects of Isoproterenol During Force Frequency Trials ...... 191

Discussion ...... 192

Hyperkalemia, Acidosis + Low O2, and Combined

Stressors ...... 192

Adrenergic Stimulation ...... 195

Conclusions ...... 197

Acknowledgements ...... 198

References ...... 199

Tables ...... 208

Figures...... 209

Supplemental Figures...... 216

vii CHAPTER VI — Conclusions ...... 218

General Discussion ...... 219

Contributions...... 220

Future Research Directions ...... 224

Final Remarks ...... 226

References ...... 227

VITA ...... 231

viii ACKNOWLEDGEMENTS

This dissertation is the culmination of a collaborative effort by a brilliant group of individuals to whom I am deeply indebted. I am grateful to my advisor, Dr. Richard Brill for his guidance and expertise throughout this foray into fish physiology. The members of my advisory committee, Drs. Mary Fabrizo, Kevin Weng, Emily Rivest, and Holly Shiels, provided invaluable perspective and encouragement throughout this process. Dr. Peter Bushnell was also an integral mentor, lending his ingenuity and wealth of knowledge to these projects throughout the summers on the Eastern Shore of Virginia. Additional guidance from Drs. Linda Schaffner, John Graves, David Dafashy, and Donna Haygood-Jackson was critical in the completion of this document.

I also must thank Drs. Jodie Rummer, James Sulikowski for welcoming me into their respective labs and offering their support in its many different forms. The opportunities to work alongside their students were essential in reminding me why I began this degree. Maggie Winchester, your assistance and persistence with the heart chapter were invaluable, and I feel lucky to have gotten a close friend out of that summer. Thank you to Maggie, Ian Bouyoucos, and Annie Schatz for their late-stage edits on this document.

The VIMS community was a tremendous source of support and motivation throughout the past five years. Thank you to everyone who pushed me, comforted me, and celebrated with me. Dan Crear, my lab cousin, thank you for putting up with all of my R questions, frustrated rants, and insistence on having the lights on in the office. To Serina Wittyngham, thank you for being a constant source of strength and humor. Danielle Tarpley and Rebecca Hailey, thank you for always providing a refuge to call “home”.

I have also been incredibly fortunate to maintain a network of friends outside of graduate school. Thank you to Juliette Rubin, Lauren Speed, Caroline Rogers, Amanda Gracia, and Laurence Ducker for fielding late-night phone calls whenever I needed them. You helped me keep this experience in perspective.

Lastly, to my family—Thank you for your unconditional support. Knowing you stand behind me through thick and thin has been essential in my ability to dream big. I wouldn’t be the person I am today without you. I love you.

ix LIST OF TABLES

1. Carbonate chemistry parameters for experimental treatments...... 73

2. Q10 values for all three species and all treatment conditions ...... 74

S1. Parameter estimates and standard error values ...... 80

CHAPTER III — Drivers of hypoxia tolerance in elasmobranch fishes: Comparing blood oxygen affinity between clearnose skate and summer flounder

1. Whole blood hematological parameters ...... 116

S1. Whole blood properties of elasmobranch fishes (Modified from Morrison et al. 2015)...... 128

S2. Metabolic Rates for selected elasmobranch fishes ...... 133

CHAPTER V — The effects of capture on the functional properties of isolated myocardial strips from three elasmobranch fishes: clearnose skate (Rostraja eglanteria), smooth dogfish (Mustelus canis), and sandbar shark (Carcharhinus plumbeus)

1. Treatment Conditions...... 208

x LIST OF FIGURES

CHAPTER I — Introduction: Elasmobranch cardiorespiratory physiology and responses to anthropogenic stress

1. Diagram of a generalized fish cardiorespiratory system ...... 35

2. Cardiac action potential in fish ...... 36

3. Diagram of the differences in the gill structure between teleost and elasmobranch fishes (Modified from Evan et al. 2005) ...... 37

1. Representative trial data showing Scrit ...... 75

2. Standard metabolic rate ...... 76

3. Maximum metabolic rate ...... 77

4. Factorial and absolute aerobic scope ...... 78

5. Hypoxia tolerance (Pcrit) ...... 79

CHAPTER III — Drivers of hypoxia tolerance in elasmobranch fishes: Comparing blood oxygen affinity between clearnose skate and summer flounder

1. Representative blood oxygen curves ...... 117

2. Hemoglobin saturation ...... 118

3. P50 values ...... 119

4. Bohr Coefficients ...... 120

5. Apparent heat of oxygenation (ΔH) ...... 121

6. pH50 values ...... 122

7. ΔpH/°C values ...... 123

8. Bicarbonate concentration ...... 124

xi 9. Hill numbers...... 125

10. Distribution of published P50 values from elasmobranch fishes ...... 126

11. Partial regression plots showing the influence of P50, temperature, and metabolic rate on Pcrit ...... 127

CHAPTER IV — A search for red blood cell swelling in five elasmobranch fishes

1. Schematic diagram of mechanisms driving oxygen in the capillaries of teleost and elasmobranch fishes ...... 165

2. Schematic diagram of Bohr and Root effects ...... 167

3. Schematic diagram of mechanisms driving red blood cell swelling in teleost fishes ...... 168

4. Changes in pHe ...... 170

5. Relative changes in hematological parameters ...... 171

6. Relationship between pHi and pHe ...... 172

7. Hematocrit and hemoglobin concentration from this study as well as from Brill et al. 2008 ...... 173

8. Mean corpuscular hemoglobin concentration (MCHC) and mean cell volume (MCV) from this study as well as from Brill et al. 2008 ...... 174

9. Schematic diagram of CO2 excretion mechanisms in the gills of teleost and elasmobranch fishes ...... 175

1. Experimental workflow ...... 209

2. Relative change in the function parameters of isolated cardiac strips during exposure to hyperkalemia, acidosis + low O2, and combined stressors ...... 210

xii 3. Relative change in the function parameters of isolated cardiac strips during exposure to hyperkalemia, acidosis + low O2, and combined stressors during force frequency trials ...... 212

4. Relative change in the function parameters of isolated cardiac strips during exposure to isoproterenol ...... 213

5. Relative change in the function parameters of isolated cardiac strips during exposure to isoproterenol during force frequency trials ...... 215

S1. Function parameters of isolated cardiac strips during exposure to hyperkalemia, acidosis + low O2, and combined stressors ...... 216

S2. Function parameters of isolated cardiac strips during exposure to isoproterenol ...... 217

xiii AUTHOR’S NOTE

Chapters II and V of this dissertation have been prepared as manuscripts for publication in peer-reviewed journals. As a result, these chapters were written in the first-person plural point of view to reflect the contributions of co-authors. Chapter II was published in 2019, and Chapter V will be prepared for publication in the near future.

The citations for Chapters II and V are:

Chapter II Schwieterman, G.D., Crear, D.P., Anderson, B.N., Lavoie, D.R., Sulikowski, J.A., Bushnell, P.G., Brill, R.W., 2019. Combined Effects of Acute Temperature Change and Elevated pCO2 on the Metabolic Rates and Hypoxia Tolerances of Clearnose Skate (Rostaraja eglanteria), Summer Flounder (Paralichthys dentatus), and Thorny Skate (Amblyraja radiata). Biology 8 (3) 56.

Chapter V Schwieterman, G.D., Winchester, M.M., Shiels, H.A., Bushnell, P.B., Bernal, D., Marshall, H.N., Brill, R.W., In preparation. The effects of elevated potassium, acidosis, reduced oxygen levels, and temperature on the functional properties of isolated myocardium from three elasmobranch fishes: clearnose skate (Rostraja eglanteria), smooth dogfish (Mustelus canis), and sandbar shark (Carcharhinus plumbeus).

xiv ABSTRACT

This dissertation examines physiological stress responses of coastal elasmobranch fishes and investigates mechanisms that maintain oxygen delivery under stress. Elasmobranch fishes are, in general, understudied despite their susceptibility (due to K-selected life histories) to unsustainably high fishing mortality and the effects of climate change. Knowledge of physiological stress responses is, therefore, necessary to understand species-specific resilience and overall susceptibility to stressors. In Chapter II, I describe the hypoxia tolerances of clearnose skate (Rostaraja eglanteria), thorny skate (Amblyraja radiata), and summer flounder (Paralichthys dentatus, a representative teleost species) under conditions of temperatures and acidification projected to occur by the end of the century due to climate change. At the least-stressful temperature, all three species exhibited significant increases in standard metabolic rate and decreases in hypoxia tolerance with a 0.4 unit drop in pH. Clearnose skate were, however, found to be among the most hypoxia-tolerant elasmobranch species known. To investigate the physiological adaptations underpinning this extreme hypoxia tolerance, I measured blood-oxygen affinity (measured as P50 - the oxygen partial pressure (pO2) at which hemoglobin is 50% saturated with O2) of clearnose skate (Chapter III). Clearnose skate exhibited a relatively high P50 (4.9 ± 0.6 kPa) at 20 °C that was not different from that of the equally hypoxia- tolerant epaulette shark (4.3 ± 0.6 kPa at 28 °C). Yet, these values are significantly higher than those of many other elasmobranch species, suggesting blood-oxygen affinity may not be correlated with hypoxia tolerance in elasmobranch fishes as it is in teleost fishes. At the cellular level, the erythrocyte intracellular pH influences hemoglobin-oxygen (Hb- O2) affinity. In teleost fishes, decreases in Hb-O2 affinity (Bohr effect) and maximum blood oxygen carrying capacity (Root effect) result from acidosis. These responses can be mitigated through red blood cell (RBC) swelling. The aim of Chapter IV was, therefore, to investigate the prevalence of RBC swelling in elasmobranchs fishes. Blood samples were taken from captive individuals of five elasmobranch species: Clearnose skate, sandbar shark (Carcharhinus plumbeus), blacktip reef shark (C. melanopterus), sicklefin lemon shark (Negaprion acutidens), and epaulette shark (Hemisyllium ocellatum) following exhaustive exercise and acute air exposure. None of the measured hematological parameters (hematocrit, blood hemoglobin concentration [Hb], RBC counts, RBC volume, and mean corpuscular hemoglobin content; MCHC) were indicative of RBC swelling, although published results show swelling occurs in sandbar and epaulette shark RBCs. Other impairments to the cardiorespiratory may result from external stressors, such as those associated with interactions with fishing gear. I hypothesized the cause of post-release mortality in elasmobranch fishes is diminished myocardial function. To test this hypothesis, I measured changes in the functional properties of isolated ventricular myocardial muscle from clearnose skate, smooth dogfish (Mustelus canis), and sandbar shark during hyperkalemia (7.4 mM K+), acidosis (a pH decline of 0.8 units), and reduced oxygen (to 31% saturation) at two temperatures (Chapter V). Stressors had relatively small and species-specific detrimental impacts on myocardial function that were only partially ameliorated adrenergic stimulation (i.e. by application of isoproterenol, an adrenaline analog). Overall, this dissertation implies that coastal elasmobranch fishes maybe highly resilient to the effects of directional climate change and the physiological stress associated with interactions with fishing gear.

xv The impacts of acute hypoxic exposure and other concomitant stressors on the cardiorespiratory physiology of coastal elasmobranch fishes

CHAPTER I

Introduction: Elasmobranch cardiorespiratory physiology and responses to stress

2 ELASMOBRANCH FISHES

Extant chondrichthyan fishes are a diverse group comprising over 1,000 species

(Carrier et al. 2012; Ebert et al. 2013) and are differentiated from teleost fishes (class

Osteichthyes) by characteristics including cartilaginous endoskeletons, internal fertilization, paired spiracles, and separate internal and external gill openings (Compagno

1990). Relatives of elasmobranch fishes include a basal vertebrate lineage dating back

400 million years (Compagno et al. 2005). This long evolutionary history has likely contributed to the success of elasmobranch fishes at colonizing almost all aquatic environments from freshwater rivers to the abyssal planes of the deep sea, although the majority of species are fully marine (Klimley 2013).

The elasmobranch subgroup includes sharks, skates, and rays, the latter two of which are collectively referred to as batoids. The batoid fishes are dorso-ventrally flattened, swim with modified pectoral fins, and are associated with a benthic, sedentary lifestyle. On the opposite end of the spectrum, lamniforme sharks show convergent evolution of morphological and physiological characteristics with tunas (e.g., fusiform body shape, large caudal keel) that enable their active, pelagic lifestyle (Bernal et al.

2001; Ebert et al. 2013). Like tunas, lamnid sharks (e.g. shortfin mako shark, Isurus oxyrhinchus and porbeagle shark, Lamna nassus; Carey et al., 1981) are regionally endothermic, employing countercurrent heat exchange to maintain red muscle tissues above ambient temperature thus enabling them to maintain elevated muscle function (and therefore high sustainable swimming speeds) at cold temperatures. Elasmobranch fishes range in size from 20 cm (dwarf lanternshark; Etmopterus perryi) to over 20 m total length (whale shark; Rhincondon typus) and are specialized to feed on prey items ranging

3 in size from plankton to marine mammals (Compagno et al. 2005; Carrier et al. 2012).

Elasmobranch fishes also occupy a broad scope of ecological niches, ranging from benthic foragers to apex predators, although they are over represented at high trophic levels (Heithaus et al. 2010). Life spans likewise vary dramatically, with some coastal sharks (e.g., blacknose shark, Carcharhinus acronotus) living only 20 years to the

Greenland shark (Somniosus microcephalus) that live to 400 years (Driggers et al. 2004;

Nielsen et al. 2016). Elasmobranch fishes display all forms of reproduction (i.e., viviparity, oviparity, and ovoviviparity), but generally have low fecundity with individual litter sizes ranging from 2-100 depending on the species (Carrier et al. 2004).

Low reproduction rates, paired with generally late ages at maturity, suggest that elasmobranch fishes are highly K-selected. Despite debate on the utility of the r- vs K- selected paradigm (Pianka 1970; Stearns 1976), the life history characteristics of elasmobranch fishes have likely contributed to a decline in global elasmobranch populations (Stevens et al. 2000; Hoffmann et al. 2010; Dulvy et al. 2014b). Fishing activities have been implicated in the decline of elasmobranch fishes globally, with 24% of species evaluated being threatened with extinction, likely an underestimate due to the data-poor nature of this group (Dulvy et al. 2014a; Dulvy et al. 2014b; Shiffman and

Hammerschlag 2016). Other causes of population decline include loss of habitat and changes in environmental conditions (Portner 2010; DiBattista et al. 2011), which may have interactive effects with industrialized fishing pressure. For example, range contraction (barring decreases in total abundance) could increase harvest rates, obscuring true trends in population size and promoting overharvesting (Burgess et al. 2017). The loss of elasmobranch species occupying predatory roles is likely to have large impacts on

4 ecosystem health, with poorly understood changes in trophic organization (Worm and

Myers 2003; Ferretti et al. 2010; Heithaus et al. 2010). Recovery rates for elasmobranch populations is generally slower than that for other groups occupying lower trophic levels

(Heithaus et al. 2010), making these declines particularly worrisome.

ANTHROPOGENIC STRESS

The impacts of humans on marine ecosystems are profound, and despite their long evolutionary history, elasmobranch fishes are not immune to anthropogenic impacts.

Increased electromagnetic fields, noise from shipping and drilling, destruction of nursery areas, pollution, artificial feeding from fishing or tourism operations, and human presence at important aggregation sites all impact elasmobranch health (Walker 2007). Here, I will focus on the two of the most pressing anthropogenic stressors encountered by elasmobranch fishes: (1) climate change, specifically interactions among rising temperatures, coastal hypoxia, and ocean acidification (Breitberg et al. 2009; Diaz and

Breitburg 2009; Najjar et al. 2010; Melzner et al. 2012; Breitburg et al. 2015), and (2) capture stress and associated post-release mortality (Hoffmayer and Parsons 2001; Frick et al. 2010b; Heberer et al. 2010; Cook et al. 2019). The negative impacts of both categories of stressors have been demonstrated through their effects on cardiorespiratory physiology (Morrison et al. 2015; Brill and Lai 2016; Lefevre 2016; McKenzie et al.

2016).

Climate Change

Climate change in the marine environment includes, but is not limited to, increasing temperatures and incidences of low oxygen (hypoxia), as well as decreases in

5 pH (acidification). In present day coastal systems, these environmental stressors often occur simultaneously, with warm summer air temperatures stabilizing stratification patterns that promote deoxygenation of bottom waters (Diaz and Rosenberg 2008;

Breitberg et al. 2015; Diaz et al. 2019), a problem compounded by increasing rates of eutrophication in coastal zones (Wallace et al. 2014). Similarly, during nighttime high tides, submerged aquatic and coastal vegetation can both deplete oxygen content and increase the partial pressure of carbon dioxide, thus increasing acidification (Melzner et al. 2013; Baumann et al. 2015). Combined, increasing temperature, hypoxia, and coastal acidification are known to have detrimental, and interactive effects on marine organisms

(Farrell et al. 2009), although the extent and nature of these impacts is difficult to determine and is likely highly variable across different ecological functional groups and among different taxa (Heithaus et al. 2010; Hare et al. 2016).

The impacts of climate change associated stressors on elasmobranch fishes are currently highly active areas of investigation, including the physiological mechanisms driving observed responses (e.g., changes in foraging success; Pistevos et al. 2015).

Increases in temperature raise standard metabolic rate in all fishes, which can drive changes in behavior, energy requirements, and both somatic and gonadal growth (Clark et al. 2013; Jutfelt et al. 2018). An elevated metabolic rate generally decreases hypoxia tolerance, as observed in sandbar shark (Carcarhinus plumbeus; Crear et al., 2019), potentially leading to changes in seasonal distributions (Rogers et al. 2016). Ocean acidification affects a suite of physiological parameters ranging from acid/base balance, metabolic rates, embryonic development, and neurosensory impairment (Di Santo 2015; reviewed by Esbaugh 2018). For some elasmobranch species (e.g., thorny skate,

6 Amblyraja radiata), population decline or lack of recovery has been attributed to indirect changes associated with environmental stressors, such as decreases in available habitat or alterations of trophic structure (Packer et al. 2003; NMFS 2017). The diversity of responses to environmental stress, as well as a lack of mechanistic understandings regarding the physiological processes undermining these responses, signifies there is still much to learn about species- and population-specific responses to anthropogenic climate change.

Capture Stress

Elasmobranch fishes are frequently a target of recreational fisheries, largely due to their reputation of providing a good fight and the associated prestige. Some fish are retained as trophies or for food, but many are released alive. Indeed, many anglers cite conservation as one of their motivations for catch-and-release fishing (Gallagher et al.

2015; French et al. 2019). Similarly, in many commercial fishing gear types, elasmobranch fishes are also frequently captured as bycatch. For example, pelagic longline and driftnet fleets are responsible for a large amount of pelagic shark bycatch

(Beerkircher et al. 2002), while trawl and dredge fisheries have high rates of benthic shark and skate bycatch (Mandelman et al. 2013). Due to their low market value, elasmobranch fishes are frequently discarded (Stevens 2000; Mandelman and Farrington

2007; Erickson and Berkeley 2008). There are, however, targeted shark fisheries, fisheries for their fins to satisfy predominantly Asian markets (Clarke et al. 2007), just livers (Suseno et al. 2014), and meat during Lent in central America (Sabbagh and

Hickey 2019). Except for the shark fin fishery, it is easier to get reliable estimates of

7 fishing mortality from targeted elasmobranch fisheries compared to those with high release or bycatch rates.

In both recreational and commercial fisheries, management measures have been put in place to aid in species- or complex- specific recovery. These include catch quotas, size and sex limitations, time-area closures, and mandated release (Skomal 2007;

Erickson and Berkeley 2008; NMFS 2008). All these policies rely on the assumption that the majority of released individuals survive and reproduce. Unfortunately, unknown numbers of released individuals later succumb to the physiological impacts of capture, thus undermining the assumed benefits of these “no take” policies (Gallagher et al. 2017;

Whitney et al. 2017). Studies have shown that in some species, post-release mortality rates are nearly 100% (e.g., lesser spotted dogfish captured in a beam trawl; S. canicula;

Revill et al., 2005; Ellis et al., 2016). While the exact causes of post-release mortality have yet to be determined, correlations with rates of post-release mortality and blood potassium concentrations ([K+]), water temperature, blood pH, behavior, and involuntary reflexes have been reported (Ellis et al. 2016). Understanding the specific physiological disruptions underpinning mortality can inform best handling practices.

Interactions with fishing gear almost universally result in one or more of the following: physical trauma from hooking or entanglement, muscle fatigue from extensive bouts of burst swimming, and rapid changes in environmental conditions as fish are brought to the surface; any of which can negatively impact the ability of a fish to maintain homeostasis (Skomal 2006; Skomal and Bernal 2010). The exhaustive exercise associated with capture is accomplished primary through anaerobic metabolism, resulting in the production of equal molar amounts of hydrogen ions (H+) and lactate in the muscle

8 (Wood 1991). These metabolic byproducts eventually leak into the blood causing metabolic acidosis (i.e., a drop in whole blood pH due to elevated concentrations of H+)

(Wood 1991; Wendelaar-Bonga 1997; Skomal and Bernal 2010). Exhaustive exercise also changes ionic concentrations in the blood (e.g., increases in potassium ion concentrations [K+], calcium ion concentrations [Ca2+], and sodium ion concentrations

[Na+]), likely due to muscle cell degradation (Cliff and Thurman 1984; Marshall et al.

2012). Interaction with fishing gear may also restrict water movement over the gills, thus limiting gas exchange and simultaneously reducing blood oxygen content and increasing the partial pressure of carbon dioxide (pCO2). This, in turn, induces a decline in whole blood pH (respiratory acidosis) (Brill and Lai 2016). Air exposure, also often associated with capture, stops gill gas exchange and causes rapid changes in body temperature, potentially compounding physiological insult (Arlinghaus et al. 2007). All of these disruptions can persist for hours, and recovery to pre-capture conditions may take up to

24 hrs (Cliff and Thurman 1984; Frick et al. 2010a; Kneebone et al. 2013).

To predict how fish populations respond to both rapid climate change and increasing fishing pressure, an understanding of species-specific tolerance levels is needed (Horodysky et al. 2015). While many physiological mechanisms are conserved across vertebrates, and especially across fishes (Kassahn et al. 2009; Berenbrink 2011), the diversity present within fishes and within the elasmobranch subclass can make extrapolations from single-species studies unreliable (Brill and Lai 2016). By viewing traditional fish physiology questions through the lens of traditional fisheries science, these two fields can provide robust information to sustainably manage marine resources in the face of climate change and overfishing (Horodysky et al. 2015; Ward et al. 2016).

9 CARDIORESPIRATORY PHYSIOLOGY OF FISHES

Fish have a unique set of cardiorespiratory adaptations that enable them to overcome the challenges associated with living in aquatic environments. Like other vertebrates, fishes must have access to oxygen to support aerobic respiration. Water is a poor respiratory medium, however, with 20-30 fold lower oxygen content per unit volume relative to air (depending on temperature and salinity; Cech and Brauner 2011).

This means that fish (at equivalent aerobic metabolic rates) require a respiratory volume

(per unit time) ≈30-40 times that of air breathers. This, in turn, results in fish being overventilated with respect to carbon dioxide excretion and exhibiting a very low circulating pCO2 (Claiborne 1998). Further, water is viscous and dense, meaning that moving water in and out of the body and across respiratory surfaces is energetically expensive.

Unlike other vertebrates, elasmobranch fishes (like teleost fishes), possess a two chambered heart and a single-loop closed cardiovascular system, with blood moving from the heart, to the gills, capillaries, and back to the heart (Figure 1) (Farrell and Pieperhoff

2011). The blood enters the heart through the sinus venosus, which collects returning venous blood. The atrium is thin walled while the ventricle, in contrast, is thick walled, with two distinct layers: (1) an inner layer made of trabeculae spanning the entire length of the chamber (spongy myocardium) and (2) an outer layer made of compact myocardium, which in elasmobranch fish is vascularized (Farrell and Pieperhoff 2011).

Upon exiting the ventricle, blood enters the conus arteriosus, which helps to smooth the flow of blood leaving the ventricle by having a delayed contraction relative to the

10 ventricle. The contribution of the conus arteriosus to overall cardiac output, however, is slight (Bushnell et al. 1992).

Contraction is caused by a series of coordinated ion exchanges across the sarcolemma, which is measured by changes in voltage (i.e., membrane potential; MP;

Figure 2). Sodium/potassium (Na+/K+) pumps maintain high extracellular [Na+] and high intracellular [K+] (Yellen 2002). When neighboring cells depolarize, they release Na+ into the extracellular environment, causing even more Na+ to enter the cell, thus raising MP above the threshold for depolarization (Vornanen 2011; Gamperl and Shiels 2014).

Depolarization causes the ion exchange of the sodium-calcium exchanger to reverse, allowing diffusion of Na+ out of the cell in exchange for Ca2+ (Bers 2002). This triggers the release of intracellular Ca2+ stores, which bind to contractile proteins (i.e., the troponin-myosin complex of the myofilaments) and initiate contraction (Vornanen 2011;

Gamperl and Shiels 2014).

From the heart, blood flows to the gills. The secondary lamellae of the gills are the respiratory surface at which gases, ions, and waste products are exchanged with the environment. Each paired gill arch has an interbranchial septum made of connective tissue that connects and supports the neighboring filaments, and gill rays that extend laterally from its base (Figure 3; Evans et al. 2005). The pathway of dissolved oxygen from the water from water to the mitochondria is driven by a sequential decline in oxygen partial pressures (pO2) which us referred to as the “oxygen cascade”. Dissolved oxygen from the water diffuses across the gill membrane into the plasma due to a difference in partial pressures. The countercurrent flow of blood and water across the gills maximizes the partial pressure difference between the two fluids, thus maximizing gas exchange.

11 Oxygen will then diffuse into the red blood cells where 98% of it will be reversibly bound to hemoglobin (Hb) (Nikinmaa 2011). Hemoglobin can be in one of two conformational states: R(elaxed) state or the T(ense) state. Changes between these states are controlled through the binding of a proton. Hemoglobin in the R-state has a high oxygen affinity, and most Hb arrives at the gills in the R-state. Each hemoglobin molecule can bind four O2 molecules; the binding of the first promotes the binding of the second, and so on -- a process known as cooperativity. This process occurs rapidly in the gills, and the oxygen remains bound to Hb as the blood is moved through the arteries to the capillaries. Once in the capillaries, elevated pCO2 results in the movement of dissolved CO2 from the plasma into the red blood cell where it is converted into bicarbonate and H+ in a reaction catalyzed by carbonic anhydrase (CA). These excess protons bind to the hemoglobin protein, prompting the release of oxygen through changes in the three dimensional shape of the hemoglobin subunits (a process known as the Bohr effect; Berenbrink 2011) thus increasing the pO2 driving gradient from the red blood cell to the plasma, and subsequently into the cells surrounding the capillaries and eventually into the mitochondria where it is used in ATP production. Changes in Hb-O2 affinity also decrease maximum blood oxygen carrying capacity, a process known as the Root effect

(Root 1931; Pelster and Randall 1998).

Recent work has shown the location of plasma-accessible CA on the capillary wall helps promote oxygen offloading in teleost fishes (Rummer and Brauner 2011;

Rummer et al. 2013). However, this process is not 100% efficient, and there is still some

O2 in the venous blood. The remaining oxygen (the venous reserve) is important as it is used to oxygenate the spongy myocardium of the ventricle, which may or may not

12 possess its own coronary circulation (Farrell et al. 2012). Elasmobranch fishes, in contrast, have a very low venous blood oxygen content (Bushnell et al. 1982). This low blood oxygen content, in turn, explains why elasmobranch fishes as vascularized compact myocardium.

The rate of oxygen delivery is described by the Fick equation (Schmidt-Nielsen

1997):

[푂2]퐷푒푙푖푣푒푟푦 = 푆푉 × 퐻푅 × ([푂2]퐴푟푡푒푟푖푎푙 − [푂2]푉푒푛표푢푠)

- Where [O2]delivery is the rate of O2 delivery by the cardiovascular system (e.g., mg O2 min

1 -1 -1 -1 -1 kg ), SV is stroke volume (e.g., ml beat kg ), HR is heart rate (beats min ), [O2]arterial

-1 is the arterial blood oxygen content (e.g., mg O2 ml ), and [O2]venous is the venous blood

-1 oxygen content (e.g., mg O2 ml ). The values of these parameters vary not only among species, but also within a given individual as metabolic demand changes (Brill and Lai

2016). In order to increase oxygen delivery to the tissues, there are both immediate and long-term modifications to the oxygen cascade that fish can employ. Assuming that water oxygen content is stable, increases in cardiac output (i.e., 푆푉 × 퐻푅) will increase the rate of oxygen delivery to the tissues. Generally, teleost fishes increase cardiac output through changes in heart rate, while elasmobranch fishes increase output through increases in stroke volume (Emery 1985; Scharold et al. 1989; Farrell 1991; Scharold and Gruber

1991; Tota and Gattuso 1996; Carlson et al. 2004; Brill and Lai 2016). Gas exchange rates at the gills can also be modified through changes in blood flow patterns within the gills, due to either active (and selective) vasoconstriction relaxation or increases in venous blood pressure accompanying changes in cardiac output. The mechanisms underlying vascular constriction and relaxation are still under investigation, but this may

13 also play a role in regulating oxygen delivery to specific tissues (Perry et al. 2016;

Sandblom and Gräns 2017).

Over longer time scales (days to weeks), fish can also alter the affinity of their hemoglobin molecules for oxygen by increasing or decreasing the intracellular concentration of allosteric molecules such as nucleoside triphosphates (NTP), calcium

+ - ion concentration ([Ca ]), or chloride ion concentrations ([Cl ]) which can affect Hb-O2 affinity. Alteration of these ion concentrations is most typically observed under prolonged exposure to low ambient oxygen conditions (i.e., hypoxia).

THE ELASMOBRANCH CARDIORESPIRATORY SYSTEM

Despite the many physiological mechanisms and structures that are highly conserved among vertebrates, and among fishes especially, the cardiorespiratory systems of elasmobranch and teleost fishes have notable differences. These differences have been extensively summarized by others (Butler and Metcalfe 1988; Tota 1999; Morrison et al.

2015; Brill and Lai 2016), so only a brief review will be provided here. Due to the diversity of body morphology and ecology within the elasmobranch group, moreover, there likely exists a diversity of cardiovascular adaptations specific to species life history, thus providing a range of functional properties to meet oxygen demands. Much of what is known about the form and function of the elasmobranch fishes’ cardiorespiratory systems is, however, from a small group of (often sedentary) species which are easily maintained in captivity (e.g., catsharks, Scyliorhinus stellaris and S. canicula; epaulette shark,

Hemiscyllium ocellatum; Port Jackson shark, Heterodontus portusjacksoni; eastern

14 shovelnose ray, Aptychotrema rostrata; and spiny dogfish, Squalus acanthias and S. suckleyi).

There are obvious anatomical differences in teleost and elasmobranch cardiorespiratory systems. Elasmobranch gills are not protected by a boney operculum as are teleost gills, but rather by individual gill slits. While teleost gills are arranged in a series of four paired rows, elasmobranch gills are arranged as pairs of five-seven (Evans et al. 2005; Olson 2011). In elasmobranch fishes, the interbranchial septum extends from the base of the gill arch to the skin, forming separated external gill slits (Wegner 2015).

Conversely, in teleost fishes the interbranchial septum is reduced, only extending to the base of the filaments (Figure 3). The lack of fused intrabronchial septum enables teleost filaments to move more freely (Evans et al. 2005). It is assumed that all oxygen uptake in elasmobranch fishes is through the gills, with negligible cutaneous gas exchange due to the thickness of dermal denticles and the non-vascularized skin of this group (Brill and

Lai 2016).

Elasmobranch fishes have Type III hearts, meaning they possess vascularization in the compact myocardium and are thus more dependent on oxygen delivery to the myocardium from arterial blood, presumably due to low venous oxygen content (Tota

1989; Tota and Gattuso 1996; Farrell et al. 2012; Brill and Lai 2016). The conus arteriosus of elasmobranchs also contains more valve-like structures compared to those of teleost fishes (Olson 2011). In teleost fishes, adrenergic stimulation (via circulating catecholamines or sympathetic innervation of the heart) increases transcellular Ca2+ movements and helps maintain myocardial function during stress (Gesser and Poupa

1979; Gesser and Jorgensen 1982; Randall 1982; Farrell et al. 1983; Farrell and Milligan

15 1986; Kieffer 2000; Hanson et al. 2006). Because elasmobranch fishes have only parasympathetic cardiac innervation (Wendelaar-Bonga 1997; Brill and Lai 2016), it remains unclear if these protective mechanisms are present. There is, however, some evidence of catecholamine storage sites within the elasmobranch myocardium (Hiatt

1943; Thompson and O'Shea 1997).

Elasmobranch erythrocytes are larger than those of teleost fishes, with higher hemoglobin content (Morrison et al. 2015). The percent of blood composed of red blood cells (hematocrit; Hct), is generally lower in elasmobranch fishes than in teleost fishes, and elasmobranch fishes generally do not exhibit the large increases in Hct under stress as do teleost fishes. The increase in Hct manifested in the latter is through a combination of RBC swelling and red blood cell ejection from the spleen (Piiper et al. 1970; Lowe et al. 1995; Gallaugher and Farrell 1998). Elasmobranch fishes also have higher circulating levels of trimethylamine oxide (TMAO) and urea, reflective of the different osmoregulation strategies between elasmobranch and teleost fishes (Weber 1983; Wood et al. 2007). Elasmobranch Hbs generally have a high Hb-O2 affinity and a higher buffering capacity (reviewed by Morrison et al. 2015).

Given the relative paucity of studies specific to the elasmobranch cardiovascular system, the degree to which oxygen delivery can be modulated over ontogeny, across phylogeny, or due to acclimation or acclimatization is still unknown. Understanding the physiological stress response of this group can not only inform best fishing practices, but also provide basic knowledge on this basal group of fishes.

16 THE CARDIORESPIRATORY STRESS RESPONSE OF FISHES

Both environmental changes associated with climate change and capture stress involve the restriction of oxygen delivery, either through reductions in ambient dissolved

(DO) concentrations (i.e., hypoxia), or through apneic asphyxia (i.e., temporary stoppage if gill-water gas exchange). Understanding how individuals respond to hypoxia at multiple physiological levels, and how they maintain oxygen delivery, is important for understanding the whole animal response (Farrell and Richards 2009; Portner 2010). To date, mechanistic explanations for observed phenomena such as species-specific post- release mortality rates of elasmobranch fishes (e.g., Musyl et al. 2011) and species- specific changes in distribution (e.g., Nye et al. 2009), are still lacking. The lack of a comprehensive understanding of these processes may be caused, in part, to the fact that reductions in oxygen delivery are frequently the result of multiple concomitant factors which have interactive, additive, antagonistic, or masking impacts (Sampaio and Rosa

2019). Multi-stressor studies are, therefore, necessary to ensure results are representative of conditions encountered in situ (McBryan et al. 2013; Sampaio and Rosa 2019).

There are known physiological mechanisms that offer a degree of tolerance to environmental and capture-associated stressors, however. In response to hypoxia, some teleost fishes are able to reduce their metabolic rate to survive periods of low to no oxygen (Sollid et al. 2005). Rising temperatures may reduce aerobic scope, or the energy available for growth, movement, and reproduction, but many fishes are able to move to avoid these areas (Lefevre 2016). Negative impacts of ocean acidification can likely be mitigated through increases in circulating bicarbonate concentrations (Sackville et al.

2018; Shartau et al. 2019). In response to capture stress, teleost species are able to

17 increase circulating levels of catecholamines which may help preserve blood-oxygen affinity and cardiac function (Farrell et al. 1983; Nikinmaa 1983; Cox et al. 2017).

Teleost fishes are also known to increase Hct levels in response to stress through splenic contraction to promote blood oxygen carrying capacity (Gallaugher and Farrell 1998).

All of the aforementioned stressors and their respective adaptations have been demonstrated to show some degree of species specificity and, more importantly, have generally been studied only in teleost fishes (Wendelaar-Bonga 1997; Hofmann and

Todgham 2010; Hannan and Rummer 2018; Cook et al. 2019). Comparatively little work has focused on elasmobranch fishes (Skomal and Mandelman 2012), likely due to the difficulties associated with maintenance in captivity for controlled experimental trials

(Bernal and Lowe 2016; Brill and Lai 2016).

The diversity of the elasmobranch group, however, likely limits generalizations based on a small number of study species, most of which occupy similar ecological niches (Brill and Lai 2016). For example, the method of carbon dioxide excretion was thought to differ dramatically from that of teleost fishes, with elasmobranch fishes relying heavily on extracellular sources of carbonic anhydrase (Gilmour and Perry 2010).

Recent work has, however, revealed that the singular study species often cited (spiny dogfish) is perhaps unique within the elasmobranch group (McMillan 2018). More comparative research would aid in understanding which physiological characteristics are widespread amongst elasmobranch fishes and which are highly species-specific.

DISSERTATION OBJECTIVES AND STRUCTURE

To further my knowledge of species-specific elasmobranch cardiorespiratory responses to stress, I conducted experiments focused on different levels of

18 cardiorespiratory physiology ranging from organismal to cellular processes. This dissertation comprises four studies focused on the use of traditional comparative physiological techniques to study the responses of coastal elasmobranch fishes to acute stress. Experiments were conducted under reduced oxygen with concomitant stressors to answer the following questions:

• How do simulated increases in temperature and decreases in pH impact

the metabolic capacity of clearnose skate (Rostaraja eglanteria), thorny

skate (Amblyraja radiata), and summer flounder (Paralicthyes dentatus)?

• How does blood oxygen affinity change under different temperature and

pCO2 conditions in clearnose skate and summer flounder?

• How does red blood cell volume change under exhaustive exercise and air

exposure in five species of elasmobranch fish?

• How does cardiac function change under capture-associated stressors in

three species of coastal elasmobranch, and is adrenergic stimulation

capable of mitigating these effects?

The general structure of this dissertation is as follows:

Chapter II details a set of experiments measuring metabolic rate and hypoxia tolerance under varying temperature and pH levels representative of the most extreme conditions experienced today. Relative to previous studies, this study uses stressors representative of present day in situ conditions. Additionally, this study incorporates comparisons across geographic range by including results from a closely related, yet allopatric, elasmobranch species and across phylogeny by including a sympatric teleost species. Results from this study have implications for future studies on environmental

19 stress associated with climate change and emphasize the importance of conducting multi- stressor experiments as physiological responses are not always additive.

Chapter III describes a study evaluating changes in blood oxygen affinity under stress, and the correlations between this metric and whole organism hypoxia tolerances of teleost and elasmobranch fishes. This study employs comparisons to published literature, with noticeable differences in trends between the teleost and elasmobranch groups.

Results from this study will inform future work on the mechanisms underpinning hypoxia tolerance in elasmobranch fishes and may provide guidance on the use of hypoxia tolerance metrics calculated from cellular, or molecular levels of biological organization.

Chapter IV examines the prevalence of RBC swelling in coastal elasmobranch fishes. This study employs comparisons to published literature, with noticeable differences in the findings regarding sandbar shark (Carcarhinus plumbeus) and epaulette shark (Hemiscyllium ocellatum). Results from this study will inform future work on the mechanisms underpinning hypoxia tolerance in elasmobranch fishes, with regard to the level of stress needed to elicit a physiological response.

Chapter V details a study focused on the impacts of simulated capture stress on the functional capacity of elasmobranch myocardial function. This study tested the impacts of hyperkalemia, acidosis, low O2, and acute temperature change on contractile properties, as well as the ability of an adrenaline analog to restore function following stress exposure. Results from this study reveal the levels of stress applied elicited few significant changes and were likely not severe enough to cause cardiac detriments which would drive post-release mortality.

20 Chapter VI summarizes findings and comments on the relevance of these findings for the greater field of fish physiology with a presentation of avenues for further research.

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37 CHAPTER II

Combined effects of acute temperature change and elevated pCO2 on the metabolic rates

and hypoxia tolerances of clearnose skate (Rostaraja elganteria), summer flounder

(Paralichthys dentatus), and thorny skate (Amblyraja radiata)

38 ABSTRACT

Understanding how rising temperatures, ocean acidification, and hypoxia affect the performance of coastal fishes is essential to predicting species-specific responses to climate change. Although a population’s habitat influences physiological performance, little work has explicitly examined the multi-stressor responses of species from habitats differing in their natural variability. Here, clearnose skate (Rostaraja eglanteria) and summer flounder (Paralichthys dentatus) from mid-Atlantic estuaries, and thorny skate

(Amblyraja radiata) from the Gulf of Maine were acutely exposed to current and projected temperatures (20, 24 or 28°C; 22 or 30°C; and 9, 13, or 15°C, respectively) and acidification conditions (pH 7.8 or 7.4). We tested metabolic rate and hypoxia tolerance using intermittent-flow respirometry. All three species exhibited increases in standard metabolic rate under an 8°C temperature increase (Q10 of 1.71, 1.07, and 2.56, respectively), although this was most pronounced in the thorny skate. At the lowest test temperature and under the low pH treatment, all three species exhibited significant increases in standard metabolic rate (22-115%; p<0.05) and decreases in hypoxia tolerance (85-150% increases in Pcrit; p<0.05). This study demonstrates the species- specific interactive effects of increasing temperature and changing ocean carbonate chemistry, the implications of which should be considered within the context of habitat.

39 INTRODUCTION

Marine climate change includes (but is not limited to) rising temperatures, increasing severity and frequency of hypoxic events, and ocean acidification

(OA)(Hoegh-Guldberg and Bruno 2010; Melzner et al. 2013; McKenzie et al. 2016;

Poloczanska et al. 2016). These three environmental changes result in interactive, yet poorly understood, impacts on both individuals and populations (Di Santo 2016; Bennett et al. 2019). Increases in temperature alone are associated with reduced aerobic scope, and thereby reduced fitness (Clark et al. 2013; Nilsson and Lefevre 2016). Hypoxia likewise reduces fitness and can cause mortality events (Claireaux and Chabot 2016), while OA can impact various aspects of a species’ biology from behavior to growth rates

(Heuer and Grosell 2014). A comprehensive understanding of the impacts of climate change on populations is, therefore, required both to manage fisheries effectively and to conserve ecologically and economically important resources (Horodysky et al. 2015;

Baumann 2019; Lyons et al. 2019).

Testing the environmental tolerances of individual species is commonly done using aerobic metabolic rate as a proxy for fitness (Clark et al. 2013; Lefevre 2016;

McKenzie et al. 2016). Intermittent flow respirometry measures rates of oxygen consumption that can be used to calculate a range of metabolic parameters including standard metabolic rate (SMR), maximum metabolic rate (MMR), absolute aerobic scope

(ASa = MMR-SMR), factorial aerobic scope (ASf = MMR/SMR), and critical oxygen level (Svendsen et al. 2016). Critical oxygen level is the lowest oxygen level at which

SMR remains stable, and below which metabolic rate declines in step with decreases in ambient oxygen (Speers-Roesch et al. 2012b). The critical oxygen level can be measured

40 in terms of percent saturation (Scrit), oxygen content (Ccrit), or partial pressure (Pcrit)

(Crear et al. 2019). Combined, these metabolic parameters can assess species- or population-specific tolerances to environmental perturbations, such as those associated with climate change (Brill 1996; Claireaux and Lagardère 1999a; Horodysky et al. 2015;

Teal et al. 2015). Because it is impossible to fully incorporate the complexity of ecological interactions (e.g., interspecific interactions, regional population distributions, seasonal variability) in models designed to predict the effects of climate change, researchers have attempted to assess vulnerability or resilience using other methods.

According to the theory of oxygen- and capacity-limited thermal tolerance (OCLTT)

(Portner 2010), aerobic scope will decline at sub-optimal temperatures because the ability of the cardio-respiratory system to supply oxygen to tissues is reduced. While the applicability of this theory is still debated (Jutfelt et al. 2018), measuring aerobic scope likely can provide information on species-specific physiological abilities and tolerances necessary to predict the effects of shifting environmental conditions (Allen et al. 2014;

Esbaugh 2018).

Most research concerning the effects of environmental stressors on marine organisms has focused on temperature or pH changes projected to occur over the next 50 to 100 years in the open ocean (Munday et al. 2012). Such studies must, however, reconcile the uncertainty in environmental forecasting with the difficulty of accounting for transgenerational effects (Rummer et al. 2013), the ability of species to alter their distributions (Morley et al. 2018), and localized adaptation (Di Santo 2016).

Additionally, while many perturbation experiments have focused on the specifics of various laboratory treatments, less attention has been given to the natural short-term

41 fluctuations in environmental conditions encountered by coastal species throughout their range or over ontogeny (Baumann and Smith 2017; Morash et al. 2018; Baumann 2019).

Estuarine environments exhibit regular, acute hypoxia and pH variability

(Breitberg 1990; Baumann et al. 2014), and species inhabiting these environments likely possess the physiological abilities necessary to withstand a broad range of environmental conditions (Norin and Metcalfe 2019), potentially providing some degree of resiliency in the face of climate change. While there is evidence that species living in variable habitats

(such as rocky pools) are already living near the limits of their physiological capabilities

(Fangue et al. 2001), little research has been done to explicitly compare the tolerances of fishes from variable estuarine environments to those from more stable habitats (e.g., higher latitudes or deeper waters) (Morash et al. 2018). Species (or populations) inhabiting variable temperate environments tend to be eurythermal, whereas species (or populations) occupying high latitudes tend to be stenothermal (Hickling et al. 2006; Nye et al. 2009), likely due to the relatively narrow range of temperatures encountered by any given individual (Schulte 2015). As temperature has a large impact on the metabolism of ectotherms (Fry 1947; Clarke and Johnston 1999; Bernal and Lowe 2016), a comparison between the thermal tolerances of species inhabiting variable and stable habitats may provide insight into the species-specific physiological abilities, and thus their capacity to withstand the impacts of climate change (Morash et al. 2018; Bennett et al. 2019).

The east coast of the United States includes habitats that differ greatly with respect to their environmental variability (Atkinson et al. 1983; Breitberg 1990; Saba et al. 2016; Baumann and Smith 2017). In the mid-Atlantic, inshore species often utilize salt marsh lagoons during the summer where daily oscillations of ± 5°C are frequently

42 accompanied by fluctuations in pH (± 0.5 pH units) and dissolved oxygen (± 4.5 mg L-1)

(Breitberg 1990; Breitberg et al. 2015). As climate change effects continue to manifest, these environments are likely to experience even greater swings in temperature, pH, and dissolved oxygen (DO) (Cochran and Burnett 1996; Breitberg et al. 2009; Diaz and

Breitburg 2009), which are likely to affect fish species such as the clearnose skate

(Rostaraja eglanteria) and summer flounder (Paralichthys dentatus). Clearnose skate range from the Gulf of Mexico to Cape Cod, USA (Packer et al. 2003) and are common in the tidal lagoons along the mid-Atlantic. They occur over a temperature range from

~9–30 °C, but prefer ~9–21°C (McEachran and Musick 1975; Schwartz 1996). Summer flounder utilize near-shore regions during the summer but migrate offshore to spawn in the fall and prefer temperatures between 9-24 °C (Scott and Scott 1988; Buchheister and

Latour 2010). In contrast, the Gulf of Maine is a more stable environment (Saba et al.

2016), with benthic temperature fluctuations limited to ~3°C (Petrie and Drinkwater

1993). Thorny skate (Amblyraja radiata) inhabit the Gulf of Maine and are most abundant between 1-5 °C (Swain and Benoit 2006), therefore occupying a habitat that is very different from that (at least during the summer months) of clearnose skate or summer flounder.

We therefore sought to quantify the effects of acute temperature change and elevated pCO2 levels on the aerobic metabolic rates and hypoxia tolerances of clearnose skate, summer flounder, and thorny skate. This approach quantified the physiological abilities of sympatric elasmobranch and teleost species (clearnose skate and summer flounder, respectively), and an allopatric elasmobranch species (i.e., thorny skate) occupying the more environmentally stable Gulf of Maine. We aimed to explore the

43 species-specific environmental tolerances that are likely to influence climate change responses in the future, and explore tolerances of species that inhabit different habitats varying in their degree of environmental fluctuation.

MATERIALS AND METHODS

All capture, handling, and experimental protocols were approved by the College of William and Mary and University of New England Institutional Animal Care and Use

Committee (IACUC-2017-03-14-11935-rwbril and IACUC- 012418-003, respectively).

Clearnose skate and summer flounder were collected from the Eastern Shore of Virginia using rod and reel during summers 2016 and 2017, and maintained in recirculating systems at the Virginia Institute of Marine Science (VIMS) Eastern Shore Laboratory at

20 - 22°C. Thorny skate were collected from the Gulf of Maine using a commercial otter trawl (Cicia et al. 2012) in February 2018, and maintained in flow through systems at the seawater laboratory at the University of New England at 5, 9, or 13°C. All individuals were given at least two weeks to acclimate to captivity before use in experimental trials and were fed ad libitum every 2-3 days. Individuals were fasted for 48 hrs prior to use in an experiment to ensure they were in a post-absorptive state (Capossela et al. 2012).

A total of 24 clearnose skates and 17 thorny skates were subjected to three to four trials each, resulting in eight trials at each of three temperatures representing the mid- to upper-range of thermal tolerances (20, 24, 28°C for clearnose skate; 5, 9, 13°C for thorny skate), under two CO2 conditions representing present day and that predicted to occur by end of century (pH of 7.8 and 7.4, respectively) (Najjar et al. 2008; Najjar et al. 2010). A total of eight summer flounder were subjected to two trials each at 22 and 30°C under

44 elevated pCO2 conditions (pH 7.4). Because the most extreme treatment (28°C and elevated pCO2) resulted in 40% mortality in preliminary trials on clearnose skate, this treatment was discontinued. The equivalent treatment (13°C and elevated pCO2) thorny skate experiments was likewise excluded.

Clearnose and thorny skates were acclimated to trial conditions for 48 hrs and one week respectively. For clearnose skate, MMR was obtained using an established chase protocol involving enforced exercise (i.e., chasing and turning individuals over to induce swimming until they no longer responded to tactile stimulus) followed by one minute of air exposure (Di Santo 2016). Thorny skate respond to being handled by curling into a ball, and thus could not be chased. Instead, this species was air exposed for eight minutes.

Respirometry protocols used with summer flounder were modified from those of

Capossela et al. (2012) in that fish were not fitted with additional sensors to measure exhalent oxygen. As Capossela et al. (2012) only measured SMR and Pcrit, those were the only variables measured from the summer flounder. These were calculated as described below. We therefore re-analyzed the data from Capossela et al. (2012) along with the elevated pCO2 data collected for this study.

Following the chase and/or air exposure protocols, individuals were placed in custom-built Plexiglas respirometers constructed to ensure the volume to animal mass ratio fell between 30:1 and 50:1. The chambers were equipped with fiber optic oxygen sensors and a recirculating pump, as recommended by Rogers et al. (2016). The respirometry chambers were placed in an outer water bath from which water used to flush the respirometer was taken. A computer program (developed in Dasylab 13.1; National

Instruments, www.ni.com) logged data and continuously controlled temperature and

45 oxygen levels for 36 hours. Each measurement cycle lasted 10 minutes and began with a

5-minute flush cycle, which was terminated when the flush pump was turned off. After allowing 2 min for the water in the measurement system to mix, the decline in oxygen over a 5-minute period was recorded before the flush pump was turned on again. At no time during normoxic trials was the chamber oxygen level allowed to fall below 80%. At the end of each data recording interval, the Dasylab software executed a call to an Excel macro routine that calculated the rate of change of O2 content (converted from percent

−1 saturation) with time ([O2]*t ) based on a linear regression of the recorded oxygen levels against elapsed time (t). The Excel macro routine subsequently calculated MO2 as:

−1 –1 MO2 = ( [O2]t )*V*W where: V = respirometer volume (l) corrected for fish volume and W = fish mass (kg). To estimate microbial oxygen consumption, rates of O2 depletion were measured both before and after the trial (i.e., when the fish was not in the chamber). We used linear regression to estimate rates of oxygen depletion due to microbial respiration occurring over the time

-1 -1 course of an experiment (generally less than 0.1 mg O2 l hr ). These values were then subtracted from the measured rates of oxygen decline.

MMR was taken as the single highest metabolic rate measured during the first 12 hours following the chase and/or air exposure protocols. For all three species, SMR was taken as the mean of the lowest 10 metabolic rates during the middle 12 hrs of the trial.

-1 Aerobic scope was calculated two ways (ASa=MMR-SMR; ASf=MMR*SMR ).

Following SMR measurements, oxygen was reduced in a step-wise fashion, with three measurements taken at 80, 60, 40, 30, 20, and 10% O2 saturation. Trials were terminated when MO2 dropped to zero and individuals were then allowed to recover in fully

46 oxygenated seawater for 1 hr before being returned to holding tanks. Scrit was defined as the point at which an individual could no longer maintain SMR. This was done following

Schurmann and Steffensen (1997), where metabolic rate measurements below SMR were evaluated to determine if all subsequent values were also below SMR. When this was true, this subset of points was fit with a linear regression. The oxygen content where this regression line and SMR intersected was defined as Scrit (Figure 1). From these, we

- calculated the critical oxygen content (Ccrit) by converting the percent saturation to mg L

1 using known temperature and salinity values. We also calculated critical oxygen partial pressure (Pcrit) by determining the partial pressure of oxygen at 1 atmosphere on the day of the start of the trial, calculating the percent saturation of seawater, and multiplying that by the temperature- and salinity-specific oxygen content of seawater at full saturation.

To better compare across different temperature ranges, we calculated Q10 values for SMR as:

10/(T1-T2) Q10 =(R2/R1) , (

where Q10 is the temperature coefficient for SMR, R1 is the SMR at T1 and R2 is the SMR at T2.

To increase pCO2, we used the standard method of bubbling CO2 gas (Michaelidis et al. 2007). A stand-alone system (TUNZE 7074; www.tunze.com/US/en), connected to a laboratory-grade glass pH probe in the outer water bath, controlled an electronic solenoid valve connected to a cylinder of CO2. The system injected a slow stream of

CO2 into the outer water bath whenever pH of the seawater rose above the set point.

Using this method, it was possible to maintain pH within ± 0.05 units of the desired level,

47 and it was unlikely that there was a biologically significant gradient in seawater pH within the tanks as the outer water bath was continuously mixed by a submersible pump.

The pH of each outer water bath in the low pH treatment was independently validated at the start and end of each trial using a SDL100 pH meter (Extech Instruments) calibrated daily with fresh pH buffers (Tunze). Additionally, water samples were taken at the start and end of each trial for dissolved inorganic carbon (DIC) and total alkalinity

(TA) AIRICA and Metrohm analyses, or for spectrophotometric determination of pH and

TA Metrohm analysis (Dickson et al. 2007). All pH values were subsequently calculated from these additional measurements using the CO2SYS software (US Geological Survey) with the constants K1 from Mehrbach et al. (1973) (refit by Dickson and Millero (1987)), and KHSO4 from Dickson et al. (2007). Data on the seawater chemistry of present day pCO2 trials were obtained from 10 samples taken from the water inflow to the seawater laboratories in Virginia and Maine (Table 1). Equivalent seawater chemistry data for the summer flounder experiments performed by Capossela et al. (2012) were not available.

All statistical analyses were conducted using SAS 9.4 (SAS Institute). Data were analyzed using a multivariate repeated measures ANOVA using the MIXED procedure to account for the correlation between metabolic indices, with individual being the random factor upon which multiple measures were made (Lapointe et al. 2014). SMR, MMR,

ASa, and Pcrit were considered response variables, and temperature, pCO2 level, and a dummy variable representing the number of repetitions being measured on a single individual were considered factors. We modeled the heterogeneity in responses among temperature treatments and specified the Kenward-Roger method for calculating the degrees of freedom (Kenward and Roger 1997). Model selection between different

48 variance/covariance structures was performed using BIC, and significant differences were determined using 95% confidence intervals derived using the LSMestimate statement in

SAS. Model data are presented from model structures using compound symmetry correlation structures. All statistics were evaluated with a significance level of = 0.05.

RESULTS

Water chemistry values (Table 1) were largely consistent with published values for both the Chesapeake Bay (Cai et al. 2011; Tzortziou et al. 2011) and the Gulf of

Maine (Cai et al. 2010; Signorini et al. 2013). The elevated pCO2 treatment had higher calculated pCO2 values than expected (Cai et al. 2010; Cai et al. 2011).

We collected data from 24 clearnose skate (1.3 ± 0.06 kg; mean mass ± SE), 17 thorny skate (1.4 ± 0.2 kg), nine summer flounder (0.36 ± 0.01 kg), and re-analyzed data from nine summer flounder reported by Capossela, et al. [53]. Despite high variability within any given parameter, models revealed differences among the metabolic response to changing environmental parameters. Model-derived estimates for all parameters can be found in Supplemental Table 1.

Standard Metabolic Rate and Q10

We observed an increase in SMR with increasing temperature in both skate species during the present day pCO2 (i.e. at high pH) experiments (Figure 2a, c; Table 1).

The effect of temperature on SMR was not apparent during the elevated pCO2 (i.e., low pH) experiments. In clearnose skate, SMR was significantly higher under the elevated pCO2 at 20°C and 24°C (Figure 2a). The SMR of summer flounder significantly increased between 22° and 30°C (p<0.01), and elevated pCO2 caused SMR to increase at

49 22° (p<0.01). There was no significant effect of temperature on SMR under elevated pCO2 (Figure 2b). In contrast, thorny skate did not demonstrate any significant differences in SMR elevated pCO2 at either test temperature (Figure 2c).

To facilitate comparisons across species, we compiled Q10 values for SMR for all three species (Table 2). Clearnose skate and summer flounder had relatively low Q10 values at the present day pCO2 treatment, while thorny skate Q10 was more similar to the expected value between 2 and 3 (Lefevre 2016; Nilsson and Lefevre 2016). Under elevated pCO2, clearnose skate and summer flounder Q10 values more than doubled, whereas thorny skate Q10 increased to a lesser degree.

Maximum Metabolic Rate and Aerobic Scope

The difference in mean MMR at 20° in clearnose skate was nearly significant between the two pCO2 conditions (Figure 3a; p=0.051). Thorny skate showed an increasing trend in MMR within a given temperature (at 9°C) at the elevated pCO2

(Figure 3b; p=0.07, p=0.07 respectively).

The ASa of clearnose skate did not vary significantly under any of the treatment conditions. The ASa of thorny skate was significantly higher at 5° under elevated pCO2.

To compare results between species, and following the recommendations of Clark et al.

(2013) and Lapointe et al. (2014), we have included plots for ASa and ASf in Figure 4.

No statistical analysis was performed on ASf as the two metrics were too similar to be fitted by the model.

Hypoxia Tolerance

The hypoxia tolerance of clearnose skate under present day pCO2 (i.e., high pH) was reduced at increased temperature, as shown by a significantly higher Pcrit at 28°

50 compared to 20° and 24° (p<0.01 for both). Under elevated pCO2, we observed a significant increase in Pcrit between 20° and 24° (p=0.04). Clearnose skate exhibited marked reductions in hypoxia tolerance under elevated pCO2 (Figure 5a), with significant elevations in Pcrit at 20° and 24° (p<0.01 for both). Summer flounder showed the expected significant increase in Pcrit under elevated temperatures at present day pCO2

(p<0.01), as well as a significant increase under elevated pCO2 at 22° (p<0.01; Figure

5b). The Pcrit of thorny skate at 13° was significantly higher compared to that measured at

5° under presnt day pCO2 (p=0.04); and Pcrit at 5° was significantly higher at elevated pCO2, than under present day pCO2 (p<0.01; Figure 5c).

DISCUSSION

Our study compares the environmental tolerances of species in two disparate environments under projected conditions (elevated temperature and elevated pCO2), focusing on how observed tolerances are impacted by multiple, concurrent stressors. In general, the physiological abilities to withstand acute exposure to environmental stressors were more similar between the sympatric species (clearnose skate and summer flounder) than to the abilities of the allopatric species (thorny skate).

Although the pCO2 values used for the elevated pCO2 treatment were somewhat variable, targeted pH values were maintained. For the purposes of interspecific comparison, moreover, the difference between the present day and elevated pCO2 treatments is likely more important than the actual values. The variability observed in the present-day conditions is likely a result of the natural conditions in near-shore water pumped in the seawater facilities; additional manipulation of the carbonate chemistry of

51 the seawater was deemed cost-prohibitive, and likely unwarranted as the modified seawater would not mimic estuarine conditions.

-1 -1 The SMR values measured at present-day pCO2 levels (38.8 ± 4.2 mg O2 kg hr

-1 -1 at 20° for clearnose skate; 45.3 ± 3.4 mg O2 kg hr at 22° for summer flounder; 15.9 ±

-1 -1 2.8 mg O2 kg hr at 5° for thorny skate; Figure 2) were lower than other studies on mid-

-1 -1 Atlantic estuarine teleost fishes at similar temperatures (e.g., 100-150 mg O2 kg hr at

-1 -1 20° (Marcek 2018); 68-84 mg O2 kg hr at 10° (Steinhausen et al. 2005)), although this could be attributed to the demersal nature of these study species. The increase in SMR under elevated pCO2 within the lowest test temperatures for all three species (105, 42, and 22% increase for clearnose skate, summer flounder, and thorny skate, respectively), and the declining difference in SMR between pCO2 treatments at elevated temperatures

(10, -16, and 16% for clearnose skate, summer flounder, and thorny skate, respectively;

Figure 2) matches trends from little skate (Leucoraja erinacea) exposed to elevated temperatures and pCO2 (Di Santo 2016), but differ from similar studies on other elasmobranch species (Esbaugh et al. 2012; Green and Jutfelt 2014; Heinrich et al. 2014;

Bouyoucos et al. 2019). This suggests there may be conserved physiological mechanisms driving this response, but much is still unknown regarding the mechanisms driving the observed patterns. The results presented here may be due to bradycardia, increased ventilatory rates, and increased blood pressure (Perry and McKendry 2001; Perry and

Gilmour 2002; Ishimatsu et al. 2004), or to increased metabolic cost of buffering against plasma pH changes (Lefevre 2016) and increased ion transport (Esbaugh 2018). These known physiological stresses are unlikely to increase metabolic rate to the extent we

52 observed in this study, however. An alternative explanation is that individuals are more active under elevated pCO2 (i.e., low pH) conditions (Nagelkerken and Munday 2016).

The effect of elevated pCO2 on SMR masks the temperature effect observed under present-day conditions, indicating that the responses to these stressors are not additive.

This may be due to an alternative version of the OCLTT hypothesis where the physiological consequences of elevated pCO2 (rather than temperature) are predicted to limit oxygen delivery (Pörtner and Knust 2006; Portner 2010). Different responses to temperature under present-day conditions and elevated pCO2 suggest there are interactive mechanisms regulating oxygen delivery in fishes (Heuer and Grosell 2014; Heuer et al.

2016; Nilsson and Lefevre 2016; Esbaugh 2018). Elevated plasma levels of CO2 (with concomitant reductions in plasma pH) reduce hemoglobin oxygen affinity (Bohr effect) and maximum blood oxygen content (Root effect); although the extent of these is unknown in the species studied here. Alternatively, the effects of one stressor could be compensating the effects of the other (Esbaugh 2018), resulting in the masking effects.

For example, increased metabolic costs of acid-base regulation under ocean acidification could be offset by reduced energetic demand elsewhere. This phenomenon as has been demonstrated with low pH-induced metabolic depression in isolated gill cells (Stapp et al.

2015). Given the large knowledge gaps concerning the mechanisms underpinning our results, we argue (as have others) that more multi-stressor studies are needed (Burnett

1997; Portner et al. 2004; Sackville et al. 2018; Baumann 2019; Lyons et al. 2019).

Clearnose skate and summer flounder exhibited lower Q10 values (Q10 = 1.62 and

1.07, respectively) at present day pCO2 than the thorny skate (Q10 = 3.87). While Q10 values lower than 2 have been associated with a decreasing ability to function

53 (Hochachka and Somero 2002), for the two Mid-Atlantic estuarine species studied here

(i.e., clearnose skate and summer flounder) the low Q10 values rather are indicative of the ability to maintain a consistent level of aerobic ATP production over a relatively broad range of temperatures (Clarke and Johnston 1999; Turingan and Sloan 2016), potentially signifying resilience to the coastal warming predicted under climate change (Allen et al.

2014). High Q10 values, in contrast, have been attributed to species from stable environments (Rummer et al. 2014). The thorny skate, therefore, may not possess isozymes (or the genetic plasticity to produce isozymes) that reduce the effects of temperature on metabolic rate over a broad range of temperatures (Hochachka and

Somero 2002; Hofmann and Todgham 2010; Norin and Metcalfe 2019), and may thus be more sensitive to temperature increases than the other two study species. The idea that the effects of temperature on metabolic rate are closely associated with native thermal range (Clarke and Johnston 1999) has received mixed support from other studies looking specifically at different populations or species. For example, Di Santo (2016) found increased sensitivity to temperature in more northern populations of little skate.

According to the evolutionary trade-off hypothesis (Clarke and Fraser 2004), the resting metabolic rate of a species (or population) at over its normal environmental temperature range represents an evolutionary optimization. In other words, species- or population- specific optimization of metabolic rates to a given temperature (or range of temperatures) might not be explained purely through the kinetic energy of sub-cellular constituents, but rather may be a suite of complex tradeoffs (Hochachka and Somero 2002; Clarke 2004;

Clarke and Fraser 2004). This becomes evident as all three species exhibited a decrease in Q10 under elevated pCO2, driven by increases in SMR and emphasizing the masking

54 impact of this additional stressor. Under projected climate change scenarios, elevated temperatures and ocean acidification are likely to have interactive effects on cellular processes (Clarke and Johnston 1999; Feder and Hofmann 1999; Kultz 2005; Bernal and

Lowe 2016). While the Q10 values presented here offer some insight into species-specific sensitivity, more research on the interactive effects of multiple, concurrent stressors on metabolism is needed.

We did not observe any significant trends in MMR between the two skate species with either temperature or pCO2 (Figure 3). This may be due to an insufficient stressor prior to the respirometry trial. Although the values presented here are lower than published values for other fish species measured at similar temperatures (Claireaux and

Lagardère 1999b; Claireaux et al. 2000; Marcek 2018), this could also be attributed to the more sedentary life style of our study species. Alternatively, the Fry paradigm (Fry 1947) for diminishing MMR values above an optimal temperature may not hold in these species

(Lefevre 2016). There is widespread dissent in the literature regarding the appropriate methods to obtain MMR (Peake and Farrell 2006; Lefevre 2016) and whether the standard Fry paradigm is valid, which limit more definitive conclusions from these data.

The aerobic scope data do not support the existence of a bell-shaped curve centered on a single optimal temperature (Topt), but rather AS being relatively temperature-independent. These results may be driven by multiple Topt values for different physiological processes (Clark et al. 2013; Schulte 2015), and are consistent with other studies (Melzner et al. 2009; Couturier et al. 2013; Rummer et al. 2013; Green and Jutfelt 2014; Lefevre 2016; Hamilton et al. 2017). The lack of significant reduction in aerobic scope under high stress conditions suggests that clearnose and thorny skates may

55 exhibit resilience to climate change in their respective environments. Given there have been conflicting reports on the capacity of elasmobranch species to acclimate to climate change conditions (Pistevos et al. 2015; Gervais et al. 2018), the findings of this study represent an important step in understanding the physiological tolerances of this understudied group (Dulvy and Reynolds 2002). An important caveat is that because we used wild-caught adults, any early life history detriments to condition and survival (Rosa et al. 2014; Di Santo 2015; Pistevos et al. 2015) remain unmeasured.

Our most significant finding, however, may be that clearnose skate (Pcrit 32 ± 2 mmHg at 20°C; mean ± SE) are as hypoxia tolerant as epaulette shark (Pcrit 38 mmHg at

28°C); and the latter has been deemed to have exceptional hypoxia tolerance (Nilsson and

Renshaw 2004; Speers-Roesch et al. 2012a). While the physiological mechanisms underlying the hypoxia tolerance of epaulette shark have received considerable attention

(Routley et al. 2002; Chapman and Renshaw 2009; Heinrich et al. 2014; Brill and Lai

2016; Heinrich et al. 2016; Johnson et al. 2016), there are no equivalent data for clearnose skate, and we encourage studies in this area. Our Pcrit data show, however, that summer flounder are also tolerant of hypoxia (Pcrit = 42 mmHg at 22°C). Considering the correlations between hypoxia tolerance and the environmental variability of a species’ native habitat, we note epaulette shark live in reef and tidal environments that experience large diel and tidal cycle fluctuations in temperature, oxygen and pH, similar to the changes occurring in estuaries along the US mid-Atlantic (Shaw et al. 2013; Baumann et al. 2014; Baumann and Smith 2017). Other species from variable environments are also hypoxia tolerant, including blue crabs (Calinectus sapidus) (Brill et al. 2015) and crucian carp (Carassius carassius), as well as many rocky tidepool fishes (Fangue et al. 2001).

56 These results are not ubiquitous, however. Sandbar shark (Carcharhinus plumbeus) have a markedly higher Pcrit value than the clearnose skate or summer flounder (Crear et al.

2019), despite being a sympatric species. As sandbar shark are an obligate ram- ventilating species (Bernal and Lowe 2016), this difference is unsurprising and is supported by findings on bonnethead shark (Sphyrna tiburo) which live in seagrass meadows likely to experience large diel cycles in dissolved oxygen. In contrast to clearnose skate and summer flounder, thorny skate are relatively intolerant of hypoxia

(Pcrit = 75 mmHg at 9°C), most likely because this species occupies the Gulf of Maine, an environment that does not exhibit wide swings in temperature and oxygen levels (Petrie and Drinkwater 1993; Saba et al. 2016). Similarly, the shovelnose ray (Aptychotrema rostrata) that occupies an environment where it rarely encounters hypoxia (Dennison et al. 2004) has a Pcrit = 54 mmHg at 28°C (Speers-Roesch et al. 2012a), indicating that this species in more intolerant of hypoxia than are clearnose skate or epaulette shark.

The increases in Pcrit under elevated pCO2 (150, 85, and 113% increases in Pcrit for clearnose skate, summer flounder, and thorny skate, respectively) may be due to the inability of the non-bicarbonate blood buffering capacity all three study species to limit reductions in plasma pH (and subsequently the intracellular environment) under elevated pCO2. To the best of our knowledge, there is no information regarding intracellular pH

(pHi) of elasmobranch red blood cells following exposure to simulated OA. Studies on brain, white muscle, and liver tissue isolated from teleost fishes and exposed to elevated pCO2 have, however, found either no change or an increases in pHi (Esbaugh et al. 2012;

Strobel et al. 2012; Heuer et al. 2016; Esbaugh 2018), suggesting OA may not have a negative impact blood oxygen transport. This is supported by a lack of increase in

57 hematocrit following exposure to elevated pCO2 (Strobel et al. 2012; Green and Jutfelt

2014; Esbaugh et al. 2016). As the differences in Pcrit were most apparent in the mid-

Atlantic species, further work on in vivo blood pH levels under changing pCO2 conditions, as well as quantification of the changes in blood oxygen affinity (Bohr shift) and maximum oxygen carrying capacity (Root effect), in these species would help elucidate the mechanisms underpinning our observed reductions in hypoxia tolerance.

Because of the high hypoxia tolerance of clearnose skate, we predict this species may also demonstrate a high blood oxygen affinity and a large Bohr effect similar to that seen in the hypoxia tolerant bat ray (Myliobatis californica) (Hopkins and Cech Jr 1994;

Speers-Roesch et al. 2012a). We also expect summer founder blood to have similar physiological characteristics to that of blood from European flounder (Platichthys flesus)

(Weber and de Wilde 1975).

These mechanisms are largely speculative, however, as there are conflicting reports of the effects of elevated pCO2 on hypoxia tolerance. For example, epaulette shark do not exhibit decreases in hypoxia tolerance under elevated pCO2 conditions

(Heinrich et al. 2014). This may be attributed to the chronic (60-day) exposure of epaulette shark to elevated pCO2 conditions, compared to the acute exposures we employed. Other studies have reported increases in Pcrit under elevated pCO2 in the

European eel (Anguilla Anguilla) (Cruz‐Neto and Steffensen 1997) and European flounder (Rogers 2015); and in acidified water for rainbow trout (Salmo gairdneri) and carp (Cyprinus carpio) (Ultsch et al. 1980). These results are, however, not universal

(Rogers et al. 2016) as croaker (Leiostomus xanthurus) and mummichog (Fundulus heteroclitus) exhibit no change in Pcrit under elevated pCO2 (Cochran and Burnett 1996;

58 Rogers et al. 2016). We note, however, these two species are common occupants of mid-

Atlantic estuaries and therefore regularly experience elevated pCO2 conditions (Cochran and Burnett 1996).

From an ecological perspective, the observed effect of elevated pCO2 on hypoxia tolerance is concerning. Currently, clearnose skate and summer flounder are unlikely to encounter waters below their Pcrit, assuming the water is at a pH of 7.8 (Baumann et al.

2014; Morash et al. 2018; Baumann 2019). Due to the effects of climate change, however, individuals in coastal waters are more likely to experience concurrent hypoxia and elevated pCO2 (Breitberg 1990; Najjar et al. 2010; Miller et al. 2016; Breitburg et al.

2018). While estuarine and coastal species may be able to tolerate current conditions, further extremes of these parameters may force populations to move to alternative habitats. While at present it is unlikely that thorny skate regularly encounter hypoxia, warming shelf waters could induce changes in dissolved oxygen distribution resulting in unfavorable habitats in areas such as the Gulf of Maine (Petrie and Drinkwater 1993;

Petrie and Yeats 2000; Saba et al. 2016). Activity patterns observed in dogfish

(Scyliorhinus canicula) suggest that sluggish benthic elasmobranch species do not increase activity under hypoxic conditions (Metcalfe and Butler 1984), although the more active bonnethead shark does (Parsons and Carlson 1998). The sedentary strategy of non- obligate ram ventilating species could, therefore, limit their ability to exploit novel habitats under unfavorable environmental conditions.

Recently, Wood (2018) argued that Pcrit as a metric of hypoxia tolerance is of limited utility due to numerous factors including the lack of repeatability and consistency and an insufficient theoretical underpinning. He proposed several alternative metrics that

59 could be used in place of Pcrit, including loss of equilibrium or measurements of ventilation. While we agree that alternative measure of hypoxia tolerance can provide useful information, for the purposes of our study of benthic flatfishes, the loss of equilibrium is not a useful metric. Further, because we standardized the calculation of

Pcrit was standardized across all three species, concerns regarding different methodology have been alleviated. We agree with Regan et al. (2019) that “Pcrit contributes to a more complete picture of an animal’s total hypoxic response by capturing the suite of aerobic contributions to hypoxic survival in a single value,” and hope the data presented here can help further our understanding of hypoxia tolerance in a range of coastal species.

CONCLUSIONS

Understanding the species- and population-specific response to the multiple environmental stressors associated with climate change is essential for managing marine sources in a changing environment. The results presented here quantify the physiological limits of clearnose skate, summer flounder, and thorny skate with respect to acute changes in temperature and elevated pCO2. All three species exhibited increases in SMR

(105, 42, and 22% for clearnose skate, summer flounder, and thorny skate respectively) at the lowest test temperature under elevated pCO2, and this increases masked increases in

SMR at the higher test temperatures. All three species also showed decreased hypoxia tolerance (150, 85, and 113% increases in Pcrit) under the most extreme combined stressors. While the clearnose skate did exhibit remarkable hypoxia tolerance under the least stressful treatment, as climate change impacts continue to increase in severity, even this species may be pushed towards or past the limits of their physiological capabilities.

60 Incorporating multi-stressor studies into future climate change research is essential to predicting how species will respond to changing environmental conditions. If conditions are near the limits of physiological abilities, individuals may choose to seek out more favorable habitats, resulting in shifting distributions, fecundities, and food web dynamics with cascading ecological and economic implications.

ACKNOWLEDGEMENTS

We would like to thank Karen Capossela for sharing her raw data. Elizabeth Shadwick,

Olivia De Meo, Annie Shatz, and Emily Rivest assisted with carbonate chemistry analysis. We would like to thank the staff of the Virginia Institute of Marine Science

Eastern Shore Laboratory and of the University of New England Marine Science Center for their help facilitating this work. Two anonymous reviewers provided useful suggestions on a previous version of this manuscript.

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72 Table 1. The carbonate chemistry parameters during the high and low pH (i.e., present day and elevated pCO2) experiments. Values are averaged across all temperature treatments. The asterisks represent values that were calculated using CO2SYS, rather than being measured directly. All values represent mean ± SD.

Alkalinity pCO2 Species pCO2 Treatment pH (μmol kg−1 SW) (matm)

Present day 7.84 ± 0.02* 2317 ± 17 703 ± 33* Clearnose skate Elevated 7.44 ± 0.04* 2285 ± 14 2290 ± 262*

Present day Unknown Unknown Unknown Summer flounder Elevated 7.46 ± 0.06* 2258 ± 11 2204 ± 301*

Present day 7.87 ± 0.04 2151 ± 17 569 ± 57* Thorny skate Elevated 7.45 ± 0.05 2155 ± 10 2111 ± 204*

73 Table 2. The effects of temperature on standard metabolic rate measured as Q10 values. For both skate species, the values are reported for two different temperature ranges due to the small or non-existent sample size at the highest temperatures and lowered pH level.

Q10 Species Temperature Present Day pCO2 Elevated pCO2 Clearnose Skate 20 ° - 28 ° 1.71 20 ° - 24 ° 1.62 0.78 Summer Flounder 22 ° - 30 ° 2.45 1.07 Thorny Skate 5 ° - 13 ° 2.56 5 ° - 9 ° 3.87 2.34

)

(

0 2

20 40 60 80 O Saturation (%) 2

Figure 1. Data from a single respirometry trial. Scrit is the intersection of the two linear regression lines.

Figure 2. SMR data from (a) clearnose skate, (b) summer flounder, and (c) thorny skate. Box and whisker plots represent raw data, with whiskers representing maximum and minimum points within 1.5 times the interquartile range above the upper quartile and below the lower quartile. Open circles denote points outside of this range. The filled circles and lines indicate the model-derived estimates and standard errors for each treatment condition. The asterisks above the boxplots represent significant differences between pH treatments within a given temperature. The letters below the boxes represent significant differences among temperatures within a given pH level.

Figure 3. MMR of (a) clearnose skate and (b) thorny skate. Box and whisker plots represent raw data, with whiskers representing maximum and minimum points within 1.5 times the interquartile range above the upper quartile and below the lower quartile. Open circles denote points outside of this range. The filled circles and lines indicate the model-derived estimates and standard errors for each treatment condition. There were no significant differences in any of the pairwise comparisons, but the “‡” symbol denotes near significance (p=0.051 in clearnose skate, and p=0.07 in thorny skate).

Figure 4. Aerobic scope of clearnose skate (panels a and c) and thorny skate (panels b and d). ASa is presented in panels a and b, and ASf in panels c and d. Box and whisker plots represent raw data, with whiskers representing maximum and minimum points within 1.5 times the interquartile range above the upper quartile and below the lower quartile. Open circles denote points outside of this range. The filled circles and lines indicate the model-derived estimates and standard errors for each treatment condition. ASa was analyzed for model analysis, but ASf was not.

Figure 5. Critical oxygen partial pressure (Pcrit) and critical oxygen content (Ccrit) of clearnose skate (panels a and d), summer flounder (panels b and e), and thorny skate (pandels c and f). Box and whisker plots represent raw data with whiskers representing maximum and minimum points within 1.5 times the interquartile range above the upper quartile and below the lower quartile. Open circles denote points outside of this range. The filled circles and lines indicate the model-derived estimates and standard errors for each treatment condition. Astericks above the boxes indicate significant differences between the pH treatments within a given temperature, while asterics and letters below the boxes indicate significance across temperatures within a pH treatment. No statistical analyses were run on Ccrit and these graphs are included only as an aid for comparison to other studies.

79 Supplementary Table 1. The estimated values ± standard error based on model output.

Species Temp pCO2 SMR MMR ASa Pcrit °C mg O2 kg-1hr-1 mg O2 kg-1hr-1 mmHg Clearnose Skate 20° Present 39 ± 4 139 ± 11 100 ± 7 33 ± 4 Elevated 80 ± 6 178 ± 17 99 ± 11 61 ± 6 24° Present 47 ± 4 141 ± 11 94 ± 7 34 ± 4 Elevated 68 ± 7 173 ± 19 105 ± 12 78 ± 7 28° Present 59 ± 4 148 ± 11 89 ± 7 51 ± 4 Elevated 65 ± 13 163 ± 35 98 ± 22 79 ± 13 Summer Flounder 22° Present 45 ± 3 - - 42 ± 3 Elevated 74 ± 9 - - 71 ± 9 30° Present 93 ± 4 - - 60 ± 4 Elevated 78 ± 9 - - 78 ± 9 Thorny Skate 5° Present 16 ± 3 40 ± 6 24 ± 3 40 ± 7 Elevated 23 ± 3 49 ± 7 27 ± 4 64 ± 9 9° Present 27 ± 5 60 ± 9 33 ± 5 75 ± 11 Elevated 32 ± 6 73 ± 11 41 ± 6 78 ± 14 13° Present 34 ± 3 53 ± 7 23 ± 4 85 ± 9 Elevated - - - -

80 CHAPTER III

Drivers of hypoxia tolerance in elasmobranch fishes: comparing blood oxygen affinity

between clearnose skate and summer flounder

81 ABSTRACT

Hypoxia tolerance requires physiological adaptations to maintain oxygen delivery to the tissues despite low environmental oxygen partial pressures (pO2). To determine if species-specific blood oxygen affinities can explain species-specific hypoxia tolerances in elasmobranch fishes, as they do in teleost fishes, I measured blood oxygen affinities

(quantified as P50, the pO2 at which hemoglobin; Hb; is 50% saturated with O2) of clearnose skate (Rostaraja eglanteria) and summer flounder (Paralichthys dentatus), sympatric elasmobranch and teleost species with similar levels of hypoxia tolerance.

Experiments were conducted at two temperatures (20 or 28°C for skate; 22 or 30°C for flounder) and two CO2 levels (0.20 and 0.37 kPa pCO2). Clearnose skate blood oxygen affinity was insensitive to pCO2. In other words, there was no decrease in P50 with an increase in pCO2 and the concomitant decrease in blood pH (i.e., there was no Bohr shift). Skate blood oxygen affinity was, however, strongly impacted by temperature. In contrast, summer flounder blood exhibited the expected decreases in P50 and carrying capacity with an increase in pCO2 and the concomitant decrease in blood pH (i.e., a combined Bohr and Root shift). Blood oxygen affinity was, however, not affected by temperature. Clearnose skate blood exhibited a high oxygen affinity (P50 = 4.9 ± 0.6 kPa at 20 °C) equivalent to published values on the hypoxia-tolerant epaulette shark

(Hemiscyllium ocellatum) (4.3 ± 0.6 kPa at 28 °C); P50 values higher than those of many other elasmobranch species. My observations do not, therefore fit the negative correlation of blood oxygen affinity (measured as P50) and hypoxia tolerance (measured as critical oxygen levels, Pcrit) observed in teleost fishes. The physiological mechanisms underlying hypoxia tolerance in elasmobranch fishes therefore warrants further investigation.

82 INTRODUCTION

Dissolved oxygen levels are not evenly distributed throughout the water column or across geographic regions; they are higher in near-surface areas where oxygen can diffuse into the water from the atmosphere or in areas where oxygen it is produced via photosynthesis (Laffoley and Baxter, 2019; Keeling et al. 2010). Where the water column is stratified due to differences in density (e.g., in estuaries), bottom water can become hypoxic when it is isolated from oxygen‐rich surface water. Decomposition of organic matter also consumes dissolved oxygen, particularly near the benthos where organic matter settles. It is common, therefore, for bottom water oxygen levels in estuaries to

-1 range from hypoxic (below 2 mg O2 l ) to anoxic (Diaz and Breitburg 2009). Climate change is increasing the severity and frequency of hypoxic events in the nearshore environment due to associated changes in water column stratification and seasonal plankton blooms (Breitberg et al. 2015). Hypoxic severity is compounded by eutrophication due to nutrient runoff (Diaz et al. 2019). Episodic hypoxia is especially evident in Chesapeake Bay and coastal lagoons along the East coast of the United States.

Here, hypoxic conditions can persist over time scales ranging from minutes to hours (on a near daily basis during the summer) to seasonal (Tyler et al. 2009; Murphy et al. 2011).

Likewise, isolated tidepools in tropical tidal flats (such as those surrounding the Great

Barrier Reef, Australia) experience similar reductions in oxygen content over diel cycles

(Kinsey and Kinsey 1967; Routley et al. 2002).

Environmental hypoxia can negatively impact fish behavior (Domenici et al.

2007; Brady and Targett 2010), distribution and habitat utilization (Schlenger et al. 2013;

Chong et al. 2018), reproductive success (Wu 2009), and overall fitness (Cheek 2011;

83 Claireaux and Chabot 2016). These impacts of hypoxia have been extensively reviewed elsewhere (Hughes 1973; Pollock et al. 2007; Farrell and Richards 2009). At the cellular level, the negative impacts of hypoxia are due to the loss of homeostasis and membrane integrity caused by the significant and rapid reduction in adenosine triphosphate (ATP) production (Lutz and Nilsson 1997). Some fish species, however, possess unique respiratory, metabolic, and cardiovascular mechanisms to maintain adequate rates of oxygen delivery to the tissues in hypoxic conditions (Nilsson and Ostlund-Nilsson 2004;

Chapman et al. 2011; Heinrich et al. 2014).

One such mechanism is a high blood oxygen affinity (Krogh and Leitch 1919;

Johansen et al. 1978; Wise et al. 1998; Nilsson and Renshaw 2004; Mandic et al. 2009;

Speers-Roesch et al. 2012b; Rogers et al. 2016); a measure of the oxygen partial pressure

(pO2, referred to as P50) at which blood achieves an oxygen content ([O2]) of 50% of its maximum carrying capacity. Blood with an elevated O2 affinity (i.e., a low P50) will achieve higher levels of oxygenation at a lower oxygen pressures, thus ensuring oxygen binding at the gills even under low partial pressure gradients between water and the arterial blood passing through the gills (i.e., hypoxia).

Teleost fishes exhibiting high blood-oxygen affinity (low P50 values) must also possess mechanisms to release oxygen bound to hemoglobin (Hb) in the capillaries, thus maintaining a series of adequate pO2 gradients from the blood to the mitochondria referred to as the oxygen cascade (Farrell and Richards 2009). The release of oxygen from Hb is largely accomplished through conformational changes in the Hb protein complex from the R(elaxed)-state (resulting in blood high affinity for oxygen), to the

T(ense)-state in which oxygen affinity is greatly reduced. In teleost fishes, changes

84 between states is driven through elevated CO2 partial pressures (pCO2) in tissue capillaries (Root 1931; Nikinmaa and Salama 1998). Carbonic anhydrase (CA) within the red blood cells or on the capillary wall will catalyze the reaction of CO2 and H2O to

- + HCO3 and H (Rummer and Brauner 2011; Rummer et al. 2013a). An increase in CO2, and subsequent reduction in intracellular pH, reduces Hb-O2 affinity by stabilizing the T- state in a phenomena knows as the Bohr effect (Berenbrink 2011). This localized change in blood gasses (and ultimately red blood cell intracellular pH) thus promotes the release of oxygen to the tissues (Nikinmaa and Salama 1998; Berenbrink 2011; Morrison et al.

2015). A reduction in red blood cell intracellular pH can also reduce maximum blood oxygen content because Hb-oxygen affinity is so reduced it cannot be fully saturated

(Root effect) (Verde et al. 2007; Rummer et al. 2013b). The conventional view holds that the Root effect evolved to enhance O2 delivery by increasing blood pO2 during passage counter-current vascular structures (“rete mirable”) associated with the retina and swim bladder (Berenbrink et al. 2005; Verde et al. 2007; Berenbrink 2011).

Blood from elasmobranch fishes lacks a Root effect (Cox et al. 2017), and only some species exhibit a Bohr effect (Morrison et al. 2015). The lack of a Root effect may be related to elasmobranch fishes’ lack of swim bladders, although the blood of two other species lacking swim bladders, sturgeon and gar, still exhibits Root effects (Berenbrink

2011). The mechanisms of oxygen delivery therefore differ between teleost and elasmobranch fishes, suggesting the physiological adaptations enabling hypoxia tolerance vary as well.

In most fishes, an increase in temperature decreases Hb-O2 affinity through changes in the quaternary structure of the Hb protein and by driving exothermic Hb-O2

85 binding reactions in the reverse direction (Rossi-Fanelli and Antonini 1960; Nikinmaa and Salama 1998). Notable exceptions to this include regionally endothermic fishes, such as shortfin mako shark (Isurus oxyrinchus) (Bernal et al. 2018), porbeagle shark (Lamna nasus) (Andersen et al. 1973), bluefin tuna (Thunnus thynnus) (Brill and Bushnell 2006), and albacore tuna (Thunnus alalunga) (Cech et al. 1984). In these species, the lack of temperature sensitivity under a closed-system temperature change serves to preserve Hb- oxygen affinity during blood transport (Brill and Bushnell 1991; Bernal et al. 2018). In contrast, non-lamniform elasmobranch fishes that behaviorally thermoregulate can exhibit the expected temperature effects on Hb-oxygen affinity (Hopkins and Cech Jr

1994; Bernal et al. 2018). The impact of temperature on Hb-oxygen affinity is quantified as the heat of oxygenation (H, or enthalpy). When examining the effects of temperature on whole blood, the term is “apparent heat of oxygenation”, as the impact of temperature changes may be modulated by concomitant changes in the red blood cell (RBC) intracellular environment.

Understanding the mechanisms underpinning whole organism hypoxia tolerance can be difficult, as phylogeny, geographic distribution, and ontogeny may impact snapshot measurements typically used to evaluate hypoxia tolerance. In a classic study of

13 species of sculpin, Mandic et al. (2009) found P50 helped explain species-specific variance in whole animal hypoxia tolerance as measured through the oxygen pressure at which an organism is no longer able to maintain standard metabolic rate (Pcrit). Other factors affecting Pcrit variability were metabolic rate (MO2) and mass-specific gill surface area (Mandic et al. 2009). In other words, as P50 decreased, so did Pcrit, signifying that blood oxygen affinity may be a critical mechanism underpinning patterns of hypoxia

86 tolerance. The association of a low P50 and low Pcrit has been seen in other hypoxia- tolerant teleost fishes, including crucian carp (Carassius carassius) (P50=0.20 kPa; Sollid et al., 2005) , common goldfish (Carassius auratus) (P50=0.35 kPa; Burggren, 1982), and tench (Tinca tinca) (P50=0.64 kPa; Jensen and Weber, 1982). P50 has frequently been referenced as a mechanism driving general hypoxia tolerance, as being able to achieve high blood oxygen saturation at low driving pressures logically would enhance rates of oxygen movement across the gills when the pO2 gradient is reduced (i.e., in hypoxia)

Hypoxia-tolerant elasmobranch fishes display a range of physiological responses to hypoxia. For example, progressive hypoxia has no effect on hematocrit (Hct; the proportion of blood comprised of red blood cells) in the shovelnose ray (Aptychotrema rostrata) (Speers-Roesch et al. 2012b) and acute hypoxia exposure had no effect on Hct or Hb concentration ([Hb]) in Atlantic stingrays (Dasyatis sabina) (Dabruzzi and Bennett

2014). Anoxic exposure does, however, result in an increase in Hct in grey carpet shark

(Chapman and Renshaw 2009). Further, anoxia exposure caused an increase in Hct in epaulette shark (Chapman and Renshaw 2009), but progressive hypoxia exposure does not (Speers-Roesch et al. 2012b). Evaluating the physiological adaptations associated with increased blood-oxygen transport is difficult, especially when treatments across studies are variable (Supplementary Table 1). Even for the well-studied epaulette shark, the only study evaluating the Bohr shift used washed red blood cells (RBCs) (Wells et al.

1992), the data from which are difficult to compare directly with results from whole blood due to the red blood cell’s ability to regulate allosteric moderators of Hb-O2 affinity and the specific constituents of the Ringers solution within which the washed red blood cells are resuspended (Dalessio et al. 1991). The diversity of methodologies used to

87 study the elasmobranch hypoxia response underscores the need for more research in this area.

Most research on the mechanisms enabling hypoxia tolerance in elasmobranch fishes has focused on the epaulette shark (Hemiscyllium ocellatum) as it regularly experiences extreme hypoxia when stranded in tidal pools (Routley et al. 2002). This species reduces its aerobic metabolism by 30% under short term repeated exposure to hypoxia ([O2] > 1.7 mg/l) (Routley et al. 2002), but also exhibit cerebral vasodilation

(Soderstrom et al. 1999) presumably to sustain aerobic metabolism in the brain. Unlike in turtles, mammals, and some teleost fishes (Bickler and Buck 2007; Buck and Pamenter

2018), there appears to be no ion channel arrest under these low oxygen circumstances

(Nilsson and Renshaw 2004). Further, epaulette sharks do not exhibit a change in their anaerobic metabolic pathways from a production of lactate to a production of non-toxic ethanol, as do crucian carp under anoxic conditions (Nilsson and Renshaw 2004;

Vornanen et al. 2009). Under longer term hypoxia exposure (>40 min), epaulette shark lose righting reflexes signifying a deeper metabolic depression (Nilsson and Renshaw

2004; Speers-Roesch et al. 2012a). To explore whether elasmobranch fishes use similar strategies as teleost fishes to enable high hypoxia tolerance, Speers-Roesch et al. (2012b) compared the P50 values between epaulette shark and shovelnose ray (Aptychotrema rostrata), the latter of which are considered to be hypoxia-sensitive. These two elasmobranch species followed a similar pattern to that of sculpin (Mandic et al. 2009), with epaulette shark P50 values being lower than those of the shovelnose ray (5.1 ± 0.4 kPa and 7.2 ± 0.4 kPa, respectively). However, Speers-Roesch et al. (2012b) also noted the relationship between Pcrit and P50 should be more thoroughly investigated before

88 making assumptions about the mechanisms enabling hypoxia tolerance across the elasmobranch group.

Another complicating factor regarding the relationship between P50 and hypoxia tolerance is how the latter is measured. Pcrit is widely accepted as a whole organism metric for hypoxia tolerance. Wood (2018) has, however, has suggested that it may not be truly representative of an individuals’ response to low oxygen conditions. Of concern are variations in the methodology used to determine Pcrit and the implications for inter- study comparisons. Regan et al. (2019) and Ultsch and Regan (2019) contend, however, this metric offers a uniquely holistic representation of hypoxia tolerance, encompassing the full suite of aerobic and anaerobic responses, with direct implications for the oxygen delivery cascade (Childress and Seibel 1998; Mandic et al. 2009). Additional metrics of hypoxia tolerance, such as loss of equilibrium, can be useful in characterizing and understanding species-specific responses, although they frequently look at only a single component of the metabolic response (Regan et al. 2019). P50 may be one such metric, and as such it is important to examine the relationships between P50 and Pcrit.

In a recent study on clearnose skate (Rostaraja eglanteria), Schwieterman et al.

(2019) found Pcrit values rivaling those of the epaulette shark. The physiological mechanisms underpinning the remarkable hypoxia tolerance of clearnose skate have not yet been studied but represents an opportunity to compare phylogenetically disparate elasmobranch species that exhibit similar Pcrit values. The purpose of this study was, therefore, to examine the blood-oxygen binding characteristics of the clearnose skate, with the goal of comparing my results to other elasmobranch species displaying a range of Pcrit values. I also measured blood-oxygen binding characteristics for summer flounder

89 (Paralichthys dentatus), a species that is sympatric to clearnose skate and that has a similar hypoxia tolerance, therefore providing a sympatric teleost species for comparison.

For both clearnose skate and summer flounder, I determined differences in blood-oxygen affinity as quantified by P50 under the two temperature treatments used when determining these species’ Pcrit values (Schwieterman et al. 2019), and two pCO2 conditions representing in vivo arterial and venous mimicking the range of arterial and venous blood pCO2 of fishes (≈0.3 – 0.4 kPa) (Perry and Tufts 1998). I predicted Pcrit would be correlated with P50, given the relationship of these two metrics demonstrated in teleost fishes (Mandic et al. 2009).

MATERIALS AND METHODS

Animal Collection and Maintenance

All experimental protocols were approved by the William & Mary Institutional

Animal Care and Use Committee (IACUC-2019-03-18-13539-rwbril). Adult clearnose skate were collected via rod and reel from the Eastern Shore of Virginia during the summers of 2018 and 2019. They were transported to the Virginia Institute of Marine

Science Eastern Shore Seawater Lab in Wachapreague, VA. Clearnose skate were maintained in recirculating tanks at 20°C for at least two weeks before experimental procedures. Summer flounder were obtained through partnerships with local recreational fishers or from the Virginia Institute of Marine Science Juvenile Fish Trawl Survey.

Individuals were brought back to the Virginia Institute of Marine Science Seawater

Research Lab in Gloucester Point, VA and maintained in recirculating tanks at 22°C. All

90 fish were fed a mixed diet of teleost and shellfish and were fasted for 48 hrs prior to blood collection.

All fish were anesthetized in a benzocaine-seawater solution (40 mg/L benzocaine) prior to experimentation. When fish were no longer responsive to tactile stimulus, they were injected with heparin diluted with saline 1:3, which was allowed to circulate for five minutes. An incision was made to expose the conus arteriosus or bulbus arteriosis, which was then catheterized with a 20-gauge Teflon catheter. Blood was collected into heparinized syringes, until mortality occurred due to exsanguination. Blood samples were then placed on ice for 12 hrs under bright light to reduce catecholamine levels (Brill et al. 2008). Because I could not obtain sufficient blood from individual summer flounder, I pooled blood samples from two individuals (Grigg 1967; Tetens and

Wells 1984b). Due to small blood sample volumes obtained from summer flounder, the number of blood oxygen curves measured from a given individual (or pooled set) ranged from 1-4.

Tonometry and Hematology

I chose temperatures based on a previous study (Schwieterman et al. 2019), in which temperatures were selected so as to mimic those experienced during the summer in the mid-Atlantic estuaries from which the fish were obtained (Capossela et al. 2012;

Marcek 2018). Clearnose skate blood was tested at 20° and 28°C, while summer flounder blood was tested at 22° and 30°. Blood from both species was exposed to two carbon dioxide levels (0.20 kPa and 0.37 kPa). Specific gas mixtures were created immediately before use by mixing air, nitrogen, and carbon dioxide using electronic gas-flow

91 controllers (MKS Instruments, Andover, MA, U.S.A.). The gas mixtures were stored in automobile tire inner tubes.

To construct oxygen equilibrium curves, I placed blood samples (≈0.5 mL) in temperature equilibrated glass tonometers flushed with six different water-vapor- saturated gas mixtures (pO2 = 0.66, 1.33, 2.67, 5.33, 10.70, or 20.00 kPa) and with one of two CO2 levels (pCO2 = 0.20 or 0.37 kPa). Blood was allowed to equilibrate under continuous shaking for two hours prior to measurement. My measurement methods follow those described in Brill et al. (2008) and Brill and Bushnell (1991). Blood-oxygen content ([O2]) was measured according to procedures described by Tucker (1967), where

30 uL of gas-equilibrated blood were injected into a degassed potassium ferricyanide solution, and the change in oxygen partial pressure (pO2) was measured by a polarographic oxygen electrode (Radiometer America, Westlake, OH, U.S.A.). Blood pH was assessed using a capillary pH electrode installed in a Radiometer MKS Mark 2 blood gas analyzer (Radiometer America) maintained at the temperatures to which blood samples were equilibrated. Using blood samples from the first and last tonometers (pO2 =

0.66 and 20 kPa), I also measured Hct, [Hb], and plasma bicarbonate concentration

- ([HCO3 ]). Hct was measured to ensure there was no hemolysis during the experimental

- protocol, and [HCO3 ] was measured to quantify blood buffering capacity. [Hb] was measured to estimate maximum blood oxygen carrying capacity ([O2]max). Hct was measured using heparinized capillary tubes centrifuged at 11,000 rpm for five minutes in an Adams Autocrit Centrifuge (Clay-Adams, Inc, New York, NY, USA). To measure

- - [HCO3 ], blood was centrifuged under mineral oil, and plasma [HCO3 ] was measured following techniques described by Cameron and Davis (1970). [Hb] was measured using

92 the standard Drabkin’s cyanomethoglobin method, where 7.5 uL of blood is vortexed with 1.5 mL of Drabkin’s solution (Sigma Aldrich), allowed to incubate for at least 15 minutes, and then absorbance read at 540 nm using either a Spectronic 200 spectrophotometer (Thomas Scientific, Swedesboro, NJ, USA) or a Shimadzu UV-

1650PC spectrophotometer (Shimadzu Scientific Instruments, Kyoto, Kyoto Prefecture,

Japan).

Data Processing and Analysis

I calculated [O2]max based on measured [Hb] and a maximum Hb O2-carrying

-1 -1 capacity corrected for temperature (1.22 ml O2 g Hb at 20°C, 1.23 ml O2 g Hb at 22°C,

-1 -1 1.27 ml O2 g Hb at 28°C, and 1.28 ml O2 g Hb at 30°C: Ganong 1973). From the resulting data, I fit the pO2 and [O2] data (expressed as % saturation) using the Hill equation in PAST 4.0 (Hammer et al. 2001). I calculated Hill numbers, a measure of cooperativity of ligand binding by performing a linear regression of log pO2 and log [% saturation * (100-% saturation)-1]. Because Hill plots are nonlinear when data from 0-

100% saturation are employed (Ikeda-Saito et al. 1983), I used mid-range values from

≈20-80% saturation in the construction of Hill plots and the calculation of Bohr coefficients. I calculated P50 values using the regression parameters from the Hill plots. I also estimated the pH of whole blood samples (pHe) at P50 (pHe 50) by linear regression of

% saturation and pHe data and then interpolating to P50. I calculated the Bohr coefficients, a measure of pH sensitivity, as:

Φ = ∆log⁡ 푃50 ÷ ∆푝퐻50

93 where P50 values are in kPa. The Root effect was calculated from [O2] of blood equilibrated to a pO2 of 20 kPa as a percentage of [O2]max. The apparent heat of oxygenation (enthalpy or H) was calculated as:

∆푙표푔푃 ∆퐻 = 2.303 × 푅 × 50 1 ∆ 푇

Where R is the gas constant and T is the absolute temperature (Wyman 1964).

Calculations and analyses were coded in R (R Team 2019). I used a linear model to compare specific metrics within species, with metric being the response variable and temperature and pCO2 as fixed effects, using the lme4 package in R (Bates et al. 2014).

Interactions between temperature and pCO2 were included when significant. Model assumptions were examined using Q-Q plots and residual plots. Assumptions of normally distributed means and homoscedasticity were deemed reasonable. Tukey’s post-hoc tests were used to determine pairwise significance using an alpha of 0.05.

To compare my findings to other studies on elasmobranch fishes, I used the P50 values given in Table 3.3 in Morrison et al. (2015), and from more recent publications

(Supplementary Table 1). I also included information on Pcrit in Supplementary Table 1 when available. To compare patterns across the limited number of elasmobranch species for which both P50 and Pcrit have been measured, I plotted these two metrics against each other and fitted a general linear regression in R (R Team 2019) using the glm function in base R. In this model, Pcrit was held as the response variable, and P50, temperature, and metabolic rate were linear factors obtained from published, species-specific values

(Supplementary Table 2). I tested models including all three factors with and without interaction terms and used Akaike’s Information Criterion (AIC) for model selection.

94 RESULTS

I collected data from 13 clearnose skate (disk width = 58.5 ± 3.2 cm, mean ± SE) and 8 pairs of summer flounder (36.8 ± 0.8 cm TL, mean ± SE). I observed a significant increase in mean MCHC in summer flounder between the two temperature treatments, but there were no other significant changes in hematological parameters due to pCO2 or temperature treatment (Table 1) and this is likely not biologically relevant.

There was overlap between blood oxygen affinity curves for clearnose skate at the two pCO2 levels tested suggest no impact of pCO2 on blood-oxygen affinity (Figure 1).

Clearnose skate blood did not display a Root effect, evidenced by the lack of difference in blood oxygen carrying capacity between blood equilibrated at the two pCO2 treatment conditions at 20 kPa O2 (F=0.84, p=0.37; Figures 1, 2). Likewise, I did not detect an influence of increasing pCO2 on blood oxygen affinity, evidenced by a lack of a significant difference in P50 values under different pCO2 levels (F=0.01, p=0.96; Figure

3a) and a lack of a significant Bohr shift at either temperature in clearnose skate (F=1.93, p=0.18; Figure 4a). The effect of temperature, however, is significant (F=24.4, p<0.05), with the mean P50 values for clearnose skate increasing 65% from 4.8 ± 0.4 to 8.0 ± 0.6 kPa from 20 to 28°C when values across pCO2 levels are combined (Figure 3a). The

-1 apparent heat of oxygenation values were -5.8 ± 1.0 kcal mol O2 at 0.2 pCO2 and -5.7 ±

-1 1.4 kcal mol O2 at 0.37 pCO2 (F=0.01, p=0.92; Figure 5).

Summer flounder showed no significant increase in P50 in response to elevated pCO2 or temperature (FpCO2=1.73, p=0.26; Ftemp=0.26, p=0.64; Figure 3b), and the calculated Bohr coefficients were unable to be statistically compared due to low sample size. The H for summer flounder was similar to that of clearnose skate (Figure 5b).

95 Elevated pCO2 and temperature caused significant differences (FpCO2=10.51, p<0.05;

Ftemp=5.57, p<0.05) between pH50 at the warm temperature treatment for clearnose skate, although temperature alone significantly impacted the pH50 of summer flounder

(F=32.92, p<0.05 Figure 6). There were no significant impacts of temperature on changes in pH50 per degree Celsius for clearnose skate (FpCO2=1.97, p=0.17; Ftemp=0.02, p=0.89) or summer flounder (FpCO2=0.02, p=0.89; Ftemp=0.25, p=0.65; Figure 7), nor were there

- significant changes in [HCO3 ] for clearnose skate (FpCO2=0.40, p=0.53; Ftemp=0.77, p=0.39) or summer flounder (FpCO2=0.01, p=0.99; Ftemp=0.47, p=0.5; Figure 8). Hill numbers showed a significant impact of temperature on cooperativity for both species

(Fskate=9.88, p<0.05; Fflounder=32.91, p<0.05; Figure 9).

The P50 values measured in clearnose skate (4.86 ± 0.58 kPa; mean ± SE) are not in the bottom percentile of those collected from the literature and were not significantly different from published values for epaulette shark (4.27 ± 0.57 kPa; Figure 10). The best fitting model for Pcrit included temperature, P50, SMR, and an interaction between P50 and

SMR. I could not interpret the main effects of SMR and P50 because there was a significant interaction (t=7.95, p<0.05). The effect of temperature on Pcrit was negative and significant (t=-6.05, p<0.05).

DISCUSSION

I sought to quantify the changes in blood-oxygen affinity for the hypoxia-tolerant clearnose skate, with comparisons to the sympatric teleost summer flounder, to help explain the physiological mechanisms enabling the extreme hypoxia tolerance of the former (Schwieterman et al. 2019). I also sought to contextualize these results with

96 comparisons to epaulette shark and other elasmobranch species. I found that increased pCO2 had no significant effect on the oxygen binding characteristics of clearnose skate blood, although it produced a Bohr and Root effect in summer flounder blood. Most importantly, I found differences in patterns of P50 and Pcrit between teleost and elasmobranch fishes, likely implying different strategies for withstanding low oxygen environments between these two groups.

While the Bohr effect is known to play an important role in oxygen offloading in teleost fishes, I did not see evidence of a Bohr effect in clearnose skate. The absence of a

Bohr effect in clearnose skate is not unique within the elasmobranch group. Other species with little to no Bohr effect include spotted ratfish (Hydrolagus colliei;  = 0) (Hanson

1967), ocellate river stingray (Potamotrygon motoro;  = -0.05) (Johansen et al. 1978); shovelnose ray (Glaucostegus typus=Rhinobatos batillum;  = -0.08) (Wells et al. 1992);

Port Jackson shark (Heterodontus portusjacksoni;  = 0) (Grigg 1974), and spiny dogfish

(Squalus suckleyi;  = 0) (Lenfant and Johansen 1966). The lack of pH sensitivity is likely due to the high histidine content of the Hbs of these species, which act as an intracellular buffer (Nikinmaa et al. 2019). Morrison et al. (2015) reviewed other elasmobranch species that do exhibit a significant Bohr effect (Supplementary Table 1), in general elasmobranch fishes are thought to exhibit moderate pH sensitivity compared to teleost fishes (Dafré and Fo 1989; Berenbrink et al. 2005).

Species-specific sensitivity of Hb isomers may be related to the activity level or routine metabolic rates, with higher metabolic rates being associated with lower hypoxia tolerance and higher Hb-O2 affinity. Although the significant interaction between SMR and P50 prohibited me from interpreting the direct effect of P50 on Pcrit, a larger sample

97 size may allow for further testing. Looking specifically at the clearnose skate, for

-1 example, it has generally low activity levels (standard metabolic rate of 39.4 mg O2 kg

-1 h at 20°C; Schwieterman et al., 2019) and exhibits high P50 values and no Bohr effect.

In this species, having a low blood-oxygen affinity would not necessarily require changes in affinity to release oxygen in the capillaries where pO2 driving gradients are high, explaining the lack of pH sensitivity. Oxygen delivery is, however, a complex process, and variation in a number of components in the oxygen cascade could facilitate both oxygen delivery and consumption (Morrison et al. 2015). More research on the role of phylogeny, evolutionary history, and environment on hypoxia tolerance and blood- oxygen affinity would help determine the validity of this relationship.

While the relationship between P50 and hypoxia tolerance has been established in teleost fishes, the correlation between the two may not hold in elasmobranch fishes. The

P50 values I measured in clearnose skate are not in the bottom percentile of published P50 values in elasmobranch fishes, but are remarkably similar to those measured in the hypoxia-tolerant epaulette shark (Speers-Roesch et al. 2012b). The overall range of P50 values in elasmobranch fishes is similar to that found in teleost fishes (Morrison et al.

2015). Further, the P50 values for clearnose skate obtained here and for the epaulette shark are an order of magnitude higher than those for hypoxia-tolerant teleost fishes (Jensen and Weber 1982; Sollid et al. 2005). Elasmobranch Hbs are generally less “fine-tune”- able than those of teleost fishes (Bonaventura et al. 1974; Weber 1983; Morrison et al.

2015), so the values presented here are likely representative of in vivo values.

One potential driver of Pcrit in elasmobranch fishes is metabolic rate. While I was unable to interpret the effect of SMR on Pcrit due to the significant interaction between

98 SMR and P50, I will discuss trends that may be important to consider for further work.

Taken as a single example, the clearnose skate P50 results contradicted the findings from the hypoxia-sensitive shovelnose ray and epaulette shark. Shovelnose ray and epaulette shark have similar resting metabolic rates, thus this variable was not considered in the analysis which found a trend of increasing Pcrit with increasing P50 in these two species

(Speers-Roesch et al. 2012b). Comparing elasmobranch species where both P50 and Pcrit have been measured, I observed an unexpected inverse relationship between P50 and Pcrit such that hypoxia intolerant species (i.e., those with a higher Pcrit value) exhibited a trend towards elevated blood oxygen affinity (i.e., blood with low P50 values). The pattern appears to be associated with metabolic rate, such that more sedentary species have lower blood-oxygen affinities but higher hypoxia tolerance compared to more active species, although this was not statistically resolved due to small sample sizes. For example, the sandbar shark (Carcarhinus plumbeus; a ram-ventilating elasmobranch with a routine

-1 -1 metabolic rate of 143 mg O2 kg h ), exhibits a P50 (2.71 kPa; measured at 0.2 kPa CO2 and 25°C) nearly half that of the clearnose skate (4.85 kPa; measured at 0.2 kPa CO2 and

20 °C) (Brill et al. 2008), which means sandbar shark have higher blood oxygen affinity.

This relationship contrasts with species-specific Pcrit values of clearnose skate and sandbar shark, with the latter having higher Pcrit value (i.e. lesser hypoxia tolerance)

(Crear et al. 2019; Schwieterman et al. 2019). This suggests that in elasmobranch fishes, routine metabolic rate is an important factor driving both P50 and hypoxia tolerance.

The potential inverse relationship between P50 and Pcrit in elasmobranch fishes

(compared to the positive correlation in teleost fishes) does not resolve the mechanisms underpinning hypoxia tolerance in elasmobranch fishes, but rather raises additional

99 questions. In epaulette shark, metabolic arrest and maintenance of blood flow to the brain are key components in withstanding anoxia (Soderstrom et al. 1999; Nilsson and

Renshaw 2004; Dowd et al. 2010). To date, there is no evidence that elasmobranch fishes are capable of switching their anaerobic metabolic pathways to produce ethanol rather than lactate, although elasmobranch fishes may be able to withstand higher levels of metabolic acidosis due to their high levels of protein-stabilizing trimethylamine oxide

(TMAO) (Wood et al. 2007). It is also possible that hypoxia-tolerant elasmobranch fishes rely on metabolic arrest (Soderstrom et al. 1999; Buck and Pamenter 2018), and this, coupled with changes in Hct and bradycardia, may be sufficient to survive hypoxic events. More sedentary, hypoxia-tolerant species may also have a greater capacity to rearrange gill blood flow to increase the effective gill surface area so as to be able to maintain aerobic metabolism in severe hypoxia, than do more active species.

Temperature had a significant impact on Pcrit when all elasmobranch species were analyzed together, which can be mechanistically explained by the decrease in Hb-oxygen binding associated with an increase in temperature. The large effect of temperature on blood-oxygen affinity of clearnose skate blood is consistent with other coastal species, including the leopard shark (Triakis semifasciata) and brown smooth-hound shark

(Mustelus henlei) (Bernal et al. 2018). The large temperature effect observed here differs from regionally endothermic lamnid species which, like tunas, exhibit reduced temperature sensitivity of blood oxygen affinity. This minimizes the effects changes in ambient temperature on oxygen delivery during rapid movements across the thermocline and avoids premature Hb-oxygen disassociation during the closed-system temperature changes to which blood is subjected as it passes from the gills (at ambient temperature) to

100 the muscle (which are maintained at significantly elevated temperatures) (Brill and

Bushnell 1991; Lowe et al. 2000; Brill and Bushnell 2006; Bernal et al. 2018). I should note, however, there were no endothermic elasmobranch species included in the analysis of factors determining Pcrit. For coastal species, diel changes in temperature may drive changes in Hb-O2 affinity (Nikinmaa et al. 2019). While diel activity patterns have often been attributed to differences in foraging efficiency under different temperatures (Sims et al. 2006; Meese and Lowe 2019) or enhanced somatic growth and digestion rates (Hight et al. 2007). Changes in affinity, and ultimately changes the rate of oxygen delivery to the tissues may also drive this behavior (Bernal et al. 2018).

The temperature effects on hypoxia tolerance are mechanistically supported by the temperature sensitivity of oxygen affinity (i.e. H values) observed here, which are similar among clearnose skate and summer flounder and are all negative. This is the expected result indicative of an exothermic reaction during Hb oxygen binding (Morrison

- et al. 2015). The apparent heat of oxygenation (H) in clearnose skate (-5.8 kCal mol O2

1 -1 ) is, however, less than that of spiny dogfish (Squalus acanthias; -10 kCal mol O2 )

(Weber et al. 1983), indicating a larger effect of temperature on blood oxygen affinity in the latter. My results are, however, consistent with those from brown smooth-hound

-1 shark (-5.7 kCal mol O2 ) (Bernal et al. 2018), which is considered to also be a benthic species that experiences significant diel temperature variations (Campos et al. 2009). This smaller absolute values of H in clearnose skate and brown smooth-hound shark are likely due to the contributions of ATP and H+ release during the change between T- and

R-states (Morrison et al. 2015).

101 Although the temperature effects on clearnose skate blood-oxygen affinity were larger than those of pCO2, the effects were small compared to published values (Bernal et al. 2018)suggesting exposure to warm waters has smaller negative implications for blood- oxygen binding in this species then in other elasmobranch species. There are, however, temperature effects on both P50 and Pcrit, suggesting these may be correlated within a given species. Schwieterman et al. (2019) found clearnose skate Pcrit values increased by

55% from 20 to 28 °C under normal pH conditions. Here, I found a 65% increase in P50 under the same temperature change. My results further support the hypothesis that metabolic suppression and metabolic rate influence hypoxia tolerance in elasmobranch fishes, given that temperature also increased clearnose skate standard metabolic rate by

51% (Schwieterman et al. 2019). One caveat to this conclusion is that blood samples were acutely warmed (over 2 hrs), while individuals for which Pcrit was measured were exposed to higher temperatures for 48 hrs.

Temperature impacts more than just Hb-oxygen affinity and may help explain decreases in hypoxia tolerance under elevated temperatures. The changes in pH50 among treatments were significantly different at the high temperature and high pCO2 treatment in clearnose skate, suggesting higher temperatures resulted in diminished buffering

-1 capacity. The mean changes in pH50 °C were similar to those of other elasmobranchs including the bat ray (Myliobatis californica) (Hopkins and Cech Jr 1994); although they were larger than those found by Bernal et al. (2018) in leopard shark, brown smooth- hound shark, shortfin mako shark, and blue shark (< 0.01 mean pH units °C−1). This is most likely due to the differences in the non-bicarbonate buffering capacity of each

- species. Measured [HCO3 ] was below 3 mM in all combinations of treatments for

102 clearnose skate and was below 4 mM in summer flounder. Compared to other fishes, these values are consistent with the dusky shark (Cliff and Thurman 1984), bonnethead shark (Harms et al. 2002), and sandbar shark (Brill et al. 2008), although they are lower than the values reported for shortfin mako shark (15.6 mM), blue shark (13.0 mM), leopard shark (10.8 mM), and brown smooth-hound shark (7.4 mM) (Bernal et al. 2018).

-1 - As both change in pH50 °C and [HCO3 ] were consistent with other elasmobranch fishes,

I assumed the non-bicarbonate buffering capacity of clearnose skate was not the driver of this species’ hypoxia tolerance.

There are also intrinsic factors within the Hb protein that govern Hb-oxygen affinity, including cooperativity or the binding of one ligand (e.g., O2) promoting the binding of subsequent ligands. The Hill numbers of clearnose skate blood (e.g., 1.2 ± 0.2 at 20°C under 0.2 kPa CO2) were relatively low, and are similar to the values for leopard shark (1.6 ± 0.1) and brown smooth-hound shark (1.36 ± 0.01) (Bernal et al. 2018), as well as other values for elasmobranch fishes (reviewed by, Butler and Metcalfe 1988;

Morrison et al. 2015). The hyperbolic blood oxygen dissociation curves of clearnose skate and summer flounder are more generally associated with better adaptation to hypoxia and high pCO2, while a more sigmoidal curve is associated with being well- suited for aerobic performance (Jensen 1991; Bernal et al. 2018). Since elasmobranch fishes are likely operating at the lower end of the saturation curve (Bushnell et al. 1982), the hyperbolic shape ensures large differences in Hb saturation even at low dissolved oxygen partial pressures. In contrast, sigmoidal curves have their steepest slopes at mid- level oxygen partial pressures and benefit fishes operating at higher Hb saturations. This difference in the shape of the blood oxygen curves is consistent with what is known about

103 these two sympatric species, with both species being benthic during warm summer months and thus potentially exposed to hypoxia in estuarine environments (Diaz et al.

2019). Adult summer flounder are lie-in-wait piscivores, although at size range of individuals used for my study (36.8 ± 0.8 cm TL), their diet also includes benthic invertebrates (Staudinger and Juanes 2010). Clearnose skate diet consists of epibenthic invertebrates and crustaceans throughout their life history (Ebert and Bizzarro 2007). The hyperbolic blood oxygen curve may therefore be an adaptation enabling these species to forage in hypoxic benthic waters, taking advantage of benthic infauna that reduce burial depth in response to low oxygen concentrations (Taylor and Eggleston 2000).

CONCLUSIONS

I found evidence that elasmobranch fishes’ P50 may not be positively correlated with whole animal hypoxia tolerance as measured by Pcrit. This observation could be explained by a lack of pH sensitivity in many elasmobranch Hbs, particularly among more sedentary species. Interestingly, both Pcrit and P50 were affected by metabolic rate, with individuals exhibiting a higher standard or routine metabolic rate also exhibiting higher blood oxygen affinities and lower hypoxia tolerances. Within a given species, temperature may be a primary factor governing metabolism, Pcrit, and P50, although studies with standardized temperature treatments for the collection of all three metrics should be done. Given the conflicting results observed for teleost and elasmobranch fishes, the mechanisms contributing to hypoxia tolerance in elasmobranch fishes warrant further attention. This work will contribute to the growing body of literature that takes a

104 mechanistic approach to anticipating the impact of anthropogenic activities on coastal fishes.

ACKNOWLEDGEMENTS

This work was supported by the National Science Foundation Graduate Research

Program (DGE-1444317). I thank the staff of the Virginia Institute of Marine Science

Eastern Shore Laboratory for their assistance facilitating this work. I would also like to thank the VIMS Juvenile Trawl Survey program, Susanna Musick and volunteer recreational anglers, and the VIMS Multi-Species Research Group for help in specimen collection. Nilanjana Das, Julie Brucker, Danielle Olive, and Serina Wittyngham all assisted with data collection.

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115 Table 1. Whole blood hematological parameters at two temperatures from clearnose skate (n = 15) and summer flounder (n = 16, but pooled for every two individuals). All values represent mean ± standard error. There were no significant differences among temperature or pCO2 treatments as determined through an analysis of variance (ANOVA). Abbreviations are as follows: Hct Hematocrit; [Hb] hemoglobin concentration; MCHC Mean Corpuscular Hemoglobin Content.

Temp* Cool Warm pCO2 (kPa) 0.20 0.37 0.20 0.37 Clearnose Skate Hct (%) 16 ± 1 16 ± 1 17 ± 1 16 ± 2 FTemp=0.01 p=0.92 FCO2=0.37 p=0.55 -1 [Hb] (g dL ) 33 ± 2 32 ± 2 33 ± 2 32 ± 3 F =0.01 p=0.94 Temp FCO2=0.19 p=0.67 MCHC (g dL-1) 34 ± 5 32 ± 7 30 ± 7 34 ± 10 FTemp=0.07 p=0.79 FCO2=0.02 p=0.90 Summer Flounder Hct (%) 23 ± 3 24 ± 5 28 ± 5 25 ± 7 FTemp=0.54 p=0.48 FCO2=0.12 p=0.73 -1 [Hb] (g dL ) 51 ± 5 54 ± 7 39 ± 8 46 ± 10 FTemp=4.44 p=0.06 FCO2=1.14 p=0.31 MCHC (g dL-1) 22 ± 4 21 ± 6 17 ± 6 17 ± 7

FTemp=7.49 p=0.02

FCO2=0.16 p=0.70

*Temperatures for clearnose skate are 20°C and 28°C; for summer flounder 22°C and 30°C

116 pO2 (kPa)

Figure 1. Representative blood oxygen curves for clearnose skate (at 20°C [a]; and 28°C [c]) and summer flounder (at 22°C [b]; and 30°C [d]). The black points and dashed line represent data from 0.2 kPa pCO2, and the grey triangles and solid lines represent data from 0.37 kPa pCO2. Note the high degree of overlap between the lines for clearnose skate, signifying no impact of increasing pCO2 on blood-oxygen affinity.

117 pCO2 (kPa)

Figure 2. Hemoglobin saturation in percent following equilibration with 20 kPa O2 at each of the two temperature and pCO2 treatment levels for clearnose skate (a) and summer flounder (b). There were no significant differences among treatment level within species, despite the evidence of a Root effect in summer flounder. The number below the bars represents the number of individuals represented for the specific treatment. Boxplots show the upper, first quartile, median, third quartile, and lower bounds of the data.

118

pCO2 (kPa)

Figure 3. The P50 of the blood at all temperature and pCO2 treatment levels for clearnose skate (a) and summer flounder (b). Significant differences within species are denoted by the lowercase letters. There were no significant differences among treatments for summer flounder, or between pCO2 treatments in clearnose skate. The number below the bars represents the number of individuals represented for the specific treatment. Boxplots show the upper, first quartile, median, third quartile, and lower bounds of the data.

119

Figure 4. The Bohr coefficient for clearnose skate (a) and summer flounder (b) at 20 and 28°C. The values for clearnose skate were not statistically different from zero, and the number of samples from summer flounder was too low to be tested statistically. Lowercase letters above the boxplots for summer flounder indicate significant differences. The number below the bars represents the number of individuals represented for the specific treatment. Boxplots show the upper, first quartile, median, third quartile, and lower bounds of the data.

120 pCO2 (kPa)

Figure 5. The apparent heat of oxygenation for clearnose skate (a) and summer flounder (b) at high and low pCO2 levels. It should be noted that data from summer flounder are from single calculations due to limitations of available data. There were no significant differences within species. The number below the bars represents the number of individuals represented for the specific treatment. Boxplots show the upper, first quartile, median, third quartile, and lower bounds of the data. Squares represent outliers beyond 1.5 of the interquartile range.

121

pCO2 (kPa)

Figure 6. pH50 of the blood at all temperature and pCO2 treatment levels for (a) clearnose skate and (b) summer flounder. Lowercase letters above the boxes represent significant differences within species. The number below the bars represents the number of individuals represented for the specific treatment. Boxplots show the upper, first quartile, median, third quartile, and lower bounds of the data. Squares represent outliers beyond 1.5 of the interquartile range.

122

pCO2 (kPa)

Figure 7. Change in pH50 per degree for clearnose skate (a) and summer flounder (b). There were no significant differences among temperature and pCO2 treatments within species. The number below the bars represents the number of individuals represented for the specific treatment. Boxplots show the upper, first quartile, median, third quartile, and lower bounds of the data. Squares represent outliers beyond 1.5 of the interquartile range.

123 pCO2 (kPa)

- Figure 8. [HCO3 ] for clearnose skate (a) and summer flounder (b). There were no significant differences among temperature and pCO2 treatments within species.

124

pCO2 (kPa)

Figure 9. The Hill numbers for clearnose skate (a) and summer flounder (b) under high and low pCO2 conditions. Lowercase letters represent significant differences within species.

125

Figure 10. A histogram of P50 values from Supplemental Table 1. The vertical black line and shaded grey area represent the P50 value for clearnose skate at 20°C with associated error. The blue line and shaded area represent the P50 value from epaulette shark and associated error from Speers-Roesch et al. (2012). The black dotted lines represent the P50 values of crucian carp and common goldfish (left to right, respectively) from Sollid et al. (2005) and Burggren et al. (1982).

126

Figure 11. Partial regression plots showing the relationship between P50 (a) and Temperature (b) with Pcrit. Values are taken from Supplementary Table 1, and regressions are taken from a generalized linear model with Pcrit as the response variable, and an interaction between metabolic rate and P50. The two values of standard metabolic rate (SMR) are the 25th and 75th quantile of the metabolic rate data, and illustrate the interaction between metabolic rate and P50. The shaded area in (b) represents the 95% confidence interval around the partial regression line.

127 Supplementary Table 1. Whole blood properties of elasmobranch fishes. Adapted and expanded from Morrison et al. 2015.

Species P50  pH pCO2 Temp Source Notes Pcrit Temp Source (kPa) (kPa) (°C) (kPa) (°C) HOLOCEPHALI Chimaeriformes Hydrolagus colliei 2.13 Absent 0.33- 11 Hanson 1967 0.37

ELASMOBRANCHII Batoidea Myliobatiformes Myliobatis 0.80 -0.45 8.37 0.03 8 Hopkins and californica 1.71 -0.45 7.63 1.01 8 Cech 1994 1 -0.47 8.33 0.03 14 2.29 -0.47 7.55 1.01 14 1.80 -0.52 7.92 0.03 20 3.20 -0.52 7.45 1.01 20 1.60 -0.47 7.99 0.03 26 2.71 -0.47 7.51 1.01 26 Potamotrygon 0.61 -0.05 7.4 Johansen et al. Washed RBCs motoro 1978 P. circularis 0.89 -0.26 7.4 Johansen et al. Washed RBCs 1978 P. laticeps 1.08 -0.25 7.4 Johansen et al. Washed RBCs 1978 Potamotrygon sp. 1.64 Present 7.7 Martin et al. 1979 Potamotrygon 1.57- -0.19- None 30 Powers et al. 1.69 -0.26 1979

128 Rajiformes Amblyraja radiata 5.3 5 Schwieterman et al. 2019 10.0 19 11.3 13 Aptychotrema 6.53 Speers- 7.2 28 Speers-Roesch rostrata Roesch et al. et al. 2012a 2012a Raia oscillata 1.47 0.13 0.2 Dill et al. 1932 2.67 0.13 10 6.00 0.13 25 12.67 0.13 38 Raia oscillata 3.68 -0.29 7.82 0.10 12 Graham et al. 1990 4.61 -0.29 7.40 1.00 12 Rhinobatos 1.97 -0.08 7.8 None 25 Wells et al. Washed RBCs batillum 1992 2.15 7.4 None 25 Raia clavata 4.03 -0.25 7.7 15 Hughes and cannulated Wood 1974 Rostaraja 5.49 Absent 7.77 0.20 20 This study 4.4 20 Schwieterman eglanteria et al. 2019 4.81 7.68 0.37 20 4.5 24 7.98 Absent 7.71 0.2 28 6.8 28 7.98 7.51 0.37 28 Torpediniformes Torpedo 2.69 -0.32 7.8 15 Hughes 1978 Cannulated, 6.6 15 Hughes 1978 marmorata respirometer 3.73 20 Selachimorpha

129 Carcharhiniformes Carcharhinus 1.48 -0.35 7.8 None 25 Wells et al. Washed RBCs melanopterus 1992 2.39 -0.35 7.4 None 25 Carcharhinus 2.71 -0.56 7.92 0.20 25 Brill et al. 8.01 24-30 Crear et al. plumbeus 2008 2019 -0.37 7.64 0.20 25 Exercise stressed

Cephaloscyllium 0.64 -0.49 7.6 5 Tetens and isabellum Wells 1984 Cephaloscyllium 1.11 -0.32 7.7 15 King 1994 Caudal isabellum venipuncture Mustelus henlei 0.97 -0.25 8.05 <0.04 10 Bernal et al. 2018 1.16 -0.28 7.99 <0.04 15 1.52 -0.33 8.08 <0.04 20 1.65 7.17 1.01 10 1.97 7.24 1.01 15 2.59 7.40 1.01 20 Negaprion 1.32 -0.24 7.8 None 25 Wells et al. acutidens 1992 1.64 -0.24 7.4 None 25

Negaprion 1.57 -0.36 7.62 None 24 Bushnell et al. 15% Tris brevirostris 1982 buffer, cannulated, rest/free swimming Prionace glauca 1.61 -0.33 8.05 <0.04 15 Bernal et al. 2018

130 2.22 -0.11 7.98 <0.04 20 3.33 -0.22 7.95 <0.04 25 2.25 7.62 1.01 15 2.45 7.48 1.01 20 4.23 7.62 1.01 25 Scyliorhinus 2.87 -0.43 7.58 0.29 17 Pleschka et al. 8.0 7 and Butler and canicular 1970 12 Taylor 1971 10.7 17 Pleschka et al. 1970 Scyliorhinus 2.26 0.20 17 Piiper and In vivo and in 7-13 15-17 Piiper and stallaris Baumgarten- vitro Baumgarten- Schumann Schumann 1968 1967 Sphyrna tiburo 1.9 28 Haggard 2000 9.4 28 Haggard 2000

Triakis 0.15- 19-22 Lai et al. 1990 cannulated semifasciata 0.20 Triakis semifasciata 1.91 -0.23 8.33 <0.04 10 Bernal et al. 2018 1.52 -0.62 8.07 <0.04 15 2.23 -0.43 8.01 <0.04 20 2.89 7.45 1.01 10 3.61 7.51 1.01 15 4.20 7.43 1.01 20 Heterodontiformes Heterodontus 0.27 -0.11 19 Cooper and cannulated portjacksoni Morris 2004 Lamniformes Isurus oxyrinchus 0.20 +0.16 7.6 None 25 Wells and Exercise Davie 1985 stressed

131 Isurus oxyrinchus 1.93 -0.74 7.93 <0.04 15 Bernal et al. 2018 1.82 -0.24 8.17 <0.04 20 2.48 -0.11 8.13 <0.04 25 2.97 7.68 1.01 15 2.93 7.33 1.01 20 2.79 7.64 1.01 25 Orectolobiformes Hemiscyllium 1.43 -0.29 7.8 None 25 Wells et al. Washed RBCs 6.5 25 Routley et al. ocellatum 1992 kPa 2006 1.89 -0.29 7.4 None 25 Hemiscyllium 4.27 28 Speers- 5.1 28 Speers-Roesch ocellatum Roesch et al kPa et al. 2012a 2012a Hemiscyllium 6.6 23 Chan and plagiosum kPa Wong 1977 Squaliformes Squalus acanthias 1.76 -0.28 7.85 0.29 15 Wells and Weber 1983 Squalus suckleyi 0.16 None 7.6 0.07 11 Lenfant and Cannulated, Hohansen free- 1966 swimming

132 Supplementary Table 2. Metabolic Rate values for elasmobranch species for which P50 and Pcrit data are also available. Measurement type refers to either standard metabolic rate (SMR) or routine metabolic rate (RMR).

Species Temp Type Metabolic Rate Source -1 -1 (°C) (mg O2 kg l ) Aptychotrema rostrata 28 SMR 96 Speers-Roesch et al. 2012 Rostaraja eglanteria 20 SMR 39 Schwieterman et al., 2019 Torpedo marmorata 23 SMR 34 Hughes 1987 Carcharhinus plumbeus 28 RMR 165 Crear et al., 2019 Scyliorhinus canicular 17 SMR 80 Hughes and Umezawa 1968 Scyliorhinus stellaris 18 SMR 51 Piiper et al., 1977 Sphyrna tiburo 29 RMR 330 Carlson and Parsons 1999 Hemiscyllium ocellatum 28 SMR 80 Speers-Roesch et al., 2012

133 CHAPTER IV

A search for red blood cell swelling in five elasmobranch fishes

134 ABSTRACT

In teleost fishes, red blood cell (RBC) swelling compensates for decreases in hemoglobin

(Hb) oxygen (O2) affinity (Bohr effect) and maximum blood oxygen carrying capacity

(Root effect) under acidosis which could compromise O2 uptake at the gill and subsequent delivery to tissues. There is general consensus elasmobranch fishes do not exhibit stress-induced RBC swelling (as do teleost fishes) to counteract the effects of acidosis. Work on exhaustively exercised juvenile sandbar sharks (Carcharhinus plumbeus) and anoxia-exposed epaulette sharks (Hemiscyllium ocellatum) has, however, revealed RBC swelling which (at least in the former) counteracts the effects of plasma acidosis on Hb-O2 affinity. The aim of this project was, therefore, to investigate the prevalence of RBC swelling in elasmobranchs fishes facing physiologically stressful conditions. Blood samples were taken from captive individuals of five different species

(clearnose skate, Rostaraja eglanteria; sandbar shark; blacktip reef shark, C. melanopterus; sicklefin lemon shark, Negaprion acutidens; and epaulette shark) exposed to either air (to induce respiratory acidosis) or chased to exhaustion (to induce metabolic acidosis). Hematological parameters (hematocrit, [Hb], red blood cell counts, red cell volume, and mean corpuscular hemoglobin content) were measured for evidence of RBC swelling. I did not find RBC swelling in any of the species examined, even though published results show RBC swelling occurs in sandbar and epaulette sharks. It appears, therefore, that these methods were insufficient to evoke the level of stress necessary to elicit RBC swelling, my sample size was too small to detect an effect, or the precision of my measurement was inadequate.

135 INTRODUCTION

Following capture, fish exhibit elevated plasma carbon dioxide partial pressure

(pCO2), decreased plasma pH, and the associated decreases in blood-oxygen affinity and maximum blood oxygen carrying capacity (Pickering, 1981; Wood, 1980; Korsmeyer et al., 1997; Brill et al. 2008; Mandelman and Skomal, 2009; Marshall et al., 2012; Skomal and Bernal, 2010; Skomal and Mandelman, 2012). The accompanying elevated plasma potassium and lactate concentrations are indicative of cellular damage and significant anaerobic metabolism (respectively). These physiological disturbances can persist for hours, potentially contributing to post-release mortality (Moyes et al. 2006; Skomal 2007;

Campana et al. 2009; Heberer et al. 2010; Whitney et al. 2016). Understanding how species overcome these physiological disturbances has implications for predicting rates of post-release mortality and best practices for handling fishes prior to release (Horodysky et al. 2015; Horodysky et al. 2016)

In teleost fishes, blood pCO2 levels increase in the capillaries causing CO2 to diffuse into the red blood cells (RBCs) where intracellular carbonic anhydrase (CA) catalyzes its hydration into carbonic acid (H2CO3), which then rapidly dissociates into

- + + HCO3 and H (Figure 1). Increasing [H ] (i.e., decreasing intracellular pH, pHi) decreases hemoglobin oxygen affinity (Hb-O2) as measured by the partial pressure of oxygen necessary for 50% saturation of Hb (P50). Decreases in affinity are caused by a conformational change in Hb from the R(elaxed) state to the T(ense) state caused by H+ binding to Hb. This rightward shift of the blood O2 dissociation curve (i.e., Bohr effect,

Figure 2) is quantified as (log p50/pH) (Jensen 2004; Berenbrink 2006; Berenbrink 2011;

Nikinmaa et al. 2019). In many teleost fishes, however, the reduction in pHi is

136 accompanied by a decrease in maximum blood O2 carrying capacity referred to as the

Root effect (Figure 2; Root 1931; Pelster and Randall 1998; Waser 2011). The Root effect is leveraged to increase oxygen delivery in the capillary through plasma-accessible

CA. The extracelluar CA essentially “short circuits” the removal of intracellular H+ via

+ + - sodium (Na ) hydrogen (H ) exchangers (NHEs) by dehydrating secreted HCO3 to CO2 which then re-diffuses into the cell promoting a decrease in pHi (Rummer and Brauner

2011; Rummer et al. 2013; Randall et al. 2014).

Similar mechanisms of gas transport exist in elasmobranch fishes (Figure 1), albeit with several notable differences. Primarily, blood from elasmobranch fishes does not exhibit a significant Root effect (Lenfant and Johansen 1966; Pennelly et al. 1975;

Weber et al. 1983a; Wells and Weber 1983; Dafré and Fo 1989; Berenbrink et al. 2005;

Brill et al. 2008), which makes reductions in pHi less of a factor in releasing oxygen in the capillaries. The lack of pH sensitivity in elasmobranch Hbs likely explains why elasmobranch capillaries do not possess plasma-accessible CA like teleost fishes (Perry and Gilmour 2006; Gilmour and Perry 2010; McMillan 2018), as “short-circuiting” the

- + NHE by re-combining HCO3 and H into CO2 in the plasma would have limited impact on Hb-O2 affinity. There is conflicting evidence as to the presence and extent of a Bohr effect within the elasmobranch group (reviewed by Morrison et al. 2015), with diverse species such as arctic skate (Amblyraja hyperborea), Eaton’s skate (Bathyraja eatonii)

(Verde et al. 2005), and shortfin mako shark (Isurus oxyrinchus) (Wells and Davie 1985) exhibiting no Bohr effect, while porbeagle shark (Lamna nassus) exhibits a Bohr effect similar in magnitude to those of teleost fishes (Bohr coefficient = -0.6) (Larsen et al.

2003). Generally, Hb-O2 binding in elasmobranch fishes is characterized by high oxygen

137 affinity and little change in Hb-O2 affinity with oxygen binding (weak cooperativity)

(Bushnell et al. 1982; Wells and Weber 1983; Brill et al. 2008). Oxygen delivery in elasmobranch fishes may be mediated by intracellular ATP and GTP concentrations

(Pennelly et al. 1975; Leray 1979; Weber 1983; Tetens and Wells 1984), intracellular Cl- concentration (Fievet et al. 1987; Nikinmaa and Salama 1998), plasma trimethylamine oxide (TMAO) and urea concentrations (Weber 1983; Weber et al. 1983a; Weber et al.

1983b; Tetens and Wells 1984), or O2 availability (Bushnell et al. 1982).

The mechanisms for maintaining blood oxygen delivery following exposure to hypoxia or exhaustive exercise are generally understood for teleost fishes. Because the influence of pHi on Hb-O2 affinity is so profound, teleost fishes have several physiological mechanisms to counteract decreases in Hb-O2 affinity during acidosis.

They may release additional red blood cells through adrenaline-mediated splenic contraction, thus increasing the [Hb] of the blood (Kuwahira et al. 1999). RBCs also have an adrenaline response, where β-adrenergic compounds bind to the β(3)-type receptors on the cell surface which, in turn, activates adenylate cyclase to form cAMP. The cAMP then binds to the βNHEs on the RBC membrane, which moves H+ out of the cell in

+ exchange for Na , thus restoring pHi and thus Hb-O2 affinity and blood O2 carrying capacity (Figure 3). The influx of osmotically active Na+ draws water into the cell, causing measurable RBC swelling (measured as an increase in RBC volume of up to

70%, Farrell 2011). Thus, as the blood passes through gills, it can still effectively bind O2 and subsequently maintain O2 delivery to the tissues through combined Bohr and Root effects (Hladky and Rink 1977; Nikinmaa 1983). RBC swelling and splenic contraction can, in combination, result in a 70-100% increases in teleost Hct in response to

138 exhaustive exercise (Yamamoto et al. 1980; Wells and Weber 1990; Gallaugher and

Farrell 1998).

It has long been thought the lack of an elasmobranch RBC swelling response was due to the lack of a significant Root effect, which makes reductions in pHi less consequential. Some elasmobranch fish do, however, exhibit a Bohr effect and thus may experience reduced blood-oxygen transport capacity following exercise or prolonged air exposure (Lowe et al. 1995; Brill et al. 2008; Wells 2009). It is unknown how these species maintain oxygen delivery under acidosis. Unlike teleost RBCs, elasmobranch

RBCs show little to no swelling response following adrenergic stimulation (Metcalfe and

Butler 1988; Lowe et al. 1995; Brill et al. 2008). This is not surprising, as currently, there is no published evidence that elasmobranch RBCs possess adrenergically mediated NHEs on the RBC surface (Lowe et al. 1995; Berenbrink et al. 2005). It is likely, however, that they possess NHEs, which assist in maintaining osmotic balance, as all fishes possess

RBC NHEs with the exception of hagfish (Nikinmaa 2011). With the exception of the mako shark (Isurus oxyrinchus) (Wells and Davie 1985), most elasmobranch Hbs also have a much higher buffering capacity than those of teleost fishes (Nikinmaa 2011), which may assist in pHi maintenance. Elasmobranch fishes also generally do not display large increases in Hct when exercised (Piiper et al. 1970; Lowe et al. 1995). The lack of change in Hct in elasmobranch fishes is likely due to a combination of the absence of both RBC swelling and splenic contraction (Opdyke and Opdyke 1971); although the latter response is still debated (Nilsson et al. 1975). Elasmobranch fishes are, therefore, thought to have less capacity to increase oxygen delivery to the tissues following exhaustive exercise (Piiper et al. 1977; Butler and Metcalfe 1988; Lowe et al. 1995).

139 The mechanism driving swelling (when present), and indeed the function of swelling, may differ between elasmobranchs species. Juvenile sandbar shark

(Carcharhinus plumbeus) and epaulette shark (Hemiscyllium ocellatum) exhibit RBC swelling following exhaustive exercise or exposure to anoxic water (Brill et al. 2008;

Chapman and Renshaw 2009). Wells and Davie (1985) likewise found evidence of RBC swelling in mako shark following rod-and-reel capture. In contrast, Graham et al. (1990) and Lowe et al. (1995) found that neither RBCs from little skates (Leucoraja ocellata) nor shovelnose ray (Rhinobatos typus) exhibit any signs of pHi regulation nor swelling.

Here, I sought to investigate the prevalence of RBC swelling in phylogenetically diverse elasmobranch fishes. Specifically, I attempt to determine if clearnose skate

(Rostaraja eglanteria), sicklefin lemon shark (Negaprion acutidens), and blacktip reef shark (C. melanopterus) exhibit RCB swelling in response to respiratory or metabolic acidosis. I also reinvestigated red blood cell swelling in sandbar and epaulette sharks to ensure that my methods were capable of inducing red blood cell swelling and that my measurement techniques were capable of detecting RBC swelling.

MATERIALS AND METHODS

All work was approved by the William & Mary Institutional Animal Care and

Use Committee (IACUC-2019-03-18-13539-rwbril) or by the James Cook University

Animal Ethics Committee (A2588 and A2394).

Clearnose skate (n=10; 61 ± 0.7 cm total length, mean ± SE) were collected by otter trawl in the lower portion of the Chesapeake Bay and were then transferred to indoor holding tanks with a recirculating seawater systems (maintained at 20 ± 1 °C) at

140 the Virginia Institute of Marine Science Eastern Shore Laboratory. Sandbar shark (n=10;

83 ± 6 cm TL) were caught via rod and reel in the coastal lagoons near Wachapreague,

VA and were also transferred to holding tanks at the Eastern Shore Laboratory equipped with a semi-open seawater system (maintained at 28 ± 1 °C). Blacktip reef (n=10; 57 ± 5 cm TL) and sicklefin lemon (n=8; 67 ± 9 cm TL) sharks were caught via gillnet (50 m long, 1.5 m tall, 5 cm stretch mesh) deployed from shore around Moorea, French

Polynesia. They were transferred to recirculating holding tanks (maintained at 29 ± 1 °C) at the Centre de Recherches Insulaires et Observatoire de l’Environnement in Moorea.

Epaulette shark (n=10; 60 ± 1 cm TL) were collected by hand near Orpheus Island,

Australia and were then transferred to holding tanks equipped with recirculating water

(maintained at 26 ± 1°C) at the Marine and Aquaculture Research Facilities Unit at James

Cook University, Townsville, Australia. Subjects were tagged with t-bar tags through the dorsal musculature for individual identification, and acclimated to captivity for at least two weeks prior to their first blood draw, except for epaulette shark which were maintained in captivity for eight months prior to use in experiments.

Blood samples were obtained through direct caudal venipuncture into heparinized syringes. Individuals were exposed to all three treatment conditions in a randomized order. Blood samples referred to as “control” were obtained from individuals that were removed from their holding tank with as little disturbance as possible. One treatment involved air exposure for five min to induce respiratory acidosis, after which a blood sample was obtained. Another treatment involved a chase protocol (enforced exercise in a round tank for five minutes) followed by a bout of air exposure for one minute to induce mixed metabolic and respiratory acidosis. In this treatment, individuals were returned to

141 their holding tanks for one hour prior to blood sampling to allow lactate and hydrogen ions to be released into the blood.

In all cases, blood samples were immediately transferred to a cooler with ice packs, and all hematological parameters were measured within 15 minutes of collection.

[Hb] was measured using the traditional Drabkin’s cyanomethoglobin method. Blood was added to a Drabkins and brij solution, allowed to incubate for at least 5 minutes, and then was read in quadruplicate at 540 nm in either a 1.5 mL cuvette using a Spectronic 200

(Thomas Scientific, Swedesboro, NJ, USA) or in a 96 well plate using a Biotek 800 TS spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA). I measured hematocrit following five minutes of centrifugation of blood samples in microbore heparinized capillary tubes in either an Adams Autocrit Centrifuge (Clay-Adams, Inc,

New York, NY, USA) or a Zipocrit haematocrit microcentrifuge (LW Scientific,

Lawrenceville, GA, USA). I measured pHe to ascertain if these methods were enough to induce acidosis. pHe was measured using whole blood and either the associated capillary pH electrode at the animal’s holding tank temperature (BMS3 Mk2, Radiometer

America, Westlake, OH, U.S.A.), or the Hanna Instruments HI 99161 pH meter (Hanna

Instruments, Woonsocket, RI, USA) at ambient air temperature (25°) following recommendations by Talwar et al. (2017). Cell counts were conducted by diluting a blood sample with plasma 1:90, a subsample of which was immediately placed on a hemocytometer slide and photographed at 40x magnification using a compound microscope. RBCs were counted using ImageJ software (Schneider et al. 2012). From these data, I was able to calculate mean cell volume (MCV; RBC countHct-1) and mean corpuscular hemoglobin content (MCHC; [Hb]Hct-1).

142 Statistical analyses were conducted base R using R statistical software (R Team

2019). The pH data were analyzed with an analysis of variance using Tukey’s post-hoc tests with the glht command in the multcomp package (Bretz et al. 2016). For the remaining metrics, I calculated relative change from the “control” treatment on the same individual and conducted t-tests on the resulting relative change data. I also compared my results to the raw data from Brill et al. (2008) to assess directly differences between my results and published values. This comparison was done using an analysis of variance

(ANOVA) with Tukey’s post-hoc tests. All statistics were evaluated with a significance level of  = 0.05.

RESULTS

In all species measured, I detected significant differences between the mean pHe of blood samples from control sharks, and blood samples collected from treatment conditions (Fsandbar=60.44, p<0.05; Flemon=4.38, p=0.05; Fepaulette=45.32, p<0.05; and

Fskate=97.11, p<0.05, Fsandbar= 4.65, p<0.05; Figure 4). The mean differences between pHe and pHi in the stress treatments were significantly different from the mean values in the control treatment in clearnose skate (F=5.40, p<0.0; Figure 5). For sandbar shark, the mean differences between pHe and pHi following air exposure were significantly different from the mean values in the control treatment (F=3.55, p<0.05; Figure 5).

I observed significant increases in both the mean Hct (tair=3.70, p<0.05; tchase=2.61, p<0.05) and mean [Hb] (tair=3.37, p<0.05; tchase=2.77, p<0.05) in blacktip reef shark. Sicklefin lemon shark also showed significant increases in mean Hct following stress (tair=3.17, p<0.05; tchase=2.96, p<0.05), and increases in mean [Hb] (tair=3.50,

143 p<0.05; tchase=3.28, p<0.05). Clearnose skate showed a significant increase in mean Hct regardless of (tair=5.18, p<0.05; tchase=4.48, p<0.05), although they exhibited an increase in mean [Hb] following air exposure only (tair=3.47, p<0.05). Epaulette shark showed no significant responses despite the change in mean pHe and sandbar shark displayed an increase in only mean Hct following both treatments (tair=2.96, p<0.05; tchase=3.32, p<0.05). Mean RBC count increased following air exposure in blacktip reef shark

(tair=3.47, p<0.05), but there were no significant changes in mean MCHC in any species

(tblacktip-air=0.83, p>0.05; tblacktip-chase=1.51, p>0.05;tlemon-air=-1.14, p>0.05; tlemon-chase=-0.67, p>0.05; tepaulette-air=-0.36, p>0.05; tepaulette-chase=0.28, p>0.05; tskate-air=0.35, p>0.05; tskate- chase=-1.32, p>0.05; tsandbar-air=-0.22, p>0.05; tsandbar-chase=0.03, p>0.05). The only significant change to mean MCV was an increase following the chase protocol in sicklefin lemon shark (tchase=2.77, pchase<0.05; Figure 6).

Comparing my results to those published by Brill et al. (2008), I found significant differences in mean Hct for stressed individuals (t=-5.25, p<0.05). I also found significantly higher mean MCHC values in this study compared to those from Brill et al.

(2008) (t= -20.51, p<0.05). Similarly, I found significantly lower mean MCV values in this study than those from Brill et al. (2008) (t=24.11, p<0.05; Figures 7 and 8). While the changes in mean pHe reported for both studies were similar, my mean Hct levels were generally lower than those reported by Brill et al. (2008) (t=1.7, p>0.05; Figure 6). The lack of significant differences in mean [Hb] (t=0.05, p>0.05) and similarities in the distributions of the mean [Hb] (Figure 7) suggest these differences in Hct are likely driving the different responses seen in mean MCHC and mean MCV between the two studies (Figure 8).

144 DISCUSSION

I observed increases in mean Hct following stress in all species. Increases in mean

Hct following capture or exercise have also been documented in sandbar shark (Brill et al. 2008), lemon shark (Negaprion brevirostrus) (Bushnell et al. 1982), mako shark

(Wells and Davie 1985), porbeagle shark (Lamna nasus), pelagic thresher shark (Alopias pelagicus), and blacktip shark (C. limbatus) (Marshall et al. 2012); and following anoxic exposure in grey carpet shark (Chapman and Renshaw 2009). It is important to note, however, that exercise did not elicit any changes in mean Hct or other hematological parameters in giant shovelnose ray (Rhinobatos typus) (Lowe et al. 1995), underscoring the species-specific nature of the elasmobranch stress responses. If increases in mean Hct were the result of RBC swelling, this should have been evident in the other metrics − notably decreases in mean MCHC with simultaneous increases in mean MCV. I only observed one instance of an increase in mean MCV, which was not coupled with a decrease in MCHC. I saw changes in the mean RBC count data only in air exposed blacktip reef shark, which coupled with the significant increases in mean Hct and mean

[Hb], may be indicative of splenic contraction in this species, although it is generally assumed this does not occur in elasmobranch fishes (Opdyke and Opdyke 1971). This may also have been evidence of a fluid shift from the plasma to the intracellular compartment, thus reducing plasma volume, and increasing mean Hct in the absence of

RBC swelling. I observed an increase in mean [Hb] without a change in mean cell counts in sicklefin lemon shark and clearnose skate, which could potentially also be evidence of a fluid shift. Alternatively, the lack of changes in mean RBC counts and mean MCV

145 could be a measurement artifact, as reliable RBC counts are difficult to achieve in elasmobranch fishes (Arnold 2005).

While my results suggest swelling did not occur in these trials, I cannot rule out the possibility, and it is more probable that my methods were insufficient to induce the physiological mechanisms that ultimately result in RBC swelling. Brill et al. (2008) and

Chapman and Renshaw (2009) have (respectively) documented RBC swelling in sandbar shark following capture and in epaulette shark following exposure to anoxia. Comparing my results to those published by Brill et al. (2008), the similarities in the distributions of the Hb levels mean these differences in Hct are likely driving the different responses seen in in MCHC and MCV between the two studies. My Hct and Hb results were similar to those found in sandbar shark previously, but I did find higher MCHC and lower MCV values, suggesting that the RBCs in this study were smaller than those from the individuals used by Brill et al. (2008), the likelihood of which is slim.

The lack of swelling responses here could be attributed to several factors, including insufficient precision in my blood analysis techniques, introduced variability in the data due to the assistance of multiple researchers in sample processing, or insufficient samples sizes to detect true effects. The techniques I used followed protocols used by

Brill et al. (2008), so the former is unlikely. I did involve undergraduate students in this research and had different people conducting Hct measurements and counting RBCs, which may have introduced variability in the data. This, coupled with the variability in an individual fish’s reaction to the stress treatments, could be the sources of variation in these data. I should note that Brill et al. (2008) had a higher samples size (n=22), compared to the sample sizes I had for a given species (n=8 or 10). The lack of sufficient

146 samples to detect a statistically significant effect may have contributed to the findings presented here.

Another reason I did not observe RBC swelling may have been a captivity effect.

Chapman and Renshaw (2009) found significant differences in the circulating lactate concentrations of unstressed wild-caught and captive epaulette sharks, and suggested that confinement alone could cause stress (Wise et al. 1998). Similarly, grey carpet shark held in captivity for two weeks had smaller hematological responses (e.g., lower percent changes in Hct) than wild-caught individuals following exposure to anoxia. This smaller treatment effect possibly evinces a potential loss of normal responses during captivity

(Chapman and Renshaw 2009). The sandbar shark used here were held for two months in captivity and were frequently dip-netted for transfer between tanks for use in another study. This may have led to a long-term physiological stress response obscuring any measurable effect of the acute stress treatments. In contrast, the individuals used by Brill et al. (2008) were held for a maximum of one week. There may have also been stress- induced changes in blood parameters measured from fish in the control treatment, thus minimizing the relative differences in my treatment blood draws. It is generally accepted, however, that elasmobranch blood parameters are not disturbed by gentle handling and restraint associated with caudal venipuncture (Cooper and Morris 1998; Brill et al. 2008), nor are elasmobranch hematological parameters unstable over short storage durations

(less than 3 hs; Schwieterman et al. 2019). It is also likely, however, that I did not observe RBC swelling in sandbar shark blood because a chase protocol does not induce the same stress response as rod-and-reel capture, even though pHe values measured were comparable to those observed from rod-and-reel captured fish (Brill et al. 2008).

147 Similarly, Chapman and Renshaw (2009) employed a 1.5 hr exposure to anoxic water, which may well be more stressful than my air exposure protocol (i.e., held out of water for <12 minutes).

The lack evidence supporting either RBC swelling or splenic contraction (e.g., a lack of significant differences in mean MCHC and mean MCV) suggests that metrics vary in their ability to detect RBC swelling. Further, I cannot dismiss the possibility that there were errors in my data collection affecting the physiologically inconsistent results of the statistical tests. For example, Chapman and Renshaw (2009) did not measure RBC counts, and thus could not calculate MCV; their assertion of RBC swelling was based on increasing Hct and changes in MCHC. Agreement of more parameters would obviously build confidence in study results.

Additional sources of variation may be inherent in the laboratory measurement methods I employed. The method for measuring Hb (using Drabkin’s solution and reading absorbance at a single wavelength- 540 nm) does not account for the relative state of the Hb dimers and thus may not be an accurate representation of the oxygen carrying capacity of the blood (Fyhn and Sullivan 1975; Morrison et al. 2015).

Elasmobranch Hbs are more likely than teleost Hbs to revert to their dimetric forms, with

94% of spiny dogfish (Squalus acanthias) Hb existing as dimers in the oxygenated state

(Fyhn and Sullivan 1975). If the absorbance values of dimers differ from tetramer forms, then measuring Hb under different oxidation states could result in different [Hb] values.

Assuming RBC swelling does occur in at least some elasmobranch fishes but was simply not induced or detected by the methods employed, two questions remain: (1) what is the function of swelling and (2) what mechanisms induce RBC swelling? I will discuss

148 potential answers to both, with postulations about other mechanisms that could affect oxygen delivery and pHi regulation. As O2 delivery and carbon dioxide (CO2) excretion are highly linked processes (Brauner 1995; Morrison et al. 2015), I will also discuss mechanisms affecting CO2 excretion in elasmobranch fishes.

Possible Functions of RBC Swelling

The sensitivity of elasmobranch Hbs to pH is diminished relative to those of teleost fishes. As seen in these clearnose skate results, elasmobranch fishes generally lack a strong Bohr shift and an observable Root shift (reviewed by Morrison et al. 2015).

When present, elasmobranch Bohr effects tend to occur over a wider pH range than those of teleost fishes, a phenomenon likely resulting from the different pKa values of elasmobranch histidine residues on various Hb isoforms (Mumm et al. 1978; Weber et al.

1983a). The diversity of Hbs may explain elasmobranch fishes’ ability to increase O2

+ loading over a broad range of pO2 values, as different isoforms will bind H (and thus switch from the T- to the R-state) at different pCO2 levels. Each isoform has a different

P50 value, signifying different levels of Hb-O2 affinity (Morrison et al. 2015). The diversity of isoforms present within a given species could, therefore, assist in oxygen delivery under stress (Fyhn and Sullivan 1975).

Another allosteric modulator of Hb-O2 affinity is TMAO. TMAO increases molecular rigidity and thereby modulates the conformation changes that underlie the

Bohr and Haldane effects (Weber 1983). Thus, TMAO may help preserve the blood oxygen affinity under decreasing pHi (for elasmobranch species which display a Bohr effect) (Weber et al. 1983a; Weber et al. 1983b). In species that do not display a Bohr effect, such as the clearnose skate, changes in intracellular TMAO concentration could

149 assist in fine-tuning Hb-O2 affinity through protein stabilization or destabilization. The mechanisms of adjusting intracellular TMAO concentration are unknown, but future work may consider measuring intracellular [TMAO] in species like the clearnose skate

(which does not exhibit a Bohr effect) and the sandbar shark (which does) (Brill et al.

2008).

Intracellular concentrations of nucleoside triphosphate (NTP) and urea may also play an important role in regulating Hb-O2 affinity. In elasmobranch RBCs, the concentration of adenosine triphosphate (ATP) is lower than in teleost RBCs (Wells and

Weber 1983; Wilhelm Filho et al. 1992). ATP concentrations further decrease under exercise (Tetens and Wells 1984), thus decreasing O2 affinity of stripped Hb (Tetens and

Wells 1984; Brill et al. 2008) and presenting the same issue as a reduction in pHi for species with a large Bohr effect: an inability to bind O2 at the gills. Urea can increase Hb-

O2 affinity by readily diffusing into RBCs (Rabinowitz and Gunther 1973) and reducing

Hb-ATP sensitivity (Weber 1983; Weber et al. 1983b). While all elasmobranch fishes have high circulating levels of urea (Wood et al. 2007), responses of elasmobranch Hb to urea are species-specific. For example, batoid and selachian Hbs are largely insensitive to urea (Bonaventura et al. 1974; Scholnick and Mangum 1991), whereas urea increases Hb oxygen affinity in draughtsboard shark (Cephaloscyllium isabella) and spiny dogfish

(Weber et al. 1983a; Weber et al. 1983b; Wells and Weber 1983; Tetens and Wells

1984). The lack of a Bohr shift in clearnose skate, a batoid, may therefore be an indication that this species uses urea to regulate Hb-O2 affinity. In isolated sandbar shark

Hbs, urea had no effect on P50 and did not ameliorate reductions in Hb-O2 affinity due to

150 ATP (Brill et al. 2008), suggesting that species exhibiting a measurable Bohr shift may regulate Hb-O2 affinity with pH changes rather than with urea.

In elasmobranch species that do exhibit a Bohr shift, the function of RBC swelling is presumed to be the same as in teleost fishes: namely, to regulate pHi and thus preserve Hb-O2 affinity under acidotic conditions. Elasmobranch fishes may not regulate pHi, however. Winter skates (Raja ocellata) exposed to hypercapnia exhibited no signs of active RBC pHi regulation (Graham et al. 1990), although clearnose skate experiencing metabolic and respiratory acidosis showed no significant change in pHe-pHi, suggesting pHi was being buffered or regulated. For batoids which do not exhibit a Bohr shift, pHi regulation may not be necessary. For species which exhibit a Bohr shift, however, it has been posited that because elasmobranch Hbs have a higher buffering capacity than teleost

+ Hbs, Hb mediates pHi and remove the necessity of H transport through the NHE when there are decreases in pHe (Nikinmaa 1997). Elasmobranch RBCs are 3-4x larger than those of teleost fishes with higher [Hb] per cell (Wilhelm Filho et al. 1992; Morrison et al. 2015), and thus a higher number of H+ binding sites per cell (Brauner et al. 1996).

Furthermore, elasmobranch RBC CA tends to function more slowly than that occurring in

+ - teleost fishes (Maren et al. 1980), reducing the rate of intracellular H and HCO3 increase. Additionally, the availability of Hb- to act as a buffer depends on its oxygenation state (the Haldane effect) (Nikinmaa et al. 2019). The generally low O2 saturation of elasmobranch arterial blood (Bushnell et al. 1982) could therefore be an adaptation to ensure intracellular buffering capacity by keeping a large store of deoxygenated Hbs available to bind H+ (Perry and Wood 1989).

151 The combined effects of Hb deoxygenation and reduced NTP levels may also regulate pHi in elasmobranch fishes. Intracellular phosphate concentrations can also affect pHi (Jensen 2004; Morrison et al. 2015). NTP levels generally decrease upon deoxygenation (Lykkeboe and Weber 1978; Jensen and Weber 1982). Because of the passive Donnan distribution of H+ across the RBC membrane (Albers and Goertz 1985), reduced intracellular concentrations of negatively charged NTPs will increase RBC pHi.

In tench (Tinca tinca), a teleost fish with RBCs insensitive to catecholamines, these mechanisms are thought to be responsible for regulating pHi (Jensen 1987).

Possible Mechanisms of RBC Swelling in Elasmobranch Fishes

The mechanism responsible for initiating RBC swelling in elasmobranch fishes is unknown (Brill et al. 2008; Chapman and Renshaw 2009), but is likely not driven through pHe changes as evidenced by my lack of significant results. The mechanisms for swelling in teleost fishes is stimulation of the beta-adrenergic NHEs. As there is no published evidence that elasmobranch RBCs have beta-adrenergic NHEs (Wood et al.

1994; Lowe et al. 1995; Berenbrink et al. 2005; Brill et al. 2008), it is unlikely that increases in circulating catecholamines are the proximate cause, as they are in teleost fishes (Yamamoto et al. 1980; Wells and Weber 1990; Gallaugher and Farrell 1998).

Further, Brill et al. (2008) were unable to induce swelling in sandbar shark RBCs with catecholamine exposure. Other potential mechanisms for swelling in elasmobranch fishes include a change in Cl-. In trout treated with propanol to block an adrenaline response, exposure to hypoxia triggered mild RBC swelling which was attributed to an increase in intracellular [Cl-] (Fievet et al. 1987). Changes in [Cl-] were likely due to H+ buffering by

Hb-, thus decreasing intracellular anion concentrations. As RBC membranes are

152 permeable to Cl-, Cl- and osmotically motivated water may be drawn into the cell in

- + response to Hb binding with H (Nikinmaa and Salama 1998). Indeed, in fishes, pHe is

- - modulated by increasing extracellular HCO3 through an exchange with Cl across the

RBC membrane (Heisler 1988). Reductions in intracellular ATP and GTP would likely also cause in influx of Cl- through the reduction of an electrical gradient further contributing to an osmotic imbalance and cell swelling (Brill et al. 2008).

Preservation of CO2 Excretion

Oxygen delivery and carbon dioxide excretion are coupled processes (Brauner et al. 1996; Brauner and Randall 1996), and while thus far discussion has focused on O2 delivery, there may be mechanisms influencing cell size and pH that are driven by CO2

- - excretion. CO2 is transported in the blood as HCO3 . HCO3 dehydration takes 25-90 seconds at physiological pH and temperature (Edsall 1969), while blood residence time in the gills is only 0.5-6.5 seconds (Bhargava et al. 1992). CA must be therefore used to increase the reaction rate up to 25,000 times (Nikinmaa et al. 2019). The method of CO2 excretion in elasmobranch fishes is characterized by membrane-bound, plasma-accessible

- CA in the gill epithelium, which contributes to the dehydration of plasma HCO3 into

CO2 (Perry and Gilmour 2006; Gilmour et al. 2007; Gilmour and Perry 2010; McMillan

2018). The high plasma buffering capacity of elasmobranch fishes (Lenfant and Johansen

1966; Graham et al. 1990) contributes H+ to this reaction, helping to decouple oxygen

+ transport and CO2 excretion relative to teleost fishes which are more reliant on H released from Hb. The resultant increase in the partial pressure of CO2 (pCO2) in the plasma can then drive CO2 excretion across the gills (Wood et al. 1994). CO2 excretion has been well studied in dogfish (S. suckleyi), which also exhibit low RBC CA activity

153 relative to teleost fishes (Maren et al. 1980; Henry et al. 1997), and lack CA inhibitors in the blood (Henry et al. 1997). Elasmobranch fishes have low levels of CA in circulation in the plasma (Wood et al. 1994), although this is thought to come from regular RBC lysis and is not likely a large contributor in CO2 excretion (Henry et al. 1997).

All of this contrasts with blood CO2 transport by teleost fishes, where plasma

- HCO3 moves into RBCs via an anion exchanger and is subsequently converted to CO2, catalyzed by intracellular CA (Randall 1982; Perry and Gilmour 2006; Nikinmaa et al.

+ - 2019). The relatively large Haldane effect in teleost fishes supplies H used in HCO3 dehydration, thus linking CO2 excretion with Hb-oxygen affinity. While dogfish and trout have been extensively studied regarding CO2 excretion and have been demonstrated to possess drastically different mechanisms, delineations between elasmobranch and teleost fishes may not be clear cut. Recent work exploring the diversity of buffering capacity and

CA density and function across 13 elasmobranch species found large interspecific variation (McMillan 2018), which is not surprising considering the diversity of the elasmobranch group (Compagno et al. 2005; Ebert et al. 2013). Given the method of CO2 extraction in elasmobranchs relies on plasma-accessible CA as well as intracellular CA

- for HCO3 dehydration, oxygen delivery and CO2 excretion are more uncoupled in elasmobranch fishes than they are in teleost fishes. This uncoupling is evidenced by unchanged CO2 excretion rates in dogfish even when Hct is artificially reduced to 5%

(Gilmour and Perry 2004). I therefore posit that preservation of CO2 excretion in elasmobranch fishes is unlikely the function of RBC swelling.

154 Future Work

Experimental work is needed to elucidate the prevalence, cause, and function of

RBC swelling in elasmobranch fishes. Although, as mentioned, it has been established that adrenaline has no impact on RBC volume, and thus is assumed to have no impact on

NHEs, more study on the presence of NHEs on elasmobranch RBC membranes could help to guide future work on RBC pHi regulation (Brill et al. 2008; Chapman and

Renshaw 2009). Evaluating the presence of NHEs on elasmobranch RBCs could be accomplished through confocal microscopy, with the use of specific primers targeting

NHEs. As these proteins are highly conserved across phylogeny, existing primers could be used for preliminary investigations into RBC membrane-bound proteins from both elasmobranchs and teleost fishes.

Additionally, future experiments could examine the impacts on Hb-O2 affinity and RBC volume of some of the aforementioned mechanisms, namely [Cl-], [ATP], and urea/TMAO on a diversity of elasmobranch species.

CONCLUSIONS

The variability in the hematological parameters I measured, as well as those previously published (e.g., Gallagher et al. 2014; Marshall 2015; Whitney et al. 2017), suggest that exhaustive exercise may not be an accurate proxy for stressors associated with capture or extended exposure to anoxia. Understanding the mechanisms driving

RBC swelling (and associated changes in the relationships between pHe, pHi, and hemoglobin O2 affinity) in elasmobranchs will increase my understanding of their physiological responses and abilities to survive capture and potential to withstand the

155 stressors associated with climate change (Esbaugh 2018). The apparent diversity of Hb responses to changes in pHi within the elasmobranch group may offer explanations of the apparent resiliency exhibited to simulated increases in pCO2 (Bouyoucos et al. 2019).

ACKNOWLEDGEMENTS

This work was supported by the National Science Foundation Graduate Research

Program (DGE-1444317) and the Graduate Research Opportunities Worldwide

Supplemental Funding Program to GDS. I thank the staff of the Centre de Recherches

Insulaires et Observatoire de l’Environnement, Marine and Aquaculture Research

Facilities Unit at James Cook University, and the Virginia Institute of Marine Science

Eastern Shore Laboratory for their assistance facilitating this work. I would also like to thank Nilanjana Das, Julie Brucker, Kim Eustache, Isabel Ender, and Bjӧrn Illing for their help with data collection.

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164

Figure 1. Schematic representation of the mechanisms driving oxygen delivery in the capillaries for teleost fishes (left panel) and elasmobranch fishes (right panel). Left Panel: In teleost fishes, CO2 produced by aerobic metabolism diffuses into the capillaries, then - + then into the RBCs (1). There, it is hydrolyzed by carbonic anhydrase (CA) into bicarbonate (HCO3 ) and H (2). Both products are moved out of the cell via ion exchangers located on the RBC cell surface (3). Because teleost fishes also possess CA on the capillary - + wall, the recombination of HCO3 and H is catalyzed (<5ms) and more molecular CO2 is produced (4). The production of + extracellular CO2 restarts the process, thus ensuring a constant production of intracellular H , which promotes O2 offloading from the hemoglobin (Hb) (5). Right Panel: Similar to the processes described in teleost fishes, CO2 produced in elasmobranch tissues will - + diffuse into the capillary and then into the RBCs (1). There, it is hydrolyzed into HCO3 and H (2), which are moved out of the cell into the plasma in exchange for other ions (3). Because elasmobranch fishes do not possess CA on the capillary wall, there is no

165 catalyzed reaction of these products (4). Elasmobranch Hbs do not exhibit a Root effect, and some may not display a Bohr effect; therefore, the increased intracellular H+ concentration may only marginally assist in oxygen offloading (5).

166

Figure 2. A schematic figure of the effects of the Bohr effect and Root effect on blood oxygen, at a set oxygen concentration or partial pressure.

167

Figure 3. The mechanisms driving RBC swelling in response to acidosis in teleost fishes. Similar to the pathways activated in the - + capillaries, here CO2 diffuses into the RBC (1) where it is hydrolyzed into HCO3 and H (2). This reaction results in an increase in + intracellular [H ], reducing intracellular pH (pHi) and negatively impacting hemoglobin (Hb) O2 affinity. Circulating catecholamines (e.g., adrenaline; AD, and noradrenaline; NA) bind to a beta-adrenergic receptors on the RBC surface, stimulating the intracellular production of cyclic-adenosine monophosphate (cAMP) via adenylate cyclase (3). cAMP will, in turn, activate the beta-adrenergic Na+-H+ exchanger (ßNHE) and cause H+ to be actively pumped out of the cell in exchange for Na+. There may be other NHEs in - - + - action here as well. Concurrently, another exchanger is passively exchanging HCO3 for Cl (4). As both Na and Cl are osmotically + - + active, while H and HCO3 are not, this increase in intracellular [Na ] will passively draw water into the cell, causing RBC swelling + by up to 70% cell volume (5). This entire process serves to reduce intracellular [H ], thus restoring pHi and Hb-oxygen affinity (6).

168 Na+/K+-ATPase (NKA) uses ATP to actively exchange intracellular Na+ for extracellular K+ at a rate of 3:2, thus restoring intracellular [Na+] (7).

169

Figure 4. The pHe of all species exposed to all three treatment conditions. Significant differences in mean pHe across treatments and within species are denoted with either an asterisks or different lowercase letters. Box and whisker plots represent raw data, with whiskers representing maximum and minimum points within 1.5 times the interquartile range above the upper quartile and below the lower quartile. N=10 for all species for all treatments excluding sicklefin lemon shark, where N=8.

170

Figure 5. The difference between plasma pH (pHe) and RBC intracellular (pHi) of blood samples from clearnose skate (a) and sandbar shark (b) for control (white), air exposed (light gray), and chased (dark gray) individuals. Box and whisker plots represent raw data, with whiskers representing maximum and minimum points within 1.5 times the interquartile range above the upper quartile and below the lower quartile. The numbers at the bottom of the panels represent the total number of individuals for which I was able to obtain both pHe and pHi measurements. Significant differences are denoted either with an asterisk or lowercase letters.

171

Shark

BlacktipReef

Shark

Sicklefin Sicklefin Lemon

Shark

Epaulette

Skate

Clearnose

Shark Sandbar

Figure 6. The relative change of all five species of coastal elasmobranch exposed to the control, and two stress treatments. Significant difference from zero is denoted with asterisks. Box and whisker plots represent raw data, with whiskers representing maximum and minimum points within 1.5 times the interquartile range above the upper quartile and below the lower quartile. Black squares denote points outside of this range.

172

Figure 7. Hemoglobin and Hct from this study as well as from Brill et al. 2008. Raw data are represented as points over the boxplots showing the median and interquartile range, with outliers shown in black filled circles. Significant differences are denoted with an asterisk. The numbers at the bottom of the panels denote the number of individuals in that treatment.

173

Figure 8. Mean corpuscular hemoglobin content (MCHC) and mean cell volume from this study as well as from Brill et al. 2008. Raw data are represented as points over the boxplots showing the median and interquartile range, with outliers shown in black filled circles. Significant differences are denoted with lowercase letters above the boxes. The numbers at the bottom of the panels denote the number of individuals in that treatment.

174

Figure 9. The mechanisms driving CO2 excretion at the gills for teleost fishes (left panel), and elasmobranch fishes (right panel). Left - - Panel: In teleost fishes, plasma HCO3 is moved into the cell in exchange for Cl (1; the rate limiting step), where intracellular carbonic + anhydrase (CA) catalyzes the reaction with H to form CO2 and water (2). CO2 then diffuses across the cell membrane the through the gill because of a partial pressure driving gradient (3). H+ promotes a conformational change in the hemoglobin (Hb) from a T state to an R state where Hb has a greater oxygen affinity. The Root and Bohr effects also promote oxygen binding through an increase in Hb affinity and carrying capacity, respectively (4). Right Panel: In elasmobranch fishes, extracellular bicarbonate can either be transported into the cell for dehydration synthesis as in teleost fishes (1), or it can be converted to CO2 using membrane-bound, plasma-accessible CA (2). In the latter scenario, H+ is provided by non-bicarbonate buffer (i.e., histidine residues on plasma proteins)

175 - + present in elasmobranch plasma. Intracellular dehydration of HCO3 utilizes intracellular H , decreasing its concentration (3). A partial - pressure gradient promotes the movement of molecular CO2 through the gills (4). Because some HCO3 is used intracellularly, the + reduction of intracellular [H ] likely also promotes the hemoglobin to change into a R state to bind O2 driven into the cell through a partial pressure gradient (5).

176

CHAPTER V

The effects of elevated potassium, acidosis, reduced oxygen levels, and temperature on

the functional properties of isolated myocardium from three elasmobranch fishes:

clearnose skate (Rostraja eglanteria), smooth dogfish (Mustelus canis), and sandbar

shark (Carcharhinus plumbeus)

177 ABSTRACT

Elevated plasma potassium levels (hyperkalemia), reduced plasma pH (acidosis), reduced blood oxygen content, and elevated temperatures are associated with high rates of at- vessel and post-release mortality in elasmobranch fishes. While the mechanism(s) linking these physiological disturbances to mortality remains undetermined, we hypothesize that the proximate cause of mortality is reduced myocardial function. To test this hypothesis, we measured changes in the functional properties of isolated ventricular myocardial muscle from clearnose skate (Rostraja eglanteria), smooth dogfish (Mustelus canis), and sandbar shark (Carcharhinus plumbeus). We selected these species based on phylogenetic distance and because they exhibit different routine activity levels. All were also known to be capable of withstanding capture and transport to experimental facilities.

Ventricular myocardial strips were subjected to the following stressors (both in isolation and in combination): hyperkalemia (7.4 mM K+), acidosis (a pH decline of 0.8 units from

7.9), and reduced oxygen (to 31% O2 saturation) applied at temperatures 5°C above and below holding temperatures. Stressors associated with capture had significant (albeit relatively small and inconsistent) species-specific detrimental impacts on myocardial function that were exacerbated by increased stimulation frequency and only partially ameliorated by isoproterenol (an adrenaline analog). The myocardium of the species examined may be less impacted by physiological disturbances than elasmobranch species that exhibit high rates of at-vessel and post-release mortality. Generalization of our results to all elasmobranch fishes may, therefore, not yet be justified.

178 INTRODUCTION

Declining populations of elasmobranch fishes have prompted management measures mandating release following capture (Skomal 2007; Erickson and Berkeley

2008). Elasmobranch fishes can, however, have high rates of post-release mortality

(Stevens et al. 2000; Campana et al. 2009; Musyl et al. 2009; Frick et al. 2010; Hoolihan et al. 2011; Musyl et al. 2011; Dulvy et al. 2014; Dapp et al. 2016; McKenzie et al.

2016). While mortality rates have been correlated with physiological disruptions observed in blood (e.g., Moyes et al. 2006; Musyl et al. 2009; Gallagher et al. 2014;

Marshall 2015; Dapp et al. 2016; Horodysky et al. 2016; Whitney et al. 2017; Musyl and

Gilman 2019), the mechanisms underlying this correlation remain unknown. Capture often includes bouts of exhaustive exercise (i.e., anaerobic metabolism) and apneic asphyxia. Both can cause: (1) measurable increases in plasma potassium (hyperkalemia, due to cellular damage), (2) metabolic and respiratory acidosis (i.e., a decrease in plasma pH ≥ 0.6 units), and (3) decreases in blood oxygen content (Hanson and Johansen 1970;

Bushnell et al. 1982; Cliff and Thurman 1984; Wells et al. 1986; Lai et al. 1990; e.g.,

Wood 1991; Lowe et al. 1995; Kieffer 2000; Manire et al. 2001; Moyes et al. 2006;

Skomal 2006; Mandelman and Farrington 2007; Brill et al. 2008; Frick et al. 2010;

Skomal and Bernal 2010; Marshall et al. 2012; Whitney et al. 2017).

Taking the fish out of the water causes further physiological disruption and may be accompanied by acute changes in body temperature (Arlinghaus et al. 2007; Schlenker et al. 2016; Vornanen 2016; Wosnick et al. 2018). Brill and Lai (2016) hypothesized that capture-associated disruption to physiological (which far exceeds natural variation) should negatively impact cardiac function and the ability of elasmobranch fishes to

179 maintain adequate cardiac output during capture or after release, although this has yet to be experimentally tested. We take this idea one step further and contend that factors limiting cardiac output exacerbate physiological disruptions, thus creating a positive feedback loop that ultimately results in at-vessel or post-release mortality.

Although mechanistic explanations linking exhaustive exercise to impaired myocardial function have been described for teleost fishes (Wood et al., 1983; Wood

1991; Hanson et al., 2006), little is known about these processes in elasmobranch fishes.

The lack of research on elasmobranch fishes is primarily due to the difficulties of capturing, transporting and maintaining them in captivity. Cardiac function has therefore been studied in fewer elasmobranch than teleost fish species (Hiatt 1943; Butler and

Taylor 1975 ; Driedzic and Gesser 1988; Van Vliet et al. 1988; Tota and Gattuso 1996;

Thompson and O'Shea 1997; Speers-Roesch et al. 2012; Larsen et al. 2017). Even fewer studies have addressed the effects of hyperkalemia (Hiatt 1943), acidosis, and reduced oxygen concentration (Speers-Roesch et al. 2012) on myocardial function of elasmobranch fishes (Brill and Lai 2016).

The physiological responses of teleost myocardial function to the disruptions associated with capture, while well studied, could be of limited utility in predicting impairment of elasmobranch myocardial function. In isolated teleost myocardium, hyperkalemia, acidosis, reduced oxygen levels, and elevated temperature (in isolation or in combination) result in membrane hyperpolarization, shortened action potentials (AP), reduced influx of sarcolemmal calcium (Ca2+), and decreased contractile force (El-Sayed and Gesser 1989; Gillis et al. 2000; Nielsen and Gesser 2001; Kalinin and Gesser 2002;

Paajanen and Vornanen 2003; Hanson et al. 2006; Shiels et al. 2010; Vornanen 2016;

180 Eliason and Anttila 2017). Decreased contractile force results from a decrease in transcellular sodium-potassium (Na+-K+) gradients and alterations of calcium-troponin binding. Although myocardial excitation-contraction mechanisms are well conserved in vertebrates (Tota et al. 2011; Shiels and Galli 2014), the morphological and physiological differences between teleost and elasmobranch fishes could result in disparate physiological disruptions in response to capture. For example, in most teleost fishes, oxygen content of the venous blood is sufficient to meet myocardial oxygen requirements, with tunas (members of the family Scombridae) being the exception (Brill and Bushnell 2001). In contrast, elasmobranch fishes have relatively low venous oxygen content (Tota 1989; Tota and Gattuso 1996; Farrell et al. 2012; Brill and Lai 2016) and are more dependent on oxygen delivery to the myocardium from arterial blood. Thus, in elasmobranch fishes, decreases in arterial oxygen concentration are likely to be detrimental to cardiac function. Another difference between these groups is the sympathetic nervous system. In teleost fishes, adrenergic stimulation (via circulating catecholamines or sympathetic innervation of the heart) increases transcellular Ca2+ movements and helps maintain myocardial function during hyperkalemia, acidosis, and hypoxia (Gesser and Poupa 1979; Gesser and Jorgensen 1982; Randall 1982; Farrell et al.

1983; Farrell and Milligan 1986; Kieffer 2000; Hanson et al. 2006). Because elasmobranch fishes have only parasympathetic cardiac innervation (Wendelaar-Bonga

1997; Brill and Lai 2016), it remains unclear if the elasmobranch myocardium possesses the adrenergic receptors and intracellular mechanisms necessary to elicit a protective effect. There is, however, some evidence of catecholamine storage sites within the elasmobranch myocardium (Hiatt 1943; Thompson and O'Shea 1997).

181 Our overall objective was to provide mechanistic explanations for species-specific rates of at-vessel and post-release mortality observed in elasmobranch fishes (Kyne and

Simpfendorfer 2010; Skomal and Bernal 2010; Stevens 2010). Specifically, we aimed to:

(1) quantify the negative impacts of the physiological disruptions accompanying capture on myocardial function, (2) determine if these impacts are species-specific or correlated with routine activity level, and (3) assess the ability of adrenergic stimulation to mitigate these responses. Isolated myocardial strips were used to examine changes in contraction properties (i.e., net force, time to peak force, time to half relaxation, rate of force development, and rate of relaxation) during exposure to stress. We studied three species that are both phylogenetically-disparate and which exhibit a range of routine activity levels: (1) clearnose skate (Rostraja eglanteria), a sedentary buccal pumping species that occupies a temperature range of ~9-30 °C (Luer and Gilbert 1985; Luer et al. 2007); (2) smooth dogfish (Mustelus canis), an active buccal pumping species that occupies a temperature range of ~5-28 °C (Larsen et al. 2017); and (3) sandbar shark (Carcharhinus plumbeus), an obligate ram-ventilating species that occupies a temperature range of 17-34

°C (Carlson 1999; Larsen et al. 2017).

MATERIALS AND METHODS

Animal Collection and Maintenance

All animal capture, care, and experimental protocols and procedures were approved by the William & Mary Institutional Animal Care and Use Committee

(protocol: IACUC-2016-02-18-10947-rwbril). All experiments were performed at the

Virginia Institute of Marine Science’s Eastern Shore Laboratory (Wachapreague,

182 Virginia, USA). Fish were caught by rod and reel using circle hooks either in estuaries near this facility or in coastal areas near Lewes, Delaware (USA). Smooth dogfish and clearnose skate were maintained together in indoor circular tanks (12 m diameter, 1 m depth) at 20 °C ± 1 °C, using a recirculating and filtered water system. Sandbar shark were maintained in a flow-through outdoor circular fiberglass tank (7 m diameter, 1.8 m depth) supplied with filtered seawater pumped from the adjacent tidal lagoon (mean temperature of 25 °C ± 3 °C). Animals were maintained in captivity for at least two weeks prior to use in an experiment and fed frozen Atlantic menhaden (Brevoortia tyrannus) ad libitum every 2-3 days.

Isolated Heart Strip Procedure

Individuals were euthanized via an overdose of sodium pentobarbital (350 mg kg-

1 or ≈10x the anesthetic dose) injected via caudal venipuncture, and hearts were immediately excised. Sixteen strips (or less, depending on heart size) of ventricular tissue

(≈1 mm x 10 mm, containing both compact and spongy myocardium) were isolated from each individual and maintained in an ice-cold elasmobranch Ringers solution

(concentrations in mM: 350 urea, 280 NaCl, 80 TMAO, 5 CaCl2, 3 MgCl2, 5 glucose, 5

o KCl, 11 NaHCO3, 0.466 Na2HPO4, 0.5 MgSO4; pH 7.9 at 20 C; modified from Driedzic

& Gesser, 1988). Calcium concentration [Ca2+] in the Ringer’s solution matched in vivo values (reviewed by Marshall et al. 2012). A 5 mM potassium concentration ([K+]) was selected because of its use in previous studies (e.g., Driedzic and Gesser 1988) and because myocardial strips in Ringers solution with [K+] ≤ 3 mM were unresponsive.

Myocardial strips were mounted between stationary tissue clips, and clips were attached to isometric force transducers (model Fort 10) in the four water-jacketed tissue baths

183 (containing elasmobranch Ringers solution) of a Myobath II system (World Precision

Instruments, Sarasota, Florida USA). Two Myobath II systems were run simultaneously for a total of eight tissue baths. Signals from the force transducers were amplified

(Transbridge 4 M amplifiers, World Precision Instruments) and recorded at 1000 Hz using either a Daqbook 120 A/D data acquisition system (National Instruments, Austin,

Texas USA) or a Measurement Computing USB-1208LS (Measurement and Computing

Corp, Norton, Massachusetts USA) data acquisition system. Both systems were controlled by custom software developed in Dasylab version 13 (National Instruments,

Austin, Texas USA).

All tissue baths were initially bubbled with 100% oxygen (pO2 84 kPa) to ensure adequate tissue oxygenation (Joyce et al. 2016; Gesser and Rodnick 2019), and the myocardial strips maintained under tension for 30 min. They were then stimulated at 0.2

Hz using 5 ms duration square waves at voltages that elicited maximum force production and further tensioned until maximal responses were measured. Myocardial strips were then stimulated for 20 min, and force data were recorded for 10 min; the force data were used as baseline values against which the fractional changes resulting the various treatments were measured. We then subjected myocardial strips to a sequence of experimental manipulations described in Figure 1, with changes in the gas used to bubble the Ringers solution and manipulation of the Ringers solution to mimic stress conditions.

Specific treatment conditions listed in Table 1 and described below. We measured force- frequency relationships where noted in Figure 1 by increasing stimulus frequency (in 0.2

Hz increments) every 60 s beginning at 0.2 Hz.

184 Treatments

We measured the effects of hyperkalemia alone, reduced oxygen and acidosis, and hyperkalemia combined with reduced oxygen and acidosis (treatments

“Hyperkalemia”, “Acidosis + Low O2”, and “Combined stressors”, respectively; Table 1) at temperatures 5°C above and 5°C below fish holding temperature (15 and 25°C for clearnose skate and smooth dogfish and 20°C and 30°C for sandbar shark). We produced the “hyperkalemia” treatment by increasing the potassium concentration [K+] from 5 to

7.4 mM through the addition 28 L of 1M KCl to two of the four tissue baths on each

Myobath II System. We selected 7.4 mM because it is slightly above the 7 mM [K+] shown to disrupt myocardial function in dusky shark (Carcharhinus obscurus) (Cliff and

Thurman, 1984). The two untreated tissue baths received equivalent volumes of Ringer’s solution. We produced the “Acidosis + Low O2” treatment by changing the gas bubbled into all tissue baths from pure oxygen to a gas mixture containing 98% air and 2% CO2

(following Joyce et al. 2016). The mixed gas lowered the oxygen content of the Ringer’s solution by 80% and mean pH by 1.00 ± 0.03, 0.76 ± 0.01, 0.85 ± 0.03, and 0.61 ± 0.03

(mean ± standard error of the mean; SEM) at 15, 20, 25, and 30 °C (respectively). The

“Combined Stressors” treatment refers to instances where we applied “Hyperkalemia” and “Acidosis + Low O2” simultaneously. To simulate adrenergic stimulation, we used isoproterenol, a beta-adrenergic antagonist that is stable under laboratory conditions. We produced the “Control + Iso” and “Combined +Iso” treatments by adding 140 L of a 10-

4 mM isoproterenol solution (made up using Ringer’s solution) to all tissue baths following the treatments described above (Table 1). This addition resulted in a nominal

10-6 mM isoproterenol concentration.

185 Data Processing and Statistical Procedure

After each experiment, length (mm) and wet mass (mg) of each muscle strip were measured, and the cross-sectional area (mm2) calculated assuming the strip was cylindrical and muscle density = 1.06 mg mm−3:

Cross-sectional area = (mass/1.06)/length (1)

Raw data were filtered and processed using custom designed software developed in Matlab (The Mathworks Inc., Natick, Massachusetts USA) as described in (Larsen et al. 2017). For each contraction, the program calculated contractile force (milliNewtons, mN), time to peak force (ms), time to half relaxation (ms), maximum rate of force

-1 -1 development (+dF/dtmax, mN ms ), and maximum rate of relaxation (-dF/dtmin, mN ms ).

Representative data under each treatment condition were selected from 10 contractions at the end of the 20 min incubation period and expressed as mean ± SEM. We used the five calculated metrics (i.e., contractile force, time to peak force, time to half relaxation,

+dF/dtmax, -dF/dtmin) from the control to estimate relative changes in myocardial function.

Data from the myocardial strips in the two control tissue baths were averaged, to serve as a single control for the two treatment strips within the same rig containing myocardial strips from the same individual.

Because myocardial strips contained both compact and spongey tissue with heterogeneous fiber orientations, they produced highly variable isometric forces, even when corrected for cross-sectional areas (Supplemental Figure 1). We therefore expressed treatment effects by calculating the relative change from the baseline values

(i.e., from the first ten minutes of recording from the same strip during which stress was not applied) as:

186 (mean treatment value – mean baseline value) / mean baseline value. (2)

We accounted for loss of force during the experiments due to fatigue (as opposed to treatment effects) by subtracting the mean percent change of the control strips from the relative change observed in the treatment strips within a given trial (Shiels et al., 2010).

In a few cases (<1% of strips), we deemed treatment effects to be biologically unreasonable (changes > 1000%) and removed these data points from further consideration. We subsequently tested mean baseline values and mean values recorded under treatment conditions for outliers using Grubb’s test (where  = 0.05), and less than

2% of total data points were removed. There were limited sample sizes and high rates of decay at elevated contraction frequencies in stress treatment (i.e., hyperkalemia, acidosis

+ low O2, and combined stressors) for smooth dogfish myocardial strips at 15°C, so all data from this species at 15°C were removed from analysis of force frequency effects.

We analyzed single-frequency data using generalized linear mixed-effects models

(GLMM), where relative change for each metric (i.e., contractile force, time to peak force, time to half relaxation, +dF/dtmax, -dF/dtmin) was the response variable, treatments

(e.g., hyperkalemia) were fixed effects, and individual strip identification was incorporated as a random effect to account for some of the non-independence of multiple measurements from a single strip. Given the presence of random effects in the models, we used restricted maximum likelihood (REML) estimation to obtain estimates of model parameters (i.e., the magnitude and direction of effects of each treatment level on the observed response). To compare differences among measured responses for all treatment levels, we fit a GLMM to data collected at temperatures 5 °C below holding temperature, and a separate GLMM to data collected at temperatures 5 °C above holding temperature.

187 With this approach, we used results from the GLMM to test for differences between measured responses and control conditions, control + isoproterenol and combined stressors + isoproterenol. We inspected the quantile-quantile plots, histograms of residuals, and Pearson-residual versus fitted plots to ensure data fit the assumed distributions and that residuals were normally distributed. Analyses were completed using the “lme4” and “mgmc” packages (Pinheiro et al. 2014) in R (R-core team 2018).

To compare mean responses from the treatment groups relative to the control, we calculated profile confidence intervals (95% confidence) for model estimates using the

“confint” command in base R with the built-in bootstrap method (n=200 bootstrap samples).

We used two-way repeated-measures ANOVAs to estimate differences between measured responses during force-frequency trials, where the dependent variables were net force, time to peak force, time to half relaxation, +dF/dtmax and -dF/dtmin, and independent variables were frequency and treatment. We specifically tested for differences in the mean values of dependent variables between control conditions, hyperkalemia, acidosis + low O2, or combined stressors at 5 °C above holding tank temperature and 5 °C below holding tank temperature (15 and 25 °C for clearnose skate and smooth dogfish, and 20 and 30 °C or sandbar shark). We likewise estimated differences between control conditions, control + isoproterenol, combined stressors, and combined stressors + isoproterenol (Table 1) at high and at low temperatures as frequency increased. Data were assessed with graphical analysis to ensure data met assumptions of normality using Sigmaplot 12.5 (Systat Software Inc. San Jose,

California). Pairwise comparisons of mean responses were examined with 95% profile

188 confidence intervals for specific combinations of frequency and treatment calculated using the “confint” command specifying the bootstrap method and using 200 bootstrap samples (Bretz et al. 2016).

RESULTS

We collected data from eight clearnose skate (disk width = 61 ± 3 cm, mean ±

SE), eight smooth dogfish (total length = 52 ± 20 cm), and ten sandbar shark (total length

= 74 ± 10 cm).

Effects of Hyperkalemia, Acidosis + Low O2, and Combined Stressors at 0.2 Hz stimulation frequency.

Hyperkalemia caused a decrease in clearnose skate mean net force at 15 °C (-27% to 0.2%; 95% confidence interval; Figure 2a), although it did not affect the mean responses (relative to control strips) of smooth dogfish and sandbar shark myocardial strips, regardless of test temperature (Figure 2b, c); mean responses for these trials were not significantly different from zero, as evidenced by the provide confidence intervals (-

38% to 11% and -18% to14% for smooth dogfish and sandbar shark, respectively).

Hyperkalemia also caused decreases in mean +dF/dtmax (-24% to -1%) and mean time to half relaxation (-53% to -23%) in smooth dogfish myocardial strips, at 15 °C (Figure 2b), and a decrease in sandbar shark mean +dF/dtmax at 20 °C. All three species decreased mean net force production under acidosis + low O2 at the warm temperatures (-68% to -

15%, -67% to -0.2%, and -43% to -4% for clearnose skate, smooth dogfish, and sandbar shark, respectively). Clearnose skate showed decreases in mean +dF/dtmin (-61% to -16%) and mean +dF/dtmax (-62% to -10%) under acidosis + low O2 at 25 °C (Figure 2a, b). We

189 also observed a decrease in mean time to half relaxation (-38% -2%) and mean -dF/dtmin

(-28%, -8%) in smooth dogfish myocardium at 15 °C, and a decrease in mean +dF/dtmax at 25 °C (-45% to -4%; Figure 2b). The significant effects of the combined stressors were apparent in decreases in mean net force (-64%, -11%), mean +dF/dtmax (-53%, -5%), and mean -dF/dtmin (-47%, -7%) in clearnose skate myocardium at 15°C (Figure 2a). Smooth dogfish myocardium also decreased mean -dF/dtmin at 15°C (-27% to -8%; Figure 2b).

Effects of Hyperkalemia, Acidosis +Low O2, and Combined Stressors During Force- frequency Trials

Hyperkalemia significantly decreased mean net force (t=3.0, p<0.05) and mean time to half relaxation (t=4.3, p<0.05) in sandbar shark myocardium at 30 °C and 1.6 Hz

(Figure 3c), but the other species were resilient to this treatment. In response to acidosis + low O2, we observed a decrease in mean net force in clearnose skate myocardium at 25

°C and 0.6-0.8 Hz (t0.6= 4.1, p<0.05; t0.8=3.6, p<0.05; Figure 3a) and an increase in mean

-dF/dtmin in sandbar shark myocardium at 30 °C and both 1.2 and 1.6 Hz (t1.2=6.0, p<0.05; t1.6= 3.8, p<0.05; Figure 3c). The responses to both hyperkalemia and acidosis + low O2 (“Combined stressors) included a decrease in mean time to peak force in clearnose skate myocardium at both 15 and 25 °C and 0.6-0.8 (t0.6=3.6, p<0.05; t0.8=4.1, p<0.05) and 0.6-1.0 Hz (t0.6=3.5, p<0.05; t0.8= 5.0, p<0.05; t1.0=4.0, p<0.05), respectfully

(Figure 3a). Combined stressors also increased mean time to half relaxation in clearnose skate myocardium at 15 °C and 0.4 Hz (t=3.5, p<0.05; Figure 3a). While there appears to be additional treatment effects at the highest frequencies, the low samples sizes at these frequencies prevented our statistical methods from resolving differences.

190 Effects of Isoproterenol at 0.2 Hz stimulation frequency.

There were three significant responses to isoproterenol alone (“Control +Iso”), primarily an increase in mean net force (40% to 125%) at 15 °C in myocardial strips from clearnose skate (Figure 4a) and at 25 °C in strips from smooth dogfish (7% to 120%;

Figure 4b). The control + Iso treatment also caused a decrease in mean time to half relaxation (-40% to -7%) at 20° C in sandbar shark myocardial strips (Figure 4c). There only response to isoproterenol in strips exposed to elevated CO2 and hyperkalemia

(“Combined +Iso”) were an increase in mean time to peak force (5% to 49%) and mean time to half relaxation (2% to 36%) in sandbar shark myocardium at 20°C (Figure 4c).

Effects of Isoprenaline During Force-frequency Trials

Isoproterenol increased mean net force in clearnose skate myocardium at 15 °C and 0.4 Hz (t=3.0, p<0.05), and increased mean time to peak force in clearnose skate strips at 15 and 25 °C and 0.4-0.8 (t0.4=3.9, p<0.05, t0.6= 3.8, p<0.05; t0.8=3.9, p<0.05) and 0.8-1.2 Hz, respectively (t0.8=3.5, p<0.05, t1.0= 5.0, p<0.05; t1.2=4.0, p<0.05; Figure

5a). It also decreased mean time to peak force and increased mean time to half relaxation in sandbar shark myocardium at 30 °C at 0.4-0.8 (t0.4=4.3, p<0.05, t0.6= 4.9, p<0.05; t0.8=4.3, p<0.05) and 1.4 Hz, respectively (t1.4=3.0, p<0.05; Figure 5c). Following exposure to combined stressors (“Combined +Iso”), isoproterenol increased mean time to peak force in clearnose skate strips at 15 and 25 °C at 0.6-0.8 (t0.6=3.4, p<0.05, t0.8= 3.9, p<0.05) and 0.8-1.0 Hz, respectively (t0.8=3.8, p<0.05, t1.0= 3.1, p<0.05; Figure 5a). It also decreased mean +dF/dtmax and mean -dF/dtmin in clearnose skate myocardium at 15

°C and 0.6 (t=4.2, p<0.05) and 0.4-0.6 Hz (t0.4=3.6, p<0.05; t0.6=3.2, p<0.05), respectively (Figure 5a). In smooth dogfish myocardium at 25° C, Control + Iso increase

191 mean net force at 0.6-1 Hz (t=3.0 p<0.05) and increased mean +dF/dtmax at 1.0 hz (t=3.9, p<0.05) and mean -dF/dtmin at 1.0 Hz (t=3.2, p<0.05; Figure 5b). Finally, the Control +

Iso treatment increased mean net force in sandbar shark myocardium at 20 °C and 0.6-0.8

Hz (t0.6=3.4, p<0.05; t0.8= 3.9, p<0.05), as well as mean time to half relaxation at 0.4-0.6

Hz (t0.4=3.1, p<0.05; t0.6=3.3, p<0.05; Figure 5c).

DISCUSSION

Hyperkalemia, Acidosis + Low O2, and Combined Stressors

We found minimal cardiac impairments in clearnose skate, smooth dogfish, and sandbar shark when isolated myocardial strips were exposed to combined stressors, even at elevated temperature and high stimulation frequencies. Although species-specific at- vessel and post-release mortality rates have been correlated with serological disruptions

(Hight et al. 2007; Mandelman and Skomal 2009; Marshall et al. 2012; Marshall 2015), our results imply that diminished myocardial function due to such disruptions may not be the main driver of mortality. Our net force results from control strips are approximately half of those published by Larsen et al. (2017) for the same study species (see supplemental Figures 1 and 2), which may reveal poor quality tissue preparations in our control values and obscure treatment effects. Our results suggest that, relative to teleost fishes (Gesser and Jorgensen 1982; Shiels et al. 2010; Joyce et al. 2016; Gesser and

Rodnick 2019), elasmobranch myocardium is more resilient to physiological disturbances. Sandbar shark and smooth dogfish myocardium appear particularly resilient to the effects of combined stressors, with no significant detriments even at high temperatures and increasing stimulation frequency. These two species regularly enter

192 estuaries on the U.S. mid-Atlantic during summer when benthic habitats can be hypoxic

(Breitburg et al. 2018) and are more active than the clearnose skate. The ability of their ventricular myocardium to maintain normal function under the combination of stressors we employed may be due to the physiological adaptations that allow them to conduct routine swimming in these seasonally hypoxic areas.

Overall, the impacts of capture-associated stressors in this study were less severe than expected. The impact of hyperkalemia on myocardial function in fish shows a reduction in contractile force in rainbow trout (Oncorhynchus mykiss), plaice

(Pleuronectes flesus), and spiny dogfish (Squalus acanthias) myocardium (Hiatt 1943), a result we did not observe even at stimulus frequencies that include normal heart rates

(0.8-1.2 Hz; Scharold and Gruber 1991; Lai et al. 1997; Dowd et al. 2006). Our results therefore differ from the response of rainbow trout myocardium, where force declines up to ~90% under severe hyperkalemia (10 mM; Nielsen and Gesser 2001; Kalinin and

Gesser 2002). The clearnose skate myocardium was most sensitive to acidosis + low O2, as evidenced by the number of significant decreases in contractile properties relative to those of the other two species exposed to this treatment. This may signify an increased reliance on sarcoplasmic reticulum Ca2+ stores (Shiels et al. 2010). Clearnose skate myocardium was the most affected by the combined stressors, exhibiting increased times to peak force and slower rates of +dF/dtmax and -dF/dtmin (Figure 3a). In both teleost and elasmobranch fishes, increased temperatures result in shorter times to peak force and half relaxation (Gamperl et al. 2004; Rodnick et al. 2014). The longer times to peak force and relaxation we recorded in clearnose skate myocardium under the combined stressors of intracellular acidosis and hyperkalemia at higher temperatures may signify a hindered

193 ability to recruit or expel sarcolemmal Ca2+ through voltage-gated ion channels or the

Na+-Ca2+ exchanger. Longer times to peak force could, however, also be due to a longer duration action potential occurring under combined stressors (Shiels et al. 2000).

The observed reductions in myocardial function in response to combined stressors were less than we initially expected. We acknowledge that, except for temperature

(Wosnick et al. 2018), the conditions used were likely conservative. For example, in longline-captured elasmobranch fishes, plasma [K+] as high as 12 mM, plasma pH values below 7.0, and arterial blood oxygen levels down to 7% saturation have been measured

(Hyatt et al. 2011; Marshall et al. 2012). Furthermore, our study species were resilient to capture and transport as they obviously remain alive post-capture and during holding in shoreside facilities. Other elasmobranchs that exhibit high rates of at-vessel or post- release mortality may exhibit more substantial changes in myocardial function. Indeed, experimental scientists are faced with a “Hobson’s choice”, in that it is difficult (if not impossible) to study myocardial function in elasmobranch species that exhibit high rates of post-release mortality because they cannot be successfully transported to, or held at, shoreside facilities.

Our results suggest the functional properties of some elasmobranch species

(including the obligate-ram ventilating sandbar shark) are minimally diminished by severe disruptions in physiological homeostasis. Although species-specific at-vessel and post-release mortality rates have been correlated with serological disruptions (Hight et al.

2007; Mandelman and Skomal 2009; Marshall et al. 2012; Marshall 2015), our results imply that diminished myocardial function due to such disruptions may not be the main driver of mortality.

194 Adrenergic Stimulation

Adrenergic stimulation can have significant effects under physiologically normal conditions. We used isoproterenol in the absence of any stressors to determine if elasmobranch myocardium possesses the receptors and intracellular mechanisms necessary to elicit an adrenergic response, which we did in both clearnose skate and smooth dogfish (Figures 4, 5). Clearnose skate myocardium did show an increase in time to peak force as stimulation frequency was increased (Figure 5). This increase may indicate a recruitment of additional intracellular Ca2+ stores that may have lengthened the contraction period and helped to maintain force at control values despite thermal stress.

Our results suggest, therefore, that elasmobranch fishes do have the adrenergic receptors and intracellular pathways necessary to affect a response to adrenergic stimulation. The myocardium from the three species we tested, however, generally appears only minimally responsive to adrenergic stimulation. Exposure of perfused whole heart preparations from spiny dogfish and common skate (Raia batis) to 10-8 M and 10-7 M adrenaline

(respectively) elicited ≈100-200% increases in net force, although the skate heart appeared less sensitive to adrenaline (Fange and Ostlund 1954). The isoproterenol concentration we used was one to two orders of magnitude higher (10-6 M), but clearnose skate myocardium likewise demonstrated an 82% increase in force under control conditions at the low temperature treatment (Figure 4a). These observations imply that an approximate doubling in force is the greatest change the skate myocardium can achieve.

This upper limit is similar to a ≈200% increase in force observed in isolated myocardium and in situ perfused heart preparations of both teleost and elasmobranch fishes (Hiatt

1943; Shiels and Farrell 1997; Nielsen and Gesser 2001; Joyce et al. 2016). A notable

195 exception is the 383% increase in force following exposure to 10-4 M adrenaline in isolated ventricle myocardium from Port Jackson shark (Thompson and O'Shea 1997).

Adrenergic stimulation is not, however, capable of fully restoring myocardial function even when detriments due to stress are minor. Though the concentration of isoproterenol we used mimicked plasma adrenaline levels in a highly stressed fish (Van

Vliet et al. 1988; Wendelaar-Bonga 1997; Hight et al. 2007), we did not see strong evidence of adrengeric ammelioration of stress effects (i.e., increases in net force under all conditions). Given the small magnitude of observed stress effects (Figures 2, 3), however, the protective role of adrenaline in elasmobranch fishes may represent an area for further research. For example, 5 .0 x10-9 M adrenaline produces an increase in amplitude of contraction in spiny dogfish atria, and addition of 10-10 M adrenaline restores contraction amplitude following extreme hyperkalemia (54 - 81 mM [K+]; Hiatt,

1943).

In all elasmobranch species studied to-date, the effects of adrenergic stimulation on force production were larger than effects on +dF/dtmax, which is the opposite of the pattern exhibited by teleost fish hearts (Hiatt 1943; Fange and Ostlund 1954; Van Vliet et al. 1988; Thompson and O'Shea 1997). The difference in the effect of adrenergic stimulation is consistent with the fact that many elasmobranch fishes primarily modulate stroke volume over heart rate to change cardiac output (Emery 1985; Scharold et al.

1989; Farrell 1991; Scharold and Gruber 1991; Tota and Gattuso 1996; Carlson et al.

2004; Brill and Lai 2016). Historically, teleost fishes (other than tunas) were thought to increase cardiac output through both cardiac output (60-40% increases) and heart rate

(40-60% increases) (Farrell 1991; Farrell and Jones 1992). Recent work, however, has

196 shown that increases in teleost cardiac output may be more due to increases in heart rate than previously thought (Altimiras and Larsen 2000; Clark et al. 2005; Iversen et al.

2010).

CONCLUSIONS

We found minor and inconsistent negative effects due to the physiological disruptions associated with capture on the functional properties of myocardial tissue isolated from clearnose skate, smooth dogfish, and sandbar shark. We also saw adrenergic stimulation is capable of inducing significant effects under physiologically normal conditions, although adrenaline is not capable of fully restoring myocardial function even when detriments due to stress are minor. We fully acknowledge, however, that our results may not be generally applicable to elasmobranch fishes. Moreover, our three study species routinely survive capture and transport to shoreside facilities, attributes which imply that their myocardial function is likely to be minimally impacted by capture.

It is also possible that other mechanisms drive post-release mortality in elasmobranch fishes. Frick et al. (2010) noted skeletal muscle tetany preceding mortality in exhaustively exercised gummy sharks (Mustelus antarcticus), and this tetany has been observed in mako shark (Isurus oxyrinchus), dusky shark, sicklefin lemon shark

(Negaprion acutidens), and clearnose skate (Bernal and Schwieterman, personal observations). Because skeletal muscle contraction and relaxation also relies on specific ion movement in and out of the cell, hyperkalemia may cause irreversible contraction

(Adams and Galvan 1986), resulting in both at-vessel and post-release mortality. Future

197 work should consider multiple physiological systems when attempting to determine drivers of capture-associated mortality.

ACKNOWLEDGEMENTS

We would like to thank T. Deemer for assistance in obtaining smooth dogfish, and D.

Crear, G. Fay, and M. Winton for their assistance with statistical analysis. We would also like to thank D. Lavoie, R. Steffan, A. Sergio, T. Bigelow and the entire staff at the

Virginia Institute of Marine Science Eastern Shore Laboratory for their unwavering logistical support and assistance in collecting specimens. Species silhouettes in Figures 1-

4 were provided by O. Shipley.

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207 Table 1. All treatments were conducted at 5°C above and 5°C below fish holding temperature (15 and 25°C clearnose skate and smooth dogfish; and 20°C and 30°C for sandbar shark). Here isoproterenol is abbreviated to “ISO” in the treatment names.

Treatment Gas [K+] (mM) Isoproteranol (mM)

Control 100% O2 5.0 0.0

Hyperkalemia 100% O2 7.4 0.0

Acidosis + Low O2 98% Air and 2% CO2 5.0 0.0

Combined Stressors 98% Air and 2% CO2 7.4 0.0

-6 Control + ISO 100% O2 7.4 10

-6 Combined + ISO 98% Air and 2% CO2 7.4 10

208 Figure 1. The experimental workflow for a given trial. This figure depicts one Myobath II System with four individual tissue baths, and during data collection, two systems would be running simultaneously with the two temperature baths set to ±5°C from holding temperature (15 and 25°C for clearnose skate and summer flounder; 20 or 30°C for sandbar shark). Note that the treatment names in grey arrows would be different if the gas used was switched to the 98% air and 2% CO2 gas mixture (e.g, “Acidosis + Low O2”, “Combined Stressors”, “Combined+Iso”). Abbreviations are as follows: stimulation (Stim), potassium chloride (KCl), isoproterenol (ISO).

209

Figure 2. The changes (%) of the functional parameters of ventricular myocardial strips isolated from clearnose skate (A), smooth dogfish (B), and sandbar shark (C) during + exposure to hyperkalemia (7.4 mM [K ]); hyperkalemia, and acidosis + low O2 (“Combined Stressors); or acidosis + low O2 at two temperatures. Values are relative to control myocardial strips not exposed to the stress treatments. Significant differences between control myocardial strips and the two treatments and are indicated by an asterisk

210 (*). The solid lines within the boxes mark the median values, the boundaries of the boxes the 25th and 75th percentiles, and the whiskers above and below the boxes the 90th and 10th percentiles, respectively. Data points between the 90th and 10th percentiles are shown as circles and those outside this interval are shown as squares. These outliers were not categorized as outliers by the Grubb’s test, which uses critical Z ratios.

211

a b c

15°C 25°C 15°C 25°C 20°C 30°C

Figure 3. The changes (%, relative to values recorded at a stimulus frequency of 0.2 Hz) of the functional parameters during force-frequency trials of ventricular myocardial strips isolated from clearnose skate (a), smooth dogfish (b), and sandbar shark (c) under control + conditions (▲), during exposure to hyperkalemia (7.4 mM [K ]) (△), acidosis + low O2 (•), or combined stressors (○) at two temperatures. Significant differences relative to control conditions are denoted for hyperkalemia (#), acidosis + low O2 (†), combined stressors (*).

212

Figure 4. The changes (%) of the functional parameters ventricular myocardial strips isolated from clearnose skate (a), smooth dogfish (b), and sandbar shark (c) during exposure to isoproterenol alone (“Control + Iso) or to hyperkalemia and acidosis + low O2 and isoproterenol (“Combined + Iso”) at two temperatures. Values are relative to control myocardial strips not exposed to these treatments. Significant differences between control myocardial strips and the two treatments and are indicated by an asterisk (*). The

213 solid lines within the boxes mark the median values, the boundaries of the boxes the 25th and 75th percentiles, and the whiskers above and below the boxes the 90th and 10th percentiles, respectively. Data points between the 90th and 10th percentiles are shown as circles and those outside this interval are shown as squares. The later values were not categorized as outliers by the Grubb’s test, which uses critical Z ratios.

214

Figure 5. The changes (%), relative to values recorded at a stimulation frequency of 0.2 Hz, of the functional parameters during force-frequency trials of ventricular myocardial strips isolated from clearnose skate (a), smooth dogfish (b), and sandbar shark (c) under control conditions (○), exposure to isoproterenol (▲; “Control + Iso”), and exposure to hyperkalemia and acidosis + low O2, and isoproterenol (•;⁡“Combined +Iso”). The decay in smooth dogfish treatment strips at 15°C at frequencies above 0.2 was deemed too high to be reasonable and those treatments were removed. Significant differences relative to control conditions are denoted for control + Iso (†), combined stressors + Iso (*)

215

Supplemental Figure 1. The raw functional parameters of ventricular myocardial strips isolated from clearnose skate (a), smooth dogfish (b), and sandbar shark (c) during + exposure to control conditions; hyperkalemia (7.4 mM [K ]); acidosis + low O2; or hyperkalemia and acidosis + low O2 (“Combined Stressors) at two temperatures. The numbers below the boxes represent the number of individuals represented in each. Each point represents an average of 10 contractions.

216

Supplemental Figure 2. The raw functional parameters ventricular myocardial strips isolated from clearnose skate (a), smooth dogfish (b), and sandbar shark (c) during exposure to control conditions, isoproterenol alone (“Control + Iso), or to hyperkalemia and acidosis + low O2, and isoproterenol (“Combined + Iso”) at two temperatures. The numbers below the boxes represent the number of individuals represented in each. Each point represents an average of 10 contractions.

217 CHAPTER VI

Conclusions

218 GENERAL DISCUSSION

The increasing severity of geographical extent of hypoxia is one of the most serious threats facing marine organisms today, as oxygen limitations have wide-ranging impacts on individuals, from behavior to fitness (Laffoley and Baxter 2019). Hypoxia rarely if ever occurs in isolation, however, and more multi-stressor research is needed to truly understand how oxygen limitation impacts marine species (Farrell et al. 2009;

McKenzie et al. 2016). The negative impacts of hypoxia and other stressors are affecting population and ecosystem health. Unfortunately, particularly vulnerable elasmobranch species remain largely understudied (Dulvy et al. 2014). To truly understand the impacts of these stressors, we must understand what is happening at the individual level

(Horodysky et al. 2015). The general aim of this dissertation was, therefore, was to undertake a functional analysis of the elasmobranch cardiorespiratory response to hypoxia and other concomitant stressors. An integrated approach was used to investigate whole organism, individual organ, and cellular responses, with comparisons across multiple species to elucidate patterns associated with activity level and phylogeny. The central aim of this final chapter is to incorporate the findings of the preceding chapters into an overview of the cardiorespiratory stress responses of coastal elasmobranch fishes.

The broader applications of this study are discussed, with comments on future research directions that build on the key findings of this dissertation.

To determine the impacts of low oxygen on the cardiorespiratory physiology of coastal elasmobranch fishes, I examined the impacts of acute increases of temperature, simulated acidification, and hypoxia on the metabolism of two skate species and one teleost as a comparison (Chapter II) as well as the blood-oxygen affinity of two species

219 (Chapter III). I also evaluated the prevalence of red blood cell swelling, a teleost response to acidosis that preserves hemoglobin (Hb)-oxygen (O2) binding under stress, in five species of elasmobranch (Chapter IV). To determine how simulated low oxygen and other stressors may disrupt cardiac function and thus oxygen delivery, I also assessed the functional properties of isolated myocardial strips from three coastal species (Chapter V).

CONTRIBUTIONS

One of the benefits of using lab-based experimental protocols is the ability to control multiple environmental parameters, thus minimizing the number of variables contributing to an observed response. While controlled experiments have great utility in determining mechanistic cause-and-effect relationships, extrapolating the results to in situ conditions may result in an oversimplification of in vivo stress responses. Recent multi- stressor studies have shown that physiological responses are not always additive, and specifically stressors associated with climate change may have antagonistic or masking effects (Di Santo 2016; Lefevre 2016). I therefore conducted a fully factorial suite of experiments on three species (clearnose skate, Rostaraja eglanteria; summer flounder,

Paralythese dentatus; and thorny skate; Amblyraja radiata), with multiple temperature and partial pressure of carbon dioxide (pCO2) conditions and to quantify interactive effects (Chapter II). The respirometry trials conducted for my research provided estimates of metabolic scope and hypoxia tolerance for clearnose skate and thorny skate, with comparisons to the metabolic rate of summer flounder. I found that the hypoxia tolerance of clearnose skate rivaled that of the hypoxia-tolerant epaulette shark (Hemiscyllium ocellatum) under the least stressful conditions tested (Routley et al. 2002). Clearnose

220 skate hypoxia tolerance significantly decreased, however, under acidification and increasing temperatures. I also found simulated acidification had a masking effect, overriding the impacts of increasing temperatures in thorny skate. These results are consistent with published studies on other skate species (Di Santo 2016), and underscore the importance of multi-stressor experiments to ensure applicability of experimental results.

Understanding hypoxia tolerance is complicated, as there are numerous ways to measure tolerance of the whole organism, as well as of lower levels of biological organization (Wood 2018). Measuring multiple parameters will increase confidence in understanding critical oxygen levels, as well as enhance the mechanistic understanding of low oxygen responses to low oxygen (Regan et al. 2019). The number of studies that measure multiple metrics of hypoxia tolerance under the same experimental conditions, is however, extremely low. This is likely due to logistic and financial constraints (Bernal and Lowe 2016; Brill and Lai 2016). To further investigate the mechanisms driving the observed hypoxia tolerance of the clearnose skate, I constructed blood-oxygen equilibrium curves (Chapter III) under the same temperature conditions used in Chapter

II. As in Chapter II, I also constructed curves for summer flounder, a sympatric teleost, for comparison. Although it appears that a high blood oxygen affinity (low P50) and an associated large Bohr shift could be one mechanism allowing hypoxia tolerance in teleost fishes (Mandic et al. 2009), my results do not support this in elasmobranch fishes. The hypoxia tolerance (as measured with critical oxygen pressure; Pcrit) and blood oxygen affinity (as measured by the pressure at which the blood is 50% saturated with oxygen;

P50) results from the clearnose skate aligned closely with that of the epaulette shark

221 (Speers-Roesch et al. 2012), which were generally high compared to other published values from elasmobranch fishes (Morrison et al. 2015). Clearnose skate also showed no evidence of a Bohr effect, suggesting the mechanisms driving oxygen delivery in this species differ greatly from those in teleost fishes (Berenbrink 2011). Comparisons to published values showed a significant interaction of the effects of metabolic rate and P50 on Pcrit. It should be noted, however, that interspecific comparisons are difficult given that small variations in test temperatures and pCO2 conditions can have large impacts on results (Brill and Lai 2016).

While the study of cardiorespiratory physiology has a long history, dating back centuries (Farrell 2011), new discoveries are constantly revising the way we think about gas transport in fishes (e.g., the discovery of plasma-accessible carbonic anhydrase in the capillaries of teleost fishes, Rummer et al., 2011; the contribution of extracellular carbonic anhydrase in elasmobranch carbon dioxide excretion, McMillin 2018). Although it is generally accepted that elasmobranch fishes do not exhibit red blood cell (RBC) swelling in response to acidosis, this may also be an area that warrants revisiting. Given the lack of a Bohr effect in clearnose skate blood (Chapter III), I was interested to see if this species exhibited RBC swelling. I also examined other coastal species (sandbar shark, Carcarhinus plumbeus; epaulette shark; blacktip reef shark, C. melanopterus; and sicklefin lemon shark, Negaprion acutidens) that span a range of hypoxia tolerances, with the idea that hypoxia tolerance, Hb-O2 affinity, and thus RBC intracellular pH regulation are all linked. I did not find evidence of RBC swelling in clearnose skate experiencing acidosis. The lack of significant swelling, must, however, be taken with consideration that my methods were insufficient to elicit RBC swelling. I was not able to demonstrate

222 RBC swelling in either epaulette shark or sandbar shark, both of which have been shown to have this response following anoxic exposure and capture on rod and reel, respectively

(Brill et al. 2008; Chapman and Renshaw 2009). Comparisons of values from sandbar shark in this study to published hematological parameters also underscore the variable nature of the physiological stress response and the difficulty in measuring red blood cell swelling in elasmobranch fishes. This phenomenon, like metabolism and blood oxygen affinity, appears to be species-specific and to require different levels of physiological disruption to trigger a response (Chapman and Renshaw 2009).

The mechanisms driving observed responses can be difficult to assess. When attempting to quantify fishing mortality, it may be helpful to understand what parameters are most important in predicting mortality events (Schlenker et al. 2016; Whitney et al.

2017). It has been postulated that the mechanisms driving post-release mortality may be linked to the serological changes associated with air exposure and exhaustive exercise

(Hanson et al. 2006), although this has yet to be explicitly studied in controlled experimental conditions in elasmobranch fishes. I used isolated myocardial strips to study the responses to simulated capture stress on cardiac function of three sympatric coastal elasmobranch fishes (clearnose skate; sandbar shark; and smooth dogfish, Mustelus canis; Chapter V). While overall the impacts on contractile parameters were minor, the significant increase in net force following the application of adrenaline suggest the species do exhibit an adrenaline response despite lacking sympathetic innervation (Brill and Lai 2016), and that there is much more to be studied about the severity of stress required to cause severe cardiac disruption. In general, all three species were less affected by all of the stressors applied (hyperkalemia, low O2 and acidosis, and the combination of

223 these) than teleost fishes (Hanson et al. 2006), suggesting that cardiac failure due to these serological changes may not be the mechanism driving post-release mortality in this group, despite the demonstrated serological changes following capture across elasmobranch species (Manire et al. 2001; Moyes et al. 2006; Mandelman and Farrington

2007; Frick et al. 2010; Marshall et al. 2012; Danylchuk et al. 2014)

FUTURE RESEARCH DIRECTIONS

While my dissertation provides some insight into the cardiorespiratory responses to hypoxia and other concomitant stressors, it is not an exhaustive study, and many questions remain regarding the responses of the species studied here, as well as the elasmobranch group as a whole. I have summarized what I view as the most pressing questions resulting from Chapters II-V, as well as some general questions that I feel are pertinent to the study of elasmobranch cardiorespiratory function.

Although I observed exceptional hypoxia tolerance in clearnose skate (Chapter

II), the drivers of whole organism level tolerance remain unclear. Research regarding the hypoxia tolerance of clearnose skate over ontogeny would help determine if exposure to hypoxia conditions during development influences the degree to which adults are able to withstand low oxygen conditions (Rosa et al. 2014; Dammerman et al. 2016). Variability in adult hypoxia tolerance resulting from ontogenetic exposure would have implications for management, as different populations of clearnose skate may have different vulnerabilities to climate change impacts. Pre-conditioning may also have implications for climate change research and would also benefit from research on individual phenotypic plasticity (Esbaugh et al. 2016; Bennett et al. 2019). Due to the limitations of

224 funding and time, I did not expose individuals to cyclic diel exposures (as occurs in situ).

I argue that future work in this area should consider diel fluctuations as short term exposures are likely to elicit different response from static conditions (Melzner et al.

2009).

Although my findings on the patterns of Pcrit and P50 in elasmobranch fishes appear to differ from those reported on teleost fishes (Mandic et al. 2009), my results are based on only a few samples and were unable to detect a significant relationship between these two metrics. My analysis also includes experiments conducted under different thermal conditions. Work with standardized measurements of metabolic rate, Pcrit, and P50 within the same species and at the same temperature treatments would be helpful in understanding the relationships of P50 and Pcrit, and a more robust data set including more of the diversity present within the elasmobranch group would aid in a comprehensive understanding of the drivers of hypoxia tolerance in elasmobranch fishes, including the possible effect of metabolic rate. Additionally, incorporating other measurements of hypoxia tolerance such as: loss of equilibrium; changes in respiratory frequency, ventilation volume, and oxygen extraction efficiency; and lactate production would help clarify the meaning of hypoxia tolerance and may alter how we think about “tolerance” vs. “intolerance” (Wood 2018). Given the lack of a Bohr effect in multiple elasmobranch

Hbs (Morrison et al. 2015), including clearnose skate, additional research on oxygen offloading in the capillaries would also prove useful in describing every step in the oxygen cascade.

It is still unclear if hyperkalemia plays a role in driving post-release mortality.

More research studying higher levels of hyperkalemia, as well as research regarding the

225 impacts of hyperkalemia on non-cardiac muscle tissue contractile properties (Frick et al.

2010) would help determine if the observed level of plasma potassium in captured individuals (Moyes et al. 2006; Musyl et al. 2009) is the driver of mortality, or merely a byproduct of tissue degradation. Evaluating the ability of adrenergic stimulation to mitigate detriments in cardiac function would also benefit from further study with more extreme stressors than those I employed (Chapter V). It would also be interesting to conduct similar myocardial strip studies on tropical or cold-water species (as opposed to the temperate species I studied), as thermal regime can impact cardiac function (Drost et al. 2016; Vornanen 2016).

FINAL REMARKS

My results are one step further in understanding the cardiorespiratory physiology of elasmobranch fishes. As climate change, industrialized fishing, and other anthropogenic stressors continue to impact this group of fishes, gaining a thorough understanding of the stress responses is critical for understanding how individuals and populations will respond (Horodysky et al. 2015). I hope the information provided herein helps to inform further research on species-specific mechanisms and tolerances, with the larger goal of providing novel information on understudied elasmobranch fishes.

226 REFERENCES

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230 VITA

Gail Danielle Schwieterman

Born in Columbus, Ohio on September 19, 1990. Graduated from Worthington Kilbourne High School, Worthington, OH in 2009. Earned a B.A. in Biology, with a minor in Chemistry, from Oberlin College in 2012. Entered into the doctoral program at William & Mary, School of Marine Science (VIMS) in 2015.

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