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On the temperature-dependence of hæmoglobin-oxygenation and blood-oxygen transport in regionally heterothermic… Morrison, Phillip Robert 2020

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ON THE TEMPERATURE-DEPENDENCE OF HÆMOGLOBIN-OXYGENATION AND BLOOD-OXYGEN TRANSPORT IN REGIONALLY HETEROTHERMIC TELEOSTS AND SHARKS by  Phillip Robert Morrison  B.Sc., Vancouver Island University, 2011  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF  THE REQUIRMENTS FOR THE DEGREE OF   DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Zoology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  December 2020  © Phillip Robert Morrison, 2020 ii  The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: On the temperature-dependence of hæmoglobin-oxygenation and blood-oxygen transport in regionally heterothermic teleosts and sharks  submitted by Phillip R. Morrison in partial fulfillment of the requirements for the degree of Doctor of Philosophy  in Zoology  Examining Committee: Colin J. Brauner, Professor, Department of Zoology, University of British Columbia  Supervisor  William K. Milsom, Emeritus, Department of Zoology, University of British Columbia Supervisory Committee Member  Dr. Jeffrey G. Richards, Professor, Department of Zoology, University of British Columbia University Examiner Dr. Robert Boushel, Director/Professor, School of Kinesiology, University of British Columbia University Examiner  Additional Supervisory Committee Members: Dr. Douglas L. Altshuler, Professor, Department of Zoology, University of British Columbia Supervisory Committee Member Dr. Robert E. Shadwick, Professor, Department of Zoology, University of British Columbia Supervisory Committee Member  iii  Abstract  Hæmoglobin (Hb)-O2 affinity in most vertebrates typically decreases with increasing blood temperature because the oxygenation enthalpy (ΔH') is usually exothermic. However, in regionally heterothermic fishes, such as tunas and some sharks, ΔH' is commonly low or even endothermic, causing a very reduced or reversed effect of temperature on Hb-O2 affinity. Regionally heterothermic fishes conserve metabolic heat with vascular heat exchangers that prevent circulatory heat loss and establish internal temperature gradients. My objective was to investigate the functional significance of reduced and reverse temperature-dependent Hb-O2 affinity in regionally heterothermic fishes. I hypothesized that temperature-independent Hb-O2 affinity conserves heat energy and matches O2 supply to O2 demand despite large internal temperature gradients, so I expected this trait to be shared by all regionally heterothermic fishes. I investigated this by (1) examining the effect of temperature on Hb-O2 affinity in three regionally heterothermic species whose Hb has not been well studied (swordfish, opah, and common thresher shark), and (2) mathematical modelling of O2 transport to quantitatively assess the relative contributions of Hb concentration and Hb-O2 affinity to O2 and heat transport.   I found that opah Hb-O2 affinity is temperature-independent, the temperature-dependence of swordfish Hb-O2 affinity is pH dependent, becoming temperature-independent at low pH, and common thresher shark Hb-O2 affinity is temperature-independent. I also found that Hb from bigeye thresher shark, a suspected regional heterotherm, is temperature-independent below 50% Hb-O2 saturation. Using a mathematical model of O2 transport I demonstrated that Hb concentration and Hb-O2 affinity are relatively more important than other factors of O2 transport in determining maximum O2 consumption in yellowfin tuna, a regional heterotherm with a iv  “high-energy demand.” I also showed that Hb with a reversed temperature-dependence diminishes temperature induced changes to blood O2 tension and prevents Hb-heat loss, as much as 13% of metabolic heat production.  These results provide insight into the functional significance of reduced and reversed temperature-dependent Hb-O2 affinity in regionally heterothermic fishes. All known lineages of regionally heterothermic fishes have Hb with a low ΔH', and increases to ΔH' have convergently evolved by different molecular mechanisms with underlying dependence on different allosteric effectors.     v  Lay Summary  Hæmoglobin is a protein found inside red blood cells of vertebrates. Inspired oxygen binds to hæmoglobin and is transported in the blood throughout the body where oxygen is unloaded to fuel metabolism. In most animals, increasing blood temperature decreases hæmoglobins binding affinity for oxygen. However, the temperature-sensitivity of hæmoglobin is unusually low in some animals, including “warm-bodied” fishes such as tunas, marlins, and some sharks that are able to keep regions of their bodies warmer than the surrounding water (i.e., regional heterothermy). In this thesis, I show that the warm-bodied opah, common thresher shark, and swordfish also have hæmoglobin with a low temperature-sensitivity. I also examined how temperature-insensitive hæmoglobin may be useful in warm-bodied fish, which I conclude is a remarkable example of a trait that has repeatedly evolved to fulfill a similar function: to transport and uniformly unload oxygen to regions of the body that are maintained at very different temperatures.       vi  Preface Chapter 2 is based on analyses that I conceived. I performed all of the in silico experiments, and analyzed the data. Chapter 3 was a collaborative project with Diego Bernal (University of Massachusetts, Dartmouth), Chugey A. Sepulveda (Pfleger Institute of Environmental Research), and Nicholas C. Wegner (National Oceanic and Atmospheric Administration), and Colin J. Brauner (University of British Columbia). Diego Bernal, Chugey Sepulveda, and Nick Wegner collected the blood samples that I used. Diego Bernal, Chugey Sepulveda, and I designed the experiments. I conducted the experiments and analyzed the data under supervision from Colin Brauner.  Chapter 4 was a collaborative project with Diego Bernal, Chugey A. Sepulveda, and Colin J. Brauner. Diego Bernal and Chugey Sepulveda collected the blood samples that I used. Diego Bernal, Chugey Sepulveda, and I designed the experiments. I conducted the experiments and analyzed the data under supervision from Colin Brauner. The experiments in this thesis followed protocols that were approved by the UBC animal care committee (animal care no: A11-0235).    vii  Table of Contents Abstract  ................................................................................................................................................. iii Lay Summary ........................................................................................................................................... v Preface  ................................................................................................................................................. vi Table of Contents.................................................................................................................................... vii List of Tables ............................................................................................................................................ ix List of Figures ........................................................................................................................................... x List of Symbols and Abbreviations ....................................................................................................... xii Acknowledgements ................................................................................................................................ xiv Chapter 1 General Introduction ............................................................................................................. 1 1.1 Hæmoglobin .................................................................................................................................... 1 1.1.1 Hæmoglobin structure and function ......................................................................................... 3 1.1.2 Blood-oxygen transport ............................................................................................................ 4 1.1.3 Allosteric regulation of Hb-O2 affinity ..................................................................................... 5 1.1.3.1 The effects of protons and PCO2: The Bohr, Haldane, and Root effects ................................. 6 1.1.3.2 The effect of temperature and thermodynamics of O2 binding ................................................ 8 1.2 Regionally heterothermic fishes and sharks .............................................................................. 10 1.2.1 Sources of heat in regionally heterothermic fishes ................................................................. 12 1.2.2 Retia mirabilia and circulatory heat exchange ....................................................................... 14 1.2.3 Elevated metabolic rates and oxygen transport ...................................................................... 18 1.3 Review and critique of Hb-O2 equilibrium studies of regionally heterothermic fishes ......... 19 1.3.1 Tuna ........................................................................................................................................ 20 1.3.2 Billfishes ................................................................................................................................. 29 1.3.3 Sharks ..................................................................................................................................... 30 1.3.4 Hypotheses for the functional significance of reduced- and reversed-temperature dependence of Hb-O2 affinity in regionally heterothermic fishes .............................................................. 31 1.4 Thesis objectives and organisation ............................................................................................. 34 1.4.1 How do Hb concentration and Hb-O2 affinity contribute to the determinants of maximal O2 transport in the yellowfin tuna? .............................................................................................. 36 1.4.2 How does the enthalpy of Hb-oxygenation influence O2 and heat transport? ........................ 37 1.4.3 How does temperature affect Hb-O2 affinity in the swordfish and the smalleye Pacific opah? ................................................................................................................................................ 38 1.4.4 How does temperature affect Hb-O2 affinity in the common thresher shark and the bigeye thresher shark? ........................................................................................................................ 38 viii  Chapter 2 Determinants of Maximal Oxygen Transport in Yellowfin Tuna (Thunnus albacares) and Rainbow Trout (Oncorhynchus mykiss), and the Functional Significance of the Enthalpy of Hb-Oxygenation on Heat and Oxygen Transport ........................................ 47 2.1 Introduction .................................................................................................................................. 47 2.2 Methods ......................................................................................................................................... 53 2.3 Results ........................................................................................................................................... 61 2.4 Discussion ...................................................................................................................................... 64 Chapter 3 The Effect of Temperature on Hæmoglobin-Oxygen Affinity of Swordfish (Xiphias gladius) and Smalleye Pacific Opah (Lampris incognitus) ................................................ 86 3.1 Introduction .................................................................................................................................. 86 3.2 Methods ......................................................................................................................................... 88 3.3 Results ........................................................................................................................................... 95 3.4 Discussion ...................................................................................................................................... 98 Chapter 4 The Effect of Temperature on Hæmoglobin-Oxygen Affinity in Sharks ...................... 114 4.1 Introduction ................................................................................................................................ 114 4.2 Methods ....................................................................................................................................... 117 4.3 Results ......................................................................................................................................... 124 4.4 Discussion .................................................................................................................................... 128 Chapter 5 General Discussion and Conclusions ................................................................................ 149 5.1 Thesis overview and major contributions ................................................................................ 149 5.2 The functional significance of an increased enthalpy of hæmoglobin-oxygenation ............. 151 5.2.1 Temperature gradients in the heat-exchanging retia mirabilia: closed-system temperature changes ................................................................................................................................. 151 5.2.2 Heat-energy conservation in species with warm red muscle ................................................ 152 5.2.3 Hæmolgobin-O2 unloading to warm and cold tissues .......................................................... 154 5.3 Matching O2 supply and O2 demand ........................................................................................ 157 5.4 Is reduced temperature-dependence of Hb-O2 affinity an example of convergent physiological function among regionally heterothermic vertebrates? ........................... 158 5.5 Future research directions ........................................................................................................ 161 5.6 Summary and final thoughts ..................................................................................................... 163 References ............................................................................................................................................. 168 Appendix: A note on the storage duration of fish blood ................................................................... 191  ix  List of Tables Table 2.1 Input data used to model ṀO2max in rainbow trout and yellowfin tuna. ................................... 84 Table 2.2 Slopes of linear regressions presented in Figure 2.3, representing the magnitude of the effect of changes in each variable on ṀO2max in rainbow trout and yellowfin tuna. ............................................... 85 Table 3.1 Fork length (FL), and blood variables for smalleye Pacific opah, swordfish, and Atlantic bluefin tuna. ........................................................................................................................................................... 111 Table 3.2 Whole blood oxygen equilibria parameters for smalleye Pacific opah and swordfish at different temperatures. ............................................................................................................................................. 112 Table 3.3 Hæmolysate oxygen equilibria parameters of smalleye Pacific opah, swordfish, and Atlantic bluefin tuna at two experimental temperatures and in the absence or presence of ATP. .......................... 113 Table 4.1 Fork length and blood variables for shortfin mako sharks, common thresher sharks, bigeye thresher sharks, blue sharks, and spiny dogfish. ....................................................................................... 145 Table 4.2 Whole blood oxygen equilibria parameters at different temperatures and pH. ........................ 146 Table 4.3 Whole blood P50 values (mmHg) and Bohr coefficients (φ) reported in the literature for shortfin mako sharks, blue sharks, and spiny dogfish. ........................................................................................... 147 Table 4.4 Hæmolysate oxygen equilibria parameters of common thresher shark at two experimental temperatures and in the absence or presence of ATP and urea. ................................................................ 148 Table 5.1 ΔH' values for known and suspected regionally heterothermic teleosts and sharks ................. 166   x  List of Figures Figure 1.1 Oxygen equilibrium curves (OECs) showing that allosteric effectors and increased temperature decrease hæmoglobin (Hb)-O2 affinity and right shift the OEC. ................................................................ 40 Figure 1.2 The interrelationships of the determinants of oxygen transport. ............................................... 41 Figure 1.3 Proposed phylogenetic relationships among regionally heterothermic fishes. .......................... 42 Figure 1.4 Endothermic tissue temperatures in regionally heterothermic teleosts and sharks. ................... 43 Figure 1.5 Hill plots of oxygen equilibria of Hb from the Atlantic bluefin tuna and the porbeagle shark . 45 Figure 1.6 Effects of closed-system temperature changes on the measured change in blood PO2 (∆PO2) in bigeye tuna, yellowfin tuna, and skipjack tuna. .......................................................................................... 46 Figure 2.1 Schematic of the fish O2 transport cascade with O2 transfer from water to the mitochondria occurring in four steps. ............................................................................................................................... 76 Figure 2.2 The change in water and blood PO2 (mmHg) along the length (l) of the exchange surface for rainbow trout and yellowfin tuna. ............................................................................................................... 77 Figure 2.3 Change in ṀO2max caused by ± 1% change in different components of the O2 transport cascade for rainbow trout and yellowfin tuna. ............................................................................................ 78 Figure 2.4 Fractional flux control coefficients (F) for the four conductances that govern flow through the O2 transport cascade and contribute to determining ṀO2max. ................................................................... 79 Figure 2.5 The effect of P50tissue on blood-O2 levels, O2 delivery, tissue O2 extraction, and maximum O2 transport. ..................................................................................................................................................... 80 Figure 2.6 Yellowfin tuna and skipjack tuna Bohr plots. ........................................................................... 81 Figure 2.7 Theoretical effect of temperature on mixed venous blood PO2 with varying enthalpy of Hb-oxygenation (ΔH'). ...................................................................................................................................... 82 Figure 2.8 The relationship between hæmoglobin (Hb)-heat transport and the enthalpy of Hb-oxygenation (ΔH' kJ mol-1). ............................................................................................................................................. 83 xi  Figure 3.1 Opah in vivo pectoral muscle temperature and swordfish in vivo cranial temperature while swimming at depth. ................................................................................................................................... 105 Figure 3.2 Whole blood oxygen equilibrium curves (OECs) for the smalleye Pacific opah and the swordfish at different pH and temperatures. ............................................................................................. 106 Figure 3.3 Predicted enthalpy of oxygenation (ΔH'WB) as a function of whole blood pH and whole blood Hb-O2 saturation for the smalleye Pacific opah and the swordfish........................................................... 107 Figure 3.4 Effects of closed-system temperature changes on the measured change in blood PO2 (∆PO2) in blood from the smalleye Pacific opah and the swordfish. ........................................................................ 108 Figure 3.5 Hill plots of Hb-O2 equilibria of smalleye Pacific opah, swordfish, and Atlantic bluefin tuna at pH 7.4. ....................................................................................................................................................... 109 Figure 3.6 Heats of oxygenation (ΔH') of stripped hæmolysates of smalleye Pacific opah, swordfish, and Atlantic bluefin tuna in the absence and presence of ATP. ...................................................................... 110 Figure 4.1 Vertical movements, and stomach and ambient temperatures for a free-swimming shortfin mako shark tracked in the Southern California Bight. .............................................................................. 137 Figure 4.2 Diurnal movements of a bigeye thresher shark captured in the Southern California Bight. ... 138 Figure 4.3 Whole blood oxygen equilibrium curves (OECs) at different pH and temperatures for shortfin mako shark, common thresher shark, bigeye thresher shark, blue shark, and spiny dogfish. ................... 139 Figure 4.4 Predicted enthalpy of oxygenation (ΔH'WB) as a function of whole blood pH and whole blood Hb-O2 saturation for shortfin mako shark, common thresher shark, bigeye thresher shark, blue shark, and spiny dogfish. ............................................................................................................................................ 141 Figure 4.5 Effects of closed-system temperature changes on the measured change in blood PO2 (Δ PO2). .................................................................................................................................................................. 142 Figure 4.6 Hill plots of Hb-O2 equilibria of common thresher shark at pH 7.3. ....................................... 143 Figure 4.7 Heats of oxygenation (ΔH') of stripped hæmolysates of common thresher shark. .................. 144 List of Symbols and Abbreviations ATP  Adenosine triphosphate βbO2  Blood-O2 capacitance coefficient  CaO2  Arterial blood-O2 content CvO2  Venous blood-O2 content Hb  Hæmoglobin [Hb]  Hæmoglobin concentration  Hct  Hæmatocrit (proportion of red blood cells in blood) ΔHO2  Enthalpy of hæme oxygenation ΔH'  Overall enthalpy of hæmoglobin oxygenation  ΔH'WB  Overall enthalpy of hæmoglobin oxygenation in whole blood L  Allosteric equilibrium constant MCHC Mean corpuscular hæmoglobin concentration  ṀO2  Moles or mass of O2 consumed (i.e., flow rate of O2) NTP  Nucleoside triphosphate  n50  Hill coefficient determined at P50 OEC  Oxygen equilibrium curve OML  Oxygen minimum layer PCO2  Partial pressure of CO2  PO2  Partial pressure of O2 PaO2  Arterial blood PO2 PvO2  Venous blood PO2 PS  The PO2 that corresponds to a given Hb-O2 saturation (S) xiii  P50  The PO2 where 50% of Hb is saturated with O2 P95  The PO2 where 95% of Hb is saturated with O2 Q̇  Cardiac output RBC  Red blood cell RM  Red muscle  R-state  High affinity hæmoglobin conformation (i.e., the relaxed state) T-state  Low affinity hæmoglobin conformation (i.e., the tense state) V̇O2  Volume of O2 consumed (i.e., flow rate of O2)  φ  Bohr coefficient       xiv  Acknowledgements  The work undertaken in this thesis would not have been possible without collaboration with Diego Bernal (University of Massachusetts, Dartmouth), Chugey Sepulveda (Pfleger Institute of Environmental Research), and Nick Wegner (National Oceanic and Atmospheric Administration). I would like to thank them for openly and enthusiastically including me in their research plans, and for supplying me with the blood samples that I experimented on. I would also like to thank my supervisor, Colin Brauner, and my committee members, Doug Altshuler, Bill Milsom, and Bob Shadwick, for their support and guidance during my time at UBC.   I also received help, advice, and guidance from Richard Brill (National Oceanic and Atmospheric Administration), Hans Malte (Aarhus University), Sally Otto (UBC), as well as many graduate students and postdocs in the Department of Zoology at UBC. 1  Chapter 1 General Introduction 1.1 Hæmoglobin  “[T]he hæmoglobin of different animals does not necessarily possess the same relative affinity for oxygen.” (August Krogh, 1910, p. 220)  Hæmoglobin (Hb) is a protein that is encapsulated in the red blood cells of jawed vertebrates (gnathostomes) where it plays an essential role in gas transport between the respiratory exchange surface (e.g., gills, lungs, or skin) and the respiring cells of an animal. The steady consumption of oxygen (O2) during mitochondrial respiration maintains a low partial pressure of O2 (PO2) in the cells relative to the inspired water or air, and metabolically produced carbon dioxide (CO2) elevates the partial pressure of cellular CO2 (PCO2). In all but a few vertebrates, Hb is pivotal to sustaining aerobic metabolism by expediting the transport of O2 down its PO2 gradient in the circulatory system from the respiratory surface to the respiring tissues (the oxygen transport cascade), while also facilitating blood-CO2 transport to the respiratory surface where CO2 is eliminated in the expired water or air. Because Hb forms a physiological link between aerobic metabolism and the environment, Hb function has been shaped by internal constraints as well as by external selective forces during vertebrate evolution, resulting in numerous examples of adaptive evolution and convergence of Hb function (e.g., Natarajan et al., 2016; Storz, 2016; Tufts et al., 2015; Weber and Campbell, 2011; Weber and Fago, 2004).  Oxygen binding to Hb is an exothermic process (i.e., the overall enthalpy, ΔH', is negative) in most vertebrates, so increasing temperature typically decreases Hbs affinity for O2; a so-called “normal” temperature dependence (Figure 1.1). However, an excellent example of 2  convergent Hb function is the multiple evolutionary origins of Hbs with a reduced sensitivity to temperature among regionally heterothermic vertebrates (Weber and Campbell, 2011). Regional heterotherms are endothermic animals since they have the physiological capacity to regulate body temperature with internally generated heat, but they maintain distinct regions of their bodies at different temperatures. This is often made possible by heat exchanging retia mirabilia that facilitate heat transfer from blood leaving to blood entering a warm body region. For example, some regionally heterothermic mammals and birds have heat exchanging retia  in their limbs or appendages that reduce heat loss to the environment (Scholander, 1955). Some teleosts and sharks have independently evolved endothermy, which is partially facilitated by heat exchanging retia that prevent internally generated heat being lost via the blood flow to the surrounding water. Because the heat exchanging retia elevate the temperatures of only select body regions warmer than ambient water temperature, and some tissues remain in thermal equilibrium with ambient water, endothermic fishes and sharks are functionally regional heterotherms. For the remainder of this thesis I will refer to these fishes as regionally heterothermic. In all but a couple of regionally heterothermic fishes investigated so far, Hb-O2 affinity is independent of temperature or even exhibits a reverse temperature-dependence (i.e., Hb-O2 affinity increases with increasing temperature, evident as a leftward shift of the black curves in Figure 1.1) (Morrison et al., 2015; Weber and Campbell, 2011). Furthermore, regional heterothermy is energetically costly, as is apparent by the high metabolic rates of those species for which it has been measured (Korsmeyer and Dewar, 2001; Sepulveda et al., 2007b). Thus, to fuel heat production and support their high metabolic rates, regionally heterothermic fishes tend to have high blood-O2 transport capacities, due in part to high Hb concentrations (e.g., Bernal et al., 2001b; Morrison et al., 2015).  3   I further review Hb function and the Hb of regionally heterothermic fishes in this chapter. Based on this information, I have developed two general hypothesis that unify this thesis: 1) temperature-independent Hb-O2 affinity conserves heat-energy, which may help maintain stably elevated body temperatures in regionally heterothermic fishes. Thus, I expected this trait to be shared by all regionally heterothermic teleosts and sharks; and 2) the Hb-oxygen-binding properties and high Hb concentrations of regionally heterothermic fishes maintain a match between O2 supply and O2 demand despite large internal temperature gradients. At the end of this chapter I outline my objectives and the research questions that I address in the subsequent chapters.  1.1.1 Hæmoglobin structure and function  Genome duplication events early in vertebrate evolution produced a functional diversification of globin genes that gave rise to the modern jawed vertebrate Hb, which is a heterotetramer composed of two α-chain subunits and two β-chain subunits (Pillai et al., 2020; Storz, 2016). Each subunit contains a hæme group with a porphyrin ring that holds an iron atom, which reversibly binds one O2 molecule when the iron atom is in the ferrous state (Fe2+); thus, one Hb tetramer can bind up to four O2 molecules. The quaternary structure of the Hb tetramer comprises an oxygenation-linked equilibrium between two distinct conformations: a high-affinity (oxygenated) R-state and a low-affinity (deoxygenated) T-state (Monod et al., 1965). Homotropic and heterotropic allostery regulate oxygenation-linked shifts in the T↔R conformational equilibrium, in such a way that interactions among the hæme binding sites instigate cooperative O2-binding (homotropic), and ligand binding to non-hæme sites regulates Hb-O2 affinity (heterotropic) (Baldwin and Chothia, 1979; Perutz, 1970).  4   The oxygen equilibrium curve (OEC) characterises the relationship between Hb-O2 saturation and PO2, and the shape and position of the OEC reflect homotropic allostery and Hb-O2 binding affinity, respectively (Figure 1.1) (Bohr et al., 1904; Wyman, 1964). August Krogh and Isabella Leitch introduced the term “tension of unloading” (tu) to denote the “O2 pressure at which the Hb is just half saturated with oxygen” and the “tension of loading (tl) as corresponding to 95 per cent. saturation” (Krogh and Leitch, 1919). Their tu term is the equilibrium constant, K, in Archibald V. Hill’s mathematical description of the OEC (i.e., Hill’s equation) (Barcroft and Hill, 1910; Hill, 1910) and is now typically referred to as P50 (i.e., the PO2 at 50% Hb-O2 saturation), which is “used as a single though incomplete characteristic” of the OEC that quantifies Hb-O2 affinity (Krogh and Leitch, 1919). Non-hæme binding of heterotropic effectors (i.e., allosteric effectors) typically increases P50 and right shifts the OEC of whole blood relative to that of stripped Hb (i.e., a Hb solution purified of endogenous allosteric effectors), and oxygenation-linked binding and dissociation of allosteric effectors shifts the T↔R conformational equilibrium, modulating blood-O2 affinity between the respiratory exchange surface and the peripheral tissues (i.e., the sites of O2 binding and offloading, respectively) (Figure 1.1). Therefore, Hb-O2 affinity is an important factor in the O2 transport cascade because it is a link between the PO2 of the inspired water or alveolar air and the tissue O2 supply.  1.1.2 Blood-oxygen transport   “In the respiratory organs proper the gases are transported from one medium to another by diffusion and, for all we know, diffusion is also the only mechanism for the transport into and out of the ultimately respiring cells, but in between we have a transport by convection in the flowing blood” (Krogh, 1941, p. 88). The oxygen capacity and the OEC are important respiratory characteristics of the blood that are central to blood-O2 transport from the respiratory organ to 5  the tissues. The diffusive steps of the O2 transport cascade can be quantified by Fick’s law of diffusion, while convection can be quantified by the Fick Principle, which is given by  ?̇?O2 = ?̇? ∙ (𝐶𝑎O2 − 𝐶𝑎O2) (1) where V̇O2 is the flow rate of O2 (i.e., O2 consumption rate), Q̇ is the cardiac output, and (CaO2-CvO2) is the arteriovenous O2 content difference. In equation 1, circulatory O2 delivery is equal to the product of Q̇ and CaO2, and the O2 extraction is equal to percentage of O2 removed from the blood [i.e., (CaO2-CvO2)/CaO2 ∙ 100%]. The arteriovenous O2 content difference can be expressed by the following   𝐶𝑎O2 − 𝐶𝑎O2 = 𝛽𝑏O2 ∙ (𝑃𝑎O2 − 𝑃𝑣O2) (2) where (PaO2-PvO2) is the arteriovenous PO2 difference and βbO2 is the O2 capacitance coefficient of the blood. βbO2 defines the gradient of the functional portion of the OEC (Figure 1.2), which is the slope of the relation between O2 content and PO2:  𝛽𝑏O2 =𝐶𝑎O2 − 𝐶𝑎O2𝑃𝑎O2 − 𝑃𝑣O2 (3) It follows that for any given cardiac output, circulatory O2 delivery and O2 extraction depend on the arterial O2 content and left or right shifts in the OEC that change βbO2 (Figure 1.2). Thus, blood-O2 transport is largely determined by Hb concentration and shifts in Hb-O2 affinity between the respiratory organ and the tissues.  1.1.3 Allosteric regulation of Hb-O2 affinity  “The affinity of blood or pure hæmoglobins for oxygen is a complex phenomenon, depending upon a number of conditions, the most important of which are temperature and hydrogen ion concentration” (Krogh, 1941, p. 95). Hb-O2 affinity is also affected by binding of chloride ions (Cl+) and organic phosphates to non-hæme sites, but rapid left and right shifts to 6  the OEC in vivo (i.e., the physiological OEC) are induced primarily by non-hæme binding of hydrogen ions (protons) and the thermodynamics of Hb-O2 binding (Figure 1.1).  1.1.3.1 The effects of protons and PCO2: The Bohr, Haldane, and Root effects  As metabolic CO2 enters the blood from the tissues there is a simultaneous and associated decline in blood pH that decreases blood-O2 affinity via negative, heterotropic interactions linked to proton binding sites. The influence of CO2 on blood-O2 equilibria was first described in the seminal paper by Bohr et al. (1904), in which Christian Bohr, Karl Hasselbalch, and August Krogh showed that increasing PCO2 right shifted the OEC. A few years later, Joseph Barcroft and Leon Orbeli (1910) reported a similar effect by adding acid to blood. The influence of CO2 and pH on Hb-O2 affinity has since become known as the “Bohr effect” (given by John Scott Haldane) and is quantified as the Bohr factor or Bohr coefficient (φ):  𝜑 =Δ log10 𝑃50ΔpH (4) The magnitude of Bohr effect directly influences Hb-O2 affinity, and the protons bound during oxygen unloading in the tissues profoundly right shifts the physiological OEC (Malte and Lykkeboe, 2018). This deoxygenation linked proton binding facilitates CO2 transport by increasing the CO2 content of the deoxygenated venous blood relative to oxygenated arterial blood, a phenomenon established by the work of Haldane and co-workers (Christiansen et al., 1914) and is now known as the “Haldane effect”. The Bohr and Haldane effects are reciprocal to one another and are considered “two sides of the same coin” because they are thermodynamically linked at the molecular level (Wyman, 1964). Consequently, CO2 that diffuses into the blood in the tissue capillaries boosts O2 offloading by decreasing Hb-O2 affinity, but elimination of CO2 at the respiratory organ promotes O2 binding by increasing Hb-O2 7  affinity. Thus, the Bohr effect plays an essential role in shifting the OEC in vivo from high to low affinity at the respiratory organ and tissues, respectively. Furthermore, because P50 and the Bohr coefficient are not independent of one another, over the course of Hb evolution structural changes that increased the magnitude of the Bohr coefficient and/or the effectiveness of a given acid load to promote Hb-O2 unloading, would have also increased whole blood P50, and vice versa (Berenbrink, 2006; Malte and Lykkeboe, 2018).   Teleosts express multiple, functionally distinct Hb isoforms, which is at least partly due to a teleost-specific genome duplication event (Opazo et al., 2013; Storz, 2016), and some teleost Hbs exhibit what has been described as an exaggerated Bohr effect (Root, 1931). Krogh and Leitch (1919) were the first to note that the blood of fishes “is very sensitive to small increases in CO2 tension which reduce the affinity for oxygen very considerably” (Berenbrink et al., 2011). The pH sensitivity of teleost Hb is known as the Root effect, named in recognition of Raymond Root (Scholander and Van Dam, 1954), and it is characterised by an exaggerated right shift of the OEC with declining pH, and a loss of cooperative Hb-O2 binding associated with the inability to fully saturate Hb below a certain pH threshold (Green and Root, 1933; Root, 1931). Root effect Hbs are part of a complex physiological system that is specialized for O2 secretion in the eye and swim bladder of teleost fishes by inducing large decreases in Hb-O2 affinity and O2 carrying capacity, which are corrected by the time the blood circulates back to the respiratory surface to replenish blood-O2 levels (e.g., Berenbrink, 2007; Berenbrink et al., 2005). Although teleost Bohr coefficients are generally numerically higher than in other vertebrates, a Bohr effect is present in all lineages of jawed vertebrate (e.g., Berenbrink, 2006; Giardina et al., 2004; Pough, 1980), but not necessarily in all species on a particular branch of the vertebrate tree (e.g., chondrichthyans; Morrison et al., 2015).  8  1.1.3.2 The effect of temperature and thermodynamics of O2 binding  Sir Joseph Barcroft and collaborators were the first to quantify the kinetics and thermodynamics of Hb-O2 equilibria, and they determined that the overall enthalpy of Hb- oxygenation (ΔH') is numerically negative (Barcroft, 1914; Barcroft and Hill, 1910). Thus, Hb-O2 binding is an exothermic process (Hb + O2 ⇋ HbO2 + heat), so increasing temperature will shift the equilibrium towards the deoxygenated form of Hb, consequently decreasing Hb-O2 affinity (Barcroft and Hill, 1910; Barcroft and King, 1909). Because the heat of O2 binding to the haeme groups (ΔHO2) is intrinsically exothermic, the overall ΔH is usually also exothermic. Values for ΔHO2 are very similar among the hæmolysates of jawed vertebrates [e.g., ~ -62 kJ mol-1 for billfish hemolysates at pH 8.0 (Weber et al., 2010)] (Morrison et al., 2015; Powers et al., 1979). However, ΔH' is typically numerically less negative than ΔHO2. This is because ΔH' comprises the sum of contributions from ΔHO2, the heat of solution of O2 (~ -12.6 kJ mol-1), the heat of conformational changes (T↔R transitions), and the heats of ionization and dissociation of allosteric effectors (e.g., protons, organic phosphates, and Cl- ions). Oxygenation linked dissociation of allosteric effectors contribute endothermically to ΔH', reducing the overall effect of temperature on Hb-O2 affinity. Values of ΔH' are typically negative in jawed vertebrates, and are quantified using the van’t Hoff isochore (Wyman, 1964):  ∆𝐻′ = ln 10 ∙ 𝑅 ∙Δ log10 𝑃50Δ1𝑇 (5) where R is the gas constant and T is the absolute temperature.   Krogh and Leitch (1919) noted the significance of the temperature sensitivity of Hb-O2 affinity for O2 uptake and transport in fishes, recognizing that fishes thermoconform to their environment (i.e., most fishes are poikilotherms) so the surrounding water temperature will 9  directly influence Hb-O2 uptake and transport. Furthermore, endothermic Hb-O2 unloading in the tissues and exothermic Hb-O2 binding at the gills cause the outward transport and loss of metabolic heat to the environment, contributing to poikilothermy in fishes (Jensen et al., 1998; Stevens and Sutterlin, 1976; Weber and Fago, 2004). Over 40 years after Krogh and Leitch reported their blood-O2 equilibrium experiments at the environmental temperatures of fishes, Alessandro Rossi Fanelli and Eraldo Antonini reported that temperature had very little effect on the O2 affinity of crystalline Hb from Atlantic bluefin tuna (Thunnus thynnus) (Rossi Fanelli and Antonini, 1960). Bluefin tuna Hb exhibited an obvious pH sensitivity, but over a wide pH range (pH 6.45 to 8.7) changing temperature between 5°C and 35°C (~5, 10, 20, 30, and 35°C) had no appreciable effect on the shape or position of the OEC. At that time, the discovery that bluefin tuna Hb was “practically unaffected by temperature” was considered novel enough that the findings were published in Nature along with a companion paper on tuna myoglobin, which was shown to be functionally similar to mammalian myoglobin (Rossi Fanelli and Antonini, 1960; Rossi Fanelli et al., 1960).   Rossi Fanelli and Antonini proposed that “the insensitivity of the oxygen equilibrium of tuna hæmoglobin to changes in temperature could represent a sort of molecular adaptation which would enable the animal to live in waters of very different temperatures without modification of the functional properties of its respiratory pigment” (Rossi Fanelli and Antonini, 1960, p. 896). In other words, they concluded that bluefin tuna should have no problem binding O2 as they move between warm and cold water above and below the thermocline, respectively. This may indeed be a benefit of temperature independent Hb-O2 affinity, but what Rossi Fanelli and Antonini may not have known, or at least did not mention, is that bluefin tuna are regionally heterothermic and can maintain the core of their body much warmer than their peripheral tissues 10  and the ambient water. It was another four years before the body temperatures of yellowfin tuna (Thunnus albacares) and skipjack tuna (Katsuwonus pelamis) were reported (Barrett and Hester, 1964), and another two more years before Francis “Frank” Carey and John Teal reported the body temperatures of bluefin tuna and described the anatomical specialisations that facilitate regional heterothermy (Carey and Teal, 1966). Reports of warm bodied tuna and descriptions of their anatomy date back to at least 1835 (reviewed by Fudge and Stevens, 1996), and although Rossi Fanelli and Antonini may not have known of these earlier publications, their discovery that bluefin tuna Hb-O2 affinity is unaffected by temperature led others to hypothesize that this trait may prevent unnecessary temperature induced shifts in the OEC between the cold and warm tissues (e.g., Carey and Gibson, 1977; Graham, 1973). Investigations into the effect of temperature on Hb-O2 affinity in other species of regionally heterothermic vertebrates has spanned the 60 years since the formative publication by Rossi Fanelli and Antonini  (Weber and Campbell, 2011), and this line of research is the basis for this PhD thesis. Before reviewing the literature on the effect of temperature on Hb-O2 affinity in regionally heterothermic teleosts and sharks, I will briefly review the anatomical and physiological specializations that conserve body heat in these disparate species.   1.2 Regionally heterothermic fishes and sharks  From the late 1960’s until his death in 1994, Frank Carey and his collaborators established that tuna and lamnid sharks are “warm-bodied,” and they began to describe the anatomical and physiological specialisations for endothermy as well as the physiological ecology of these fishes (e.g., Block and Carey, 1985; Carey, 1973; Carey and Lawson, 1973; Carey and Teal, 1966; Carey and Teal, 1969a; Carey and Teal, 1969b; Carey et al., 1971; Carey et al., 1981; Carey et al., 1981; Carey et al., 1982; Carey et al., 1984; Carey et al., 1985; Linthicum and 11  Carey, 1972; Wolf et al., 1988). In the 1980’s and early 1990’s, Frank Carey and Barbara Block would also describe the “brain heaters” and aspects of the ecology of billfishes (e.g., Block, 1986; Block, 1991b; Block et al., 1992a; Block et al., 1992b; Carey, 1982b; Carey, 1990). Carey’s pioneering research on tuna has been reviewed by Graham and Dickson (2001), and research on fish endothermy and the physiological ecology of large pelagic fishes is carried on by others to this day. Since Carey and Teal (1966) described the endothermic capacity of the Atlantic bluefin tuna, it has been well established that endothermy has evolved independently in several teleost and elasmobranch lineages, including tunas (Scombridae: Euthynnus, Auxis, Katsuwonus, Thunnus), billfishes (Istiophoridae and Xiphiidae), the smalleye Pacific opah (Lampris incognitus), the common thresher shark (Alopias vulpinus), and sharks in the family Lamnidae (salmon shark, Lamna ditropis, porbeagle shark, Lamna nasus, shortfin mako, Isurus oxyrinchus, longfin mako, Isurus paucus, white shark, Carcharodon Carcharias) (Figure 1.3) (Bernal and Sepulveda, 2005; Block, 1986; Block and Carey, 1985; Carey, 1982b; Carey and Teal, 1966; Carey and Teal, 1969a; Carey and Teal, 1969b; Carey et al., 1971; Carey et al., 1985; Runcie et al., 2009; Sepulveda et al., 2008; Wegner et al., 2015).  Endothermic fishes are functionally regional heterotherms (sometimes referred to as regional endotherms) since they conserve metabolic heat to maintain only select tissues or regions of their bodies warmer than the surrounding water. In contrast, most other fishes are poikilothermic and thermoconform with their environment because metabolic heat is transferred to the environment through conduction across the body wall, as well as by convective transfer in the blood to the gill circulation where heat is lost to the inspired water (Brill et al., 1994; Stevens and Sutterlin, 1976). Thus, for regionally heterothermic fishes to maintain elevated body temperatures in their aquatic environments they need a source of metabolic heat and a 12  mechanism to retain that heat (Graham and Dickson, 2001). Although the source of heat varies among species, all regionally heterothermic fishes have retia mirabilia that function as countercurrent heat exchangers, like those of mammals (Scholander and Krog, 1957), and these retia are situated between the warm tissues and the gills to mitigate circulatory heat loss. The anatomical and physiological specialisations that facilitate thermoconservation in fishes are well reviewed (e.g., Bernal and Lowe, 2015; Bernal et al., 2001b; Bernal et al., 2012; Block, 1991a; Block, 1991b; Carey, 1973; Dickson and Graham, 2004; Fudge and Stevens, 1996; Graham and Dickson, 2000; Graham and Dickson, 2001; Graham and Dickson, 2004), and another in depth analysis could easily take up too much of this thesis. So here I will provide a brief overview of the features that are most pertinent to Hb function and blood-O2 transport.  1.2.1 Sources of heat in regionally heterothermic fishes   Tunas, sharks in the family Lamnidae, and the common thresher shark are able to maintain a warm body core (Figure 1.4) by retaining the heat generated by the continuous contraction of their red myotomal swimming muscles (RM) (Bernal and Sepulveda, 2005; Carey and Teal, 1966; Carey and Teal, 1969a; Carey and Teal, 1969b; Carey et al., 1985; Sepulveda et al., 2008). Unlike most other fishes in which the RM is positioned subcutaneously, the warm RM of regional heterothermic tunas and sharks is located medially and more anteriorly in the body, close to the vertebral column (Carey and Teal, 1966; Carey and Teal, 1969a; Carey et al., 1985; Sepulveda et al., 2005; Sepulveda et al., 2008). Because these species must swim to ram ventilate their gills, the continuous contraction of their internalized RM provides a constant source of heat that is somewhat insulated from the environment by the surrounding white muscle. Additionally, tunas and lamnid sharks are also capable of elevating cranial (brain and eyes) and visceral temperatures (Figure 1.4) (Anderson and Goldman, 2001; Bernal et al., 2001b; Block and Carey, 13  1985; Carey et al., 1981; Carey et al., 1984; Goldman, 1997; Goldman et al., 2004; Sepulveda et al., 2004; Sepulveda et al., 2007a; Wolf et al., 1988).   Billfishes (Istiophoridae) and the swordfish (Xiphias gladius) are also capable of cranial endothermy and warm their heads with heat generating organs that evolved from the superior rectus extraocular muscles (Figure 1.4) (Block, 1986; Block, 1991b; Carey, 1982b). A putative heater organ also independently evolved in the butterfly kingfish (also known as the butterfly mackerel, Gasterochisma melampus), a scombrid fish closely related to tunas, but cranial endothermy has yet to be confirmed in butterfly kingfish (Tullis et al., 1991). Although the ocular muscles of some other regionally heterothermic fishes likely also contribute to cranial endothermy (Bernal et al., 2001b; Block and Finnerty, 1994; Runcie et al., 2009; Tubbesing and Block, 2000; Wolf et al., 1988), the heater organs of billfishes and the butterfly kingfish are unique because they are the only examples in fishes of tissues that are specialised for heat production (Block, 1994; Tullis et al., 1991).  The smalleye Pacific opah stands out among regionally heterothermic fishes because it has evolved what has been referred to as a form of whole-body endothermy (Wegner et al., 2015). The opah heats its cranium and most of its body core including the heart (Wegner et al., 2015), which is unique among fishes because the hearts of other fishes, including regional heterotherms, remain near ambient water temperature (e.g., Bernal et al., 2001b; Brill and Bushnell, 2001; Brill and Lai, 2015). The opahs heart receives warm blood from both the arterial and venous circulations, and is insulated from the opercular cavity by a fat layer. The lateral rectus extraocular muscle appears to be involved in heating the opahs cranium (Runcie et al., 2009), but most of the metabolic heat that warms the body is produced by the dark red, aerobic pectoral muscles that power swimming by the continuous “flapping” of their pectoral fins. A fat 14  layer also insulates the body core from the surrounding water, preventing heat loss from the peripherally situated pectoral muscles (Wegner et al., 2015). The system of heat exchanging retia is also quite different in the opah because the retia are associated with the gill vasculature and send warm blood to the entire body (Wegner et al., 2015), whereas the retia of other regional heterotherms are associated with the blood supply to the warm tissues only (see next section).  1.2.2 Retia mirabilia and circulatory heat exchange  The heat exchanging rete mirabile, or “wonderful net,” is a tissue of closely intermingled and countercurrent running arterioles and venules (e.g., Carey et al., 1985; Lemons et al., 1987; Stevens et al., 1974), which conserves heat in a warm tissue by enabling the inflowing arterial blood to be warmed by the outflowing venous blood that is returning to the gills (Bernal et al., 2001b; Block, 1986; Carey, 1982b; Carey and Teal, 1966; Carey and Teal, 1969a; Fudge and Stevens, 1996; Graham and Dickson, 2001; Runcie et al., 2009; Wegner et al., 2015). Because thermal diffusion occurs about ten times faster than molecular diffusion, the blood of fishes thermally equilibrates with water in the time it takes for the blood to transit through the gill lamellae and exchange gases with the inspired water (Brill et al., 1994; Graham and Dickson, 2001; Stevens and Sutterlin, 1976). Thus, heat exchanging retia mitigate excessive metabolic heat loss from the warm tissues by cooling the outflowing venous blood near to ambient temperature before it reaches the gills, which creates a stable thermal gradient or thermal excess between the warm tissues and the ambient water (Figure 1.4) (Graham and Dickson, 2001).  The systemic circulation of regionally heterothermic sharks and most tunas are highly modified to route the bulk of the systemic blood flow through subcutaneous arteries and veins, which direct the RM blood supply through lateral heat exchanging retia (Bernal et al., 2001b; Burne, 1924; Carey and Teal, 1966; Carey and Teal, 1969a; Carey et al., 1985; Patterson et al., 15  2011). This contrasts with most other fishes, in which the major conduits of the systemic circulation are situated medially, just ventral to the vertebral column. However, some tunas have only a central circulation that supplies blood to the RM via a central heat exchanging retia, and some Thunnus species lack a central rete and appear capable of adjusting the relative amount of systemic blood flow between the central circulation and the lateral heat exchanging retia, which should increase their capacity to physiologically thermoregulate (Bernal et al., 2001b; Bernal et al., 2017; Graham and Dickson, 2001; Sepulveda et al., 2008). Lamnid sharks may also be able to physiologically thermoregulate by redistributing the systemic blood flow in a similar way (Bernal and Lowe, 2015; Bernal et al., 2001a; Bernal et al., 2009). The RM heat exchanging retia establish elevated and relatively stable operating temperatures for the RM, which in some species creates a thermal excess of 10 to 20°C even in frigid, sub-polar water (Bernal and Lowe, 2015; Bernal et al., 2001b; Bernal et al., 2005; Carey and Teal, 1969b; Carey et al., 1971; Patterson et al., 2011; Sepulveda et al., 2008). The swordfish also has putative heat exchanging retia that supply blood to the medially located red aerobic swimming muscles, but these retia are not as efficient at heat retention as the red muscle retia of tunas and sharks and likely serve to just slow down cooling of the red muscle (Carey, 1990; Stoehr et al., 2018).  Tunas and lamnid sharks also possess heat exchanging retia in the orbital and visceral circulations, and orbital retia also conserve heat generated by the thermogenic organs of billfishes (Bernal et al., 2001b; Block, 1986; Block and Carey, 1985; Burne, 1924; Carey, 1982b; Carey et al., 1981; Fudge and Stevens, 1996; Linthicum and Carey, 1972; Wolf et al., 1988). In the case of the swordfish, the orbital rete maintains cranial temperature within a narrow range that is elevated as much as 12°C above ambient water temperature when swordfish are in deep, cool water during daylight hours (< 10°C) (Carey, 1982b; Carey, 1990). Likewise, the orbital 16  retia of tuna and lamnid sharks efficiently elevate eye and brain temperatures, as much as 18°C above ambient in Atlantic bluefin tuna, and 10 to 13°C above ambient in lamnid sharks (Figure 1.4) (Bernal and Lowe, 2015; Bernal et al., 2001b; Block and Carey, 1985; Linthicum and Carey, 1972). The ability to maintain warm brain and eyes should increase integration and processing of sensory information, and previous work on swordfish eyes has shown that the elevated temperature increases temporal resolution (Fritsches et al., 2005), which should provide regionally heterothermic fishes with crucial advantages over their thermoconforming prey (Block, 1986).  The smalleye Pacific opah is quite different from other regionally heterothermic fishes because it has a heat exchanging rete mirabile located inside each gill arch, each of which is relatively thick and insulated with fat (Wegner et al., 2015). These gill retia warm the arterial blood immediately after being oxygenated at the gills, warming the blood that then perfuses the entire body, including the heart (Wegner et al., 2015). Furthermore, an orbital rete enables further heat localization in the cranial region, causing it to be even warmer than the rest of the body (Runcie et al., 2009; Wegner et al., 2015). Cranial temperatures in the opah are also relatively constant and elevated at least 6°C above the ambient environment, while body and heart temperature are elevated at least 3 to 5°C (Figure 1.4) (Wegner et al., 2015).  The bigeye thresher shark (Alopias superciliosus) has been suspected to be capable of cranial endothermy because it has unusually large eyes and a putative orbital rete (Block and Carey, 1985; Block and Finnerty, 1994; Dickson and Graham, 2004; Weng and Block, 2004). However, two of my collaborators that were involved in the work included in Chapters 3 and 4 of this thesis, have made opportunist measurements of the eye temperature of bigeye threshers and found them to be no warmer than ambient sea surface temperature (Diego Bernal and 17  Chugey A. Sepulveda, personal communication). Furthermore, the pelagic thresher shark (Alopias pelagicus) also has an orbital rete (Block and Carey, 1985), the common thresher shark has a rete associated with the viscera, there are RM associated retia in the pectoral fins of the Chilean devil ray (Mobula tarapacana), and orbital retia are present in some species of mobulid rays (Alexander, 1995; Alexander, 1996; Fudge and Stevens, 1996), but it has yet to be confirmed if these retia have a thermoconserving function (Bernal and Lowe, 2015).  The heat exchanging retia of regionally heterothermic fishes as well as other regionally heterothermic vertebrates (e.g., some mammals and birds) cause blood-O2 uptake to occur at a very different temperature to O2 unloading in the tissues that are thermally isolated by a rete. In regionally heterothermic fishes, when blood flows through a rete into or out of the warmer tissues it is subjected to what has been described as “closed-system” temperature changes (Brill and Bushnell, 1991a). This is because the change in blood temperature can potentially affect blood PO2 and PCO2, but the content of blood gases should remain constant due to the size and thickness of rete vessels (i.e., arterioles and venules) that ought to preclude diffusion of gases out of the blood (Carey et al., 1985; Graham and Dickson, 2001; Stevens et al., 1974). Furthermore, most regionally heterothermic fishes are pelagic predators that subject themselves to varying water temperatures while moving above and below the thermocline or during latitudinal migrations (e.g., Bernal et al., 2009; Bernal et al., 2017; Block et al., 1992b; Block et al., 2001; Carey, 1990; Dewar et al., 2011; Sepulveda et al., 2010; Wegner et al., 2015; Weng et al., 2005). Thus, blood-O2 uptake must occur over the range of temperatures encountered by a species, but the blood must also transport O2 over steep internal temperature gradients, and O2 offloading must occur from the lowest (usually at the gills) to the highest body temperatures (usually at the muscle).     18  1.2.3 Elevated metabolic rates and oxygen transport   Tunas and lamnid sharks exhibit elevated metabolic rates and a remarkable evolutionary convergence for “thunniform” swimming, which is due to a number of shared morphological and physiological traits, including internalized red muscle (Bernal et al., 2001b; Donley et al., 2004). Maintaining elevated red muscle temperatures also elevates metabolic rates, and tuna are often referred to as “high-energy-demand” or “high-performance” fishes because of their high metabolic rates and large aerobic scopes (Brill and Bushnell, 1991b; Korsmeyer and Dewar, 2001). Lamnid sharks also appear to have elevated metabolic rates compared to other sharks (Bernal et al., 2012; Ezcurra et al., 2012; Sepulveda et al., 2007b), and in both tunas and lamnids, the increased metabolic demand for O2 has been matched by parallel increases in the structural and physiological factors of the O2 transport cascade (Bernal et al., 2001b; Brill and Lai, 2015; Wegner, 2015; Wegner et al., 2010). This is especially apparent in the cardiovascular system of tunas (Brill and Bushnell, 2001).  Tunas have very high blood-O2 carrying capacities due to high Hb concentrations that are comparable to mammalian values (Bernal et al., 2001b; Brill and Bushnell, 2001; Gallaugher and Farrell, 1998). Tunas also have gills with a large surface area and thin diffusion distances to increase O2 extraction from the water, and large powerful hearts to circulate blood at high rates (Brill and Bushnell, 2001; Muir and Hughes, 1969; Wegner et al., 2006; Wegner et al., 2010). Thus, rates of circulatory O2 delivery are exceptionally high in tuna. High Hb concentration in tunas and other regionally heterothermic fishes has been proposed to be necessary to supply O2 at high rates while minimizing blood flow, which should match the high O2 demands of heat production and help maintain elevated tissue temperatures by reducing circulatory heat loss (Carey and Gibson, 1983; Gibson and Carey, 1982). However, heat production and heat loss may 19  be independent of blood flow (Brill et al., 1994; Graham and Dickson, 2004), and Brill and Bushnell have proposed that the high Hb concentration facilitates high rates of circulatory O2 delivery without unnecessarily increasing cardiac output when aerobic metabolism is elevated (Brill and Bushnell, 1991b; Brill and Bushnell, 2001). I tend to agree with Brill and Bushnell because tuna hearts appear to be extremely “specialised” for pumping large volumes of blood, and tuna Hb concentrations may have reached a level that cannot be surpassed without greatly increasing blood viscosity and causing cardiovascular impairment. Therefore, high blood-O2 carrying capacities should match O2 supply to O2 demand while also maintaining sufficient O2 stores to recover from strenuous swimming without imposing exhaustive demands on cardiac function. Furthermore, tuna have evolved certain traits to facilitate high rates of O2 diffusion into the muscles (Korsmeyer and Dewar, 2001), and all studied species of  tuna have a large Bohr coefficient (Brill and Bushnell, 1991a; Cech et al., 1984; Jones et al., 1986). The latter is very likely necessary to right-shift the OEC in the tissue capillaries to unload O2 at high enough rates to meet the demand for O2 without increased blood flow.  1.3 Review and critique of Hb-O2 equilibrium studies of regionally heterothermic fishes “Bluefin tuna hemoglobin, in contrast to all other vertebrate hemoglobins has an affinity for oxygen which is almost independent of temperature . . . Such low thermal coefficients make it seem possible that in the warm fishes part of the integration of life processes has been achieved by evolving systems with low sensitivity to temperature with the bluefin having in addition a fairly well-controlled temperature.” (Carey and Lawson, 1973, p. 390)  It was not until 1973 that further research was published on the hæmoglobins of regionally heterothermic fishes (Andersen et al., 1973), 13 years after Rossi Fanelli and Antonini reported their findings on bluefin tuna hæmoglobin. This research was led by Quentin Gibson 20  and over the next decade Gibson and co-workers, often in collaboration with Frank Carey, further investigated the effect of temperature on hæmoglobin function in tunas and lamnid sharks. Since then, hæmoglobins with reduced thermal sensitivity have been reported from many species of regionally heterothermic vertebrates with heat-exchanging retia, including fishes and mammals (Weber and Campbell, 2011). Among these animals, relative increases in the enthalpy of hæmoglobin-oxygenation have convergently evolved by different molecular mechanisms. Here I will review the literature on the effect of temperature on Hb-O2 affinity in tunas, billfishes, and lamnid sharks. I am not aware of any published studies on the temperature sensitivity of Hb from opah or thresher sharks.  1.3.1 Tuna   Atlantic bluefin tuna Hb is one of the most well studied fish Hbs, including structural and functional studies of ligand binding, as well as studies on the effect of temperature on Hb-O2 affinity and the mechanism of the Root effect (Brunori, 1966; Carey and Gibson, 1977; Ikeda-Saito et al., 1983; Jensen, 2001; Morris and Gibson, 1982; Morris et al., 1981; Rodewald et al., 1987; Yokoyama et al., 2004). Seventeen years after the first report that Atlantic bluefin tuna Hb-O2 affinity was unaffected by temperature, Carey and Gibson (1977) reported that the shape of the OEC for bluefin tuna Hb was unusually temperature dependent (Carey and Gibson, 1977; Rossi Fanelli et al., 1960). Carey and Gibson found that below 20% Hb-O2 saturation, bluefin tuna hæmolysate had a higher O2 affinity at 14°C than at 22°C, but above 20% saturation O2 affinity was higher at 22°C causing the OECs for the two temperatures to crossover (i.e., switching from a normal to a reverse temperature-dependence). Similar reverse temperature effects were later reported for whole blood from Atlantic bluefin tuna (Carey and Gibson, 1983). In a study on the primary Hb isoform of Atlantic bluefin tuna (Hb I) at various pH levels and 21  temperatures, a saturation dependent reverse temperature effect (i.e., positive ΔH') was also observed, but the OECs at different temperatures crossed over around 50% saturation for Hb I (Figure 1.5) (Ikeda-Saito et al., 1983). The positive ΔH' is due to endothermic contributions from oxygenation-dependent Bohr protons released at high Hb-O2 saturation (Ikeda-Saito et al., 1983). Organic phosphates (i.e., adenosine triphosphate, ATP) probably do not have an appreciable endothermic contribution to ΔH' since organic phosphates do not appear to influence Hb from both bluefin tuna and bigeye tuna (Thunnus obesus) (Andersen et al., 1973; Ikeda-Saito et al., 1983). The increased Hb-O2 affinity at high saturation with increasing temperature is linked to an unusually high temperature dependency of the allosteric equilibrium constant, L, which defines the proportions of the T- and R-states (𝐿 ∝  𝑇𝑅) (Carey and Gibson, 1977; Carey and Gibson, 1983; Ikeda-Saito et al., 1983; Morris and Gibson, 1982). Low temperatures stabilize the low affinity T-state conformation (high L), preventing the T→R transition even at high O2 saturation, owing to shifts between multiple T-state conformations that can be explained by an extended form of the MWC two-state model (Ikeda-Saito et al., 1983; Morris and Gibson, 1982; Morris et al., 1981). The large number of Bohr groups stabilize the T-state, but increasing temperature causes the release of Bohr protons that are bound at high O2 saturation, decreasing L and increasing Hb-O2 affinity (Ikeda-Saito et al., 1983).  Prior to most of this research on Atlantic bluefin tuna Hb, Andersen et al. (1973) investigated the temperature dependence of Hb-O2 affinity in several regionally heterothermic teleosts and elasmobranchs, including the bigeye tuna. However, it is not clear if Andersen and coworkers experimented on whole blood or hæmolysates since they used both terms to describe the same set of experiments, and they did not report the sample pH for their OEC experiments. Nonetheless, they reported that the shape of bigeye tuna OECs were affected by temperature 22  (5°C, 15°C, 25°C, and 35°C), where the upper portion of the OECs at 5 and 15°C were relatively close together. This reduced effect of temperature at high O2 saturation between 5 and 15°C was likely due to endothermic contributions to ΔH' from Bohr proton release and a decrease in L with increasing temperature, but temperature does not have as large an effect on L as it does on bluefin tuna Hb. Furthermore, the effect of temperature on L appears to be temperature dependent because the OECs at 25 and 35°C are quite right shifted compared to the 15°C OEC. Like bluefin tuna Hb, organic phosphate (ATP) did not influence bigeye tuna Hb (Andersen et al., 1973).   Also in 1973, Jeffrey Graham reported that preliminary experiments showed no pronounced effect of temperature on the O2 affinity of whole blood from black skipjack tuna (Euthynnus lineatus), although he provided no data or description of the experiments (Graham, 1973).  A couple of years later Gary Sharp reported O2 equilibrium experiments for red blood cell (RBC) suspensions from yellowfin tuna (Thunnus albacares), albacore tuna (Thunnus alalunga), bigeye tuna, and a closely related ectothermic species, wahoo (Acanthocybium solandri) (Sharp, 1975). Sharp used relatively fresh blood samples from yellowfin and albacore tuna, but the bigeye tuna and wahoo samples were taken from deceased fish obtained at a fish market. The experiments were conducted on RBCs removed from blood plasma, rinsed, and resuspended in buffered glycerol solutions (i.e., not whole blood). Sharp provided complete OECs for a range of temperatures and pH levels, as well as P50 values and precise pH measurements made at 50% Hb-O2 saturation (pH50). However, the experimental pH treatments were very low, with pH50 values ranging from 7.44 to 6.69, which are now known to be well below physiologically routine arterial and venous levels for tuna (~7.7-7.8) (Bushnell and Brill, 1992; Jones et al., 1986; Korsmeyer et al., 1997a). The low pH levels for bigeye tuna and wahoo 23  RBC suspensions were attributed to the poor quality and relatively acidotic state of the blood (pH 6.7-7.0) sampled from fish caught at least 24 hours prior to blood withdrawal. The results of the O2 equilibrium experiments showed that P50 was temperature independent for RBCs from yellowfin and albacore tuna, whereas increasing temperature increased P50 (i.e., decreased Hb-O2 affinity) for bigeye tuna and wahoo. The yellowfin tuna OECs also crossed over around 50% Hb-O2 saturation, whereby Hb-O2 affinity increased with increased temperature (i.e., a reverse temperature-dependency) above 50% saturation, similar to what would be observed a few years later for bluefin tuna Hb. This observation prompted Sharp to note that “the effects of changes of parameters such as temperature on the dissociation properties of yellowfin hemoglobin could be misleading if only P50 values are evaluated” (Sharp, 1975, p. 684). The effect of temperature on the position of the OEC at O2 tensions above and below P50 was an important observation that has been overlooked in more recent studies. Furthermore, although Sharp did not quantitatively assess the effect of temperature on the entire OEC he did provide enough figures and information that the appropriate data can be extracted from his study for further analysis.   Reduced and reversed effects of temperature on whole blood-O2 affinity have been reported for quite a few species of tuna. A lot of the work on the blood respiratory properties of tuna has been led by Richard Brill and Peter Bushnell who, along with David Jones, were able to develop techniques to sample blood from resting and cannulated tuna, which is the best procedure for O2 equilibrium experiments on whole blood from fish. In the following studies the effect of temperature on blood-O2 affinity was determined by comparing only P50 values from OECs constructed with whole blood at different temperatures. Cech et al. (1984) reported a reverse temperature-dependency for albacore P50, which appeared to be saturation dependent  (Thunnus alalonga; 5, 10, 15, 20, and 25°C); Brill and Bushnell (1991a) reported that P50 was 24  independent of temperature for skipjack and yellowfin tuna (20 and 30°C); Lowe et al. (Lowe et al., 2000) reported that bigeye tuna P50 was “essentially unaffected” by temperature (15 and 25°C); Brill and Bushnell (2006) reported a slight reverse effect of temperature on P50 for Atlantic bluefin tuna (15 and 25°C); Clark et al. (2008a) studied blood from southern bluefin tuna (Thunnus maccoyii), and they found that temperature had a reverse effect on P50 between 10 and 23°C, and P50 was independent of temperature between 23 and 36°C; Lilly et al. (2015) reported temperature-independence and reverse temperature-dependence of P50 for Pacific bluefin tuna (Thunnus orientalis ), and variable effect of temperature on yellowfin tuna P50, including normal temperature-dependence, temperature-independence, and reverse temperature-dependence (RBCs rinsed and resuspended in buffered saline; 15, 20, 25, and 30°C).   The studies described in the previous paragraph provide good evidence (qualitative) that all tunas probably have Hbs with a reduced thermal sensitivity, albeit to varying degrees among species. However, the effect of temperature on blood-O2 affinity was not quantitatively assessed in an appropriate way in any of those studies. Furthermore, except for the study by Brill and Bushnell (1991), the data presented by the authors offer limited opportunities for further analysis. I will outline the major reasons for these shortcomings because they are relevant to the results that I present in Chapters 3 and 4: 1. The authors of each study did not assess the effect of temperature on the shape of the OEC (i.e., above and below 50% Hb-O2 saturation), even though previous studies have shown that the effect of temperature on Hb-O2 affinity is dependent on Hb-O2 saturation (Andersen et al., 1973; Carey and Gibson, 1977; Carey and Gibson, 1983; Ikeda-Saito et al., 1983; Sharp, 1975). The saturation dependence of the effect of temperature is largely due to the non-linear release of Bohr protons, with most proton dissociation occurring 25  between 50 and 100% Hb-O2 saturation in teleosts (Brauner et al., 1996; Ikeda-Saito et al., 1983). Some of the authors listed above have even shown that in yellowfin tuna blood most of the oxygenation-dependent release of Bohr protons occurs between 40 and 100% O2 saturation (Lowe et al., 1998). 2. Exemplary or representative OECs are presented in each study, but in some it is obviously apparent that the OEC is not a good fit to the data. This is likely due to imprecise measurements of Hb-O2 saturation and/or blood PO2, and not a poor choice of OEC model. Precise O2 equilibrium measurements should yield data that take the form of an OEC, so an appropriate OEC model should exactly fit to the data, or at least fit reasonably well enough that most of the data points lie on the curve. However, in some of the representative OECs very few, and in some cases none, of the data points lie on the fitted OEC (e.g., Lowe et al., 2000). 3. The analyses were not appropriate for comparing P50 values at different temperatures. To compare among temperature treatments, the influence of blood pH on P50 must be controlled somehow. In all but one of the studies the authors reported that pH greatly affected P50, as to be expected in teleosts. However, the authors reported a mean P50 for all blood samples included in each temperature and PCO2 treatment (OECs were constructed at different PCO2 treatments to manipulate blood pH), but the P50 values for all individuals in a treatment had different blood pH values. Cech and co-workers even stated that “[t]he variation in P50 seemed to stem primarily from small differences in blood pH measured for individual fish” (Cech et al., 1984, p. 25), yet they and others did not determine P50 at specific pH values from plots of logP50 vs pH (i.e., Bohr plots) as is standard practice (e.g., Fago et al., 1997; Hall, 1966; Lahiri, 1975; Larsen et al., 2003; 26  Reeves, 1980; Riegel et al., 1966; Weber et al., 1976; Weber et al., 2010). The “small differences in blood pH” that Cech et al. referred to were actually large differences, with blood pH ranging from 7.61 to 7.76, which is typical of most studies on fish blood. Lilly et al. (2015) did not measure the pH of their samples.  Because of these shortcomings, the summary data presented in these studies probably do not accurately describe the blood respiratory properties of each species, and additional information is needed to use the data for further analysis of the effect of temperature on blood P50. The exception is Brill and Bushnell (1991a) who included a table of all P50 and pH values determined for each individual yellowfin and skipjack tuna that they studied. I have further analyzed these data in Chapter 2.   The effect of closed-system warming and cooling on blood PO2 were assessed in some of the tuna whole blood studies, and these data are interesting and are worth brief discussion here because I present the same type of measurements in Chapters 3 and 4. In the absence of Hb, increasing temperature will considerably increase PO2 in a closed-system due to a reduction in plasma O2 solubility, and vice versa (i.e., Henry’s law). If the effect of temperature on plasma O2 solubility is removed from the total change in blood PO2 (ΔPO2), then it can be reasonably assumed that any change to blood PO2 beyond what would be expected solely in plasma is due to temperature induced Hb-O2 unloading or binding. In blood with a normal temperature-dependence, warming will decrease Hb-O2 affinity and cause O2 to dissociate from Hb and diffuse across the RBC membrane into the plasma, which will increase blood PO2 beyond what would be expected due to the temperature dependence of plasma O2 solubility. Cooling will have the opposite effect; Hb-O2 affinity will increase and promote Hb-O2 binding, which will decrease blood PO2 because O2 will diffuse form plasma into the RBCs. Temperature-independent Hb-O2 27  affinity would cause ΔPO2 to be dependent only on the temperature-dependence of plasma O2 solubility, while reverse-temperature dependent Hb-O2 affinity would cause blood PO2 to decrease with warming, but increase with cooling. In species where the temperature-dependence of Hb-O2 affinity is saturation dependent (i.e., when the OECs crossover), ΔPO2 would also be expected to be saturation dependent.  Joseph Cech and coworkers initiated these closed-system experiments to test Jeffrey Graham’s hypothesis that “temperature-insensitive hemoglobin has evolved in tuna to eliminate the problem of premature oxygen dissociation as blood crosses a thermal gradient [in a heat exchanging rete mirabile] en route to the muscles” (Graham, 1973, p. 1967). They reported that albacore blood equilibrated at different PO2’s and at 10 or 25°C then rapidly warmed to 30°C in a closed-system caused a reduction of blood PO2, the opposite effect for blood equilibrated at 30°C then cooled to 10°C. These results are not unexpected given that Cech and coworkers observed a reverse temperature-dependence in their whole blood OECs. David Jones and coworkers did similar experiments with blood from kawakawa (also known as mackerel tuna; Euthynnus affinis), but they used dorsal aortic blood from fish maintained at 25°C and then warmed the blood to 35°C (Jones et al., 1986). They reported that warming dorsal aortic blood caused PO2 to increase by about 25%, so Jones et al. concluded that “our tuna can live quite happily in the absence of any advantage conferred by reversed thermal effects” (Jones et al., 1986, p. 210). Brill and Bushnell (1991a) reported that skipjack tuna blood had a low sensitivity to closed-system temperature changes, but yellowfin tuna blood had a normal temperature-dependence. Bigeye tuna blood also exhibited a normal temperature-dependence during closed-system temperature changes, whereas Atlantic bluefin tuna blood exhibited a reverse temperature-dependence like albacore blood (Brill and Bushnell, 2006; Lowe et al., 2000). The 28  different closed-system results for skipjack, yellowfin, and bigeye tuna seem to have perplexed the researchers that made those observations (Brill and Bushnell, 1991a; Brill and Bushnell, 2006; Lowe et al., 2000). However, in those studies the effect of temperature on plasma O2 solubility and thus, plasma PO2 was not considered.   In Figure 1.6, I have replotted curves that were fit to the closed-system PO2 data for skipjack tuna, yellowfin tuna, and bigeye tuna, as reported by Brill and Bushnell (2006), but I have also included the expected change to plasma PO2 due to the temperature dependence of plasma O2 solubility. Skipjack blood PO2 changed less than would be expected due to the temperature dependence of plasma O2 solubility, yellowfin tuna plasma PO2 closely follows the expected change, and bigeye tuna PO2 exceeds the expected change. This indicates a reverse temperature-dependence of O2 affinity in skipjack blood, temperature-independence in yellowfin tuna blood, and normal temperature-dependence in bigeye tuna blood. Whole blood-O2 affinity (i.e., P50) exhibits a reverse temperature-dependence in skipjack tuna, and is temperature-independent in yellowfin tuna (Brill and Bushnell, 1991a). Therefore, closed-system temperature changes to blood PO2 in skipjack and yellowfin tuna are qualitatively like the temperature-dependence of blood P50. The same is true of bigeye tuna blood, although further explanation is justified here.   Bigeye tuna blood P50 was reported to be almost temperature-independent and quite low (i.e., high Hb-O2 affinity), which was interpreted to benefit O2 loading during dives to deep, cold and hypoxic water (Lowe et al., 2000). However, the large change to blood PO2 with closed-system warming was interpreted to benefit O2 unloading to the red muscle, despite the finding of temperature-independent Hb (Lowe et al., 2000). This interpretation would require large changes of ΔH' between the gills and the systemic circulation, although this is not consistent with the data 29  and the physiological mechanisms have never been proposed. Lowe et al. (2000) presumed that bigeye tuna has a high blood-O2 affinity based on their reported P50 values of 12.5 mmHg (pH 8.05) at 15°C, and 15.9 mmHg (pH 8.08) at 25°C. However, these P50 values were determined at a high blood pH. The authors also reported values at more physiologically relevant pH values: 26.7 mmHg (pH 7.710) at 15°C, and 29.7 (pH 7.780) mmHg at 25°C. If these values are adjusted with their reported Bohr coefficients to a common pH of 7.71, then the P50 at 25°C is 34.2 mmHg. The ΔH' value for this temperature change is -18 kJ mol-1, which is relatively reduced compared to many other vertebrates, but is normal relative to other tuna (i.e., increasing temperature increases P50). Therefore, the effect of closed-system temperature changes on bigeye tuna blood PO2 is similar to the effect of temperature on whole blood-O2 equilibria.  1.3.2 Billfishes  Andersen et al. (1973) reported that increased temperature considerably right-shifted swordfish OECs (5, 25, and 35°C), and ATP had a marked effect on swordfish hæmolysates. The major Hb component from striped marlin (Kajikia audax) also exhibited a normal temperature-dependence (Brittain, 1986), but Roy Weber and coworkers found that adding ATP to hæmolysates of striped marlin caused temperature-independence of P50 (10 and 25°C) within a physiologically relevant pH range. The presence of ATP had a similar effect on the temperature-dependence of shortbill spearfish (Tetrapturus angustirostris) hæmolysate P50, but reversed the temperature-dependence of blue marlin (Makaira nigricans) hæmolysate P50 (10 and 25°C). However, Weber et al. did not assess the effect of temperature on the shape of the OEC. Like tuna, the temperature-dependence of billfish Hb is reduced by oxygenation-linked dissociation of allosteric effectors that contribute endothermically to ΔH'. Unlike tuna, however, the primary 30  effector of billfish Hb is ATP, with minor enthalpic contributions from other allosteric effectors (likely protons) (Weber et al., 2010).  1.3.3 Sharks  Hæmoglobins from the shortfin mako shark and the porbeagle shark were also included in the study by Andersen et al (1973). Mako OECs showed a reduced temperature-dependence with no effect of temperature on the shape of the OEC (5, 15, 25, and 35°C), whereas porbeagle OECs showed a reduced temperature-dependence at low O2 saturations, but a reverse temperature-dependence at high O2 saturations causing the OECs to crossover around 50% O2 saturation, like those of Atlantic bluefin tuna. Dickinson and Gibson (1981) reported similar temperature-independence for Hb from the salmon shark (Lamna ditropus; described as the Pacific porbeagle shark, Lamna ditrotus), and for both porbeagle and salmon shark Hb temperature-independence is due to a decrease in the allosteric equilibrium constant, L, with increasing temperature (Andersen et al., 1973; Dickinson and Gibson, 1981). Further work in Roy Weber’s lab by Larsen et al. (2003) showed that ATP induced the reverse temperature-dependence of porbeagle Hb (Figure 1.5). Porbeagle Hb has a high intrinsic O2-affinity with a normal temperature-dependence, as appears to be the case for most elasmobranch Hbs (Morrison et al., 2015), but the addition of ATP to stripped Hb reduces the O2-affinity and reverses the effect of temperature. Like bluefin tuna Hb, the reverse temperature-dependence of porbeagle Hb stems from a reduction in L with increasing temperature and a stabilization of the T-state at low temperature. In porbeagle Hb, the presence of ATP decreases both the T-state association constant (KT) and the R-state association constant (KR), but causes KT to become temperature invariant while KR decreases with increasing temperature. Stabilization of the T-state at low temperature is due to preferential binding of ATP to the T-state relative to the R-state, but as 31  temperature increases the T-state becomes less stable and a T→R transition occurs, promoting the release of ATP and protons. The release of these allosteric effectors contributes endothermically to ΔH', causing it to become positive.  I am aware of only one study that has investigated the effect of temperature on whole blood-O2 affinity of a regionally heterothermic shark. Bernal and coworkers reported a very reduced effect of temperature on the blood-O2 affinity, almost temperature-independent, for the shortfin mako shark (15, 20, and 25°C). The effect of temperature on blood-O2 affinity appeared to be saturation dependent, but not to the same extent as previously reported for the porbeagle shark and the bluefin tuna, and closed-system warming of mako blood caused a slight reduction in blood PO2 (Bernal et al., 2018). Bernal et al. also reported sizeable Bohr coefficients from mako blood, which contrasts with the pH-independence of blood-O2 affinity reported by Wells and Davie (1985) for mako blood at 25°C.. 1.3.4 Hypotheses for the functional significance of reduced- and reversed-temperature dependence of Hb-O2 affinity in regionally heterothermic fishes  Reductions or reversals in the temperature-dependence of Hb-O2 affinity have been reported for many regionally heterothermic vertebrates with heat-exchanging retia, including fishes, mammals, and birds (Weber and Campbell, 2011). Among these animals, relative increases in the enthalpy of hæmoglobin-oxygenation have convergently evolved by different molecular mechanisms. However, some non-heterothermic vertebrates also have Hbs with a reduced-temperature dependency, including some poikilothermic teleosts and sharks (e.g., Bernal et al., 2018; Clark et al., 2010; Hopkins and Cech, 1994). Although this trait is not exclusive to regionally heterothermic vertebrates, it does seem to be shared by them. Several 32  hypotheses have been proposed for the functional significance of reduced- and reversed-temperature dependent Hb-O2 affinity, which I will review.  Environment temperature and O2 loading  As I explained in section 1.1.3.2, the first report of temperature-independent Hb-O2 affinity was made by Rossi Fanelli and Antonini (1960), and they proposed that this trait may enable O2 uptake as tuna swim through varying water temperatures. This trait is not exclusive to regionally heterothermic vertebrates as some non-heterothermic vertebrates also have Hbs with a reduced-temperature dependence, including some pelagic teleosts and sharks (e.g., Bernal et al., 2018; Clark et al., 2010; Hopkins and Cech, 1994). This trait may therefore benefit O2 loading at different environmental temperatures, but this does not explain why this trait is possibly shared by all regionally heterothermic vertebrates, including mammals and birds. Furthermore, some species such as the circumtropical yellowfin tuna infrequently descend below the thermocline, so their realized thermal niche is warmer and narrower (~ 17-24°C) relative to that of the Atlantic bluefin tuna, which subjects itself to a wide range of water temperatures during migrations and descents below the thermocline (Bernal et al., 2009; Bernal et al., 2017; Block et al., 2001). Thus, the functional convergence for reduced temperature-dependent Hb-O2 affinity among regional heterotherms is likely related to the internal temperature gradients caused by heat-exchanging retia. Premature O2 unloading in the retia causing an arterio-venous PO2 gradient  Jeffrey Graham “proposed that the function of temperature-independent hemoglobin is to ensure the efficient delivery of oxygen to muscle tissue by preventing the premature unloading of this gas in the rete or outer layers of the muscle mass” (Graham, 1973, p. 1964). He hypothesized 33  that Hb with a normal temperature-dependence would progressively unload O2 as the blood is warmed in a rete, possibly lowering the amount of O2 in blood before its reaches the muscles (Graham, 1973). Carey and Gibson further explained that if tuna Hb had a normal temperature dependence, then “as the cold arterial blood coming from the gills was warmed in the heat exchanger the partial pressure of oxygen would rise while at the same time the partial pressure in the venous blood would fall as it cooled, both effects operating to increase the head of oxygen pressure transferring oxygen from the arterial to the venous blood and so to decrease respiratory efficiency” (Carey and Gibson, 1977, p. 1377). It seems unlikely that arterio-venous O2 diffusion would occur because the heat-exchanging retia are composed of vessels that resemble arterioles and venules. The size of the arterioles and venules and the diffusion distance between them are about an order of magnitude greater than the vessels of a rete specialized for gas exchange (e.g., the swim bladder rete) (Carey et al., 1985; Graham and Dickson, 2001; Lemons et al., 1987; Stevens et al., 1974). However, even though arterio-venous O2 diffusion likely does not occur, reverse temperature-dependence may be important for preventing premature O2 unloading in species that maintain a large thermal excess between the gills and warm muscles (~10-20°C); species such as the bluefin tunas, the porbeagle sharks, and the salmon shark. However, it should only take seconds for the blood to move from a rete to the tissue capillaries (Brill and Bushnell, 2001), and the amount of O2 that would be unloaded from Hb with a normal temperature-dependence and warmed by 10°C corresponds to a decreases in Hb-O2 saturation of less than 0.3% [calculated from closed-system warming of big-eye tuna blood from 15 to 25°C (Lowe et al., 2000)], which seems unlikely to negatively affect circulatory O2 delivery.  Energy conservation and maintenance of O2-supply to tissues of different temperatures 34   Reductions in the temperature-dependence of Hb-O2 affinity have also been proposed to save energy and prevent excessive leftward shifts of the OEC, and thus maintain O2 unloading, as blood flows from the warm body to the colder legs or appendages of regionally heterothermic mammals (e.g., Brix et al., 1989a; Brix et al., 1989b; Giardina et al., 1989a; Giardina et al., 1989b). An increased ΔH' would require less energy to bind and unload O2, which has been regarded as a potential energy saving mechanism in arctic mammals (Brix et al., 1989a).   It may seem intuitive that a normal temperature-dependence would enhance O2 transport since a left shifted OEC at the cold gills should benefit O2 uptake, and a right shifted OEC in the warm tissues should benefit O2 unloading. However, a normal temperature-dependence could impair O2 unloading to the colder tissues in a regional heterotherm. Temperature-independence of Hb-O2 affinity would prevent temperature from directly influencing P50 in the warm and cold tissues of regionally heterothermic fishes, and a reverse temperature-dependence may actually enhance O2 unloading from the venous return to the myocardium in the cold hearts of tunas, billfishes, and sharks (Clark et al., 2008a). An increased ΔH' would also reduce the energy required for O2 unloading in the cold peripheral tissues of regionally heterothermic fishes, and temperature independence of P50 should prevent liberation of heat upon O2 binding at the gills (Weber and Fago, 2004; Weber and Wells, 1989). These ideas are further explored in the following chapters.  1.4 Thesis objectives and organisation  I have developed two hypotheses that underlie the objectives of this thesis: 1) temperature-independent Hb-O2 affinity conserves heat-energy, which may help maintain stably elevated body temperatures in regionally heterothermic fishes. Thus, I expected this trait to be shared by all regionally heterothermic teleosts and sharks; and 2) the Hb-oxygen-binding 35  properties and high Hb concentrations of regionally heterothermic fishes maintain matching between O2 supply and O2 demand despite large internal temperature gradients. I will briefly explain the justification for these hypotheses before stating my research objectives and questions.  Hæmoglobin of regionally heterothermic fishes must transport O2 despite internal temperature gradients. Heat is also transported by Hb, and because heat absorbed during O2 unloading is liberated upon O2 binding at the gills, Hb contributes to heat loss to the environment. However, temperature-independent Hb-O2 affinity would eliminate heat absorption by Hb upon O2 unloading, preventing Hb-heat transport away from the warm tissues. Moreover, reductions and reversals in the temperature dependence of Hb-O2 affinity may prevent unwanted temperature-induced shifts of the OEC, maintaining O2 unloading to cold tissues with a high metabolic demand such as the heart. Therefore, it would be expected that all regionally heterothermic fishes would have Hbs with a reduced or reverse temperature-dependence to conserve metabolic heat in the warm tissues (hypothesis 1), and match O2 supply with O2 demand in their warm and cold tissues (hypothesis 2). Furthermore, blood-O2 transport depends on Hb concentration and Hb-O2 affinity, both of which influence the O2 capacitance of the blood (βbO2). The evolution of endothermy is associated with increased metabolic rates (Block and Finnerty, 1994; Brill, 1996; Ciezarek et al., 2019; Sepulveda et al., 2007b), and regionally heterothermic fishes have high Hb concentrations that are necessary to achieve high rates of circulatory O2 delivery without unnecessarily increasing cardiac output (Brill and Bushnell, 1991b; Gibson and Carey, 1982). Tunas exhibit exceptionally high metabolic rates (Korsmeyer and Dewar, 2001), and I hypothesize that during aerobically sustained exercise they depend on a high βbO2 to unload O2 to the swimming muscles (hypothesis 2). In this thesis, the general objectives that are addressed in the subsequent four chapters are outlined by the following 36  questions: 1) How do Hb concentration and Hb-O2 affinity contribute to the determinants of maximal O2 consumption, circulatory O2 delivery and tissue O2 extraction, in the yellowfin tuna? 2) How does the enthalpy of Hb-oxygenation influence O2 and heat transport? 3) How does temperature affect Hb-O2 affinity in the swordfish and the smalleye Pacific opah? And 4) How does temperature affect Hb-O2 affinity in the common thresher shark and the bigeye thresher shark? 1.4.1 How do Hb concentration and Hb-O2 affinity contribute to the determinants of maximal O2 transport in the yellowfin tuna?  Sensitivity analyses of the structural and physiological factors that determine maximal O2 transport (ṀO2max) have been conducted for species representing all the major extant tetrapod clades, but no analysis of the O2 transport system of fishes has been published. The yellowfin tuna is an excellent model to test for O2 transport limitations to maximal O2 flux, because the yellowfin tuna has a relatively high ṀO2max and all components of the O2 transport cascade appear to have evolved in parallel to maintain matching between O2 supply and demand (Brill and Bushnell, 2001; Korsmeyer and Dewar, 2001; Wegner et al., 2010). Furthermore, the yellowfin tuna has high Hb concentration and a relatively large Bohr effect (Brill and Bushnell, 1991a), both of which will contribute to a high βbO2 and are likely important for enhancing circulatory O2 delivery and tissue O2 extraction.   In Chapter 2 I have used a mathematical model of the fish O2 transport cascade to quantitatively assess the possible contributing factors to O2 supply limitation at ṀO2max in yellowfin tuna. In doing this, I have also quantified how Hb concentration and Hb-O2 affinity influence circulatory O2 delivery and tissue O2 extraction. Because the yellowfin tuna has a relatively large mixed venous O2 reserve at ṀO2max, I hypothesized that tissue O2 extraction 37  may be constrained by βbO2, which will cause an apparent tissue O2-diffusion limitation in the model. 1.4.2 How does the enthalpy of Hb-oxygenation influence O2 and heat transport?  The thermodynamics of Hb-oxygenation mandate that heat energy is absorbed and released by the circulating Hb. Reductions and reversals of ΔH' should reduce or eliminate the heat absorbed during Hb-O2 unloading in the warm tissues of regionally heterothermic fishes (Weber and Wells, 1989). Furthermore, temperature-independence and reverse temperature-dependence of Hb-O2 affinity should prevent excessive temperature induced shifts to the physiological OEC between tissues maintained at very different temperatures (e.g., >10°C difference), possibly enhancing O2 unloading to cold organs such as the heart. Although these concepts are intuitively obvious, the effect of the temperature-dependence of Hb-O2 affinity on heat-conservation and blood PO2 over a large internal temperature gradient have not been quantitatively evaluated.   In the second part of Chapter 2, I address the question of how does the enthalpy of Hb-oxygenation influence O2 and heat transport? This was investigated using previously published data on tuna blood-O2 affinity to calculate the heat-energy transported by Hb, and to calculate blood PO2 (~P50) over a range of temperatures and ΔH' values. I hypothesized that since temperature-independent Hb-O2 affinity will prevent a left-ward shift of the OEC with decreasing temperature, then blood PO2 at approximately 50% Hb-O2 saturation, and thus Hb-O2 unloading, will remain relatively constant over a wide range of body temperatures (15-30°C).   38  1.4.3 How does temperature affect Hb-O2 affinity in the swordfish and the smalleye Pacific opah?  Hæmoglobins from regionally heterothermic tunas and billfishes (Istiophoridae) exhibit reduced temperature-dependence, temperature-independence, or reverse temperature-dependence. The swordfish is closely related to billfishes in the family Istiophoridae, but the limited available evidence indicates that swordfish Hb may have a normal temperature-dependence (Andersen et al., 1973). However, since Weber et al. (Weber et al., 2010) found that ATP induced temperature-independence of Hb-O2 affinity in billfishes, further investigations of swordfish Hb are warranted. The smalleye Pacific opah is another regionally heterothermic teleost, but I am not aware of any published studies on the effect of temperature on Hb-O2 affinity in the opah.    In Chapter 3 I address the following question: How does temperature affect Hb-O2 affinity in the swordfish and the smalleye Pacific opah? If temperature-independent Hb-O2 affinity is necessary to maintain regional heterothermy, then I expect that both the swordfish and the opah should have Hbs that are insensitive to temperature. I address this by constructing OECs at different temperatures in whole blood and hæmolysates, and by measuring the effect of closed-system temperature changes on blood PO2.  1.4.4 How does temperature affect Hb-O2 affinity in the common thresher shark and the bigeye thresher shark?  At least three species of regionally heterothermic sharks in the family Lamnidae have Hbs with a reduced or reversed temperature-dependence. However, the effect of temperature on Hb-O2 affinity has not been investigated in the common thresher shark or the bigeye thresher 39  shark. Moreover, the temperature sensitivity of shortfin mako Hb has been previously studied, but a more thorough investigation is warranted to be able to quantify the effect of temperature on mako Hb-O2 affinity.    In Chapter 4, I address the question of how does temperature affect Hb-O2 affinity in the common thresher shark and the bigeye thresher shark? If temperature-independent Hb-O2 affinity is necessary for sharks to maintain regional heterothermy, then like my expectations for the regionally heterothermic teleosts in Chapter 3, I also expect that the common thresher shark will have Hbs that are insensitive to temperature. The ecology and vascular anatomy of the bigeye thresher shark make for a curious case. The bigeye thresher makes diel movements from warm water above the thermocline to cold water deep below thermocline, and it also has an orbital rete, although its function is unclear. Therefore, the temperature-dependence of Hb from the bigeye thresher may provide some insight into the functional significance of temperature-independent Hb-O2 affinity in regionally heterothermic fishes.  40           Figure 1.1 Oxygen equilibrium curves (OECs) showing that allosteric effectors and increased temperature decrease hæmoglobin (Hb)-O2 affinity and right shift the OEC.  Hb-O2 affinity is quantified as P50, the partial pressure of O2 at 50% Hb-O2 saturation. The red OEC farthest to the left represents the intrinsic O2 affinity (pH 7.4) of a stripped hæmolysate (i.e., purified of endogenous allosteric effectors). The two black OECs in the middle are for whole blood, and are right shifted relative to stripped Hb due to binding of allosteric effectors (heterotropic interactions), which include hydrogen ions (H+), chloride ions (Cl-), and organic phosphates (P). Left and right shifts of the physiological OEC are primarily due to the Bohr effect (i.e., oxygenation linked binding of H+ ions) and changes in temperature (T). Some teleost Hbs exhibit a Root effect, which is represented by the blue OEC to the far right. Below a threshold pH level, the Root effect is generally characterized by a very right-shifted OEC, a loss of cooperative O2-binding (homotropic interactions), and the inability to fully saturate Hb.  41  Figure 1.2 The interrelationships of the determinants of oxygen transport.  Circulatory O2 transport and tissue O2 extraction govern rates of O2 consumption. Circulatory O2 delivery is the product of the arterial O2 content and cardiac output. The O2 capacitance of the blood (βbO2) is the slope of the functional portion of the oxygen equilibrium curve, and is an important determinant of tissue O2 extraction (the O2 content removed from the blood expressed as a percentage of arterial O2 content). Hæmoglobin is an important determinant of both circulatory O2 delivery and tissue O2 extraction because blood Hb concentration determines arterial O2 content, and left and right shifts of the OEC influence βbO2. For a given cardiac output, O2 transport depends on arterial O2 content and βbO2. The plots were constructed with data from humans at rest and exercising at their maximum rates of O2 consumption (Sun et al., 2000). 42              Figure 1.3 Proposed phylogenetic relationships among regionally heterothermic fishes. Red arrows indicate orders (plus tetrapod) that have evolved endothermy. Red circles indicate the ability of a species to elevate the temperature of the specified tissue; black circles indicate a species suspected or assumed to be able to elevate tissue temperature; white circles indicate ectotherms. Green circles indicate species studied in this thesis. The phylogenetic relationships are based on those of Kumar et al. (2017), Santini and Sorenson (2013), Santini et al. (2013), Sorenson et al. (2014), and Underkoffler et al. (2018). 43   Figure 1.4 Endothermic tissue temperatures in regionally heterothermic teleosts and sharks. (A) Red muscle (RM) and cranial temperatures of representative regionally heterothermic fishes described in Chapter 1. Tissue temperatures are plotted as function of sea surface temperature (tunas and sharks) or ambient temperature (opah). (B) Heater organ temperature relative to sea surface temperature in billfishes. The white regions represent temperatures above ambient water temperature. Figures A and B recreated from Bernal et al. (2009) and Block (1990), respectively, with data from the following: Barrett and Hester (1964) (yellowfin tuna, Thunnus albacares, RM); Carey and Teal (1969b) (Atlantic bluefin tuna, Thunnus thynnus, and skipjack tuna, Katsuwonus pelamis, RM); Linthicum and Carey (1972) (Bluefin tuna eye and brain); Block and Carey (1985) and 44  Bernal et al. (2001b) (porbeagle shark, Lamna nasus, and shortfin mako shark, Isurus oxyrinchus); Bernal and Sepulveda (2005) and Patterson et al. (2011) (common thresher shark, Alopias vulpinus); Anderson and Goldman (2001) (salmon shark, Isurus oxyrinchus); Wegner et al. (2015) (smalleye Pacific opah, Lampris incognitus); Carey (1982) (swordfish, Xiphias gladius); Block (1990) (white marlin, Kajikia albida, blue marlin, Makaira nigricans, spearfish, Tetrapturus angustirostris).            45            Figure 1.5 Hill plots of oxygen equilibria of Hb from the Atlantic bluefin tuna and the porbeagle shark Atlantic bluefin-tuna O2 equilibria for Hb component I were at pH 7.0, and show the temperature-independence at 50% Hb-O2 saturation and a reverse temperature-dependence above 50% saturation (modified after Ikeda-Saito et al., 1983). Porbeagle shark O2 equilibria for Hb component III were at pH 7.4 in the presence of ATP, and show a reverse temperature-dependence below ~75% Hb-O2 saturation and the crossing over of the OECs and almost temperature independence above ~75% saturation (constructed with parameters reported by Larsen et al., 2003).   46  Figure 1.6 Effects of closed-system temperature changes on the measured change in blood PO2 (∆PO2) in bigeye tuna, yellowfin tuna, and skipjack tuna. This figure was created from a figure in Brill and Bushnell (Brill and Bushnell, 2006), with data from Brill and Bushnell (1991a) and Lowe at al. (2000). Tuna blood was equilibrated at a range of O2 tensions (Equilibration PO2) and then heated (red curves) or cooled (blue curves). Bigeye tuna blood temperature was changed between either 15 and 25°C, and skipjack and yellowfin tuna blood temperatures were changed between 20 and 30°C (circles). Black lines indicate the temperature induced ∆PO2 expected due to changes in solubility of blood plasma alone at a given equilibration PO2 (i.e., Henry’s Law) between 15 and 25°C (dashed), and 20 and 30°C (dotted).47  Chapter 2 Determinants of Maximal Oxygen Transport in Yellowfin Tuna (Thunnus albacares) and Rainbow Trout (Oncorhynchus mykiss), and the Functional Significance of the Enthalpy of Hb-Oxygenation on Heat and Oxygen Transport “The circulatory system of man and the vertebrate animals can be considered as made up of a small number of organs or subordinate systems, which are easy to recognize anatomically, and the functions of which are on the whole quite distinct. We have a propulsive organ: the heart; a distributing organ: the system of arteries; an organ for interchanges of substances between the blood and the tissues: the capillaries; an organ for collecting the blood and carrying it back to the heart: the venous system.” (August Krogh, 1922, p. 1) 2.1 Introduction  As August Krogh so eloquently explained, “diffusion and convection are the only processes responsible for the oxygen transport into respiratory organs and within organisms” (Krogh, 1941, p. 20). These diffusive and convective processes that form the O2 transport cascade can be simplified into four steps: 1) convective flow of water or air to the respiratory organ by ventilation; 2) diffusion of O2 across the respiratory barrier into the blood; 3) convective O2 transport in the blood to the tissue capillaries; 4) diffusion of O2 from the blood into the cells where O2 is consumed by the mitochondria during oxidative phosphorylation of adenosine triphosphate (ATP). Because each O2 transport step can be described by relatively simple and well-known equations (e.g., Rahn and Fenn, 1955; Taylor and Weibel, 1981), O2 transport can be quite easily mathematically modelled. At each step, the flow rate of O2 (expressed in mols, ṀO2, or as a volume, V̇O2, and from herein referred to as ṀO2) can be 48  expressed as the product of an O2 partial pressure gradient (ΔPO2) and a conductance (G) that is defined by structural and physiological factors (Taylor and Weibel, 1981; Taylor et al., 1987; Wang and Malte, 2011). Therefore, if the O2 transport cascade is modelled as an integrated system with the conductances arranged in series (Figure 2.1), the relative contribution of each structural or physiological factor in determining the maximum rate of O2 transport (ṀO2max) can be quantitatively evaluated (di Prampero, 1985; Jones and Karas, 1988; Shephard, 1969; Wagner, 1988; Wang and Malte, 2011).  During exercise, ṀO2 increases linearly with exercise intensity to a point where ṀO2max can be experimentally defined (Hillman et al., 1979; Norin and Clark, 2016; Robergs et al., 2010; Seeherman et al., 1981; Zhang et al., 2019). In healthy individuals, it is typically only when exercising to ṀO2max that circulatory O2 transport may be maximized and O2 supply may limit ATP synthesis (Robergs, 2001; Wagner, 2000). The factors that contribute to circulatory O2 transport include ventilation (V̇), respiratory diffusion conductance (Gd), hæmoglobin (Hb) concentration, Hb-O2 affinity, cardiac output (Q̇), and tissue O2 conductance (Gdt). Importantly, these factors determine arterial-O2 saturation, arterial-O2 concentration, circulatory O2 delivery (the product of Q̇ and arterial-O2 concentration), and tissue O2 extraction (i.e., all processes included in the diffusion of O2 from the blood to the mitochondria) (Wagner, 2000). ṀO2max is determined by the interactions of all the factors mentioned above, and experimental or evolutionary increases in the relative magnitude of any one factor must generally be matched with an increase in some or all of the other factors to result in an increase in ṀO2max (di Prampero, 1985; Jones and Karas, 1988; Jones and Lindstedt, 1993; Taylor and Weibel, 1981; Taylor et al., 1987; Wagner, 1996a; Wagner, 2011). Mathematical models of O2 transport and the relative importance of each physiological factor in determining ṀO2max have been 49  investigated in a limited range of vertebrates representing mammals, birds, reptiles, and amphibians (di Prampero and Ferretti, 1990; Jones, 1998; Jones and Karas, 1988; Scott and Milsom, 2006; Wagner, 1993; Wagner, 1996b; Wagner, 2011; Wang and Hicks, 2002; Wang and Hicks, 2004; Withers and Hillman, 1988).   Comparative biologists are interested in ṀO2max because it determines an animal’s maximal aerobic performance, and the traits that determine ṀO2max are heritable and thus are very likely subject to natural and sexual selection (e.g. Garland and Bennett, 1990; Hayes and O’Connor, 1999; Nespolo et al., 2016; Storz et al., 2019). Across species, ṀO2max is generally correlated with the oxidative capacity of the active muscles (Hoppeler, 1990), and matching in the O2 supply-demand relationship is heavily dependent on circulatory O2 delivery, making the heart and Hb important targets for evolutionary increases to ṀO2max (e.g., Dohm et al., 1994; Gallaugher et al., 2001; Garland and Bennett, 1990; Gonzalez et al., 2006). In support of this, arterial O2 delivery is strongly correlated with ṀO2max in a variety of vertebrates (Gallaugher et al., 2001; Hillman et al., 2013; Kayar et al., 1994), and integrative models of the O2 transport cascade have consistently shown Q̇ to be one of the more important factors determining ṀO2max (di Prampero and Ferretti, 1990; Jones, 1998; Scott and Milsom, 2006; Wagner, 2011; Wang and Hicks, 2002; Withers and Hillman, 1988). However, an increased capacity for circulatory O2 delivery requires corresponding increases in respiratory and muscle diffusive conductance. Accordingly, animals with a relatively high ṀO2max have so-called adaptive changes to increase O2 conductance at each step of the O2 transport cascade (Bennett et al., 1984; Bernal et al., 2001b; Bishop, 1999; Brill and Bushnell, 2001; Bushnell and Jones, 1994; Henderson et al., 2002; Jones et al., 1989; Lindstedt et al., 1991; Longworth et al., 1989; Taylor et al., 1987).  50   The fish O2 transport cascade has not yet been modeled as an integrated system to assess the relative contribution of each factor to determining ṀO2max, even though extensive studies indicate that circulatory O2 delivery is very likely maximized during maximal aerobic exercise, at least in Pacific salmonids (Oncorhynchus spp.). A three-fold increase in Q̇ supports increased circulatory O2 delivery in salmonids swimming at their ṀO2max, but there is good experimental evidence to suggest that Q̇ and ṀO2 reach a maximum and plateau prior to a fish achieving its maximum aerobically sustained swimming speed (Eliason et al., 2013; Gallaugher et al., 2001; Kiceniuk and Jones, 1977; Lee et al., 2003; Thorarensen et al., 1996). Furthermore, hæmatocrit and intraspecific variability in maximum Q̇ are both correlated with ṀO2max in rainbow trout (Oncorhynchus mykiss) (Claireaux et al., 2005; Gallaugher et al., 1995). Although, when trout were made anemic there was no compensatory increase in Q̇ to maintain circulatory O2 delivery (Gallaugher et al., 1995). Thus, during maximal aerobic exercise, Q̇ may reach a maximum causing arterial-O2 content to predominantly determine maximum circulatory O2 delivery. Then again, reduced arterial-O2 content may prevent a compensatory increase in Q̇ by causing myocardial ischemia, and it has been suggested that peripheral diffusion limitations may maintain a venous O2 threshold that protects cardiac O2 supply (Farrell, 2002; Farrell and Clutterham, 2003; Steffensen and Farrell, 1998). However, rainbow trout ṀO2max was not increased with hyperoxia (Duthie and Hughes, 1987), which contrasts results in mammals (Knight et al., 1993; Wagner et al., 1996) and may indicate that in rainbow trout, both O2 and substrate supply are maximized to meet the demand of oxidative phosphorylation at ṀO2max. This may be the case because as rainbow trout approach ṀO2max, oxidative substrates are depleted in the red-oxidative muscle fibers concomitant with an increased reliance on white-51  glycolytic muscle fibers to power swimming and potentially supply substrate, as lactate, to the red muscle (Richards et al., 2002).    The high performance tunas have some of the highest ṀO2 values measured among fishes, which is at least in part related to the high metabolic demands of endothermy and their exceptional swimming performance (Korsmeyer and Dewar, 2001). To sustain high rates of circulatory O2 delivery and tissue O2 extraction, all factors of the tuna O2 transport cascade seem to have evolved in conjunction with their high metabolic rates (Bernal et al., 2001b; Brill and Bushnell, 2001; Bushnell and Jones, 1994). A likely hypothesis proposes that the high-performance physiology of tunas, including exceptionally large hearts and high cardiac output, evolved to supply O2 at high enough rates to fuel multiple metabolically demanding activities, not exclusively for exceptionally high swimming performance (Brill, 1996; Korsmeyer et al., 1996a). Nevertheless, the cardiovascular systems of tunas are relatively well studied and seem adequately equipped to support high levels of aerobic exercise (Brill and Bushnell, 1991b; Brill and Bushnell, 2001; Bushnell and Jones, 1994). Although there are limited data on the swimming ṀO2max of tunas (Blank et al., 2007; Korsmeyer and Dewar, 2001), Korsmeyer et al. (1997a) were able to measure cardiac performance and arterial and venous blood gases in yellowfin tuna swum to fatigue. Thus, the yellowfin tuna is the only regionally heterothermic or high-energy demand fish that has been studied to a reasonable degree to quantify how Hb concentration and Hb-O2 affinity affect βbO2, circulatory O2 delivery, and tissue O2 extraction at ṀO2max.  Tuna are regionally heterothermic, so blood must transport and unload O2 to tissues of widely different temperatures. Yellowfin tuna blood-O2 affinity is temperature-independent, but among other species of tuna, the temperature-dependence of blood-O2 affinity ranges from 52  slightly reduced to greatly reversed, so hypotheses for the functional significance of this trait in tuna are equivocal (Brill and Bushnell, 1991a; Brill and Bushnell, 2006; Cech et al., 1984; Clark et al., 2008a; Jones et al., 1986; Lowe et al., 2000). However, this trait likely maintains a relatively stable blood PO2 in tissues of different temperatures, and probably also conserves metabolic heat energy in the warm tissues (Weber and Wells, 1989).   In this study I have assessed the possible contributing factors to O2 supply limitation at ṀO2max in two fish models: the rainbow trout and the yellowfin tuna. For each model, I quantitively evaluated the relative importance of each factor to determining ṀO2max. To do this, I have used published data for each species (Kiceniuk and Jones, 1977; Korsmeyer et al., 1997a), and a mathematical model of the O2 transport cascade as described by Wang and Malte (2011), which includes a model of countercurrent exchange of O2 in the gill (Malte and Weber, 1985). In doing this, I have also quantified how Hb concentration and Hb-O2 affinity influence circulatory O2 delivery and tissue O2 extraction. Because the yellowfin tuna has a relatively large mixed venous O2 reserve at ṀO2max, I hypothesized that tissue O2 extraction is dependent on βbO2 (i.e., the magnitude of the Bohr shift), which will cause an apparent tissue O2-diffusion limitation in the model. I have also evaluated how the temperature-dependence of Hb-O2 affinity influences venous PO2 in tuna. I hypothesized that since temperature-independent Hb-O2 affinity will prevent a left-ward shift of the OEC with decreasing temperature, then blood PO2 will remain relatively constant over a wide range of body temperatures (15-30°C). Lastly, I have calculated what the energetic savings are relative to the oxycalorific equivalent of glucose metabolism over a range of ΔH' values.   53  2.2 Methods Mathematical model of fish O2 transport   The following system of equations conserve O2 mass at each step as O2 molecules are passed from water to the blood, and from the blood to the tissues. The equations used to model O2 transport, and the procedure to simultaneously solve the system of equations was described by Wang and Malte (2011). The countercurrent exchange of O2 at the respiratory surface of the gill, as formulated by Malte and Weber (1985), is described by two coupled differential equations, one for water and one for blood:  𝑑𝑃𝑤𝑑𝑙=𝐺𝑑𝐺𝑣∙ (𝑃𝑤 − 𝑃𝑏) (1a)  𝑑𝑃𝑏𝑑𝑙=𝐺𝑑𝐺𝑝∙ (𝑃𝑤 − 𝑃𝑏) (1b) In these equations Pw and Pb are the PO2 in water and blood, respectively, as a function of length (l) along the exchange surface (i.e., Pw = Pw(l), and Pb = Pb(l)). Gd, Gv, and Gp, are the diffusion, ventilation, and perfusion conductances, respectively. Gd includes conductances for the gill membrane (Gdm) and the erythrocytes (Gde) (Weibel et al., 1993), and these are given by  𝐺𝑑−1 = 𝐺𝑑𝑚−1 + 𝐺𝑑𝑒−1 (2)  𝐺𝑑𝑚 = 𝐾 ∙𝑆𝐴𝑡 (3)  𝐺𝑑𝑒 = 𝜃O2 ∙ 𝑉𝑐  (4) where K is the Krogh diffusion constant, SA is the area of the exchange surface, t is its thickness, 𝜃O2 is the rate of O2 binding by whole blood (i.e., Hb), and Vc is the volume of the capillaries in 54  the gill lamellae. In this model, Gd is fixed at a constant value that is calculated as the conductance required to match O2 exchange in the gill and tissues, respectively (i.e., an assumed maximal value). The ventilation conductance is calculated by   𝐺𝑣 = ?̇?𝑤 ∙ 𝛼𝑤O2 (5) where V̇w is the ventilation volume and 𝛼𝑤O2 is the solubility of O2 in water (i.e., the capacitance coefficient of O2 in water). The perfusion conductance is calculated by   𝐺𝑝 = ?̇? ∙ 𝛽𝑏O2 (6) where Q̇ is cardiac output and 𝛽𝑏O2 is the O2 capacitance coefficient in blood. Due to the nonlinearity of the OEC, which describes the relationship between Hb-O2 saturation and PO2, 𝛽𝑏O2 is not a constant and depends on blood PO2 (i.e., Pb). 𝛽𝑏O2 is defined from the slope of the relationship between blood-O2 content (Cb) and Pb  𝛽𝑏O2 =𝑑𝐶𝑏𝑑𝑃𝑏 (7) In this model the OEC is described by the Hill equation, which requires only two parameters and gives a reasonable approximation of the relationship between fractional Hb-O2 saturation (SHb) and Pb if the parameters for other OEC models are not available. The Hill equation is given by    𝑆Hb =𝑃𝑏𝑛𝑃50𝑛 + 𝑃𝑏𝑛 (8) where P50 is the PO2 at which 50% of Hb is saturated, and n is the Hill coefficient (i.e., a parameter that describes the sigmoidicity of the OEC, and quantifies cooperativity among the ligand binding sites). The blood-O2 content (i.e., Cb) is determined from SHb, the tetrameric Hb concentration (CHb), and the O2 that is physically dissolved in the blood plasma. The relationship between Cb and Pb then becomes 55   𝐶𝑏 = 𝑃𝑏 ∙ 𝛼𝑏O2 + 4 ∙ 𝐶Hb ∙𝑃𝑏𝑛𝑃50𝑛 + 𝑃𝑏𝑛 (9) where 𝛼𝑏O2 is the solubility of O2 in plasma.   Gas exchange in the tissues is described by a third differential equation  𝑑𝑃𝑏𝑑𝑙=𝐺𝑑𝑡𝐺𝑝∙ (𝑃𝑏 − 𝑃𝑡) (10) where Gdt is the diffusion conductance of the tissues, and Pt is the tissue PO2. Gdt is fixed at a constant value that is calculated as the conductance required to match O2 exchange to the reported ṀO2max value (Table 2.1). Here, Pt is treated as the mitochondrial PO2 (PmitO2) and is assumed to be zero at ṀO2max, as justified by Cano et al. (Cano et al., 2013). Finally, ṀO2 is determined by the Fick principle  ?̇?O2 = ?̇? ∙ (𝐶𝑎 − 𝐶𝑣) (11)  where Ca – Cv is the arteriovenous O2 content difference.  Solution of the equations  The system of equations (Eqns. 1a, 1b, and 10) are simultaneously solved numerically by an iterative method. The equations describing countercurrent O2 exchange at the gill (Eqns. 1a and 1b) form a boundary value problem that requires initial inputs of Pw and Pb at the same point on the exchange surface [i.e., Pb(l = 0) and Pw(l = 0)]. The mixed venous PO2 (Pv) and the inspired PO2 (PI) are the independent values and are initial inputs of the model at Pb(l = 0) and Pw(l = l), respectively. The dependent values are the arterial PO2 (Pa) and expired PO2 (PE), which are the model outputs at Pb(l = l) and Pw(l = 0), respectively, but because Pw(l = 0) needs to be known initially, an estimate of PE is input. This results in values for Pa and PE, and the Pa value is then input into the equation for O2 exchange in the tissues (Eqn. 10), which results in a 56  new Pv value that is used to initiate a new cycle of the model. This is repeated until the values for Pa, Pv (and PE) no longer change. The parameters required to determine Gv and Gp are taken from the literature (see below).  Data  The experimental data used in this study are summarized in Table 2.1. Rainbow trout data are from Kinceniuk and Jones (1977), and yellowfin tuna data are from Korsmeyer et al. (1997). As described by Korsmeyer et al (1997), the yellowfin tuna ṀO2max was estimated from the relationship between swim speed and ṀO2 from another study on similar sized yellowfin tuna (Dewar and Graham, 1994). The corresponding cardiac output was calculated with the Fick principle and is in line with the heart rate measurements made by Korsmeyer et al. (1997) and previous measurements of stroke volume (Brill and Bushnell, 2001; Korsmeyer et al., 1997a). Tuna ventilation volume was determined as  ?̇?𝑤 =?̇?O2𝑃𝐼 ∙ 𝛼𝑤O2 ∙ 𝑈 (12) where U is the fraction of O2 extracted from the incurrent/inspired water and is often referred to as O2 utilization. Bushnell and Brill (Bushnell and Brill, 1991) determined a U of 0.553 in swimming yellowfin tuna, which was used to estimate a V̇w of 3.3 L min-1 kg-1 for the tuna studied by Korsmeyer et al. (1997), close to previous determinations of V̇w in yellowfin tuna (Bushnell and Brill, 1991; Jones et al., 1990).  The P50 values at the gill (P50gill) were calculated using published studies on blood from rainbow trout and yellowfin tuna (Brill and Bushnell, 1991a; Weber et al., 1976). For yellowfin tuna, Bohr plots (i.e., plots of log10P50 vs pH) were constructed with published P50 values and accompanying pH values at 20 and 30°C (Brill and Bushnell, 1991a). The slope of the 57  relationship between log10P50 and pH is the Bohr coefficient, which was used to determine P50 at the reported blood pH of swimming yellowfin tuna. The rainbow trout Bohr coefficient reported by Weber et al. (1976) was used to adjust rainbow trout P50gill to in vivo blood pH. Estimates of P50gill are likely physiologically applicable because in vitro O2 equilibria experiments can be designed to closely match in vivo conditions in the gill or lung. However, they do not necessarily match condition in the tissue capillaries, where metabolic CO2 diffuses into the blood (Figure 2.1) and acidifies the blood relative to arterial pH, which decreases Hb-O2 affinity and right shifts the OEC via the Bohr effect (i.e., Hb-O2 dissociation linked binding of hydrogen ions, commonly referred to as Bohr protons). Although the concentration of hydrogen ions is greater in venous blood than in arterial blood, mixed venous pH is often close to or even relatively more alkaline than arterial pH (e.g., Korsmeyer et al., 1997a) because Hb acts as a pH buffer. Hb-proton binding prevents large changes to blood pH while also decreasing venous blood-O2 affinity by stabilising the low affinity T-state Hb confirmation, so it is Bohr proton saturation and not pH that is relevant to venous blood-O2 affinity. Consequently, mixed venous blood pH cannot be treated as an independent variable because it is dependent on blood-tissue gas exchange and non-bicarbonate buffering (e.g., Hb-H+ binding). Thus, measurements of venous blood pH are not useful to estimate P50 in the tissue capillaries (P50tissue). Here, P50tissue was estimated by fitting an OEC to the in vivo Pv and Cv values, and the P50 of that OEC was used as an approximation of P50tissue. In this model, P50gill was used to determine Gp in equations 1a and 1b, and P50tissue was used to determine Gp in equation 10.   Tetrameric Hb concentrations (i.e., CHb) were calculated to match the reported values for arterial O2 content, and the venous O2 contents (i.e., Cv) were calculated from the reported 58  values for Ca and Ca-Cv. Values for the solubilities of O2 in plasma and water were taken from Boutilier et al. (1984). Sensitivity analysis to assess the possible contributing factors to O2 supply limitation at ṀO2max  After initially determining values of Gd and Gdt that resulted in model outputs of Pa, Pv, Ca, Cv, and ṀO2max that closely matched in vivo values (Table 1), the model was used to test the sensitivity of the system for each species data set. Following a similar procedure to Jones (1998), each of the four conductances was individually changed by increasing or decreasing by 1% the values of the diffusion conductances, Gd and Gdt, and the factors that determine Gv and Gp (V̇w, Q̇, CHb, and P50tissue). For each change of a variable (i.e., Gd, Gdt, V̇w, Q̇, CHb, and P50tissue), the corresponding change in ṀO2max was expressed as a fractional change relative to the reported ṀO2max value. The sensitivity of flux through the system to changes of each variable (i) was quantified as the slope of the line (bi) fit to the fractional ṀO2max values calculated for each variable at -1%, control (i.e., no change), and +1% values. The slopes were standardized to fractional flux control coefficients (Fi) for each of the four conductances by dividing the slope for each variable by the sum of the slopes for all the variables in the system (Eqns. 13 and 14).   𝐹𝑖 =𝑏𝑖∑ 𝑏𝑖 (13)  𝐹𝑠𝑦𝑠𝑡𝑒𝑚 = 1.0 = 𝐹𝐺𝑣 + 𝐹𝐺𝑑 + 𝐹𝐺𝑝 + 𝐹𝐺𝑑𝑡 (14) In equation 14, changes in V̇w and Q̇ were used to change Gv and Gp, respectively. By doing this, the sum of the slopes (Eqn. 13) for V̇w, Q̇, Gd, and Gdt are equal to one.  59   To assess the extent to which P50tissue affects O2 unloading, tissue O2 extraction, and ṀO2max, P50tissue was decreased stepwise until it was equal to P50gill (i.e., no Bohr shift). At each step, the effect of decreasing P50tissue was determined by comparing model output values to the respective values with no change to the model.  Assumptions for the O2 transport model  In this model it is assumed that ṀO2max is O2 supply limited, that the entire system is in a steady state of O2 exchange (i.e., ṀO2 transients at the onset and cessation of exercise are not considered), and that the gills can be represented each by a single compartment (i.e., an exchange surface of length l). There are also several implicit assumptions made in this analysis, as described by Malte and Weber (1985) and Wagner (1993). At the gill, it is assumed that diffusion limitation is the primary factor affecting O2 exchange, that there is no ventilation/perfusion inhomogeneity in the gills, that V̇w and Q̇ are not cyclic processes, and that there is no gill metabolism. In the tissues it is assumed that tissue-O2 extraction is diffusion limited, and that there is no perfusion/metabolism inhomogeneity, and that there are no intramuscular limitations to O2 transport. The tissue diffusion conductance, Gdt, is assumed to comprise all components of peripheral diffusion and mitochondrial O2 uptake, including the kinetics of cellular O2 diffusion and intracellular O2 flux. It is assumed that there are no shunts for blood flow or diffusion. The blood is assumed to be a homogenous medium (i.e., no plasma or cells) that O2 diffuses to and from without any hinderance by Hb-O2 binding and dissociation or any other chemical reactions.  Calculation of Hb-heat transport  Metabolic heat production (HP kJ min-1) is given by  60   𝐻𝑃 = 𝑂2𝑘𝐽 ∙ (𝐶𝑎 − 𝐶𝑣) ∙ ?̇? (15) where O2 kJ is the oxycalorific equivalent for glucose metabolism and is equal to 473 kJ mol-1. The heat transferred by Hb is given by   𝐻𝐻𝑏 =  ∆𝐻′ ∙ (𝐶𝑎 − 𝐶𝑣) ∙ ?̇? (16) where ΔH' (kJ mol-1) is the overall enthalpy of Hb-oxygenation. If HHb is quantified as a percentage of HP, then the percentage of heat transport by Hb is independent of O2 extraction and cardiac output, and is a relatively constant ratio of ΔH'/O2kJ. The heat potentially transported to the environment during Hb-oxygenation at the gills was modeled as a function of ΔH', assuming that ΔH' is constant across the entire OEC.  Temperature dependence of blood PO2  The effect of temperature on yellowfin tuna blood PO2 was evaluated at four different values of ΔH' from 15°C to 30°C. The results of this model are meant to predict the blood PO2 in the peripheral tissues that are not protected by heat exchanging retia, and are thus influenced by ambient water temperature. Venous PO2 values for the tissue capillaries are not known, so mixed venous blood PO2 of slowly swimming yellowfin tuna was used as a starting reference. Although this may be considered a limitation of the model, it allowed me to estimate the effect of temperature on in vivo P50 since mixed venous blood is close to 50% saturated, which also validates using ΔH' values determined at P50 (i.e., 50% Hb-O2). Starting values, as reported by Korsmeyer et al. (1997a), were: PO2 = 40mmHg; pH = 7.800; temperature = 25°C. Blood PO2 was predicted at the different temperatures using the van’t Hoff isochore (Wyman, 1964) 61   ∆𝐻′ =  ln 10 ∙ 𝑅 ∙Δ log10 𝑃50Δ1𝑇 (17) where R is the gas constant, and T is the absolute temperature. Similar to a previous model of O2 transport in regionally heterothermic mammals (Brix et al., 1990), blood PO2 was determined as a function of blood pH at each temperature, and at a constant pH of 7.800. Blood pH was adjusted by -0.017 pH units/°C according to the results of Brill and Bushnell (1991a), and the blood PO2 was then adjusted to that pH using equation 18 and the Bohr coefficient (φ) determined for yellowfin tuna (as described above):  𝑃O2(𝑝𝐻,𝑇) = 𝑃O2(𝑇) ∙ 10(𝜑∙∆𝑝𝐻) (18) ΔH' values were calculated for yellowfin tuna and skipjack tuna using the van’t Hoff isochore. As blood temperature changes, the degree of relative alkalinity to pH of neutrality should remain fairly constant (Kim and Milsom, 2019; Rahn, 1967; Reeves, 1972), so blood PO2 was also modeled as a function of blood temperature only and assuming a constant pH of 7.800. Skipjack tuna Bohr plots were constructed with data from Brill and Bushnell (1991a) as described above for yellowfin tuna. A third ΔH of -19 kJ mol-1 was calculated for Bigeye tuna (Thunnus obesus) using data reported in Lowe et al. (2000), and a fourth ΔH of -35 kJ mol-1 for rainbow trout blood at pH 7.8 was taken from Weber et al. (Weber et al., 1976).  2.3 Results O2 transport    For both the tuna and trout models, the mean partial pressure gradient between PI and Pb (ΔPO2gill), the mean gill capillary Pb (P̄cgill), and the mean tissue capillary PO2 (P̄ctissue) are reported in Table 2.1 and illustrated in Figure 2.2. The estimates of Gd and Gdt (Table 2.1) were 62  calculated to match model predictions of Pa and Pv to the reported values, which resulted in estimated ṀO2max values that were within 0.1% of the reported values. The model estimates of ṀO2max were then used as the “control” values for the sensitivity analyses. The relative changes in ṀO2max caused by ± 1% changes in model variables (i.e., Gd, Gdt, V̇w, Q̇, CHb, and P50tissue) are shown in Figure 2.3. The three points (-1, 0, and +1% change) were collinear (R2 > 0.999) for each variable in both models, and the slopes of linear regressions fit to the three points for each variable are reported in Table 2.2. For rainbow trout, altering Q̇ had the greatest affect on ṀO2max followed by CHb, P50tissue, Gdt, Gd, and V̇w. For yellowfin tuna, altering P50tissue had the greatest affect on ṀO2max followed by Q̇, CHb, Gdt, Gd, and V̇w. The resulting fractional flux control coefficients for the four conductances are shown in Figure 2.4.  H-O2 affinity and Hb-heat transport  Figure 2.5 shows the effect of reducing P50tissue on ṀO2, circulatory O2 delivery, tissue O2 extraction and blood O2 levels. In the absence of a right shifted OEC in the tissue compartment (i.e., P50gill = P50tissue), yellowfin tuna ṀO2 was reduced by 44% whereas rainbow trout ṀO2 decreased by 16%. The relatively larger decrease of yellowfin tuna ṀO2 was primarily due to reduced tissue-O2 extraction with decreasing P50tissue (Figure 2.5).   The relationship between log transformed P50 and blood pH for both yellowfin tuna and skipjack tuna are shown in Figure 2.6 (the regression equations are given in the caption of Figure 2.6). The Y-intercept values from least-squares linear regressions were not different between the two temperatures for yellowfin tuna blood [F = 0.260, d.f. = (1, 19), P = 0.616], indicating that P50 is independent of temperature, so a single linear regression was fit to the combined 20°C and 30°C data. A reverse temperature effect was evident in skipjack tuna blood, as indicated by a lower Y-intercept at 30°C than at 20°C [F = 9.439, d.f. = (1, 19), P = 0.006]. Both species 63  exhibited a marked Bohr effect (i.e., an inverse relationship between log10P50 and pH). A Bohr coefficient of -0.625 (Figure 2.6) was determined for yellowfin tuna, which was then used to adjust PO2 over a range of temperatures and associated pH values shown. The resulting effect of temperature on blood PO2 is shown in Figure 2.7. Over a temperature gradient from 30°C to 15°C and the concomitant pH change, Hb with a reversed temperature effect (i.e., skipjack tuna; ΔH' = 16 kJ mol-1; Figure 2.5) causes no change to blood PO2, whereas Hb with an O2 affinity that is independent of temperature (i.e., yellowfin tuna; ΔH' = 0 kJ mol-1; Figure 5) causes a pH dependent decline in PO2 with declining temperature (ΔPO2 ≈ -9 mmHg from 25 to 15°C).  The ΔH' values calculated for yellowfin tuna and skipjack tuna blood were used to determine the amount of heat transported by Hb. The enthalpy of Hb-O2 binding expressed as a percentage of the oxycaloric equivalent of glucose catabolism (~473 kJ mol-1) for a range of enthalpy values is shown in Figure 2.7. In rainbow trout blood between pH 7.8 and pH 7.5 (ΔH' values of -35 and -11 kJ mol-1, respectively), about 7% to 2% of the heat produced is transported by Hb and potentially lost to the environment in conjunction with Hb-O2 binding at the gills. Yellowfin tuna blood shows no effect of temperature on Hb-O2 affinity (Figure 2.6) (Bushnell and Brill, 1991), which should avert the absorption and transport of heat away from the warm tissues by Hb (Figure 2.8). A reverse temperature-dependence like that observed by Brill and Bushnell (1991) in skipjack tuna blood, may cause Hb-oxygenation linked retention of heat equivalent of up to 4% or more of the energy produced during glucose catabolism.      64  2.4 Discussion Main findings  The main objectives of this study were twofold: 1) to quantitatively evaluate the factors determining the maximal rate of O2 transport in the rainbow trout and the yellowfin tuna; and 2) to determine the influence of the enthalpy of Hb-O2 binding on Hb-heat transport and blood PO2. In both the yellowfin tuna and rainbow trout O2 transport models, circulatory O2 delivery and tissue diffusing capacity (i.e., perfusion conductance and tissue diffusion conductance, respectively) were relatively more important determinants of ṀO2max than were V̇w and gill diffusing capacity (Figures 2.3 and 2.4). Thus, when rainbow trout and yellowfin tuna are forced to swim in controlled and normoxic conditions (Kiceniuk and Jones, 1977; Korsmeyer et al., 1997a), gill O2 uptake is not a major determinant of O2-supply limitation. The model results of this study and the results of many others give good reason to suggest that ṀO2max is governed by the interactions between central and peripheral cardiovascular O2 transport (i.e., circulatory O2 delivery and tissue O2 extraction). Under exercise conditions when O2 supply limits ṀO2max in fish, the anatomical separation of muscle fiber types and a need to secure venous O2 supply to the heart may contribute to circulatory O2 delivery and muscle O2 extraction apparently limiting O2 supply to the muscles. These ideas are further explored below. Temperature independent and endothermic ΔH' values that are characteristic of most tuna have been hypothesized by others to conserve metabolic heat and avert disruptions to Hb-O2 unloading over internal temperature gradients, and my model results support these hypotheses.    In mathematical analyses of the O2 transport systems of air-breathing vertebrates, the sensitivity of the system has often been tested by using large changes in model variables (i.e., 20-100%) (Jones and Karas, 1988; Scott and Milsom, 2006; Wagner, 1996b; Wagner, 2011; Withers 65  and Hillman, 1988). However, because of the sigmoidal shape of the OEC, large changes to variables can cause large and unrealistic changes to model outputs of blood O2 content and ṀO2max, which precludes quantitative determination of the sensitivity of the system. To test the sensitivity of the O2 transport systems modeled in this study, the model variables were perturbed by 1%, ceteris paribus. This approach was adopted from the elegant sensitivity analysis of the equine O2 transport system by Jones (1998), and a similar approach by diPrampero (1985) who analysed the factors determining maximum O2 transport in humans using the results of experimental perturbations of variables in the O2 transport system. Limiting the variable perturbations to 1% allows for “fine-tuning” of the system within a range of realistic deviations of the model variables. For the fish O2 transport models, qualitatively similar results were achieved by perturbing the model variables by 1, 5, 10, and 20%, but the effects on ṀO2max deviated from linearity at the higher relative perturbations. However, the results of this analysis are likely qualitatively comparable to other analyses that used larger variable perturbations (i.e., the conclusions from analyses that used >20% changes would likely not have changed if smaller model perturbations were used).   Tests of the O2 transport system have also often tested the hypothesis of symmorphosis (Garland and Huey, 1987; Jones, 1998; Weibel and Taylor, 1981). As originally formulated, symmorphosis proposed that regulated morphogenesis should result in changes to the structural and functional factors of the O2 transport cascade that are proportional to changes to maximal flux through the system, such that no one factor or component of the system should be more limiting than the others (Taylor and Weibel, 1981). Thus, symmorphosis has been suggested as null hypothesis in empirical tests for rate-matching of O2 flux through each step of the O2 transport cascade (Jones et al., 1990; Weibel et al., 1991). However, perfect rate matching at 66  each step of the O2 transport cascade should not necessarily be an a priori hypothesis because symmorphosis may not be a likely evolutionary outcome (Dudley and Gans, 1991; Garland, 1998; Garland and Huey, 1987). An important reason why it is unlikely that the physiology of all animals has been “optimized” for ṀO2max (e.g., Jones, 1998; Jones and Lindstedt, 1993) is that  biological structures (e.g., proteins, tissues, or organs) may not evolve in parallel (e.g., Dalziel et al., 2012), because each may play important roles in multiple physiological processes (Dudley and Gans, 1991; Garland, 1998). This is exemplified by the fish gill, which is a multifunctional, metabolic organ that has central roles in acid-base regulation, osmotic and ionic regulation, and excretion of nitrogenous wastes (Evans et al., 2005). Nevertheless, aquatic gas exchange is a dominant function of the gill and the gill morphometrics that govern gas diffusion (i.e., lamellar surface area and the thickness of the blood-water barrier) seem to be correlated with metabolic capacity among fishes, with tunas having the highest relative gill surface areas (Bigman et al., 2018; Hughes and Morgan, 1973; Wegner, 2015; Wegner et al., 2006; Wegner et al., 2010).   Gill diffusion conductance was not a relatively important determinant of O2 supply limitation in either fish model, in contradiction to symmorphosis, at least regarding maximal O2 transport in normoxia. Comparably, even though the lungs of air-breathing tetrapods are relatively unmalleable (e.g., Henderson et al., 2002; Weibel, 2000) pulmonary diffusing capacity is not a major limiting factor to O2 flux in normoxia (Scott and Milsom, 2006; Wagner, 1996b; Withers and Hillman, 1988). However, in the Thoroughbred racehorse, ventilation and pulmonary diffusing capacity are the most important factors determining maximum O2 flux, which has likely resulted from strong selection for aerobic power that has pushed the capacity for O2 transport to the physiological limits of the equine lung (Jones, 1998). Interestingly, highly aerobic human athletes experience similar pulmonary limitations to exercise (Hopkins, 2005; 67  Hopkins and Harms, 2004; Hopkins et al., 1996). When swimming at ṀO2max, rainbow trout likely approach the functional limits of gill O2 uptake, but yellowfin tuna do not seem to use the entire functional capacity of the gill. Experimental manipulations of rainbow trout gill surface area provide good evidence that most of the gill lamellae are perfused to increase O2 uptake during maximal aerobic swimming (Duthie and Hughes, 1987). Increased blood pressure in the ventral aorta and increased blood flow will decrease the blood-water barrier and increase the functional gill surface area by recruiting distal lamellae, which maintains a high arterial O2 saturation at ṀO2max that is comparable to resting values (Kiceniuk and Jones, 1977). In comparison, yellowfin tuna arterial PO2 and O2 saturation increase with increasing swimming speed but are still relatively low during exercise (Korsmeyer et al., 1997a). Higher blood pressure and blood flow likely cause recruitment of lamellar surface area (Korsmeyer et al., 1997a), but the high vascular resistance reported in yellowfin tuna (Bushnell and Brill, 1992) may limit or cause heterogeneous lamellar perfusion. In general, when considering total O2 exchange between the environment and the metabolizing tissues, it is not advantageous to decrease blood flow even if it increases O2 extraction efficiency (Malte, 2011). In tuna, decreased blood flow would probably not increase O2 extraction since the functional area for diffusion would also decrease. Even though tuna arterial saturation was low, it did not seem to hinder total O2 flux. Since tuna ram-ventilate their gills it seems likely that as tuna swim faster, and increase ventilation and perfusion, that arterial blood saturation may increase in proportion to O2 demand.    A digression concerning the determination of ṀO2max in fishes is necessary before discussing peripheral cardiovascular O2 transport limitations. To assess the ṀO2max of mammals and some other tetrapods (usually reported as V̇O2max in air breathing animals), researchers 68  typically use a graded exercise test and determine ṀO2max as the point where ṀO2 plateaus and does not increase further with increasing exercise intensity (e.g., Seeherman et al., 1981). Testing protocols often include other criteria to assess ṀO2max, including but not limited to measurements of heart rate, blood lactate, the respiratory exchange ratio, and the rate of perceived exertion (humans). Graded exercise protocols have also been developed for fish (Beamish, 1978; Brett, 1964), in which the highest or peak ṀO2 (ṀO2peak) measured during swimming is used to approximate ṀO2max, sometimes also supported with measurements of heart rate or blood lactate (Norin and Clark, 2016; Zhang et al., 2019). However, due to methodological constraints of measuring aquatic respiration, and the typical recruitment of glycolytic muscles that accompany dynamic swimming gait changes when a fish nears fatigue (Peake and Farrell, 2004; Richards et al., 2002), ṀO2max may not always characterize the same thing in fish as it does in mammals (Zhang et al., 2019). However, in many cases ṀO2peak is likely a good enough approximation of ṀO2max in fish (e.g., Lee et al., 2003; Norin and Clark, 2016; Zhang et al., 2020).   Kiceniuk and Jones (1977) and Korsmeyer et al. (1997) used the same graded exercise protocol for their studies on rainbow trout and yellowfin tuna, respectively, which included velocity increments every 60 minutes until fish fatigued. The rainbow trout showed a plateau in heart rate at the penultimate velocity increment, which is good evidence that ṀO2max was likely achieved at the fatiguing swimming velocity (Kiceniuk and Jones, 1977). Yellowfin tuna were reported to have been swum to fatigue, as characterized by dynamic changes to swimming gait and elevated blood lactate levels, although their maximal swimming velocities, heart rates, and levels of O2 extraction were lower than previously observed or predicted (Brill and Bushnell, 1991b; Dewar and Graham, 1994; Korsmeyer et al., 1996b; Korsmeyer et al., 1997a; Korsmeyer 69  et al., 1997b). During exercise, tuna rely almost entirely on changes in heart rate to modulate cardiac output, whereas trout increase cardiac output by increasing both heart rate and stroke volume (Brill and Bushnell, 2001). The lower than expected heart rate reported for yellowfin tuna may indicate that cardiac output could have been increased further, which would increase circulatory O2 delivery (Korsmeyer et al., 1997a). The arteriovenous O2 content difference was similar between rainbow trout and yellowfin tuna, but tissue O2 extraction reached 86% in rainbow trout and only 49% in yellowfin tuna, with resulting mixed venous saturation of around 13% and 44%, respectively (Kiceniuk and Jones, 1977; Korsmeyer et al., 1997a). Thus, it is possible that the large venous O2 reserve in yellowfin tuna indicates that they did not reach their maximal aerobic swimming limits or ṀO2max, but Korsmeyer et al. (1997) explained that the mixed venous O2 content may not reflect the O2 extracted by the swimming muscles (i.e., the loins of red muscle), which are anatomically separated from the other skeletal muscles in fishes and have their own vascular supply (Bone, 1978).  Systemic blood flow is redistributed during swimming exercise, causing a disproportionate increase in blood flow to the red muscle (Q̇RM) to support the increased metabolic demand. At rest, rainbow trout Q̇RM is about 9% of cardiac output, but when swimming at 80% of maximum effort Q̇RM increases to 42% of cardiac output, which supports a 12-fold increase in red muscle ṀO2 (Randall and Daxboeck, 1982). Randall and Daxboeck (1982) estimated red muscle ṀO2 to be 87% of total ṀO2, based on a red muscle O2 extraction of 96%. Thus, the muscles powering swimming can be assumed to account for most of the increased ṀO2, so the results of the rainbow trout model can be argued to reflect limiting conditions at ṀO2max. Yellowfin tuna Q̇RM has not been determined, but based on measurements made in lightly anesthetized albacore tuna (Thunnus allalunga) (White et al., 70  1988), Korsmeyer et al. (1996b) conservatively estimated that Q̇RM would be 36% of maximum cardiac output, which they used to model red muscle ṀO2 during maximal aerobic swimming. They assumed that if red muscle extracts 90% of the O2 in the blood, then the red muscle ṀO2 would be 67% of ṀO2max in a 2.1 kg yellowfin tuna with an estimated ṀO2max of 1600 mg O2 kg-1 hr-1 (0.834 mmol O2 kg-1 min-2). Taking their model a step further, I calculated a total O2 extraction of 48% for the entire circulation, which is almost identical to their in vivo measurements that were used for this study’s model (Korsmeyer et al., 1997a). Thus, yellowfin tuna red muscle O2 extraction likely exceeds 49%, but an O2 extraction of 49% may be reasonable for the entire cardiac output at ṀO2max. Therefore, the relative importance of Gdt in this study’s yellowfin tuna model likely reflects whole body O2 extraction, and not necessarily a limitation for O2 diffusion across the red muscle capillaries. Tissue specific PO2 and blood O2 content values would permit determination of O2 extraction in each tissue compartment, but to the best of my knowledge those measurements have not been reported.    A major assumption of the model that I used is that the tissue diffusion conductance, Gdt, comprises everything that may affect O2 diffusion and transport from the blood to the mitochondria. In models for humans and birds, Gdt has been shown to be a relatively important determinant of ṀO2max, particularly in elite athletes or in hypoxia (di Prampero, 1985; Jones and Karas, 1988; Scott and Milsom, 2006; Wagner, 1996a; Wagner, 1996b). This seems intuitive because if all factors contributed equally to O2 flux then F would equal 0.25 for each of the four conductances. So, if Gv and Gd are relatively non-limiting, then it would be expected that the resistance to O2 flux would be shared by the factors determining circulatory O2 delivery and tissue O2 extraction (i.e., Q̇, [Hb], and Gdt). However, it has been argued that tissue diffusion cannot be the primary limitation to ṀO2max  for at least two reasons: 1) because not all 71  capillaries are perfused during maximal exercise, so if there is unused diffusing capacity then tissue diffusion cannot be limiting to O2 transport; and 2) because in ectothermic vertebrates that experience different body temperatures, temperature has a greater affect on ṀO2max than it does on gas diffusion coefficients (Hedrick et al., 2015; Hillman et al., 2013). This argument is from the perspective that there is a single rate-limiting step to O2 transport, whereas I have adopted the well-established hypothesis that ṀO2max is set by all steps in the O2 transport cascade (Jones and Karas, 1988; Wagner, 1988; Wagner, 1993). Put differently, ṀO2max is governed by the interaction between circulatory O2 delivery and tissue O2 extraction, the two components of O2 transport to the mitochondria. Moreover, there is good experimental evidence showing that raising the PO2 gradient for O2 diffusion into exercising muscle without increasing circulatory O2 delivery increases O2 extraction and ṀO2max (Richardson et al., 1998; Wagner et al., 1990), which emphasizes the relative importance of peripheral O2 diffusion to determining ṀO2max.  In salmonids, a peripheral O2 diffusion limitation has been proposed as a possible mechanism to prevent venous PO2 falling below a threshold that protects cardiac O2 supply, thus preventing myocardial ischemia during exercise (Farrell, 2002; Farrell and Clutterham, 2003; Steffensen and Farrell, 1998). The relative importance of Gp and Gdt to determining ṀO2max in the rainbow trout model likely reflects a limited capacity for O2 extraction and diffusion from the blood to the tissues other than red muscle, so the mixed venous PO2 is most likely dependent on the remainder of the systemic blood flow. The situation is different in tuna, which have hearts with large ventricles that are well-endowed with a coronary circulation, so tuna hearts may not be as dependent on venous O2 supply to the myocardium (Brill and Bushnell, 2001).   The intrinsic metabolic capacity of tuna red muscle is similar to that of other active pelagic teleosts, but the elevated temperatures of tuna red muscle increases its aerobic metabolic 72  capacity and also warms the adjacent white muscle, which has higher anaerobic and aerobic metabolic capacity relative to most other fishes (Dickson, 1995; Dickson, 1996; Korsmeyer and Dewar, 2001). The gills and cardiovascular systems of tuna are capable of high rates of circulatory O2 delivery (Brill and Bushnell, 2001; Muir and Hughes, 1969; Wegner et al., 2006; Wegner et al., 2010), and the muscles have evolved certain traits that facilitate high rates O2 diffusion, including: relatively small red muscle fibers with high capillarity, relatively high amounts of mitochondrial protein due to densely packed mitochondrial cristae, and high concentrations of myoglobin in both the red and white muscle (Dickson, 1996; Korsmeyer and Dewar, 2001; Mathieu-Costello et al., 1992; Mathieu-Costello et al., 1996; Moyes et al., 1992). Collectively, these traits plus elevated muscle temperatures will increase O2 extraction and may ensure that the red muscle is never hypoxic during exhaustive exercise (Brill, 1996). Thus, it seems that muscle O2 extraction and circulatory O2 delivery are well matched in tunas (reviewed by: Brill and Bushnell, 2001; Korsmeyer and Dewar, 2001), which is supported by the model results of this study (Figures 2.3 and 2.4). Therefore, it is not unreasonable to propose that at ṀO2max, red muscle O2 extraction may even exceed the 90% extraction predicted by Korsmeyer et al. (1997a).   Hæmoglobin concentration and shifts in Hb-O2 affinity had relatively important contributions to determining ṀO2max in both the rainbow trout and yellowfin tuna models (Figure 2.3). As outlined by Brill and Bushnell (1991b), the high Hb concentrations and blood-O2 carrying capacity of tunas likely contribute to high circulatory O2 delivery without excessive increases in cardiac output. A high Hb concentration may also be important for maintaining a high mixed venous O2 content in the blood flow through the non-red muscle portion of the systemic circulation, which has a lower O2 extraction than the red muscle. Thus, high Hb 73  concentrations are likely necessary to match O2 supply with the high metabolic capacity of the warmed red muscle, while also securing a high O2 supply to fuel other metabolically demanding processes, as well as to facilitate rapid recovery after exhaustive exercise in the pelagic environment where there is nowhere to hide and rest (Brill, 1996; Korsmeyer et al., 1996a).   In the tissue capillaries, an increased P50 due to the Bohr and Root effects appears to be necessary to maintain O2 supply by enhancing O2 offloading relative to Hbs that lack a Bohr or Root effect. Reducing P50tissue, which would be equivalent to reducing the Bohr coefficient or βbO2, reduced ṀO2 more so in yellowfin than in rainbow trout. As described above, tuna have evolved several traits that should enhance tissue O2 diffusion, and it seems likely that natural selection has pushed the cardiovascular system of tunas close to the functional limits for O2 transport. Thus, the numerically large Bohr coefficients are probably necessary to increase O2 unloading and tissue O2 extraction to match O2 supply to the high O2 demand of the red muscle. This dependence on large right shifts of the OEC to unload O2 likely contributed to the relative importance of Gdt in the yellowfin tuna model. As is evident in Figure 2.5, decreasing P50tissue appreciably decreased O2 extraction, which would influence Gdt in the model. An emerging model for teleosts with pH sensitive Hbs proposes that plasma accessible carbonic anhydrase in the tissue capillaries accelerates CO2 hydration, which rapidly acidifies the red blood cells and decreases Hb-O2 affinity via the Bohr and Root shifts (Brauner and Harter, 2017; Rummer et al., 2013). There is good evidence to suggest that in salmonids this system maintains rates of O2 unloading high enough that cardiac output is not unnecessarily increased to possibly unsustainable levels to maintain circulatory O2 transport (e.g., Harter et al., 2019). Although it is not clear if all tuna have Root effect Hbs, tuna blood-O2 affinity does show a pronounced sensitivity to pH (Brill and Bushnell, 1991a; Brill and Bushnell, 2006; Cech et al., 1984; Clark et 74  al., 2008a; Jones et al., 1986; Lowe et al., 2000). Therefore, it seems likely that tunas would be highly dependent on such a system during strenuous swimming to unload O2 to the exercising red muscle without unnecessarily large increase in cardiac output, which is in line with my results (e.g., Figures 2.3 and 2.5) and the extensive research by Brill and Bushnell (Brill and Bushnell, 1991b; Brill and Bushnell, 2001). Moreover, temperature-independent Hb-O2 affinity could be interpreted to impair O2 unloading to the warm tissues such as the RM during exercise, but plasma accessible carbonic anhydrase in the tissue capillaries would overcome this by promoting Hb-O2 unloading in the absence of temperature induced shift of the OEC.   Hæmoglobin may also serve an important role in thermoregulation, because Hb-O2 transport is linked to heat transport. The intrinsic enthalpy of hæme-oxygenation (i.e., ΔHO2) is around 13% of the oxycalorific equivalent for glucose metabolism (473 kJ mol-1; Figure 6), but the oxygenation dependent dissociation of allosteric effectors contributes endothermically to ΔH', so in vivo Hb-heat transport will typically be lower than 13% of metabolic heat production. In humans about 9% of the heat produced is transported by Hb, and in rainbow trout about 2-8% may be transported to the gills by Hb and subsequently lost to the environment upon Hb-oxygenation (Figure 2.8) (Coates, 1975; Weber and Wells, 1989; Weber et al., 1976). Temperature-independent Hb-O2 affinity eliminates Hb-heat transport (yellowfin tuna in Figure 2.8), and a reversed temperature-dependency, where ΔH' is positive, would cause heat to be released to the tissues upon Hb-O2 unloading (skipjack tuna in Figure 2.8), which may contribute to maintaining the RM warm.   Reduced and reverse temperature-dependent Hb-O2 affinity may also avert possible impaired Hb-O2 unloading to the cold peripheral tissues, which are separated from the warm body regions by heat exchanging retia (Brix et al., 1989b; Clark et al., 2008a; Giardina et al., 75  1989a). For example, in Figure 2.7 it is evident that temperature-independence stabilizes mixed venous PO2 over a wide range of temperatures. This is further discussed in Chapter 5 (5.3.3). Summary and conclusions  Oxygen transport from the environment to the mitochondria is governed by the interactions between circulatory O2 delivery and tissue O2 extraction. All factors of the O2 transport cascade contribute to determining ṀO2max, although their relative importance may vary. In both the yellowfin tuna and the rainbow trout models, perfusion conductance had the greatest relative contribution to determining ṀO2max, with F values of 0.47 and 0.50 for tuna and trout, respectively. Tissue diffusion conductance also had a relatively important contribution to overall O2 flux, but more so for yellowfin tuna than for rainbow trout (F values of 0.39 and 0.28, respectively). This likely reflects the anatomical separation of skeletal muscle fiber types, and a lower O2 extraction in the blood flow to the tissues other than the exercising red muscle. Hæmoglobin concentration and cardiac output contributed almost equally to determining Gp in yellowfin tuna. A high P50 in the tissues (i.e., a right shift OEC due to proton binding) was an important determinant of ṀO2max due to its influence on Hb-O2 unloading and tissue O2 extraction. Therefore, in the absence of temperature-induced right shift of the OEC in the warm and exercising RM, a large Bohr effect is required to unload O2. High Hb concentrations and numerically high Bohr coefficients in tuna are very likely important for them to achieve such high rates of O2 consumption, by increasing circulatory O2 delivery and tissue O2 extraction without unnecessarily large increases in cardiac output. Reductions in the temperature sensitivity of tuna hæmoglobin probably has an important heat conserving function, and may avert impaired to O2 unloading to cold tissues by preventing leftward shifts of the OEC. 76  Figure 2.1 Schematic of the fish O2 transport cascade with O2 transfer from water to the mitochondria occurring in four steps. At each step, the flow rate of O2 (ṀO2) can be expressed as the product of an O2 partial pressure gradient (ΔPO2) and a conductance (G). The ventilation conductance (Gv) is a product of the ventilation volume (V̇w) and the solubility of O2 in water (𝛼𝑤O2), and diffusion from water to blood is characterized by a diffusion conductance (Gd). The perfusion conductance (Gp) is a product of the cardiac output (Q̇) and the blood-O2 capacitance (𝛽𝑏O2), and diffusion from blood to the tissues is characterized by a diffusion conductance (Gdt). PIO2, inspired PO2 (PI); PEO2, expired PO2 (PE); PaO2, arterial PO2 (Pa); Pv̄O2, mixed venous PO2 (Pv); PmitO2, mitochondrial or tissue PO2 (Pt). 77              Figure 2.2 The change in water and blood PO2 (mmHg) along the length (l) of the exchange surface for rainbow trout and yellowfin tuna.  Arrows indicate the direction of flow of water (blue) or blood (red). The solid red curve represents blood along the gill capillary exchange surface, and the dashed red curve is the blood along the tissue capillary exchange surface. The inspired PO2 (PI), expired PO2 (PE), arterial PO2 (Pa), and the mixed venous PO2 (Pv) are indicated at the point where the curves meet the vertical axes. The mean PO2 in the gill capillary (Pcgill; solid circle) and in the tissue capillary (Pctissue; open circle) are indicated at their respective positions along the exchange surface. 78                 Figure 2.3 Change in ṀO2max caused by ± 1% change in different components of the O2 transport cascade for rainbow trout and yellowfin tuna.  The changes in ṀO2max were determined by solving the mathematical model described in the text. The lines are linear regressions for the three data points for each variable: ventilation (V̇w), hæmoglobin concentration (Hb), cardiac output (Q̇), P50 in the tissue capillaries (P50tissue),  gill diffusion conductance (Gd), and tissue diffusion conductance (Gdt).79      Figure 2.4 Fractional flux control coefficients (F) for the four conductances that govern flow through the O2 transport cascade and contribute to determining ṀO2max.  Solid yellow bars to the left of each pair are those for the yellowfin tuna model, and diagonal striped bars to the right are those for the rainbow trout model. Gp, perfusion conductance; Gdt, tissue diffusion conductance; Gd, gill diffusion conductance; Gv, ventilation conductance. The F values were calculated as the slopes reported in Table 2.2 divided by the sum of the slopes of the four conductances. Changes to cardiac output (Q̇) and ventilation (V̇w) were used to determine F values for Gp and Gv, respectively.80             Figure 2.5 The effect of P50tissue on blood-O2 levels, O2 delivery, tissue O2 extraction, and maximum O2 transport.  P50tissue was decreased stepwise until it was equal to P50gill (i.e., no Bohr shift), and at each step the effect of decreasing P50tissue on O2 transport was determined. Closed symbols correspond to the left y-axis, and open symbols correspond to the right y-axis. Top panel: venous O2 content (CvO2; closed squares), the arteriovenous O2 content difference (CaO2-CvO2; closed diamonds), venous PO2 (PvO2; closed circles). Bottom panel: Circulatory O2 delivery (closed squares), relative O2 consumption (MO2; closed triangles), venous Hb-O2 saturation (open diamonds), tissues O2 extraction (open circles).  81              Figure 2.6 Yellowfin tuna and skipjack tuna Bohr plots. Linear regressions (± 95% CIs) were fit to data reported by Brill and Bushnell (1991a) and measured at 20°C (blue circles) and 30°C (red diamonds). Yellow fin tuna P50 was temperature-independent, as indicated by similar regression intercepts [F = 0.260, d.f. = (1, 19), P = 0.616], so a single regression (black line) was fit to the combined data. Skipjack tuna P50 showed a reversed temperature-dependence, as indicated by a lower intercept at 30°C than at 20°C [F = 9.439, d.f. = (1, 19), P = 0.006]. Yellowfin tuna 20°C: combined data [log10P50 = -0.625 (± 0.230) ∙ pH + 6.244 (± 1.782)]. Skipjack tuna 20°C: log10P50 = -0.717 (± 0.337) ∙ pH + 7.096 (± 2.638); 30°C: log10P50 = -0.626 (± 0.219) ∙ pH + 6.294 (± 1.693). Parameters ± 95% CIs.  82  Figure 2.7 Theoretical effect of temperature on mixed venous blood PO2 with varying enthalpy of Hb-oxygenation (ΔH').  The venous blood PO2 of slowly swimming yellowfin tuna was used as a starting reference: PO2 = 40 mmHg; Hb-O2 ~ 50%; pH = 7.800; temperature = 25°C (Korsmeyer et al., 1997a). The blood PO2 was adjusted to each temperature using varying ΔH' values and the van’t Hoff isocore (see methods). It was assumed that yellowfin tuna blood pH changes -0.017 pH units/°C, according to Brill and Bushnell (1991a). (A) The blood PO2 was then adjusted to the pH at each temperature using a Bohr coefficient of -0.625, or (B) it was assumed that blood pH was constant at pH 7.800 since the degree of relative alkalinity to pH of neutrality should remain constant as temperature changes (e.g., Rahn, 1967). ΔH' values: 16 kJ mol-1, calculated for skipjack tuna (Katsuwonus pelamis) (Figure 2.5); 0 kJ mol-1, calculated for yellowfin tuna (Thunnus albacares) (Figure 5); -19 kJ mol-1, estimated for bigeye tuna (Thunnus obesus) using P50 values and Bohr coefficients presented in Lowe et al. (2000); -35 kJ mol-1 for rainbow trout (Oncorhynchus mykiss) at pH 7.8 (Weber et al., 1976).   83           Figure 2.8 The relationship between hæmoglobin (Hb)-heat transport and the enthalpy of Hb-oxygenation (ΔH' kJ mol-1).  Hb-heat transport is ΔH' expressed as a percentage of the oxycalorific equivalent for glucose metabolism (473 kJ mol-1). Values for specific species are indicated by solid circles. Rainbow trout values are from Weber et al. (1976), human values at pH 7.4 were determined with data reported by Reeves (1980), and yellowfin and skipjack tuna values were determined with data from Brill and Bushnell (1991; Figure 5). ΔH' values were determined with P50 values, although in some species ΔH' is saturation dependent. The intrinsic enthalpy of hæme oxygenation (ΔHO2) for blue marlin and human HbA were determined in purified Hb solutions and were taken from Weber at al. (2010) and Atha and Ackers (1974), respectively. Negative Hb-heat transport values indicate that heat absorbed upon deoxygenation in the tissues is released upon oxygenation in the lungs or gills, whereas positive values indicate no heat loss associated with Hb-oxygenation.  84    Table 2.1 Input data used to model ṀO2max in rainbow trout and yellowfin tuna. 1 Variable determined with the model.  2 Variable taken from or calculated from literature data: rainbow trout, Kiceniuk and Jones (1977); yellowfin tuna, Korsmeyer et al. (1997a). 3 Rainbow trout P50, Hill coefficient, and Bohr coefficient are from (Weber et al., 1976). Physiological variable Symbol Unit Rainbow trout Yellowfin tuna Temperature T °C 10 25 1Diffusion conductance, gill Gd µmol min-1 kg-1 mmHg-1 2.07 2.66 1Diffusion conductance, tissue Gdt µmol min-1 kg-1 mmHg-1 4.82 4.70 2Ventilation  V̇w mL min-1 kg-1 1730 3300 2Cardiac output Q̇ mL min-1 kg-1 52.6 116.6 O2 solubility, water 𝛼𝑤O2 µmol L-1 mmHg-1 2.240 1.368 O2 solubility, blood plasma 𝛼𝑏O2 µmol L-1 mmHg-1 1.986 1.510 Inspired PO2 PI mmHg 150 150 2Arterial PO2 Pa mmHg 126 89 2Mixed venous PO2 Pv mmHg 16 39 2Hb concentration (tetrameric) CHb mmol L-1 1.06 1.83 2Arterial O2 content Ca mmol L-1 4.33 6.60 2Arteriovenous O2 content difference Ca – Cv mmol L-1 3.70 3.21 3Blood/Hb-O2 affinity, gill P50gill mmHg 27 25 Blood/Hb-O2 affinity, tissue P50tissue mmHg 40 44 3Hill coefficient  n  2 1.6 3Bohr coefficient  ϕ  -0.52 -0.62 Enthalpy of Hb-oxygenation ΔH' kJ mol-1 -11.3 0 1Mean PO2 gradient, gill Δ(PO2gill) mmHg 79.4 70.7 1Mean capillary PO2, gill P̄cgill mmHg 51.4 58.6 1Mean capillary PO2, tissue P̄ctissue mmHg 39.9 56.8 2Maximum rate of O2 transport ṀO2max mmol min-1 kg-1 0.194 0.375 85  Table 2.2 Slopes of linear regressions presented in Figure 2.3, representing the magnitude of the effect of changes in each variable on ṀO2max in rainbow trout and yellowfin tuna.  Variable Rainbow trout Yellowfin tuna V̇w 0.039 0.033 Gd 0.185 0.115 Gdt 0.277 0.388 P50tissue 0.365 0.800 CHb 0.474 0.461 Q̇ 0.499 0.465 86  Chapter 3 The Effect of Temperature on Hæmoglobin-Oxygen Affinity of Swordfish (Xiphias gladius) and Smalleye Pacific Opah (Lampris incognitus) “[T]emperature invariance of oxygen binding is not some second-order effect, but is of primary importance to these fish.” (Francis G. Carey, 1982a, p. 228) 3.1 Introduction  The swordfish (Xiphias gladius) and the smalleye Pacific opah (Lampris incognitus) are both highly active and regionally heterothermic species. While the opah is capable of a whole-body form of endothermy and can heat the cranial region and entire body core including the heart, the swordfish (family Xiphiidae) and other billfishes (family Istiophoridae) are often referred to as “regional endotherms” because they heat the eye and brain region only (i.e., cranial endothermy) (Block, 1986; Block, 1991b; Carey, 1982b; Runcie et al., 2009; Wegner et al., 2015). Endothermy probably provides the opah and the swordfish with crucial advantages over their thermoconforming prey, since both species spend most of the day in cold water deep below the thermocline (Carey, 1990; Carey and Robinson, 1981; Dewar et al., 2011; Sepulveda et al., 2010; Wegner et al., 2015), where warm eyes will enhance temporal resolution (Fritsches et al., 2005) and the opahs warm heart and swimming muscles likely enhance aerobic performance.   In opah and billfishes, metabolic heat generated by the extraocular muscles is conserved with a heat exchanging rete that arises from the carotid artery, warming the eyes and brain (Block, 1986; Carey, 1982b; Runcie et al., 2009). Warm blood perfuses the entire body of the opah, including the heart, due to a series of heat exchanging retia within the gills as well as insulting fat layers around the gill retia and the body core, which conserve the heat produced by the continuous contraction of the swimming muscles (Wegner et al., 2015). The cranial retia 87  effectively maintain swordfish cranial temperatures relatively constant and elevated as much as 12°C above ambient water temperature when swordfish are in deep cold water (Figure 3.1) (Carey, 1990). Cranial temperatures in the opah are also relatively constant and elevated at least 6°C above the surrounding water, while body and heart temperature are elevated at least 3 to 5°C (Figure 3.1) (Wegner et al., 2015).   A consequence of efficient heat exchange within the circulation is the establishment of large internal temperature gradients (i.e., regional heterothermy), which are exacerbated when opah and swordfish dive below the thermocline into cold water (Figure 3.1). During daily sojourns between warm and cold waters above and below the thermocline, respectively, swordfish may experience water temperatures from as low as 4°C to as high as 30°C in less than a few hours, in addition to possibly low environmental oxygen levels in deep water (Carey, 1990; Carey and Robinson, 1981; Dewar et al., 2011; Sepulveda et al., 2010). Therefore, blood-O2 uptake at the gills must occur over the range of environmental temperatures that are encountered by opah and swordfish, but blood-O2 transport occurs over the steep internal temperature gradients between the cold gills and warmest tissues.  As reviewed in Chapter 1, the O2 affinity of most jawed vertebrate Hbs typically decreases with increasing temperature, but regional heterotherms tend to have Hbs with a reduced or even reverse temperature-dependence (Weber and Campbell, 2011). In one of the first comparative studies of Hb from regionally heterothermic fishes, Andersen et al. (1973) reported that increasing temperature greatly decreased the O2-affinity of swordfish Hb (i.e., a normal temperature-dependence), but the blood pH and detailed methods were not reported in that study. However, Weber et al. (2010) reported an ATP-induced temperature-independence of P50 in hæmolysates from three billfishes that are closely related to swordfish: striped marlin (Kajikia 88  audax = Tetrapturus audax), blue marlin (Makaira nigricans), and shortbill spearfish (Tetrapturus angustirostris) (reviewed in Chapter 1, section 1.3.2). Thus, a more thorough investigation of the effect of temperature on blood and Hb-O2 affinity in swordfish is warranted. I am not aware of any O2 equilibria studies on Hb or blood from any of the opah species.    In this Chapter I address the following question: How does temperature affect Hb-O2 affinity in the swordfish and the smalleye Pacific opah? If temperature-independent Hb-O2 affinity is necessary to maintain regional heterothermy, then I expect that both the swordfish and the opah should have Hbs that are insensitive to temperature. I address this by constructing OECs at different temperatures in whole blood and hæmolysates, and by measuring the effect of closed-system temperature changes on blood PO2. Furthermore, since swordfish and other billfishes are closely related and share similar anatomy and physiology related to their capacity for endothermy, I expect that swordfish Hb would exhibit an ATP-induced temperature independence like previously studied billfish Hbs. I also present data from Atlantic bluefin tuna hæmolysates to compare the enthalpic contributions of oxygenation linked effector dissociation among the different lineages of regionally heterothermic teleosts.  3.2 Methods  All capture, handling, and experimental procedures followed guidelines approved by the University of Massachusetts (animal care protocol no. 13-06), the California Department of Fish and Wildlife (Scientific Collection permit no. SC-2471), and the University of British Columbia (UBC) Animal Care Committee (animal care no. A11-0235 and A15-0266). Blood collection 89   Swordfish (n = 7) were captured by deep-set buoy gear (Sepulveda et al., 2014), opah (n = 4) were captured by hook and line in the coastal waters off Southern California (i.e., the Southern California Bight), and Atlantic bluefin tuna (n = 2) were captured by hook and line off Massachusetts (fork lengths are reported in Table 3.1). Blood was drawn by caudal puncture into heparinized syringes. Blood samples were kept on ice and shipped by courier to the UBC campus in Vancouver, Canada, where experiments were conducted within 1 to 4 days after the blood was collected. Preliminary experiments with swordfish blood showed no changes in Hb concentration, hæmatocrit (Hct; the proportion of red blood cells in blood), plasma pH, or Hb-O2 affinity (i.e., P50, the PO2 at 50% Hb-O2 saturation), and no evidence of red blood cell (RBC) lysis for up to 6 days after blood was collected, provided blood was refrigerated during this time (see Appendix).  Experimental protocol   Immediately after blood samples arrived at UBC, Hb concentration and Hct were measured, and subsamples of blood were centrifuged to separate the plasma from the red blood cells (RBCs) for measurement of plasma osmolality. The packed RBCs and remaining plasma were frozen at -80°C for determination of RBC intracellular ATP concentration and plasma lactate concentration. Whole blood oxygen equilibrium curves (OECs) were constructed by quantifying the relative Hb-O2 saturation at a range of equilibration PO2’s at two CO2 levels and two (swordfish) or three (opah) temperatures. Blood pH, and PO2 were measured in subsamples of blood equilibrated with gas mixes at each of the OEC temperature treatments. After completing the whole blood experiments, RBCs were separated from blood plasma by centrifugation, then the RBCs were thrice rinsed in ice cold saline and frozen at -80°C for 90  experiments on stripped hæmolysates. OECs were constructed for stripped hæmolysates in the presence and absence of effector ions at two different temperatures for each species (see below).  Hæmatological parameters  Hæmoglobin concentration was measured by the cyanmethæmoglobin method using Drabkin’s reagent and a hæm-based extinction coefficient of 11 mmol-1 cm-1 (Völkel, and Berenbrink, 2000). All Hb concentrations are expressed as tetrameric Hb ([Hb], in mM). Hct was measured as the percentage of packed RBCs relative to total blood volume after centrifuging samples at 11,500 rpm for five minutes. Mean corpuscular hæmoglobin concentration (MCHC, in mM) was calculated by dividing [Hb] by Hct. Plasma osmolality (mOsm kg-1) was measured in 10µL of undiluted plasma with a vapour pressure osmometer (VAPRO 5520, Wescor, Logan, Utah). ATP was assayed with a colourimetric assay kit (SIGMA-ALDRICH MAK190, Sigma-Aldrich Co. LLC, St. Louis, Missouri), and plasma lactate was measured spectrophotometrically using  the  LDH-catalyzed  reaction  converting  lactate  to pyruvate, where the reduction of NAD+ to NADH was measured at 340 nm  (Bergmeyer et al., 1983). Whole blood oxygen equilibria, pH, and PO2  Oxygen equilibria experiments were conducted at temperatures that correspond near to the coldest environmental temperatures and the warmest tissue temperatures in the opah and the swordfish. The coldest experimental temperature was 10°C, which is close to the coldest water temperature regularly encountered by both the swordfish and the opah (Sepulveda et al., 2010; Wegner et al., 2015). The warmest experimental temperature was 25°C for swordfish (i.e., near the warmest cranial and water temperatures), and 15°C and 20°C for the opah (near the warmest body and cranial/water temperatures, respectively) (Carey, 1990; Sepulveda et al., 2010; Wegner 91  et al., 2015). At each temperature treatment, experiments were conducted at two physiologically relevant CO2 levels, 0.25% CO2 and 1.00% CO2, to manipulate blood pH at a high and low level to quantify the Bohr coefficient (i.e., Δ𝑃50Δ𝑝𝐻).   The relationship between Hb-O2 saturation and PO2 (i.e., an OEC) was assessed on replicate samples using a custom microplate-based, parallel assay, multi-cuvette tonometry cell as described by Lilly et al. (2013). Cuvettes were formed by sandwiching blood samples (~ 3μL) between two sheets of low density polyethylene (Glad® ClingWrap) that were secured on an aluminum ring with two plastic O-rings, which were then placed in a gas tight tonometry cell modified to fit into a SpectraMax 190 microplate reader (Molecular Devices, Sunnyvale, USA). Optical density (OD) was measured every 20 to 30 seconds at 390nm (an isosbestic point where OD is independent of Hb-O2 saturation), and at 430 nm and 436 nm (wavelengths that typically correspond to a maximum absorbance for deoxygenated Hb). Initially, blood was equilibrated with pure N2 for a minimum of 30 minutes until OD at 430/436 nm was stable, which was assumed to indicate full Hb deoxygenation. After deoxygenation, the Hb-O2 saturation was increased with stepwise increments of the O2 tension, balanced with N2, up to 21% O2. Full Hb-O2 saturation was assumed after a final increment to 30% O2 in the absence of CO2. Gas mixtures of O2, CO2, and N2 were obtained using a Wӧsthoff DIGAMIX® gas mixing pump (H. Wösthoff Messtechnik, Bochum, Germany). Fractional Hb-O2 saturations were calculated for each equilibration step as the change in ΔOD (ΔOD = 430 nm or 436 nm - 390 nm) from full deoxygenation, relative to that between full deoxygenation (pure N2) and full oxygenation (30% O2).   Whole blood pH was measured in approximately 500 μL of blood equilibrated for 1 h with either 0.25 or 1.00% CO2 and a range of O2 tensions (balanced with N2) in rotating glass 92  tonometers thermostated to either 10, 15, 20, or 25°C. Blood was drawn into a gas tight syringe pre-flushed with the gas mixture, and pH was measured by drawing the blood through a Microelectrodes 16-705 flow-thru pH electrode in combination with a 16-702 flow-thru reference electrode (Microelectrodes Inc., Bedford, NH, USA) thermostated to the experimental temperature.  To mimic the closed-system temperature changes that blood experiences in the arterioles and venules of a heat exchanging rete mirabile, approximately 500 μL blood samples equilibrated at either 10, 20, or 25°C were injected into a pH electrode (as described above) and a Radiometer E5046 PO2 electrode thermostated at the equilibration temperature as well as another pair of electrodes thermostated to a warmer or cooler experimental temperature according to Cech et al. (1984) and Brill and Bushnell (1991a). Although the blood was static within the electrode chamber, the blood was rapidly heated or cooled in a system where there is minimal exchange of gases and ions between the blood and another medium. Prior to injecting the blood, each PO2 electrode was flushed with the experimental gas mixture to minimise electrode response time to the respective PO2. Temperature induced changes in pH and PO2 were monitored using data acquisition software, and when it appeared that pH and PO2 traces had stabilized, the respective values of each were recorded.  Hæmolysates   Frozen and packed RBCs were slowly thawed on ice (>24 h) then mixed with an equal volume of cold 0.1 mM Hepes buffer (pH 8.0) and centrifuged at 10,000 RCF to remove cell debris. The resulting erythrolysates were stripped of endogenous ionic effectors by passage through mixed bed ion exchange resin (Amberlite® MB-20). MetHb (Hb+) levels were assessed by oxygenating 10-20 µL of the hæmolysates in 1000 µL of 100 mM Hepes buffer (pH 7.4) that 93  was bubbled with 100% O2, and a spectral scan was made from 500-700 nm (i.e., an oxyHb spectrum). If there was an evident peak or unusually high absorbance at 630 nm, an absorption maximum for metHb, then the Hb+ was reduced by adding a molar excess of sodium hydrosulfite to the hæmolysate, followed by passage through mixed bed ion exchange resin. Hæmolysates were concentrated with centrifugal filters (30 kDa). Oxygen equilibria were determined in 0.1 M Hepes buffer at a Hb concentration of 0.6 mM in the absence and presence of saturating levels of ATP (ratio of the concentration of ATP/Hb = 30). OECs were generated at 10 and 20°C for opah hæmolysates, and 10 and 25°C for swordfish hæmolysates, and 15 and 25° bluefin tuna hæmolysates following the procedures described above, except without CO2, and the final O2 equilibration step (i.e., full saturation) was with 100% O2. The pH of the hæmolysate solutions was measured at the experimental temperature with a thermostated Mettler Toledo InLab Micro glass pH electrode (Mettler-Toledo LLC, Columbus, OH, USA).  Data analysis   All statistical analyses and curve fitting were performed in R v 3.5.2 (R Core Team, 2017). An oxygen equilibrium curve (OEC) was constructed for each blood or hæmolysate sample by fitting a three-parameter logistic (3PL) model to paired data of fractional Hb-O2 saturation (response variable) and PO2 (explanatory variable). The R-language formula for the OEC model was ‘HbO2 ~ d/(1 + exp(b*(log10(PO2) - log10(e)))’. The best-fit parameter values (b, d, and e) were used to calculate the PO2 values corresponding to specific Hb-O2 saturations (PS; i.e., P10,  P20, P30, P40, P50, P60, P70, P80, P90,and P95). Hill cooperativity coefficients were determined at P50 by differentiating the 3PL equation at P50. Because teleost blood pH is typically dependent on Hb-O2 saturation (Brauner et al., 1996; Lowe et al., 1998), OEC parameters for each individual were used to calculate Hb-O2 saturation at the equilibration O2 94  tensions, and the pH at a specific Hb-O2 saturation (pHS) was then estimated from plots of %Hb-O2 vs pH. The effects of pH and temperature on Hb-O2 affinity were assessed with linear mixed models, where the response variable was log10 PS (e.g., log10P50) and the explanatory variables were pHS (continuous), assay temperature (as a factor), the interaction term between pHS and assay temperature, and individual (id) as a random effect (R-language formula, ‘log10(PS) ~ pHS*temperature + (1|id)’). Mixed models were fit at each saturation from P10 to P95, and for each model a Likelihood Ratio Test (LRT) of fixed effects, fit with maximum likelihood estimation using a Chi square distribution, was used to assess the relative importance of temperature in the model (i.e., to test the null hypothesis that temperature is a significant effector of Hb-O2 affinity). Nonlinear least-squares curve fitting by the Levenberg-Marquardt algorithm was performed using the nlsLM function from the ‘minpack.lm’ package for R (Elzhov et al., 2010), and linear mixed models were fit using the lmer function from the ‘lme4’ package with the ‘lmerTest’ package (Bates et al., 2014; Kuznetsova et al., 2017).  The mixed model fits were used to predict PS values with bootstrap estimated standard errors (500 replications), and these were used to construct whole blood OECs at constant pH for each species temperature treatments. Predicted P50 values were used as a proxy for whole blood-O2 affinity. Hæmolysate PS values were calculated at specific pH values from mixed linear models fit to data for each temperature and effector treatment. The temperature-dependence of whole blood and hæmolysate O2 affinities were quantified by calculating ΔH' values using the van’t Hoff equation (Wyman, 1964): ∆𝐻′ = 2.303 ∙ 𝑅 ∙Δ𝑙𝑜𝑔𝑃SΔ1𝑇, 95  where R is the gas constant and T is the absolute temperature (Kelvin). Because ΔH' calculations requires that the concentration of allosteric effectors be known and the experimental conditions for Hb are carefully controlled (e.g., stripped hæmolysates), I consider the whole blood calculations as estimates at best. I denote the whole blood calculations as ΔH'WB, and for their calculation I determined PS values at constant pH and I assumed that RBC intracellular concentrations of other allosteric effector were constant. The heat of solution of O2 (~12.6 kJ mol-1) is included in whole blood ΔH'WB values, but excluded from hæmolysate ΔH' values. The pH dependency of Hb-O2 affinity was determined by calculating Bohr coefficients at different %Hb-O2 saturations (PS): 𝜑 =Δ log10 𝑃SΔpH where φ values are the slopes (± 95% confidence intervals) from the fitted models of log10PS vs pH values. 3.3 Results  Species lengths and blood parameters are summarized in Table 3.1. Whole blood OEC’s were successfully constructed for four opah and five swordfish. Hæmolysate experiments were conducted on samples from three opah, six swordfish, and the two bluefin tuna.  Whole-blood experiments   Whole blood P50 values at pH 7.7 [an approximation of arterial blood pH from measurements reported from swimming yellowfin tuna (Korsmeyer et al., 1997a)], Hill coefficients, and Bohr coefficients are reported in Table 3.2. Both the opah and the swordfish have relatively high blood-O2 affinities (i.e., low P50 values), and large Bohr coefficients. Hill coefficients tended to decline with decreasing pH, and although temperature did not influence n50 96  values for opah, they were highest at 10°C in swordfish blood (χ2 = 4.209 df = 1, P = 0.0402). Whole blood OECs at pH 7.7 and lower pH levels that correspond to temperate dependent pH shifts are presented in Figure 3.2.   In opah whole blood, temperature had a negligible effect on blood-O2 affinity below 90% saturation. At 90% saturation temperature was an important model factor (χ2 = 10.101 df = 4, P = 0.039), which is evident as a significant increase in blood-O2 affinity (decreasing P90) as temperature increased from 10 to 15°C, and no evident effect of temperature between 15 and 20°C. In opah blood at pH 7.7 and between 10 and 15°C, ΔH'WB values were negative (exothermic) at low saturation, but increased with increasing saturation, becoming positive (endothermic) above ~44% saturation. This transition from exothermic to endothermic ΔH'WB values occurred at lower saturations with lower blood pH (Figure 3.3). Between 15 and 20°C, ΔH'WB values were negative but near to zero at pH 7.7. At lower blood pH, ΔH'WB values were negative and quite exothermic below ~80% saturation (Figure 3.3), although below 90% saturation there were no apparent differences between PS values at 15 and 20°C (Figure 3.2).   In swordfish whole blood temperature had a negligible effect on blood-O2 affinity below 50% saturation, but above 50% saturation temperature and the interaction between temperature and pH were important model factors (P50: χ2 = 7.284, df = 2, P = 0.026; P60: χ2 = 12.532, df = 2, P = 0.002; P70: χ2 = 18.790, df = 2, P = 0.0000831; P80: χ2 = 17.621, df =2, P = 0.000149; P90: χ2 = 9.7449, df = 2, P = 0.007654; P95: χ2 = 7.605, df = 2, P = 0.02232). The effect of temperature on blood-O2 affinity was pH dependent in swordfish blood. At high pH, an increase in temperature decreased blood-O2 affinity above 50% saturation, evident as a right shift of the upper portion of the OECs (pH 7.7) between 10 and 25°C. The effect of temperature was reduced as pH declined, and around pH 7.4 temperature had a negligible effect on blood-O2 affinity. The 97  pH dependency of the effect of temperature on whole blood-O2 affinity is evident in plots of ΔH'WB as a function of saturation, where ΔH'WB becomes more endothermic with decreasing saturation and pH (Figure 3.3).  The effects of closed-system temperature changes on blood PO2 are shown in Figure 3.4. Closed-system warming of opah blood generally decreased blood PO2, due to increased Hb-O2 affinity with increasing temperature (i.e., a reverse temperature-dependence). Closed-system cooling of opah blood tended to increase blood PO2, but this effect was variable. In contrast, closed-system temperature changes of swordfish blood changed blood PO2 beyond that predicted by Henry’s law [i.e., increasing temperature will increase PO2 in a closed system due to a reduction in plasma O2 solubility and vice versa]. The change in PO2 due to the change in solubility between 10 and 25°C was subtracted from the total change in blood PO2 (ΔPO2) to estimate the contribution to ΔPO2 from temperature induced O2 unloading or binding to Hb (ΔPO2Hb). Values of ΔPO2Hb with warming did not show any evident trend with initial pH or equilibration PO2 (i.e., initial PO2), and averaged (± 95% CI) 12.2 (± 6.5) mmHg. This increase in PO2 was assumed to be due to temperature induced Hb-O2 unloading and would be caused by < 1% decrease in Hb-O2 saturation. With closed-system cooling of swordfish blood, ΔPO2Hb ranged from -34 to 6.5 and was correlated with the initial equilibration PO2 (Pearson’s r = -0.603, P = 0.0004) with greater decreases at higher equilibration PO2.   Hæmolysate experiments   Hill plots of the hæmolysate experiments are presented in Figure 3.5, and P50 and enthalpy values are summarized in Table 3.3. Opah stripped hæmolysates showed a very slight reduction in Hb-O2 affinity by increasing temperature from 10 to 20°C, whereas the O2 affinity of swordfish hæmolysates was greatly reduced by increasing temperature from 10 to 25°C. 98  Bluefin tuna hæmolysates showed a reverse temperature effect. The intrinsic O2 affinity of Swordfish hæmolysate was higher (i.e., lower P50) than those of opah and bluefin tuna. ATP greatly reduced the O2 affinities and temperature sensitivities of both opah and swordfish hæmolysates. The presence of ATP caused a reverse temperature-dependency of O2 affinity in Opah hæmolysates, resulting in positive ΔH' values (i.e., endothermic) (Figure 3.6 and Table 3.3). For swordfish hæmolysates, the effect of temperature on O2 affinity was both pH and ATP dependent, with ΔH' becoming increasingly more endothermic with declining pH (Figure 3.6). At pH 7.4 [an approximation of RBC intracellular pH (Weber et al., 2010)] and in the presence of ATP, the effect of temperature on swordfish hæmolysate O2 affinity was reversed above 30% saturation (Figure 3.6B).   Bohr factors were similar between temperature treatments for stripped hæmolysates of both opah and swordfish. The addition of ATP increased the effect of pH on Hb-O2 affinity, and caused Bohr factors to be higher at 10°C than at higher temperatures (Table 3.3).  3.4 Discussion Main findings  The purpose of this study was to investigate the temperature-dependence of Hb-O2 affinity in blood and stripped hæmolysates of two regionally heterothermic teleosts, the opah and the swordfish. I hypothesized that temperature-independent Hb-O2 affinity is associated with the evolution of regional heterothermy, so I expected that both the swordfish and the opah should have Hbs that are insensitive to temperature. I also conducted experiments on Atlantic bluefin tuna hæmolysates to compare the enthalpic contributions of oxygenation linked effector dissociation among the different lineages of regionally heterothermic teleosts.  99   The results show temperature-independence of Hb-O2 affinity in the opah, and a pH and saturation dependent effect of temperature on Hb-O2 affinity in the swordfish (Figures 3.2-3.6). Opah whole blood-O2 affinity showed a reverse temperature-dependence, which caused a reduction in PO2 when blood was warmed in a closed system (Figure 3.4). These findings were supported by the results of experiments on hæmolysates, which showed that opah Hb-O2 affinity was temperature-independent within a physiologically relevant pH range, and the addition of ATP caused a reverse temperature-dependence (Figures 3.5 and 3.6). Swordfish blood-O2 affinity decreased with increasing temperature, but the effect of temperature was dependent on O2 saturation and blood pH (Figures 3.2 and 3.3). These results were paralleled in closed system warming of swordfish blood, which caused blood PO2 to increase beyond what would be expected due to the temperature dependence of the O2 solubility of blood plasma (Figure 3.4). Experiments on swordfish hæmolysates showed an ATP induced temperature independence of Hb-O2 affinity below pH 7.5, like other billfishes (Figures 3.5 and 3.6) (Weber et al., 2010). Bluefin tuna hæmolysates showed a reverse temperature-dependence (Figures 3.5 and 3.6), in accordance with previous studies (Carey and Gibson, 1977; Ikeda-Saito et al., 1983). The results of this study exemplify the importance of evaluating how temperature influences the shape and position of the entire OEC, and that generalized conclusions “could be misleading if only P50 values are evaluated” (Sharp, 1975). Whole blood and closed-system temperature changes   Opah and swordfish have relatively high Hb-O2 affinities, with whole blood P50 values around 18 mmHg in the opah and 12 mmHg in the swordfish at pH 7.7 and 10°C. These relatively low P50 values may ensure adequate O2 uptake when opah and swordfish are at depths near the oxygen minimum layer (Figure 3.1) (Sepulveda et al., 2010; Wegner et al., 2015). The 100  ΔH'WB values for opah and swordfish blood P50 at pH 7.7 were around 1 kJ mol-1 O2 and -23 kJ mol-1 O2, respectively.   Temperature induced shifts of the swordfish OEC were observed during closed-system warming and cooling, which caused respective increases and decreases to the blood PO2 (Figure 3.4) that were similar to previous reports from bigeye tuna blood (Figure 1.6) (Lowe et al., 2000). The closed-system PO2 changes observed in bigeye tuna and swordfish blood exceed the PO2 changes expected in plasma, indicating that O2 is being released or bound to Hb with warming and cooling, respectively. The swordfish ΔH'WB value is close to that for bigeye tuna (~ -18 kJ mol-1 O2), which likely underlies the similar closed-system results for these species (Lowe et al., 2000). Furthermore, if swordfish blood is returning to the gills around 50% saturated and leaving the gills around 95% saturated, then the average enthalpy of oxygenation might be around -37 kJ mol-1, potentially causing about 8% of the heat produced during glucose metabolism to be liberated and lost to the inspired water during Hb-oxygenation. This heat loss may prevent efficient heat retention in a large tissue mass such as the red muscle, which may contribute to why the red muscle retia of swordfish are not as efficient at heat retention as those of tunas and sharks and likely serve to just slow down cooling of the red muscle (Carey, 1990; Stoehr et al., 2018).   The temperature-independence and reverse temperature-dependence exhibited by opah blood during closed system warming and cooling is probably necessary for O2 transport to the tissues, which may range from cold ambient temperatures as low as 8°C to the warmest tissues that are around 17°C. Over this temperature range, opah blood PO2 should remain relatively stable throughout the circulation. Furthermore, the enthalpy of oxygenation for opah Hb-O2 101  binding from 50% to 95% saturation would be about +22 kJ mol-1, which should eliminate any loss of metabolic heat during Hb-oxygenation.  Molecular mechanisms of reduced temperature dependence of Hb-O2 affinity  Reductions in the thermal sensitivities of Hb-O2 affinity results from oxygenation-linked effector dissociation that contribute endothermically to ΔH', but the relative contributions of different effectors vary among lineages of regional heterotherms. In swordfish, the primary effector is ATP with secondary contributions from protons, which is the same as other billfishes in the family Istiophoridae (Weber et al., 2010). In billfishes, ATP binding reduces Hb-O2 affinity below pH 8.0 and causes Hb-O2 affinity to be temperature-independent above 50% O2 saturation and within the physiological RBC intracellular pH range (~ pH 7.0 – 7.5; Figure 3.6) (Weber et al., 2010). At low temperature ATP is likely preferential bound at high O2 saturations, but rising temperature causes dissociation of ATP and protons from Hb, stabilizing the high affinity conformation and causing ΔH' to approach zero or become positive. Although ATP had large enthalpic contributions to ΔH' in opah hæmolysates, temperature appears to have no effect on stripped hæmolysate O2 affinity in opah, at least within a physiologically relevant pH range (Figure 3.6). Thus, the decreased effect of temperature on Hb-O2 affinity is likely due to temperature-dependent dissociation of protons that stabilizes the high-affinity conformation at higher temperatures and increases ΔH' to 0 kJmol-1. Preferential binding of ATP at low temperature probably stabilizes the low affinity conformation, but increasing temperature causes dissociation of ATP, probably decreasing the allosteric constant, L, by stabilizing the high affinity R-state conformation. The enthalpic contribution of ATP dissociation will cause ΔH' to become positive. Protons are the main effector of Atlantic bluefin tuna Hbs, consistent with previous work (Ikeda-Saito et al., 1983). Therefore, although the molecular mechanisms 102  underlying reductions in the temperature sensitivity of Hb-O2 affinity are similar among regionally heterothermic teleosts, the varying roles of allosteric effectors on reducing ΔH' indicate the independent evolution of temperature insensitivity among tunas, billfishes, opah. Elucidation of the effector binding sites and the amino acid arrangements and mutations that gave rise to them among different lineages of regionally heterothermic teleosts is worthy of future research. Possible limitations  My main objective was to construct OECs at temperatures close to the minimum and maximum temperatures that the blood would experience in vivo. Swordfish make diel, vertical movements from the warm upper mixed layer to cold waters below the thermocline, subjecting themselves to ambient temperatures from 4 to 30°C while maintaining relatively stable cranial temperatures from around 19 to 24°C (Carey, 1990; Dewar et al., 2011; Sepulveda et al., 2010). Although opah occasionally swim into warm water of around 20°C in the warm upper mixed layer, they tend to remain in cooler water below the thermocline where they maintain pectoral muscle temperatures around 14 to 17°C, or around 5°C above ambient (Wegner et al., 2015). Cranial temperatures measured in decked opah have been reported to average around 21°C, although this may be warmer than experienced in free swimming fish (Runcie et al., 2009). The experimental temperatures (10, 15 and 20°C for opah, and 10 and 25°C for swordfish) encompass the low and high ambient and body temperatures reported for these two species, and these temperatures are also  comparable to previous studies on closely related or ecologically similar species (Andersen et al., 1973; Brill and Bushnell, 2006; Lowe et al., 2000; Weber et al., 2010). The experimental temperatures used for the Atlantic bluefin tuna hæmolysates matched those used for bluefin tuna blood by Brill and Bushnell (2006). A potential limitation is that fish 103  were captured at-sea because it was unrealistic to sample resting and cannulated swordfish and opah. The individuals included in this study had likely experienced varying levels of exercise induced fatigue and consequent respiratory and metabolic acidosis, which are indicated by relatively high plasma lactate levels (Table 3.1). However, the blood pH levels achieved with the CO2 exposures were within physiologically relevant ranges, which allowed me to estimate blood PS at physiologically relevant pH values. The main objective of this chapter was to investigate the effect of temperature on Hb-O2 affinity, and it is very unlikely that any exercise or stress induced physiologically changes would have compromised the thermodynamics of Hb-O2 binding in whole blood. However, my measurements of RBC intracellular ATP levels were relatively low, and if in vivo ATP:Hb ratios are higher in swordfish blood, then the effect of temperature on Hb-O2 affinity would likely be reduced at higher pH than I measured in vitro. Nonetheless, our results from whole blood experiments were corroborated by the results of experiments on hæmolysates purified of endogenous ionic effectors.  Summary   The results presented here show temperature-independent and reversed temperature-dependence of Hb-O2 affinity in blood and hæmolysates of opah, and temperature-independence at low pH in swordfish blood and hæmolysates. Reductions and reversals of the temperature sensitivity of Hb are likely associated with the evolution of heat exchanging retia mirabilia (Weber and Campbell, 2011) that facilitated the evolution of regional heterothermy among disparate lineages of teleosts and sharks (Block and Finnerty, 1994; Carey, 1982a; Dickson and Graham, 2004). Among regionally heterothermic teleosts, the effector ions that underlie the molecular mechanism of modulations to the enthalpy of Hb-O2 binding differ among the opah, 104  billfishes, and tunas, and this striking functional convergence is paralleled in the Hbs of other regionally heterothermic vertebrates with heat exchanging retia (Weber and Campbell, 2011).105                       Figure 3.1 Opah in vivo pectoral muscle temperature and swordfish in vivo cranial temperature while swimming at depth. Depth (black), water temperature (blue) and tissue temperature (red) profiles were redrawn with data from Wegner et al. (2015; Opah) and Carey (1990; Swordfish).   106                 Figure 3.2 Whole blood oxygen equilibrium curves (OECs) for the smalleye Pacific opah and the swordfish at different pH and temperatures. Blood PO2 values were interpolated (± bootstrap standard errors) from linear mixed models of logPO2 vs pH at specific Hb-O2 saturation levels (see Methods section). Opah OECs were constructed at 10°C (circles), 15°C (squares), and 20°C (triangles) at pH 7.7 (filled symbols and solid curves) and pH 7.5 (open symbols and dashed curves).  Swordfish OECs were constructed at 10°C (circles) and 25°C (squares) at pH 7.7 (filled symbols and solid curves), pH 7.5 (open symbols and dashed curves) , and pH 7.4 (open symbols plus a dot, and dash-dotted curves).107                       Figure 3.3 Predicted enthalpy of oxygenation (ΔH'WB) as a function of whole blood pH and whole blood Hb-O2 saturation for the smalleye Pacific opah and the swordfish. ΔH'WB values were calculated with the van’t Hoff isochore (see methods) at constant pH between 10-15°C and 15-20°C for opah blood, and between 10-25°C for swordfish blood. The blood-O2 tensions (PO2) at specific blood-O2 saturation levels at a given pH and temperature were determined from the data presented in Figure 3.1. 108                   Figure 3.4 Effects of closed-system temperature changes on the measured change in blood PO2 (∆PO2) in blood from the smalleye Pacific opah and the swordfish.  Opah and swordfish blood was equilibrated at a range of O2 tensions (Equilibration PO2) and then heated (filled symbols) or cooled (open symbols). Opah blood temperature was changed between either 10 and 15°C (triangles) or 15 and 20°C (circles), and swordfish blood was changed between 10 and 25°C. Dotted lines indicate the temperature induced ∆PO2 expected due to changes in solubility of blood plasma at a given equilibration PO2 (i.e., Henry’s Law) between 10 and 15°C (dashed), and 15 and 20°C (dotted) for opah blood, and between 10 and 25°C (dashed) for swordfish blood109           Figure 3.5 Hill plots of Hb-O2 equilibria of smalleye Pacific opah, swordfish, and Atlantic bluefin tuna at pH 7.4. Experiments were conducted on stripped hæmolysates in the absence of ATP (open symbols), and in the presence of ATP (filled symbols) at two temperatures. Low temperature data (circles) are at 10°C for opah and swordfish, and 15°C for bluefin tuna. Warm temperature data (squares) are at 20°C for opah, and 25°C for swordfish and bluefin tuna. Data points (± bootstrap standard errors) were predicted at pH 7.4 from linear mixed models of logPS vs pH (see Methods section). Experiments were conducted in 0.1 M Hepes buffer at Hb concentrations of 0.6 mM, and ATP was added at saturating concentrations (18 mM; ATP/Hb = 30). 110          Figure 3.6 Heats of oxygenation (ΔH') of stripped hæmolysates of smalleye Pacific opah, swordfish, and Atlantic bluefin tuna in the absence and presence of ATP. In panel A, ΔH' values were calculated with the van’t Hoff equation (see Methods) with logP50 values that were interpolated from logP50/pH relationships. ΔH' values in panel B were calculated from data reported in Figure 3.4, and plotted as function of Hb-O2 fractional saturation at pH 7.4. Experiments were conducted in 0.1 M Hepes buffer at Hb concentrations of 0.6 mM, and ATP was added at saturating concentrations (18 mM; ATP/Hb = 30). ΔH' values exclude the heat of O2 in solution (-12.6 kJ mol-1). 111  Table 3.1 Fork length (FL), and blood variables for smalleye Pacific opah, swordfish, and Atlantic bluefin tuna. Adenosine triphosphate (ATP) concentration (mM) is reported relative to mean corpuscular hæmoglobin concentration (MCHC). Values are means ± standard error with samples sizes in parentheses. If values were measured in only one or two individuals, then the individual measurements are reported.     Opah Swordfish Bluefin Tuna FL (cm) 116 149.5 ± 12.4 (4) 107, 109 Hæmatocrit (%) 59.1 ± 2.5 (4) 46.3 ± 4.7 (7) 57.8, 53.5 Hæmoglobin (mM) 2.21 ± 0.12 (4) 1.81 ± 0.15 (7) 2.53, 2.36 MCHC (mM) 3.74 ± 0.14 (4) 3.96 ± 0.20 (7) 4.37, 4.41 ATP:Hb 1.32, 0.68 0.67 ± 0.16 (7) 0.40, 0.54 Plasma osmolality (mOsm/kg) 565.2 ± 6.4 (3) 437.5 ± 19.9 (7) 410, 379 Plasma lactate (mM) 14.7 ± 0.79 (3) 12.0 ± 1.53 (6) 18.7, 18.6 112  Table 3.2 Whole blood oxygen equilibria parameters for smalleye Pacific opah and swordfish at different temperatures.  Whole blood P50 (PO2 at 50% Hb-O2 saturation) is reported at pH 7.7, a proxy for arterial blood pH, and was predicted from linear mixed models of logP50 vs pH (see Methods section). Bohr coefficients were determined as the the model slopes (i.e., ΔlogP50/ΔpH). Hill cooperativity coefficients (n50) were determined at P50 and estimated at pH 7.7 from mixed linear models of n50 vs pH. P50 and n50 values are reported with bootstrap estimated standard errors, and Bohr coefficients are reported with the 95% confidence intervals for the slopes of the linear mixed models.   Opah  Swordfish  10°C 15°C 20°C  10°C 25°C P50 (mmHg) at pH 7.7 (SE) 17.9 (15.7 to 20.4) 17.7 (15.5 to 20.2) 17.9 (15.7 to 20.4)  12.1 (10.3 to 14.3) 19.6 (17.1 to 22.5) n50 at pH 7.7 (SE) 2.03 (0.15) 2.14 (0.15) 2.26 (0.15)  1.57 (0.10) 1.36 (0.09) Bohr coefficient (95% CI) -0.49 (-0.77 to -0.20) -0.46 (-0.75 to -0.11) -0.64 (-1.09 to -0.49)  -1.29 (-1.80 to -0.80) -0.65 (-1.23 to -0.01) 113  Table 3.3 Hæmolysate oxygen equilibria parameters of smalleye Pacific opah, swordfish, and Atlantic bluefin tuna at two experimental temperatures and in the absence or presence of ATP. P50 values (± bootstrap standard errors) were predicted at pH 7.4 from linear mixed models of logP50 vs pH (see Methods section), and Bohr coefficients are the model slopes (i.e., ΔlogP50/ΔpH) with their 95% confidence intervals. ΔH' values were calculated with logP50 values at pH 7.4, and excludes the heat of O2 in solution (-12.6 kJ mol-1 O2).   Opah  Swordfish  Bluefin tuna  10°C 20°C  10°C 25°C  15°C 25°C P50 (mmHg), pH 7.4         Stripped Hb  6.13 (± 1.40) 7.32 (± 1.45)  2.02 (± 0.30) 4.39 (± 0.73)  20.58 (± 2.31) 13.57 (± 1.57) Hb + ATP 102.45 (± 4.57) 30.31 (± 12.74)  28.71 (± 1.54) 34.26 (± 2.37)    Bohr coefficients         Stripped Hb  -0.50 (-0.69 to –0.34) -0.40 (-0.55 to -0.25)  -0.26 (-0.37 to -0.15) -0.12 (-0.24 to 0.02)  -0.71 (-0.91 to -0.51) -0.66 (-0.89 to -0.45) Hb + ATP -2.78 (-3.15 to -2.42) -2.04 (-2.45 to -1.65)  -1.28 (-1.44 to -1.12) -0.98 (-1.15 to -0.79)    ΔH' (kJ mol-1), pH 7.4         Stripped Hb  0.28  -23  42 Hb + ATP 97  4    114  Chapter 4 The Effect of Temperature on Hæmoglobin-Oxygen Affinity in Sharks “The ability to swim swiftly, achieved through the extra power available from warm muscles, must have been a powerful selective advantage in the evolution of tuna and lamnid sharks. Starting with the basic features of a fish vascular system the stringent requirements of speed have resulted in the remarkable parallel evolution of almost identical systems in two completely unrelated groups of fish.” (Francis G. Carey and John M. Teal, 1969a, p. 203) “[A]lthough the lamnid sharks are powerful and vigorous predators, they are also delicate animals.” (Francis G. Carey et al., 1985, p. 93) 4.1 Introduction  Sharks in the family Lamnidae (salmon shark, Lamna ditropis, porbeagle shark, Lamna nasus, shortfin mako, Isurus oxyrinchus, longfin mako, Isurus paucus, white shark, Carcharodon Carcharias), as well as the common thresher shark (Alopias vulpinus) have evolved specialised circulatory systems that retain the heat produced by their RM, which are internalized relative to other sharks (Figure 1.3) (Bernal et al., 2001b; Bone and Chubb, 1983; Burne, 1924; Carey and Teal, 1969a; Carey et al., 1985; Patterson et al., 2011). In these regionally heterothermic sharks, the bulk of the systemic circulation is directed laterally through subcutaneous arteries and veins, which redirect the blood supply to the medially situated RM through a heat exchanging rete mirabile on either side of the body. Lamnid sharks also have warm brains, eyes, and viscera that are all supplied with blood that passes through additional heat exchanging retia in transit to those organs (Alexander, 1998; Bernal et al., 2001b; Block and Carey, 1985; Burne, 1924; Carey et al., 1981; Carey et al., 1985; Tubbesing and Block, 2000). Interestingly, the bigeye thresher shark 115  (Alopias superciliosus), a close relative of the common thresher shark, has unusually large eyes that are supplied with blood through an orbital rete suspected of having a heat exchanging function (Block and Carey, 1985; Block and Finnerty, 1994; Dickson and Graham, 2004; Weng and Block, 2004).   In the temperately distributed shortfin mako and common thresher shark, RM temperatures are elevated around 5 to 10°C over ambient in the mako, and around 2 to 5°C in the common thresher shark (Figure 1.4) (Bernal and Sepulveda, 2005; Carey and Teal, 1969a; Carey et al., 1985; Patterson et al., 2011). It is not clear if the bigeye thresher shark has warm eyes, since opportunistic measurements of bigeye thresher eye temperatures have shown them to be no warmer than ambient sea surface temperature (Diego Bernal and Chugey A. Sepulveda, personal communication). However, mako eye, brain, and visceral temperatures are also much warmer than ambient (Anderson and Goldman, 2001; Block and Carey, 1985; Carey et al., 1981). For example, mako stomach temperatures remain warmer than ambient water, even during repeated descents below the thermocline, and are on average about 4°C warmer than the surrounding water (Figure 4.1) (Sepulveda et al., 2004).   Like many other pelagic species, regionally heterothermic sharks encounter a wide range of water temperature during vertical movements through the water column or over long latitudinal migrations into temperate and subpolar waters (Bernal et al., 2009; Weng et al., 2005). The shortfin mako and the common thresher routinely descend below the thermocline into water that may be more than 10°C cooler than surface waters (Figure 4.1) (Cartamil et al., 2011; Cartamil et al., 2016; Holts and Bedford, 1993; Sepulveda et al., 2004), and bigeye thresher sharks subject themselves to an extremely wide range of water temperatures (~ 6 to 25°C) during diel vertical migrations (Figure 4.2) (Coelho et al., 2015; Nakano et al., 2003; Sepulveda et al., 116  2019; Weng and Block, 2004). Consequently, blood-O2 uptake at the gills must occur over the range of environmental temperatures that these sharks encounter, and movements into cold water increase internal temperature gradients. Furthermore, the bigeye thresher shark spends most of the day in deep, cold water near the oxygen minimum layer (OML) where the concentration of dissolved oxygen may be less than 2 mg L-1 (Figure 4.2), so a high Hb-O2 affinity may be necessary to extract O2 from the hypoxic water.    Since ΔH' is negative (i.e., exothermic) for most vertebrate Hbs, increasing blood temperature typically decreases Hb-O2 affinity. This can potentially benefit O2 unloading to warm active tissues, or O2 uptake in fishes in cold hypoxic water. However, most studied regionally heterothermic fishes have Hb with a greatly reduced or even reversed temperature-dependence (reviewed in Chapter 1, section 1.3). It may seem unlikely that Hbs from regionally heterothermic sharks would have evolved temperature-independent O2-affinty linked to enthalpic contributions from allosteric effectors, since sharks in general tend to have high Hb-O2 affinities, small Bohr effects, and lower concentrations of nucleoside triphosphates (NTPs) in their RBCs relative to teleosts (Morrison et al., 2015). However, ATP does affect the O2 affinity, the Bohr effect, and temperature sensitivity of shark Hbs (Larsen et al., 2003; Weber et al., 1983a), and Larsen et al. (2003) have shown that ATP is responsible for reversing the temperature-dependence of O2 affinity in porbeagle shark Hb. There are not many whole blood O2 equilibria studies of lamnid sharks, although Bernal et al. (2018) reported that shortfin mako whole blood-O2 affinity was almost temperature-independent. However, Bernal et al. (2018) reported sizable Bohr coefficients, in contrast to a previous report of pH independence in mako blood (Wells and Davie, 1985), and the measures of blood-O2 affinity were not reported at precise blood pH values, making it difficult to quantify the effect of temperature on blood-O2 affinity in mako 117  blood. I am not aware of any published studies on the Hb or blood of the common thresher shark or the bigeye thresher shark.  In this chapter I address the question of how does temperature affect Hb-O2 affinity in the common thresher shark and the bigeye thresher shark? If temperature-independent Hb-O2 affinity is necessary for sharks to maintain regional heterothermy, then like my expectations for the regionally heterothermic teleosts in Chapter 3, I also expect that the common thresher shark will have Hbs that are insensitive to temperature. The bigeye thresher makes diel movements from warm water above the thermocline to cold water deep below thermocline, and it also has an orbital rete, although its function is unclear. Therefore, I expect the bigeye thresher shark to have a high Hb-O2 affinity, and the temperature-dependence of bigeye thresher shark Hb may provide some insight into the functional significance of temperature-independent Hb-O2 affinity in regionally heterothermic fishes. Additionally, I have also conducted experiments of blood from shortfin mako sharks to add to the results of Bernal et al. (2018) on blood from three makos, to corroborate the findings of that study with a larger sample size. For comparison, experiments were also conducted on blood from two previously studied ectothermic sharks, the blue shark (Prionace glauca) and the spiny dogfish (Squalus acanthias) (Bernal et al., 2018; Wells and Weber, 1983).   4.2 Methods  All capture, handling, and experimental procedures followed guidelines approved by the University of Massachusetts (animal care protocol no. 13-06), the California Department of Fish and Wildlife (Scientific Collection permit no. SC-2471), and the University of British Columbia (UBC) Animal Care Committee (animal care no. A11-0235 and A15-0266). 118  Blood collection  Bigeye thresher sharks (n = 9) were captured by deep-set buoy gear (Sepulveda et al., 2014) in the coastal waters off Southern California (i.e., the Southern California Bight). Common thresher sharks (n = 2) and spiny dogfish (n = 8) were captured by hook and line off Massachusetts. Shortfin mako (n = 9) and blue sharks (n = 8) were captured by hook and line off Southern California or off Massachusetts. Blood was immediately drawn by caudal puncture into heparinized syringes. Blood samples were kept on ice and shipped by courier to the UBC campus in Vancouver, Canada, where experiments were conducted within 1 to 4 days after the blood was collected. Preliminary experiments with swordfish blood (Xiphias gladius) showed no changes in Hb concentration, hæmatocrit (Hct), or blood P50, and no evidence of RBC lysis for up to 6 days after blood was collected, provided blood was refrigerated during this time (see Appendix). Experimental protocol   Immediately after blood samples arrived at UBC, Hb concentration and Hct were measured, and subsamples of blood were centrifuged to separate the plasma from the RBCs for measurements of plasma osmolality. The packed RBCs and remaining plasma were frozen at -80°C for determination of RBC intracellular ATP concentration and plasma lactate concentration. Whole blood was kept in the fridge at 4°C and OECs were constructed by quantifying the relative Hb-O2 saturation at a range of equilibration PO2’s at two CO2 levels and two temperatures. Blood pH, and PO2 were measured in subsamples of blood equilibrated with gas mixes at each of the CO2 and temperature treatments.  Hæmatological parameters 119   Hæmoglobin concentration was measured by the cyanmethæmoglobin method using Drabkin’s reagent and a hæm-based extinction coefficient of 11 mmol-1 cm-1. All Hb concentrations are expressed as tetrameric Hb ([Hb], in mM). Hct was measured as the percentage of packed RBCs relative to total blood volume after centrifuging samples at 11,500 rpm for five minutes. Mean corpuscular hæmoglobin concentration (MCHC, in mM) was calculated by dividing [Hb] by Hct. Plasma osmolality (mOsm kg-1) was measured in 10µL of undiluted plasma with a vapour pressure osmometer (VAPRO 5520, Wescor, Logan, Utah). ATP was assayed with a colourimetric assay kit (SIGMA-ALDRICH MAK190, Sigma-Aldrich Co. LLC, St. Louis, Missouri), and plasma lactate was measured spectrophotometrically using  the  LDH-catalyzed  reaction  converting  lactate  to pyruvate, where the reduction of NAD+ to NADH was measured at 340 nm  (Bergmeyer et al., 1983). Whole blood oxygen equilibria, pH, and PO2  Oxygen equilibria experiments were conducted at temperatures that correspond near to the coldest environmental temperatures and the warmest tissue or environmental temperatures for each species. The coldest experimental temperature was 10°C for the bigeye thresher shark, and 15°C for the common thresher shark and the mako, which are within the range of the colder water temperatures regularly encountered by these species (Cartamil et al., 2016; Sepulveda et al., 2004; Sepulveda et al., 2019). The warmest experimental temperatures corresponded to the warmest water temperatures encountered by bigeye thresher sharks (25°C), and the warmest RM temperatures in the mako (25°C) and the common thresher sharks (22°C) (Bernal and Sepulveda, 2005; Carey and Teal, 1969a; Patterson et al., 2011; Sepulveda et al., 2019). At each temperature treatment, experiments were conducted at two physiologically relevant CO2 levels, 0.25% CO2 120  and 1.00% CO2 (0.25% and 0.50% CO2 for spiny dogfish), to manipulate blood pH at a high and low level to quantify the effect of pH on blood-O2 affinity (i.e., the Bohr effect).   The relationship between Hb-O2 saturation and PO2 (i.e., an OEC) was assessed on replicate samples using a custom microplate-based, parallel assay, multi-cuvette tonometry cell as described by Lilly et al. (2013). Cuvettes were formed by sandwiching blood samples (~ 3μL) between two sheets of low density polyethylene (Glad® ClingWrap) that were secured on an aluminum ring with two plastic O-rings, which were then placed in a gas tight tonometry cell modified to fit into a SpectraMax 190 microplate reader (Molecular Devices, Sunnyvale, USA). Optical density (OD) was measured every 20 to 30 seconds at 390nm (an isosbestic point where OD is independent of Hb-O2 saturation), and at 430 nm and 436 nm (wavelengths that typically correspond to a maximum absorbance for deoxygenated Hb). Initially, blood was equilibrated with pure N2 for a minimum of 30 minutes until OD at 430/436 nm was stable, which was assumed to indicate full Hb deoxygenation. After deoxygenation, the Hb-O2 saturation was increased with stepwise increments of the O2 tension, balanced with N2, up to 21% O2. Full Hb-O2 saturation was assumed after a final increment to 30% O2 in the absence of CO2. Gas mixtures of O2, CO2, and N2 were obtained using a Wӧsthoff DIGAMIX® gas mixing pump (H. Wösthoff Messtechnik, Bochum, Germany). Fractional Hb-O2 saturations were calculated for each equilibration step as the change in ΔOD (ΔOD = 430 nm or 436 nm - 390 nm) from full deoxygenation, relative to that between full deoxygenation (pure N2) and full oxygenation (30% O2).   Whole blood pH and PO2 were measured in approximately 100-200 μL of blood equilibrated for 1 h with either 0.25 or 1.00% CO2 (0.50% for spiny dogfish) and a range of O2 tensions (balanced with N2) in rotating glass tonometers thermostatted to either 10, 15, 22, or 121  25°C. Blood was drawn into a gas tight syringe pre-flushed with the gas mixture, and then the blood was injected into pH and PO2 electrodes thermostatted to the respective experimental temperature. Blood pH was measured by drawing the blood through a Microelectrodes 16-705 flow-thru pH electrode in combination with a 16-702 flow-thru reference electrode (Microelectrodes Inc., Bedford, NH, USA), and PO2 was measured with a Radiometer E5046 oxygen electrode.   To mimic the closed-system temperature changes that blood experiences in the arterioles and venules of heat exchanging retia mirabilia, approximately 100-200 μL blood samples equilibrated at either 10, 15, 22 or 25°C were injected into both a pH microelectrode and an oxygen electrode (as described above) thermostatted to a warmer or cooler experimental temperature according to previous studies (Bernal et al., 2018; Brill and Bushnell, 1991a; Cech et al., 1984). Although the blood was static within the electrode chamber, the blood was rapidly heated or cooled in a system where there is minimal exchange of gases and ions between the blood and another medium. Immediately prior to blood injection, each oxygen electrode was flushed and thus pre-equilibrated with the respective experimental gas mixture to minimise electrode equilibration time with the blood. Temperature induced changes in pH and PO2 (ΔPO2) were monitored using data acquisition software, and when it appeared that pH and PO2 traces had stabilized, the respective values of each were recorded.  Common thresher shark hæmolysate   Frozen and packed RBCs from one common thresher shark were slowly thawed on ice (>24 h) then mixed with an equal volume of cold 0.1 mM Hepes buffer (pH 8.0) and centrifuged at 10,000 RCF to remove cell debris. The resulting erythrolysate was stripped of endogenous ionic effectors by passage through mixed bed ion exchange resin (Amberlite® MB-20). MetHb 122  (Hb+) levels were assessed by oxygenating 10-20 µL of the hæmolysates in 1000 µL of 100 mM Hepes buffer (pH 7.4) that was bubbled with 100% O2, and a spectral scan was made from 500-700 nm (i.e., an oxyHb spectrum). The hæmolysate was concentrated with a centrifugal filter (30 kDa). Oxygen equilibria at 15 and 22°C were determined in 0.1 M Hepes buffer at a Hb concentration of 0.6 mM in the absence and presence of saturating levels of ATP (ratio of the concentration of ATP/Hb = 30). The experiments were also conducted in the absence or presence of 500 mM of urea, because elasmobranch tissues have high urea concentrations, and urea can have an antagonistic effect on the ATP sensitivity of elasmobranch Hbs (reviewed by Morrison et al., 2015). OECs were generated following the procedures described above, except without CO2, and the final O2 equilibration step (i.e., full saturation) was with 100% O2. The pH of the hæmolysate solutions was measured at the experimental temperature with a thermostated Mettler Toledo InLab Micro glass pH electrode (Mettler-Toledo LLC, Columbus, OH, USA).  Data analysis   All statistical analyses, curve fitting, and linear mixed model fitting were performed in R v 3.5.2 (R Core Team, 2017). Linear regressions were performed with GraphPad Prism version 6.01 (GraphPad Software, La Jolla California, USA). Nonlinear least-squares curve fitting by the Levenberg-Marquardt algorithm was performed using the nlsLM function from the ‘minpack.lm’ package for R (Elzhov et al., 2010), linear mixed models were fit using the lmer function from the ‘lme4’ package with the ‘lmerTest’ package (Bates et al., 2014; Kuznetsova et al., 2017).   Hæmoglobin concentration, Hct, and MCHC were compared among species by one-way analysis of variance, and differences among the species means were assessed by Tukey’s multiple comparison test. An oxygen equilibrium curve (OEC) was constructed for each blood or hæmolysate sample by fitting a three-parameter logistic (3PL) model to paired data of fractional 123  Hb-O2 saturation (response variable) and PO2 (explanatory variable). The R-language formula for the OEC model was ‘HbO2 ~ d/(1 + exp(b*(log10(PO2) - log10(e)))’.The best-fit parameter values (b, d, and e) were used to calculate the PO2 values corresponding to specific %Hb-O2 saturations (PS; i.e., P10,  P20, P30, P40, P50, P60, P70, P80, P90,and P95). Hill cooperativity coefficients (n50) were determined at P50 by differentiating the 3PL equation at P50.   The effects of pH and temperature on whole blood-O2 affinity were assessed with linear mixed models, where the response variable was log10 PS (e.g., log10P50) and the explanatory variables were pH (continuous), assay temperature (as a factor), the interaction term between pH and assay temperature, and individual (id) as a random effect (R-language formula, ‘log10(PS) ~ pH*temperature + (1|id)’). Mixed models were fit at each saturation from P10 to P95, and for each model a Likelihood Ratio Test (LRT) of fixed effects, fit with maximum likelihood estimation using a Chi square distribution, was used to assess the relative importance of temperature in the model (i.e., to test the null hypothesis that temperature is a significant effector of Hb-O2 affinity). Linear models were used to assess the relationship between common thresher shark hæmolysate-O2 affinity (response variable; log10 PS) and pH (explanatory variable) for each experimental treatment.   The mixed model fits (whole blood) were used to predict PS values with bootstrap estimated standard errors (500 replications), and these were used to construct OECs (whole blood) at constant pH (i.e., isohydric OECs) for each species temperature treatment. Hæmolysate PS values were calculated at specific pH values from mixed linear models fit to data for each temperature and effector treatment, and the curve fitting function of GraphPad Prism was used to determine the 95% CI’s of the interpolated values. The temperature-dependence of whole blood 124  and hæmolysate O2 affinities were quantified by calculating ΔH' values using the van’t Hoff equation (Wyman, 1964): ∆𝐻′ = 2.303 ∙ 𝑅 ∙Δ𝑙𝑜𝑔𝑃SΔ1𝑇, where R is the gas constant and T is the absolute temperature (Kelvin). Because ΔH' calculations requires that the concentration of allosteric effectors be known and the experimental conditions for Hb are carefully controlled (e.g., stripped hæmolysates), I consider the whole blood calculations as estimates at best. However, they are useful for comparisons among species and studies. I denote the whole blood calculations as ΔH'WB, and for their calculation I determined PS values at constant pH, and I assumed that RBC intracellular concentrations of allosteric effectors were constant. The heat of solution of O2 (~12.6 kJ mol-1) is included in whole blood ΔH'WB values, and excluded from hæmolysate ΔH' values. The pH dependency of Hb-O2 affinity was determined by calculating Bohr coefficients at different %Hb-O2 saturations (PS): 𝜑 =Δ log10 𝑃SΔpH where φ values are the slopes (± 95% confidence intervals) from the fitted models of log10PS vs pH values. 4.3 Results  Species lengths and blood parameters are summarized in Table 4.1. Hb concentration and Hct were not different between blue sharks and spiny dogfish, but were lower in these species compared to bigeye threshers and shortfin makos, and bigeye thresher values were lower than those for shortfin mako (Hb: F(3, 31) = 27.00, P < 0.0001; Hct: F(3, 31) = 17.72, P < 0.0001). MCHC was not different between shortfin makos and bigeye threshers, which both had higher 125  MCHC than blue sharks and spiny dogfish (F(3, 31) = 18.76, P < 0.0001). Hb concentration, Hct, and MCHC for the two common thresher sharks were within the range of values measured in shortfin makos. Blood pH was low in some individuals (at 0.25% CO2), and plasma lactate levels were generally high except for dogfish (Table 4.1).  Whole-blood experiments   Whole blood OEC’s were successfully constructed for seven shortfin mako sharks, two common thresher sharks, five bigeye thresher sharks, four blue sharks, and seven spiny dogfish. Whole blood OECs and P50 values for the shortfin mako, the bigeye thresher, and the blue shark were predicted at pH 7.7 and pH 7.5, approximated arterial pH for the blue shark and the mako shark, respectively (Lai et al., 1997). Spiny dogfish OECs and P50 values are reported at an arterial pH of 7.85 (Swenson and Maren, 1987; Wells and Weber, 1983), as well as pH 7.7 for comparison among species. Common thresher shark OECs and P50 values are reported at pH 7.3 due to the relatively acidotic state of the blood from the two individuals that we sampled (Table 4.1 and inset of Figure 4.3).   Oxygen equilibria parameters are reported in Table 4.2 and OECs are presented in Figure 4.3. All shark species had relatively low P50 values (i.e., high blood-O2 affinities), but those of the mako shark and the bigeye thresher shark were exceptionally low. Blood pH and PCO2 had little influence on P50, as indicated by low Bohr coefficients or Bohr coefficients with 95% confidence intervals that included zero (e.g., common thresher shark blood at 22°C). Hill coefficients were relatively low and were generally independent of blood pH (Figure 4.3 and Table 4.3). Temperature was an important predictor of n50, but the response was relatively negligible and inconsistent with lower n50 values at higher temperatures for the shortfin mako (χ2 = 7.450, df = 2, P = 0.024) and the bigeye thresher (χ2 = 18.922, df = 2, P = 0.00008), but higher 126  n50 values at higher temperatures for the common thresher (χ2 = 10.358, df = 2, P = 0.006), the blue shark (χ2 = 7.118, df = 2, P = 0.028), and the spiny dogfish (χ2 = 18.922, df = 2, P = 0.00008).   In shortfin mako blood, temperature negligibly affected blood-O2 affinity below 90% saturation. At 95% saturation blood PO2 was 9-10 mmHg greater at 25°C than at 15°C, but the P95 standard errors overlap (Figure 4.3) and temperature was not an important model factor for P95 (χ2 = 5.520, df = 2, P = 0.063). Mako ΔH'WB values were positive (endothermic) at low saturation, but decreased to negative values (exothermic) with increasing saturation and at high pH, although ΔH'WB values were still relatively temperature independent (< 10 kJ mol-1 O2) (Figure 4.4). In common thresher shark blood, a slight reverse effect of temperature on blood-O2 affinity was evident (Figures 4.3 and 4.4), although temperature was not an important model factor. Bigeye thresher blood-O2 affinity was unaffected by temperature below 60% O2 saturation, but from P60 to P95 temperature was an important factor (P50: χ2 = 5.258, df = 2, P = 0.073; P60: χ2 = 10.970, df = 2, P = 0.004; P70: χ2 = 17.511, df = 2, P = 0.0002; P80: χ2 = 24.426, df =2, P = 0.000005; P90: χ2 = 32.580, df = 2, P = 0.00000008; P95: χ2 = 31.867, df = 2, P = 0.0000001). The saturation dependency of ΔH'WB in bigeye thresher shark blood is evident as positive ΔH'WB values below 60% saturation, but decreasingly negative values above 60% saturation (Figure 4.4), as well a right shift of the upper portion of the OEC with increasing temperature (Figure 4.3). Increasing temperature decreased blood-O2 affinity in both blue shark and spiny dogfish blood across most of the OEC (Figure 4.3) due to exothermic ΔH'WB at all saturations (Figure 4.4). In blue shark blood, temperature did not significantly affect P10 or P20, but temperature was an important model factor from P30 to P95 (χ2 = 8.481 to 25.748, df = 2, P = 0.014 to 0.000009).  127   The effects of closed-system temperature changes on blood PO2 are shown in Figure 4.5. Closed-system warming of mako and common thresher blood generally increased blood PO2, but less so than that predicted by Henry’s law (i.e., increasing temperature will increase PO2 in a closed system due to a reduction in plasma O2 solubility, and vice versa). Closed-system warming of blood from bigeye threshers, blue sharks, and spiny dogfish increased PO2 beyond that predicted by the change in solubility of O2, indicating that temperature induced Hb-O2 offloading likely contributed to ΔPO2. The greatest ΔPO2 occurred in bigeye thresher blood. Closed-system cooling caused blood PO2 to decrease, but less so in mako and common thresher blood than in the other species.  Common thresher shark hæmolysate experiments   Hill plots of the hæmolysate experiments are presented in Figure 4.6 and Figure 4.7. Summary data at pH 7.3 are presented in Table 4.4. Common thresher shark stripped hæmolysates had a low intrinsic O2 affinity that was independent of temperature and pH. The addition of ATP reduced O2 affinity and induced a pH dependency at 15°C that was not evident at 25°C. This pH dependency is evident as large Bohr coefficients with confidence intervals that do not include zero (Table 4.4). At 25°C urea reduced the effect of ATP on Hb-O2 affinity causing a reverse temperature-dependence (Figure 4.7), although the confidence intervals for the data at each temperature overlap (Figure 4.6). At pH 7.3 and in the presence of ATP and urea, ΔH' was +35 kJ mol-1 O2 (excluding the heat of O2 in solution).     128  4.4 Discussion Main findings  The main objective of this study was to investigate the effect of temperature on whole blood Hb-O2 affinity in the regionally heterothermic common thresher shark, and a closely related species that is a suspected of having warm eyes, the bigeye thresher shark. The effect of temperature on blood-O2 affinity in shortfin mako shark was also investigated, although this species has been studied previously (Bernal et al., 2018). This study tested the hypothesis that the blood-O2 affinity of the common thresher is less affected by temperature than most non-heterothermic species, similar to the blood and Hbs of the shortfin mako, the porbeagle shark, and the salmon shark (Andersen et al., 1973; Bernal et al., 2018; Dickinson and Gibson, 1981; Larsen et al., 2003). Additionally, this study also tested the hypothesis that the bigeye thresher shark has a relatively high blood-O2 affinity due to the low environmental oxygen levels that this species encounters daily (Figure 4. 2). Whole blood Hb-O2 affinity was independent of temperature for both common thresher shark and shortfin mako (Figure 4.3). When blood from these two species was warmed in a closed-system meant to mimic warming of blood in a heat exchanging rete mirabile, blood PO2 changed less than would be expected due to temperature dependence of the solubility of O2 in plasma (Figure 4.4). Bigeye thresher blood had a high O2 affinity (i.e., low P50; Table 4.2) that exhibited a temperature-dependence that was dependent on Hb-O2 saturation. At low saturation, bigeye thresher blood-O2 affinity was independent of temperature (Figure 4.3), but above ~50% saturation O2 affinity decreased with increasing temperature due to an increasingly exothermic ΔH'WB (Figure 4.4). Closed-system warming and cooling of bigeye thresher blood caused large changes to blood PO2.  Blood-O2 carrying capacity and O2 affinity  129   Hæmatocrits and Hb concentrations reported in this study (Table 4.1) are similar to previous reports for shortfin mako, common thresher, blue shark, and spiny dogfish (reviewed by Morrison et al., 2015). Shortfin mako and common thresher Hct and Hb concentrations are higher than typical values for ectothermic sharks, indicating a higher blood-O2 carrying capacity that is consistent with previous studies of sharks capable of RM endothermy (Bernal et al., 2001b; Emery, 1986). Those for bigeye thresher shark are similar to hammerhead sharks in the genus Sphyrna, which are intermediate between typical values for other ectothermic sharks and those for regionally heterothermic sharks (Morrison et al., 2015). This may indicate that bigeye thresher sharks as well as some other large pelagic sharks have higher oxygen demands (i.e., higher metabolic rates) than most non-heterothermic sharks, so they have relatively higher blood-O2 carrying capacities to match. Curiously, the blue shark occupies an ecological niche that overlaps with those of the shortfin mako, the common thresher, and the bigeye thresher (Bernal et al., 2009; Cartamil et al., 2011; Kinney et al., 2020), but unlike these sharks, the blue sharks cardiovascular system (i.e., the relative size and physiology of the heart, and blood-O2 carrying capacity) does not appear capable of higher rates of circulatory O2 delivery compared to other ectothermic sharks (Bernal et al., 2001b; Brill and Lai, 2015; Morrison et al., 2015).     This study’s results for shortfin mako and blue shark blood are qualitatively like those reported by Bernal et al. (2018), although their shortfin mako Bohr coefficients and P50 values are larger than those reported here (Table 4.3). However, the low P50 value and small Bohr coefficients reported by Wells and Davie (1985) for shortfin mako blood at 25°C (Table 4.3) are close to those in this study (Table 4.2). Blue shark P50 values are similar between this study and that by Bernal et al. (2018), and the slightly larger Bohr coefficients that they reported (Table 4.3) fall within the 95% confidence intervals for the Bohr coefficients reported here (Tables 4.2). 130  The Spiny dogfish P50 value (pH 7.85) reported by Wells and Weber (1983) at 15°C is slightly lower than the value that I determined, and they reported a Bohr coefficient of -0.28 (Table 4.3), whereas I observed no significant effect of pH on P50 at 15°C (Table 4.2).  Temperature had little to no effect on blood P50 of the shortfin mako, the common thresher shark, or the bigeye thresher shark (Table 4.2). In contrast, increasing temperature increased P50 (i.e., decreased blood-O2 affinity) in blue shark and spiny dogfish blood (Table 4.2), and as expected for these two species, the temperature dependence of blood-O2 affinity was consistent across most of the OEC (Figure 4.3). Temperature did not uniformly affect the bigeye thresher OEC, which is evident as a right shift of the 25°C OEC above 50% Hb-O2 saturation (Figure 4.3). At low O2 saturations, bigeye thresher shark ΔH'WB values were positive (endothermic) and are comparable to the those of the shortfin mako and the common thresher shark, but ΔH'WB became decreasingly negative (exothermic) with increasing saturation. Around 90% O2 saturation, the ΔH'WB values are comparable to those of the blue shark and the spiny dogfish.   The bigeye thresher shark whole blood P50 values are among some of the lowest that I have measured, and one of the lowest reported from elasmobranchs (Morrison et al., 2015). I suspect that such a low P50 that is temperature-independent likely benefits O2 uptake over the range of environmental conditions that bigeye thresher sharks encounter due to vastly different daytime and nocturnal distributions. Bigeye thresher sharks spend most of the day well below the thermocline near the upper reaches of the oxygen minimum layer (OML), but ascend to the warmer upper mixed layer at night (Figure 4.2) (Sepulveda et al., 2019). While near the OML, a low P50 combined with relatively thin lamellar diffusion distances and a gill surface area that is larger than that of any other studied elasmobranch (Wootton et al., 2015), should facilitate better 131  O2 diffusion into the blood from the relatively cold hypoxic water. Swordfish make similar diel vertical migrations (Sepulveda et al., 2010), and also like bigeye thresher sharks, swordfish have relatively large gill surface areas and a low P50. Thus, the respiratory characteristics of the bigeye thresher shark and the swordfish seem to be well adapted for extracting O2 from the hypoxic water near the OML.   It is notable that at 10°C the blue shark’s P50 is the same as the bigeye thresher’s, which are both only slightly lower than the shortfin mako’s P50 at 15°C (Table 2). However, blue sharks and shortfin makos do not routinely enter water low in O2 (Bernal et al., 2009). Elasmobranchs in general tend to have high Hb-O2 affinities and small Bohr coefficients (Morrison et al., 2015), so a low P50 may not be remarkable in the bigeye thresher. Alternatively, if bigeye thresher stripped hæmolysate has a lower intrinsic O2 affinity than its congeners, the common thresher and the pelagic thresher (Alopias pelagicus), then it is reasonable to hypothesize that a low P50 is adaptive to low environmental O2 in the bigeye thresher shark. To that end, I have completed some preliminary experiments on bigeye thresher shark stripped hæmolysates, and at 10°C and 25°C the intrinsic P50 values (pH 7.3) are 1.5 mmHg and 3.2 mmHg, respectively. These values are not too dissimilar to the common thresher shark values reported in Table 4.4, or the swordfish values reported in Table 3.3. Furthermore, although the intrinsic Hb-O2 affinities of both the bigeye and the common thresher sharks are much lower than many other elasmobranchs (Morrison et al., 2015), they are similar to some, including the porbeagle shark and the spiny dogfish (Larsen et al., 2003; Weber et al., 1983a). Therefore, the high whole blood-O2 affinity of the bigeye thresher shark does not appear to involve evolutionary changes in the intrinsic Hb-O2 affinity, and is likely due to differences in the 132  sensitivity of Hb to allosteric effectors (i.e., heterotropic allostery) and/or the physicochemical working environment for Hb within the red blood cells.   The absence of a Bohr shift in shortfin mako blood was also reported by (Wells and Davie, 1985) and is interesting because the Bohr effect is generally considered beneficial for O2 offloading in muscle capillaries (e.g., Jensen, 2004). The shortfin mako has the highest measured rates of oxygen consumption among sharks (Sepulveda et al., 2007b), so it would be expected that a Bohr shift would benefit O2 unloading to the swimming muscles. It is not clear if there is an adaptive benefit to a low P50 that is insensitive to pH in the shortfin mako, or if these are general traits of elasmobranch Hbs that may potentially contribute to the upper limit of aerobically sustained exercise. Closed-system temperature changes   Temperature-independent Hb-O2 affinity in blood from the shortfin mako and the common thresher shark was reflected in closed-system changes to blood temperature, when blood PO2 changed less than would be expected due to the temperature dependence of plasma O2 solubility (Figure 4.5). In comparison, closed-system temperature changes caused large changes to blood PO2 for the bigeye thresher, blue shark, and the spiny dogfish (Figure 4.5), indicating that warming and cooling of blood caused Hb-O2 unloading and binding, respectively. In bigeye thresher blood, the magnitude of temperature induced changes to blood PO2 exceeded those measured in blue shark and spiny dogfish blood. Because bigeye thresher blood has such a high O2 affinity, most of the equilibration O2 tensions used in this study would have caused high Hb-O2 saturation levels where increasing temperature greatly right shifts the bigeye thresher OEC (Figure 4.3), causing excessive Hb-O2 unloading as the blood is warmed. In mako and common thresher blood, temperature-independent Hb-O2 affinity and possible reverse temperature-133  dependence likely caused the relatively decreased effect of closed-system temperature changes on blood PO2. Similar results have been previously reported for the shortfin mako and some tunas (Bernal et al., 2018; Brill and Bushnell, 1991a; Brill and Bushnell, 2006; Cech et al., 1984).   During closed-system warming, the average change in blood pH of the shortfin mako and the blue shark was around -0.023 U/°C (Table 4.2), which is at the higher end of previous reports from tunas (-0.021 to -0.010 U/°C) (Brill and Bushnell, 1991a; Brill and Bushnell, 2006; Lowe et al., 2000). However, bigeye thresher and common thresher blood pH was unaffected by closed system temperature changes. It is not clear what caused this lack of a shift in blood pH during temperature changes, but it is worthy of future research as are studies on the effect of temperature on acid-base state in sharks in general (Heisler, 1988). Molecular mechanisms of temperature-independent Hb-O2 affinity in the common thresher shark  Common thresher shark intrinsic Hb-O2 affinity is temperature-independent, and in the presence of ATP and urea, temperature has a reversed effect on Hb-O2 affinity. It is not clear what underlies the temperature-independence since pH had no effect on Hb-O2 affinity in the absence of urea and ATP. It seems unlikely that I failed to strip all endogenous allosteric effectors from the hæmolysates since the P50 was low, 2.5 mmHg. Urea reduces the O2 affinity of many vertebrate Hbs, including those of some, but not all elasmobranchs (reviewed by Morrison et al., 2015). Urea did not affect common thresher shark Hb-O2 affinity, but it did decrease the effect of ATP on Hb-O2 affinity, which has also been reported for Hbs from some sharks (e.g., spiny dogfish) but not others (e.g., sandbar shark, Carcharhinus plumbeus) (Brill et al., 2008; Weber et al., 1983a; Weber et al., 1983b). Urea insensitivity may somehow be linked to the intrinsic temperature-independence, but I can offer no mechanistic explanation for this. 134  Alternatively, the common thresher shark may have multiple Hb components that are differentially affected by proton binding, in which case the combined hæmolysate may appear to be intrinsically temperature-independent due to proton linked increases to ΔH' for some Hb components but not others. The major Hb components of porbeagle shark show a normal temperature-dependence in the absence of ATP, but a reverse temperature-dependence in the presence of ATP (Larsen et al., 2003). Thus, it seems that ATP is the major effector that underlies reverse-temperature-dependence in lamnid sharks and the common thresher shark, but further research is warranted to elucidate any structural or functional differences and the possible independent evolution of this trait in common thresher shark Hb.  Possible limitations  The primary objective of this study was to construct whole blood OECs at two different but physiologically relevant temperatures for the shortfin mako, common thresher, and bigeye thresher. Our experimental temperatures encompass the low and high ambient and body temperatures reported for these species (Carey and Teal, 1969a; Cartamil et al., 2016; Patterson et al., 2011; Sepulveda et al., 2004; Sepulveda et al., 2019), and these temperatures are also comparable to previous studies (Andersen et al., 1973; Bernal et al., 2018). The individuals included in this study had likely experienced varying levels of exercise induced fatigue and consequent respiratory and metabolic acidosis. This is indicated by relatively high plasma lactate levels in shortfin mako, common thresher, and blue sharks (Table 4.1), which are comparable to lactate levels reported from capture-stressed shortfin mako and blue sharks (Wells et al., 1986). However, bigeye thresher and spiny dogfish plasma lactate levels were relatively low. Furthermore, Hct and Hb concentration values (Table 4.1) were similar to previously published values (Morrison et al., 2015), indicating that capture stress did not cause lasting RBC swelling. 135  It is not possible or safe to obtain blood samples from resting and cannulated large sharks held in laboratory aquaria, so collecting blood samples by caudal peduncle puncture is an accepted method for obtaining shark blood for oxygen equilibria experiments (Brill et al., 2008; Cooper and Morris, 1998). Except for the two common thresher sharks, the blood pH levels that we achieved with the CO2 exposures were within physiologically relevant ranges, which allowed us to estimate blood PS at physiologically relevant pH values. The common thresher shark blood was, however, in good shape and showed no signs of RBC lysis or RBC swelling [i.e., Hcts were close to previously published values (Emery, 1986; Filho et al., 1992)]. The main objective of this study was to investigate the effect of temperature on Hb-O2 affinity, and it is very unlikely that any exercise or stress induced physiologically changes would have compromised the allosteric effect of temperature on Hb function in whole blood. This is supported by the experiments on common thresher shark stripped hæmolysates that showed temperature effects that are comparable to those observed in whole blood. Lastly, although only two common thresher sharks were included in this study, the results were consistent between the two individuals and with the stripped hæmolysate experiments, and since this studies results on the effect of temperature on shortfin mako (n = 7) are consistent with the limited sample size (n = 3) of Bernal et al. (2018), it is unlikely that the common thresher shark data are not representative of the species.  Conclusion  This study shows for the first time that Hb-O2 affinity is independent of temperature in blood and hæmolysates from the regionally heterothermic common thresher shark. Temperature independent Hb-O2 affinity was previously shown in blood from the shortfin mako and was corroborated in this study. It was also shown that the bigeye thresher shark has a low P50 that is 136  insensitive to temperature, which indicates an exceptional tolerance to hypoxia and likely allows this species to exploit the OML and a wide range of water temperatures.137       Figure 4.1 Vertical movements, and stomach and ambient temperatures for a free-swimming shortfin mako shark tracked in the Southern California Bight. The mako was 18.0 kg and 120 cm fork length, and was tracked in 2002 by Chugey Sepulveda (Pfleger Institute of Environmental Research). Shaded area represents night, depth is shown in black, stomach temperature in red, and water temperature in blue. Figure created from data and figures in Sepulveda et al. (2004).138    Figure 4.2 Diurnal movements of a bigeye thresher shark captured in the Southern California Bight. The bigeye thresher shark was tagged with a pop-up satellite archival transmitter and tracked for 30 days by Sepulveda et al. (2019).  The panel on the left shows a 3-day portion of the 30-day track. The shaded area represents night, depth is shown in black (dashed line), and water temperature in blue. The panel on the right shows the typical temperature (blue) and dissolved oxygen concentration (black) as a function of depth in the Southern California Bight, where the bigeye thresher sharks were tagged. Daytime depths are near the upper reaches of the oxygen minimum layer (< 2 mg O2 L-1). Figure created from data and figures in Sepulveda et al. (2019). 139  Figure 4.3 Whole blood oxygen equilibrium curves (OECs) at different pH and temperatures for shortfin mako shark, common thresher shark, bigeye thresher shark, blue shark, and spiny dogfish. OECs were constructed at either 10°C (black triangles), 15°C (blue circles), 22°C (green diamonds), or 25°C (red squares). Blood PO2 values were interpolated (± bootstrap standard errors) from linear mixed models of logPO2 vs pH at specific Hb-O2 saturation levels (see Methods section). Shortfin mako, common thresher, bigeye thresher, and blue shark (15 and 25°C) OECs are at pH 7.7 (filled symbols and solid curves) and pH 7.5 (open symbols and 140  dashed curves). The blue shark OEC at 10°C is at pH 7.45 (filled triangles and solid curve), and spiny dogfish OECs are at pH 7.85 (filled symbols and solid curves), and pH 7.7 (open symbols and dashed curves). Inset figure show the relationships between log10PS vs pH at 50 and 95% Hb-O2 saturation (P50 and P95, respectively), and n50 vs pH, plotted with the line from the linear mixed models (see methods). 141   Figure 4.4 Predicted enthalpy of oxygenation (ΔH'WB) as a function of whole blood pH and whole blood Hb-O2 saturation for shortfin mako shark, common thresher shark, bigeye thresher shark, blue shark, and spiny dogfish. ΔH'WB values were calculated with the van’t Hoff isochore (see methods) at constant pH between the indicated temperatures. The blood-O2 tensions (PO2) at specific blood-O2 saturation levels at a given pH and temperature were determined from the data presented in Figure 4.3. 142  Figure 4.5 Effects of closed-system temperature changes on the measured change in blood PO2 (Δ PO2). Shark blood was equilibrated at a range of O2 tensions and a low CO2 tension (circles; PCO2 = 1.9 mmHg) or a high CO2 tension (triangles; PCO2 = 3.8 mmHg in spiny dogfish, and 7.6 mmHg in the other sharks), was heated (filled red symbols) or cooled (open blue symbols) between the temperatures specified above. Dotted lines indicate the temperature induced ΔPO2 expected due to changes in solubility of blood plasma at a given equilibration PO2 (i.e., Henry’s Law).143  Figure 4.6 Hill plots of Hb-O2 equilibria of common thresher shark at pH 7.3. Experiments were conducted on stripped hæmolysates in the absence or presence of ATP and urea at two temperatures (15°C and 22°C). Data points 95% confidence intervals were predicted at pH 7.3 from linear models of logPS vs pH (see Methods section). Experiments were conducted in 0.1 M Hepes buffer at Hb concentrations of 0.6 mM, ATP was added at saturating concentrations (18 mM; ATP/Hb = 30), and urea was added at a concentraion of 500 mM. The data points for Hb + Urea at 15°C overlapped with those of Stripped Hb, so were omitted for clairty.144  Figure 4.7 Heats of oxygenation (ΔH') of stripped hæmolysates of common thresher shark.  In panel A, ΔH' values were calculated with the van’t Hoff equation (see Methods) with logP50 values that were interpolated from logP50/pH relationships. In panel B, ΔH' values were calculated from data reported in Figure 4.6, and plotted as function of Hb-O2 fractional saturation at pH 7.3. Experiments were conducted at 15°C and 22°C in 0.1 M Hepes buffer at Hb concentrations of 0.6 mM, and ATP was added at saturating concentrations (18 mM; ATP/Hb = 30), and urea was added at a concentration of 500 mM. ΔH' values exclude the heat of O2 in solution (-12.6 kJ mol-1).145  Table 4.1 Fork length and blood variables for shortfin mako sharks, common thresher sharks, bigeye thresher sharks, blue sharks, and spiny dogfish. Values are means ± standard error with samples sizes in parentheses. Mean blood pH is reported with the range of values. If values were measured in only one or two individuals, then the individual measurements are reported.  4 Measured in blood equilibrated to 0.25% CO2 and saturating levels of O2.  Shortfin mako shark Common thresher shark Bigeye thresher shark Blue shark Spiny dogfish Fork length (cm) 128 ± 8 (9) 135, 175  135 ± 13 (5) 52 ± 3 (8) Hæmatocrit (%) 31.6 ± 2.0 (10) 29.7, 36.8 25.5 ± 1.0 (9) 19.2 ± 1.2 (8) 17.9 ± 1.5 (8) Hæmoglobin (mM) 1.48 ± 0.12 (10) 1.25, 1.65 1.13 ± 0.04 (9) 0.69 ± 0.03 (8) 0.63 ± 0.06 (8) MCHC (mM) 4.65 ± 0.19 (10) 4.20, 4.49 4.45 ± 0.08 (9) 3.61 ± 0.12 (8) 3.52 ± 0.08 (8) Plasma osmolality (mOsm/kg) 959 ± 6 (7) 1004 931 ± 27 (9) 914 ± 32 (6) 937 Plasma lactate (mM) 8.8 ± 4.2 (4) 7.14 3.4 ± 0.35 (7) 6.6 ± 1.7 (5) 1.4 ± 0.2 (6) Blood pH (range)4 15°C: 7.73 (7.35-8.13) 25°C: 7.60 (7.34-8.04) 15°C: 7.30, 7.38 22°C: 7.36, 7.27 10°C: 7.51 (7.42-7.70) 25°C: 7.54 (7.43-7.70) 15°C: 7.70 (7.52-7.86) 25°C: 7.54 (7.27-7.69) 15°C: 7.72 (7.64-7.88) 25°C: 7.69 (7.59-7.83) 146  Table 4.2 Whole blood oxygen equilibria parameters at different temperatures and pH.     Shortfin mako shark 15°C (n = 7) 25°C (n = 7) P50 mmHg (SE), pH 7.7 10.3 (9.3 to 11.4) 10.7 (9.5 to 12.1) pH 7.5 10.7 (9.6 to 11.8) 10.7 (9.7 to 11.8) n50 (± SE), pH 7.7 2.07 (± 0.11) 1.83 (± 0.12) pH 7.5 2.09 (± 0.11) 1.85 (± 0.11) Bohr coefficients (95% CI) pH range -0.08 (-0.30 to 0.14) pH 7.264 to 8.134 0.01 (-0.20 to 0.40) pH 7.112 to 8.037 ΔpH/ΔT°C (± 95% CI) CS temperature change -0.023 (± 0.011) 15 → 25°C 0.029 (± 0.013) 25 → 15°C Common thresher shark 15°C (n = 2) 22°C (n = 2) P50 mmHg (SE), pH 7.3 20.0 (18.4 to 21.7) 17.7 (16.2 to 19.3) n50 (± SE), pH 7.3 1.43 (± 0.08) 1.72 (± 0.08) Bohr coefficients (95% CI) pH range -0.13 (-0.60 to 0.33) pH 7.089 to 7.378 -0.88 (-1.56 to 0.06) pH 7.165 to 7.360 ΔpH/ΔT°C (± 95% CI) CS temperature change -0.0003 (± 0.007) 15 → 22°C -0.002 (± 0.006) 22 → 15°C Bigeye thresher shark 10°C (n = 5) 25°C (n = 5) P50 mmHg (SE), pH 7.7 7.9 (6.9 to 9.2) 8.5 (7.2 to 9.9) pH 7.5 7.5 (6.7 to 8.2) 8.7 (8.0 to 9.5) n50 (± SE), pH 7.7 2.42 (± 0.19) 1.80 (± 0.22) pH 7.5 2.17 (± 0.13) 1.51 (± 0.11) Bohr coefficients (95% CI) pH range 0.14 (-0.16 to 0.42) pH 7.139 to 7.695 -0.06 (-0.74 to 0.33) pH 7.289 to 7.697 ΔpH/ΔT°C (± 95% CI) CS temperature change -0.003 (± 0.003) 10 → 25°C -0.0002 (± 0.005) 25 → 10°C Spiny dogfish 15°C (n = 7) 25°C (n = 7) P50 mmHg (SE), pH 7.85 22.9 (21.1 to 24.8) 33.8 (31.1 to 36.7) pH 7.7 22.5 (20.9 to 24.3) 35.6 (32.9 to 38.5) n50 (± SE), pH 7.85 1.17 (± 0.03) 1.26 (± 0.04) pH 7.7 1.14 (± 0.02) 1.23 (± 0.02) Bohr coefficients (95% CI) pH range 0.04 (-0.12 to 0.19) pH 7.497 to 8.045 -0.15 (-0.41 to -0.03) pH 7.551 to 7.944 Blue shark 10°C (n = 3) 15°C (n = 4) 25°C (n = 4) P50 mmHg (SE), pH 7.7  17.0 (15.6 to 18.6) 24.7 (21.8 to 27.9) pH 7.5/7.45 8.2 (6.7 to 9.7), pH 7.45 17.7 (16.4 to 19.0), pH 7.5 26.7 (24.9 to 28.7), pH 7.5 n50 (± SE), pH 7.7  1.51 (± 0.07) 1.50 (± 0.09) pH 7.5/7.45 1.39 (± 0.13), pH 7.45 1.58 (± 0.06), pH 7.5 1.42 (± 0.06), pH 7.5 Bohr coefficients (95% CI) pH range -0.14 (-0.54 to 0.26) pH 7.013 to 7.456 -0.08 (-0.41 to 0.53) pH 7.128 to 7.863 -0.17 (-0.52 to 0.45) pH 7.163 to 7.689 ΔpH/ΔT°C (± 95% CI) CS temperature change  -0.022 (± 0.013) 15→25°C 0.023 (± 0.010) 25→15°C 147     Table 4.3 Whole blood P50 values (mmHg) and Bohr coefficients (φ) reported in the literature for shortfin mako sharks, blue sharks, and spiny dogfish.                        Species T°C pH P50 (mmHg) ϕ Reference Shortfin mako shark 15 7.93 14.5 -0.74 Bernal et al. (2018)  15 7.68 22.3    25 8.13 18.6 -0.11   25 7.64 20.9    25 7.6 10.6 0.16 Wells and Davie (1985) Blue shark 15 8.05 12.1 -0.33 Bernal et al. (2018)  15 7.45 19.1    25 7.95 25.0 -0.22   25 7.48 31.7   Spiny dogfish 15 7.85 17.9 -0.28 Wells and Weber (1983) 148  Table 4.4 Hæmolysate oxygen equilibria parameters of common thresher shark at two experimental temperatures and in the absence or presence of ATP and urea.  P50 values (± 95% confidence intervals) were predicted at pH 7.3 from linear models of logP50 vs pH (see Methods section), and Bohr coefficients are the model slopes (i.e., ΔlogP50/ΔpH) with their 95% confidence intervals. ΔH' values were calculated with logP50 values at pH 7.3, and excludes the heat of O2 in solution (-12.6 kJ mol-1 O2).   15°C  22°C P50, pH 7.3 Log10 P50 ± 95% CI P50 (mmHg)  Log10 P50 ± 95% CI P50 (mmHg) Stripped Hb 0.397 ± 0.080 2.49  0.395 ± 0.110 2.48 Hb + Urea 0.352 ± 0.322 2.25  0.456 ± 0.786 2.86 Hb + ATP 1.322 ± 0.044 21.01  1.394 ± 0.103 24.76 Hb + ATP + Urea 1.232 ± 0.028 17.04  1.136 ± 0.310 13.69 Bohr coefficients (ϕ) ϕ ± 95% CI  ϕ ± 95% CI Stripped Hb -0.006 ± 0.226  0.105 ± 0.346 Hb + Urea -0.498 ± 1.263  -0.273 ± 4.132 Hb + ATP -0.451 ± 0.198  -0.081 ± 0.302 Hb + ATP + Urea -0.388 ± 0.122  -0.184 ± 0.738 ΔH' (kJ mol-1), pH 7.3  Stripped Hb +13 Hb + Urea -12 Hb + ATP -4 Hb + ATP + Urea +35  149   Chapter 5 General Discussion and Conclusions “I look upon controversy especially as one of the chief ways in which truth is approached. We may fondly imagine that we are impartial seekers after truth, but with a few exceptions, to which I know that I do not belong, we are influenced and sometimes strongly by our personal bias and we give our best thoughts to those ideas which we have to defend. Nevertheless we should of course all do our best to avoid controversy, in the sense that we should take every possible care to verify our facts and substantiate our conclusions before publishing our results.” (August Krogh, 1929, p. 248) 5.1 Thesis overview and major contributions  My objective for this thesis was to investigate the functional significance of reduced and reverse temperature-dependent Hb-O2 affinity in regionally heterothermic teleost and sharks. There are two underlying hypotheses that guided this work: 1) temperature-independent Hb-O2 affinity conserves heat-energy, which may help maintain stably elevated body temperatures in regionally heterothermic fishes, so I expected this trait to be shared by all regionally heterothermic teleosts and sharks capable of maintaining internal temperature gradients; and 2) the Hb-oxygen binding properties and high Hb concentrations of regionally heterothermic fishes maintain matching between O2 supply and O2 demand despite large internal temperature gradients. To address these hypotheses, I developed four specific research questions: 1) how do Hb concentration and Hb-O2 affinity contribute to the determinants of maximal O2 consumption, circulatory O2 delivery and tissue O2 extraction, in the yellowfin tuna? 2) How does the enthalpy of Hb-oxygenation influence O2 and heat transport by Hb? 3) How does temperature affect Hb-O2 affinity in two regionally heterothermic teleosts, the swordfish and the smalleye Pacific opah? 150  4) How does temperature affect Hb-O2 affinity in the regionally heterothermic common thresher shark, and a suspected regional heterotherm, the bigeye thresher shark?  Addressing the first question, using a mathematical model of O2 transport I demonstrated that Hb concentration and Hb-O2 affinity are relatively important factors determining ṀO2max in the yellowfin tuna, a regional heterotherm with a “high-energy demand” (Chapter 2). Addressing the second question, I used published data to determine a range of enthalpy values for different species of tuna, which I used to model the potential heat-energy savings and influence of temperature on blood PO2. I showed that Hb with a reversed temperature dependence should prevent Hb-heat loss, as much as 13% of metabolic heat production, and prevent any changes to blood PO2 over a wide temperature range (Chapter 2). Addressing the third question, I showed that smalleye Pacific opah Hb-O2 affinity was independent of temperature, and the temperature-dependence of swordfish Hb-O2 affinity was pH dependent, becoming temperature-independent at low pH (Chapter 3). Addressing the fourth question, I showed that common thresher shark Hb-O2 affinity was independent of temperature, and bigeye thresher shark Hb has a high O2 affinity that is temperature-independent at low saturation, but has a normal temperature-dependence at high saturation (Chapter 4).  These results provide insight into the functional significance of reduced and reversed temperature-dependent Hb-O2 affinity in regionally heterothermic fishes (Table 5.1). In this chapter I offer my interpretation of these results and those of previous studies to provide some context for the evolution and functional significance of this Hb trait in regionally heterothermic teleosts and sharks.   151  5.2 The functional significance of an increased enthalpy of hæmoglobin-oxygenation 5.2.1 Temperature gradients in the heat-exchanging retia mirabilia: closed-system temperature changes  Hæmoglobin of regionally heterothermic fishes must transport O2 across internal temperature gradients established by the heat exchanging retia. Experiments on the effects of closed-system temperature changes on blood PO2 were initiated to test the hypothesis that temperature-independent Hb-O2 affinity prevents premature Hb-O2 unloading during closed-system warming in the heat exchanging retia (Carey and Gibson, 1977; Cech et al., 1984; Graham, 1973). Although the relative importance of closed-system changes to blood PO2 in the heat exchanging retia has likely been overstated, and it is probably more important that P50 is not too different in the capillaries of warm and cold tissues, the results of closed-system experiments are good for qualitative comparisons of the temperature-dependence of blood-O2 affinity among species.    Blood from smalleye Pacific opah, shortfin mako shark, and common thresher shark shows reverse temperature-dependence during closed-system warming, when blood PO2 changed less than and/or in the opposite direction as would be expected due to the temperature dependence of the solubility of O2 in plasma (Figure 3.4). These results are similar to previous reports for Atlantic bluefin tuna, albacore tuna, and skipjack tuna (section 1.3.1 and Figure 1.6) (Bernal et al., 2018; Brill and Bushnell, 1991a; Brill and Bushnell, 2006; Cech et al., 1984). In contrast, swordfish and bigeye thresher shark blood PO2 increased with closed-system warming (Figure 3.4 and Figure 4.5), more than would be expected due to the temperature dependence of the solubility of O2 in plasma, indicating that increasing temperature caused Hb-O2 unloading due to decreased Hb-O2 affinity. The unloaded O2 diffuses from the RBCs into the plasma, 152  which increases plasma PO2. These results are similar to those previously reported for bigeye tuna blood (Figure 1.6) (Lowe et al., 1998). Furthermore, all of the closed-system effects of temperature on blood PO2 described above correspond to the effect of temperature on whole blood-O2 affinity for each species, in contrast to previous speculation that the effect of temperature on blood-O2 affinity may differ between the gills and systemic circulation of some species (Lowe et al., 2000).   If warming of arterial blood in a heat exchanging rete causes premature Hb-O2 unloading that is detrimental to circulatory O2 delivery, then it would be expected that temperature-independent Hb-O2 affinity would be a trait common to all regionally heterothermic fishes. Since closed-system warming decreased blood-O2 affinity and increased blood PO2 in both bigeye tuna and swordfish, it seems unlikely that premature O2 unloading in a heat exchanging rete adversely affects circulatory O2 delivery. Moreover, relatively short blood transit times through the heat exchanging retia may limit excessive temperature induced O2 unloading (Brill and Bushnell, 2001), and even if closed-system warming induced rapid Hb-O2 unloading, the reduction in saturation is probably inconsequential to circulatory O2 delivery. 5.2.2 Heat-energy conservation in species with warm red muscle  An increased ΔH' will reduce the energy required for O2 unloading in the cold peripheral tissues of regionally heterothermic fishes, and prevent liberation of heat upon O2 binding at the gills (Giardina et al., 1989b; Weber and Fago, 2004; Weber and Wells, 1989). In most tunas and regionally heterothermic sharks, ΔH' values are near zero or positive (i.e., temperature-independent and reverse-temperature dependent Hb-O2 affinity, respectively; Table 5.1) eliminating any outward heat transport linked to Hb-O2 unloading and binding. This could potentially save up to 13% of metabolic heat production compared to the intrinsic ΔH' (i.e, ΔHO2 153  ≈ -61 kJ mol-1 O2; Figure 2.8). Therefore, it seems reasonable to hypothesize that the heat-energy savings of this trait may be important for preventing Hb-heat transport away from the warm red muscle, increasing the efficiency of heat retention. In contrast to this hypothesis, bigeye tuna and swordfish ΔH' values are negative, and therefore exothermic, so it is not clear if the energy savings of a numerically low ΔH' contributes appreciably to thermoconservation in regionally heterothermic fishes. There are, however, important differences between bigeye tuna and other cold tolerant tunas, and between billfishes and other regionally heterothermic fishes, which are worthwhile considering and are briefly discussed below.  Red muscle temperatures are not stable in either bigeye tuna or swordfish, and are influenced by ambient water temperature more so than in cold tolerant tunas and lamnid sharks.  During daily sojourns into cold water deep below the thermocline, bigeye tuna routinely return to warmer surface waters before their RM temperature falls below about 17°C, and while swordfish are at depth their RM cools close to ambient temperature (Bernal et al., 2009; Holland et al., 1992; Malte et al., 2007; Stoehr et al., 2018). However, the behavioural thermoregulation exhibited by the bigeye tuna prevents the warm tissues from becoming hypothermic, and warming-up in surface waters is accelerated by redistributing blood flow between the central circulation and lateral heat-exchanging retia (i.e., physiological thermoregulation) (Holland and Sibert, 1994; Holland et al., 1992). In comparison, some large tunas and lamnid sharks that inhabit cold temperate or polar waters for months at a time with no warm refuges, maintain RM temperatures stably elevated by as much as 10 to 20°C above ambient (Anderson and Goldman, 2001; Bernal and Lowe, 2015; Bernal et al., 2017; Block et al., 2001; Carey and Teal, 1969b; Goldman et al., 2004; Gunn and Block, 2001). In the blood of these cold-tolerant tunas and sharks the temperature-dependence of Hb-O2 affinity is reversed (Brill and Bushnell, 2006; 154  Carey and Gibson, 1977; Cech et al., 1984; Clark et al., 2008a; Dickinson and Gibson, 1981; Larsen et al., 2003; Lilly et al., 2015).   Billfish cranial endothermy is quite remarkable, but the warm tissues make up a relatively small proportion of the entire body compared to regionally heterothermic fishes capable of RM endothermy. Since most of the systemic circulation perfuses tissues and organs that are near ambient water temperature, reversed Hb-oxygenation enthalpy would probably not greatly affect thermoregulation and heat conservation in billfishes, including swordfish. Furthermore, in at least swordfish, the orbital rete effectively retains heat generated by the heater organ, keeping cranial temperature elevated well above cold ambient temperatures (Figure 3.1) (Carey, 1990).   Although numerically low or positive ΔH' values will conserve heat-energy, it is not clear if these savings significantly influence body temperatures in regionally heterothermic fishes. It seems unlikely that reversed Hb-oxygenation enthalpy helps maintain stable cranial temperatures in billfishes, and the absence of temperature-independent Hb-O2 affinity in bigeye tuna may justify rejecting the hypothesis that this trait has an appreciable contribution to thermoconservation. If a regional heterothermic fish had a ΔH' equivalent to rainbow trout, -35kJ mol-1, then about 7.4% of the metabolic heat production would be lost from Hb to the environment (Figure 2.8). In a slowly swimming yellowfin tuna, this may increase blood heat-energy loss from 0.105 to 0.112 kJ kg-1 min-1. This may be inconsequential over short periods, but tuna are considered “energy speculators” (Brill, 1996; Korsmeyer and Dewar, 2001), so any energy savings may have long term value, but may not be essential for thermoconservation. 5.2.3 Hæmolgobin-O2 unloading to warm and cold tissues  Reduced and reverse temperature-dependent Hb-O2 affinity should prevent excessive temperature induced shifts to the physiological OEC, matching O2 supply to O2 demand in 155  tissues maintained at very different temperatures (Giardina et al., 1989a; Weber and Campbell, 2011). Reverse temperature-dependent Hb-O2 affinity is probably most important in cold-tolerant species like Atlantic bluefin tuna, because blood-O2 affinity and PO2 will not drastically change as peripheral tissue temperature changes with environmental temperature, and as blood flows from the gills to warmer tissues. In other words, blood PO2 would not be greatly affected by temperature changes in the body or environment of a regionally heterothermic fish, which I showed in Chapter 2. Using yellowfin tuna mixed venous blood gas data at 25°C (Korsmeyer et al., 1997a) and applying different ΔH' values to adjust blood PO2 to temperatures ranging from 30°C down to 15°C, it becomes evident that temperature-independence stabilizes mixed venous PO2 over the temperature range, and reverse temperature-dependence may enhance O2 unloading at cold temperatures (Figure 2.7).   When cold tolerant regional heterotherms enter cold water, a reverse temperature-dependence probably protects Hb-O2 unloading to the cold peripheral tissues by preventing blood PO2 from decreasing so low that it may impair Hb-O2 unloading and tissue O2 extraction. This may be particularly important for cardiac O2 delivery (Clark et al., 2008a) because tuna, shark, and billfish hearts remain near ambient water temperature due to their anatomical position and a coronary circulation that supplies arterial blood directly from the gills (Bernal et al., 2001b; Brill, 1987; Brill and Bushnell, 2001; Cox, 2015; Daxboeck and Davie, 1986). Thus, ambient temperature directly influences cardiac function (Clark et al., 2008b; Clark et al., 2013). Although the hearts of most lamnid sharks are not too different from ectothermic sharks (Brill and Lai, 2015), tuna have large powerful hearts compared to other teleosts (Brill and Bushnell, 1991b; Brill and Bushnell, 2001; Farrell et al., 1992). This is exemplified by the relatively large hearts of bigeye tuna, which also appear to have enhanced performance at colder temperatures 156  relative to some tuna with warmer distributions (Bernal et al., 2017; Brill and Bushnell, 1991b; Galli et al., 2009). The hearts of some other cold-tolerant tuna, swordfish, and salmon shark also appear to have evolved a greater tolerance to functioning in cold water (Bernal et al., 2017; Galli et al., 2009; Weng et al., 2005). Thus, in species such as Atlantic bluefin tuna, a reverse temperature-dependence has been proposed to enhance O2 unloading to cold organs with a high metabolic demand, such as the heart (Clark et al., 2008a).   At approximate arterial pH values, swordfish ΔH'WB (-23 mol-1 O2) is more exothermic than other regionally heterothermic fishes, but it is more endothermic (less numerically negative) than values for some regionally heterothermic mammals that have heat exchanging retia in their limbs, which can function to retain heat or dissipate heat. For example, ΔH'WB values (including the heat of O2 in solution) for both reindeer and musk-ox are about -27 kJ mol-1 O2, which is much less than for humans (~ -52 kJ to -44 kJ mol-1 O2) (Brix et al., 1990; Reeves, 1980). The temperature-dependence of reindeer Hb is also saturation dependent, but unlike swordfish Hb, the effect of temperature on Hb-O2 affinity decreases with increasing saturation in reindeer blood. At high O2 saturations that are similar to arterial levels, reindeer Hb-O2 affinity is almost independent of temperature, which likely prevents temperature induced shifts to Hb-O2 affinity as the arterial blood is cooled in the heat exchanging retia en route to the cold legs (Giardina et al., 1989a). Swordfish Hb, however, has a “normal” temperature dependence at high O2 saturations and arterial blood pH values, but at low pH, Hb-O2 affinity becomes temperature-independent. This is due to proton and ATP binding that is more favourable at low than at high temperature, which may also avert temperature induced shifts to Hb-O2 affinity in the capillaries of the cold tissues.  157   Smalleye Pacific Opah are quite different than other regionally heterothermic fishes because they have warm hearts and maintain most of their body warmer than ambient (Wegner et al., 2010). However, temperature varies within the body, so temperature-independent Hb-O2 affinity within the range of body temperatures should maintain matching between O2 supply and O2 demand to all the tissues and organs even though they vary in temperature.  5.3 Matching O2 supply and O2 demand   Hæmoglobin concentration and O2 affinity both have important contributions to blood-O2 transport because they determine blood-O2 carrying capacity and the O2 capacitance of the blood (i.e., βbO2). Thus, Hb is an important determinant of circulatory O2 delivery and tissue O2 extraction. Furthermore, tuna cardiac outputs are quite low during routine swimming (Clark et al., 2008b; Clark et al., 2013) but routine metabolic rates are relatively high (Korsmeyer and Dewar, 2001), so Hb likely plays an essential role in sustaining aerobic metabolic rates and heat production. In Chapter 2, I showed that Hb concentration and an increased P50 in the tissues (i.e., a Bohr shift) are important determinants of maximum O2 transport in yellowfin tuna.   Sharks, however, differ. In general, cardiac performance does not appear to be exceptional in regionally heterothermic sharks compared to ectothermic sharks, quite unlike the situation in tuna (Brill and Lai, 2015). Although shortfin mako routine and maximum (i.e., the highest measured) O2 consumption rates are higher than other sharks (Sepulveda et al., 2007b), the routine rates are about half that of yellowfin tuna (Brill and Lai, 2015). Because mako shark cardiac output does not seem to increase considerably with increased swimming activity, circulatory O2 delivery also will not greatly increase, so more O2 will have to be extracted from the blood to match O2 supply to the elevated O2 demand. High Hb-O2 affinity and high Hb 158  concentration maintain elevated levels of arterial saturation and blood-O2 carrying capacity. However, mako blood, and that of other large active sharks, does not exhibit a significant Bohr shift. Therefore, the maximum aerobic metabolic rates of regionally heterothermic sharks may be constrained by reduced circulatory O2 delivery due to low cardiac outputs, and relatively low tissue O2 extraction due to their inability to alter Hb-O2 affinity, and thus βbO2, between the gills and the tissues.  5.4 Is reduced temperature-dependence of Hb-O2 affinity an example of convergent physiological function among regionally heterothermic vertebrates?  Many cold-tolerant mammals, aquatic mammals, and birds have heat exchanging retia mirabilia that supply blood to limbs or appendages (Figure 1.3) (e.g., Cutright and McKean, 1979; Kahl, 1963; Øritsland, 1970; Rommel and Caplan, 2003; Scholander, 1955; Scholander and Krog, 1957; Scholander and Schevill, 1955). In some regionally heterothermic mammals and birds, the limbs are colder than the rest of the body so the heat exchanging retia cool and warm the blood flowing into and out of the appendages, respectively. However, the retia can also function to dissipate heat and are important for whole animal temperature regulation (e.g., Steen and Steen, 1965). Furthermore, carnivores and artiodactyls (Cetartiodactyla) have a carotid rete that cools the brain, which is thought to have contributed to niche expansion and diversification of artiodactyls relative to similar mammals like perissodactyls that lack a carotid rete (Baker, 1972; Lust et al., 2007; Mitchell and Lust, 2008).    It is generally regarded that reductions in the temperature dependence of Hb-O2 affinity averts impaired blood-O2 transport across internal temperature gradients, which exist between tissues or organs that are much warmer or colder than the respiratory organs of regionally heterothermic vertebrates with heat exchanging retia (Weber and Campbell, 2011). I did not 159  question this hypothesis until I started writing this thesis during the “lockdown” imposed due to the COVID-19 pandemic, even though a few years ago I amassed all of the mammal blood P50 data that I could find, but I found few studies on the effect of temperature on Hb-O2 affinity in mammals. Most of these studies investigated the effect of temperature on Hb-O2 affinity in regionally heterothermic mammals with limited comparisons to non-heterotherms, so I find the results of these studies equivocal regarding this hypothesis.   Low temperature-dependence of Hb-O2 affinity may be a trait shared by cetartiodactyls, with good evidence for this from reindeer/caribou (Rangifer tarandus), red deer (Cervus elaphus), musk-ox (Ovibos moschatus), common minke whale (Balaenoptera acutorostrata), and pig (Sus sp.) (Brix et al., 1989a; Brix et al., 1989b; Brix et al., 1989a; Pellegrini et al., 1999; Willford and Hill, 1986). Among carnivores, cat (Felis catus) and harbour seal (Phoca vitulina) Hbs also have a low temperature-dependence (Cambier et al., 2004; Willford and Hill, 1986), but there are not many other published studies on the temperature-dependence of carnivore Hbs, including limited data on Hb from the polar bear (Ursus maritimus), which has heat exchanging retia in its legs (Øritsland, 1970). Furthermore, there is a dearth of information on the temperature-dependence of Hb from heterothermic birds (reviewed by Weber and Campbell, 2011). Lastly, in all of these examples of Hb with a low temperature-dependence, including other examples provided by Weber and Campbell (2011), the conclusion of a reduced temperature-dependence is based on limited comparisons to few other mammalian Hbs aside from human HbA. It seems that the O2 affinity of HbA may be particularly sensitive to temperature (ΔH' ≈ -45kJ mol-1 O2), so further comparisons among regionally heterothermic and non-heterothermic mammals are warranted to investigate whether reductions in the temperature-dependence of Hb-O2 affinity are adaptive to animals with heat exchanging retia.  160   Reduced temperature-dependence of Hb-O2 affinity is not a trait that is exclusive to regional heterotherms. For example, this trait has been reported for several ectothermic fishes, including some elasmobranchs and teleosts (e.g., Barlow et al., 2017; Bernal et al., 2018; Cech et al., 1994; Clark et al., 2010; Hopkins and Cech, 1994; Morrison et al., 2015; Weber et al., 1976). However, in most studies of the effect of temperature on Hb-O2 affinity of fishes, the effect of pH and temperature on Hb-O2 affinity were not appropriately quantitively assessed (except for studies by Roy Weber or Angela Fago and colleagues), and the reported summary data provide limited opportunities for further analyses. Furthermore, in a couple of studies on Hb of Atlantic Cod (Gadus morhua), the authors reported reduced and reverse temperature-dependence, but the figures of their data clearly show a normal temperature dependence (e.g., Barlow et al., 2017; Nelson et al., 2019). Therefore, there is reasonable evidence that reduced temperature-dependent Hb-O2 affinity may be a trait that is quite widespread among fishes, and is worthy of further research since the apparent existence of this trait in some species may be a result of inadequate data analysis. Furthermore, it is not clear if this trait has any functional significance in ectotherms or if it is a consequence of effector binding that was selected for reasons unrelated to the temperature sensitivity of Hb. Interestingly, reduced and reversed temperature-dependent blood-O2 affinity has also been reported in chub mackerel (Scomber japonicus), an ectothermic scombrid that is closely related to tuna (Clark et al., 2010), so it reasonable to propose this trait was present in the common ancestor to tuna. However, it does not seem to be a coincidence that all regionally heterothermic teleosts and sharks investigated to date have Hb with reduced temperature sensitivities (Table 5.1).   Hæmoglobins with reduced or reversed temperature-dependence are present in blood of common thresher shark, lamnid sharks, smalleye Pacific opah, tunas, swordfish, and istiophorid 161  billfishes. Among studied tunas, all species exhibit either temperature-independent or reverse temperature-dependent Hb-O2 affinity, except for bigeye tuna (Brill and Bushnell, 1991a; Brill and Bushnell, 2006; Cech et al., 1984; Clark et al., 2008a; Graham, 1973; Lilly et al., 2015; Lowe et al., 2000). The temperature-dependence of bigeye tuna Hb-O2 affinity is, however, relatively reduced compared to that of most other vertebrates.   I do not disagree with the proposal that reductions in the temperature-dependence of Hb-O2 affinity are an example of convergent physiological function among regionally heterothermic vertebrates, but more research and thought to this topic is needed.  5.5 Future research directions  The findings of this thesis add to the list of regional heterotherms that posses Hb with a numerically low ΔH' (Table 5.1), and provides some insight into the functional significance of this trait. However, there are still a lot of unanswered questions and work to be done to better understand the evolution and functional significance of reduced and reverse temperature-dependent Hb-O2 affinity.   Does the butterfly kingfish also have Hb with a reduced temperature-dependence? The butterfly kingfish is a close relative of the tunas in the family Scombridae and is likely capable of elevating cranial temperature (Tullis et al., 1991), although endothermy has yet to be confirmed with temperature measurements (Figure 1.3; Table 5.1). Investigations into cranial temperature and the effect of temperature on blood- and Hb-O2 affinity are needed in the butterfly kingfish.   Hoes does the temperature-dependence of Hb-O2 affinity differ among the tunas, and how do the tunas compare to ectothermic scombrid fishes? There appears to be differences among tunas that may be related to internal temperature gradients that are increased by the 162  depths and water temperatures that each species exploits, as well as species specific capacities to maintain stably elevated RM temperatures. Furthermore, at least one ectothermic scombrid fish exhibits reduced and reverse temperature dependence. I think that the family Scombridae are an excellent model for functional, structural, and molecular studies to further investigate the evolution and significance of reduced and reverse temperature-dependent Hb-O2 affinity. Although the Atlantic bluefin tuna is well studied, no other species in the family Scombridae has been studied in such detail (see Chapter 1, section 1.3.1).   Has temperature-independent Hb-O2 affinity evolved independently in lamnid sharks and the common thresher shark? There is good evidence to suggest that RM endothermy evolved independently in lamnid sharks and the common thresher shark. Structural and molecular studies of Hbs from these sharks, as well as closely related ectothermic sharks, would provide insight into the evolution of this trait.   Is the temperature-dependence of Hbs from regionally heterothermic mammals and birds reduced compared to non-heterothermic species? Although it is assumed that in regionally heterothermic mammals ΔH' is relatively low, it is not well known how the temperature-dependence of Hb compares to closely related non-heterothermic mammals. The order Cetartiodactyla (even-toed ungulates and whales) is an excellent group to investigate the evolution and functional significance of this trait among regionally heterothermic mammals. Some species Hbs are already well characterized (e.g., reindeer Brix et al., 1990; Giardina et al., 1989b; Giardina et al., 1989b), phylogenetic relationships are quite well established (Zurano et al., 2019), and the heat exchanging carotid rete is thought to have been important to the diversification and niche expansion of cetartiodactyls (Lust et al., 2007; Mitchell and Lust, 2008).  163   Does the bigeye thresher shark have warm eyes? Although the bigeye thresher shark has an orbital rete suspected of having a heat exchanging function (Block and Carey, 1985; Weng and Block, 2004), cranial temperatures are not warmer than sea surface temperature (Diego Bernal and Chugey Sepulveda, personal communication). However, I suspect that ocular muscles are capable of elevating eye temperatures above ambient temperature when bigeye thresher sharks are in deep cold water, but like opah, eye temperatures are probably not warmer than surface waters (Wegner et al., 2015). Temperature measurements of the eyes and ambient water, while bigeye thresher sharks are in cold water below the thermocline, are needed to establish if this shark is regionally heterothermic. 5.6 Summary and final thoughts  When I first started thinking about applying for NSERC funding to start a PhD thesis, my objective was to develop a project where I could study sharks and respiratory physiology. That seems to have worked out. I cannot remember what led me to start searching the literature for papers on Hb function in regionally heterothermic sharks, but my introduction to the topic of the temperature-dependence of Hb-O2 affinity in regional heterotherms was through papers by Roy Weber and colleagues (Larsen et al., 2003; Weber and Campbell, 2011; Weber et al., 2010). With these papers, and others (Andersen et al., 1973; Brill and Bushnell, 1991a; Brill and Bushnell, 2006; Carey and Gibson, 1977; Clark et al., 2008a; Clark et al., 2010; Lowe et al., 2000; Rossi Fanelli and Antonini, 1960), I adopted the hypothesis that the temperature-dependence of Hb-O2 affinity will be reduced or reversed in all regionally heterothermic fishes, so as to prevent premature O2 unloading as blood is warmed in a heat exchanging retia. This was at odds with what some others thought, but seemed like a testable hypothesis if I could get blood from enough unstudied species. Since then, I have amended that hypothesis and learned enough 164  that I am confident that I can critically assess and synthesize the literature on this topic. I hope I have done that reasonably well.   In this thesis I have provided evidence that all lineages of known regionally heterothermic teleosts and sharks have Hb with a low overall ΔH'. Specifically, I have shown temperature-independence in smalleye Pacific opah, common thresher shark, and mako shark, a pH-dependent reduced temperature-dependence in swordfish, and a saturation-dependent reduced temperature-dependence in bigeye thresher shark. The oxygenation-dependent release of allosteric effectors contributes endothermically to ΔH' and stabilizes the high affinity Hb conformation at high relative to cold temperature. The main effector of tuna Hb is protons (Ikeda-Saito et al., 1983), whereas in Hbs of billfishes it is pH-dependent binding of ATP (Weber et al., 2010), and ATP is the primary effector of lamnid shark Hbs (Larsen et al., 2003). I have shown that ATP and protons are the main effectors of swordfish Hb, like other billfishes. In opah Hb, proton binding and dissociation cause temperature-independence, but ATP reverses it. Common thresher shark Hb seems to have an intrinsic temperature-independence that is reversed in the presence of ATP. Thus, increases to ΔH' appear to have repeatedly evolved by different molecular mechanisms with underlying dependence on different allosteric effectors.   I have also attempted to explain the functional significance of high Hb concentration in regionally heterothermic fishes, as well as the functional significance of low ΔH'. In all regional heterotherms, high Hb concentration is probably essential to matching O2 supply to O2 demand without unnecessarily increasing cardiac output. There are two major effects of an increased ΔH': it will reduce the energy used during the Hb-O2 binding and unloading cycle, and it will reduce the effect of temperature on Hb-O2 affinity. Presumably, the functional significance of reduced and reverse temperature dependence is associated with at least one of these effects. 165  Swordfish and billfishes heat only the cranial region, and swordfish have a normal temperature dependence at high pH and high saturation, which I suspect is also the case for other billfishes. Any Hb linked energy savings are probably inconsequential to billfishes, but a right shifted OEC at low temperature due to temperature dependent ATP and proton binding should promote O2 unloading uniformly to all tissues despite the tissue temperature. In species that heat their body core (tuna, lamnid sharks, common thresher shark, and opah), Hb-O2 affinity is temperature-independent or exhibits a reverse temperature-dependence, except for the bigeye tuna. In these species, other than bigeye tuna, reduced and reverse temperature-dependence probably conserves heat-energy and prevents Hb-O2 affinity from being too high to unload O2 to the cold tissues and organs, especially those with a high metabolic demand like the heart. That these disparate lineages of teleosts and sharks have independently evolved regional heterothermy is quite remarkable, but the evolution of temperature insensitive Hbs through different molecular strategies to modulate ΔH' is an exceptional example of convergent physiological function.    166  Table 5.1 ΔH' values for known and suspected regionally heterothermic teleosts and sharks  1 WB = whole blood; Hb = hæmolysate or Hb component 2 Graham (1973) reported no effect of temperature on blood-O2 affinity, bot no other details were given. 3 ΔH' cannot be calculated with the data provided in the publication.    Endothermy    Taxon Common name Swimming muscle Eye / brain Viscera ΔH' (kJ mol-1) ΔH' conditions1 ΔH' reference Teleostei        Order Istiophoriformes        Family Xiphiidae        Xiphias gladius Swordfish  X  -23 WB; pH 7.7; 10-25°C 1      +4 Hb+ATP; pH 7.4; 10-25°C 1 Family Istiophoridae        Istiophorus albicans Atlantic sailfish  X     Istiophorus platypterus Indo-Pacific sailfish  X     Makaira mazara Indo-Pacific blue marlin  X  +26 Hb+ATP; pH 7.4; 10-25°C 2 Makaira nigricans Blue marlin  X     Istiompax indica Black marlin  X     Kajikia albida Atlantic white marlin  X     Kajikia audax Striped marlin  X  +4 Hb+ATP; pH 7.4; 10-25°C 2 Tetrapturus angustirostris Shortbill spearfish  X  -7 Hb+ATP; pH 7.4; 10-25°C 2 Tetrapturus belone Mediterranean spearfish  X     Tetrapturus georgii Roundscale spearfish  X     Tetrapturus pfluegeri Longbill spearfish  X     Order Lampriformes        Family Lamdridae        Lampris australensis Southern spotted opah Assumed Assumed     Lampris guttatus North Atlantic opah Assumed Assumed     Lampris immaculatus Southern opah Assumed Assumed     Lampris incognitus Smalleye Pacific opah X X  +1 WB; pH 7.7; 10-20°C 1 Lampris lauta East Atlantic opah Assumed Assumed     Lampris megalopsis Bigeye Pacific opah Assumed Assumed     Order Scombriformes        Family Scombridae        Gasterochisma melampus Butterfly kingfish  Assumed     Scomber japonicus Pacific chub mackerel Ectotherm Ectotherm Ectotherm +42 WB; pH 7.7; 10-20°C 3 Allothunnus fallai Slender tuna X X     Auxis rochei Bullet tuna X Assumed     Auxis thazard Frigate tuna X X     Euthynnus affinis Kawakawa tuna X X     Euthynnus alletteratus Little tunny X X     Euthynnus lineatus Black skipjack tuna X X  ≈0 WB2 4 Katsuwonus pelamis Skipjack tuna X X  +18 WB; pH 7.7; 20-30°C 1, 5 Thunnus alalunga Albacore tuna X X X +7 WB; pH not given; 5-25° 6 Thunnus albacares Yellowfin tuna X X  +4 WB; pH 7.7; 20-30°C 1, 5 Thunnus atlanticus Blackfin tuna X Assumed     Thunnus maccoyii Southern bluefin tuna X Assumed X >0 WB3 7 Thunnus obesus Bigeye tuna X X ? -18 WB; pH 7.7; 15-25°C 8 Thunnus orientalis Pacific bluefin tuna X X X >0 WB3 9 Thunnus thynnus Atlantic bluefin tuna X X X +21 WB; pH 7.7; 15-25°C 10 Thunnus tonggol Longtail tuna X Assumed             Elasmobranchii        Order Lamniformes        Family Alopiidae        Alopias pelagicus  Pelagic thresher shark  ?     Alopias superciliosus Bigeye thresher shark  ?  -3 WB; pH 7.7; 10-25°C 1 Alopias vulpinus Common thresher shark X  ? +12 WB; pH 7.3; 15-22°C 1 Family Lamnidae        Carcharodon carcharias White shark X X X    Isurus oxyrinchus Shortfin mako shark X X X -3 WB; pH 7.7; 15-25°C 1 Isurus paucus Longfin mako shark ? X X    Lamna ditropis Salmon shark X X X ≥0 Hb 11 Lamna nasus Porbeagle shark X X X +12 Hb+ATP; pH 7.3; 10-26°C 12 167  This table was adapted from Dickson and Graham (2004). X = evidence of endothermy including measurements of elevated tissue temperatures; ? = putative heat exchanging retia mirabilia reported, but temperature measurements are lacking; Assumed = endothermy is assumed due to morphological characteristics and/or phylogenetic relationships to known endothermic species. References for the evidence for endothermy: (Bernal and Sepulveda, 2005; Block, 1990; Block, 1991a; Block and Carey, 1985; Dickson and Graham, 2004; Patterson et al., 2011; Runcie et al., 2009; Sepulveda et al., 2007a; Sepulveda et al., 2008; Underkoffler et al., 2018; Wegner et al., 2015). ΔH' values are from the following: 1 = this thesis; 2 = (Weber and Campbell, 2011); 3 = (Clark et al., 2010); 4 = (Graham, 1973); 5 = (Brill and Bushnell, 1991a); 6 = (Cech et al., 1984); 7 = (Clark et al., 2008a); 8 = (Lowe et al., 2000); 9 = (Lilly et al., 2015); 10 = (Brill and Bushnell, 2006); 11 = (Dickinson and Gibson, 1981); 12 = (Larsen et al., 2003).  168  References Alexander, R. L. (1995). Evidence of a counter-current heat exchanger in the ray, Mobula tarapacana (Chondrichthyes: Elasmobranchii: Batoidea: Myliobatiformes). J. Zool. 237, 377–384. Alexander, R. L. (1996). 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Evol. 133, 256–262.  191  Appendix: A note on the storage duration of fish blood  In Chapter 3 and Chapter 4, oxygen equilibrium curves were constructed on whole blood from fish and sharks captured off Southern California (i.e., the Southern California Bight) or off Massachusetts. Blood samples were drawn by caudal puncture into heparinized syringes, and were then shipped, on ice, by courier to the UBC campus in Vancouver, Canada, where experiments were conducted within 1 to 4 days after the blood was collected. This sampling technique was  previously used for a study on chub mackerel blood-O2 affinity (Clark et al., 2010). In that study, experiments were conducted at the UBC Vancouver campus, but blood was collected from mackerel captured off Southern California by the same researcher (Dr. Chugey Sepulveda) that sampled blood for my thesis research. Clark et al. (2010) concluded that fish blood is viable for up to 6 days if stored at 4°C. To verify this sampling technique, we conducted preliminary experiments on swordfish blood.   Swordfish (n = 3) were captured by deep-set buoy gear (Sepulveda et al., 2014) off Southern California. Blood was drawn by caudal puncture into heparinized syringes. Blood samples were kept on ice and shipped by courier to the UBC campus in Vancouver, Canada, where experiments were conducted over 11 days after the blood was collected. Blood was refrigerated at 4°C during this time. Each day after blood samples arrived at UBC (2 days post-capture) hæmoglobin (Hb) concentration (mM) and hæmatocrit (Hct; the ratio of the volume of red bloods cells to the total volume of blood) were measured (Figure A.1). Whole blood pH, plasma Hb concentration, and whole blood P50 (mmHg; the partial pressure of oxygen that corresponds to 50% Hb-O2 saturation) were also determined, but not every day (see Figure A.1). Hb concentration, Hct, pH, and P50 were determined as described in Chapter 3 (section 3.2). To assess if considerable red blood cell lysis occurred during the storage duration, plasma Hb 192  concentration was measured in triplicate 10 µl samples of blood plasma that were separated from the red blood cells by centrifugation. Mean corpuscular hæmoglobin concentration (MCHC, in mM) was calculated by dividing [Hb] by Hct.   To assess if Hb concentration, plasma Hb concentration, Hct, MCHC, and pH changed over time, linear models were fit to the data for each individual swordfish. For each of these variables, the slopes of the fitted lines were not significantly greater than zero, indicating that there was no biologically relevant change in each variable. The P50 data were inspected visually, and based on these data and a previous study by Clark et al. (2010), it was concluded that OECs could be constructed on blood stored for up to 6 days after blood was collected, provided blood was refrigerated at 4°C during this time. For the work reported in Chapters 3 and 4, OECs were constructed within 4 days post-capture, less than the proposed maximum of 6 days.   193  Figure A.1 The effect of storage duration on blood from swordfish (Xiphias gladius). Blood from swordfish (n = 3, represented by different shaped symbols) was stored at 4°C for 11 days post-capture. Hb concentration (A), plasma Hb concentration (B), hæmatocrit (C), mean corpuscular Hb concentration (D), whole blood pH (E), and whole blood P50 (F) were not appreciably changed over the respective sampling periods.   

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