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On the teleost system for enhanced oxygen unloading : time course following catecholamine removal and… Shu, Jacelyn 2019

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  ON THE TELEOST SYSTEM FOR ENHANCED OXYGEN UNLOADING:  TIME COURSE FOLLOWING CATECHOLAMINE REMOVAL AND AN INVESTIGATION INTO OTHER TELEOSTS     by   Jacelyn Shu  B.Sc., The University of British Columbia, 2016     A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF  THE REQUIREMENTS FOR THE DEGREE OF  Master of Science   in  THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES  (Zoology)   THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)    May 2019  © Jacelyn Shu, 2019   ii  The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis/dissertation entitled:  On the teleost system for enhanced oxygen unloading: time course following catecholamine removal and an investigation into other teleosts  submitted by Jacelyn Shu  in partial fulfillment of the requirements for the degree of Master of Science in the Faculty of Graduate and Postdoctoral Studies (Zoology)  Examining Committee: Colin Brauner, Department of Zoology Supervisor  Eric Taylor, Department of Zoology Supervisory Committee Member  Anthony Farrell, Department of Zoology Additional Examiner     Additional Supervisory Committee Members: Jeffrey Richards, Department of Zoology Supervisory Committee Member    iii  Abstract Recent studies suggest that teleost fishes may be able to greatly enhance the amount of oxygen (O2) unloaded to the tissues during a generalized acidosis associated with stress. This mechanism relies on pH-sensitive hemoglobin, a red blood cell (RBC) β-adrenergic Na+/H+ exchanger (β-NHE) activated by catecholamines to protect RBC pHi, and plasma-accessible carbonic anhydrase (paCA) at the tissues, but not at the gills, to short-circuit the β-NHE, acidify the RBC, and unload O2 from hemoglobin. This system has been shown to increase tissue PO2 (∆PO2) by up to 30 Torr and has been proposed to play an important role both during early teleost evolution, as well as in modern teleost physiology. To date, most studies regarding this system have been conducted in the context of high stress and circulating catecholamines; little is known about this system under low catecholamine levels, such as following stress. In addition, despite being often extrapolated to all teleosts, this system has only been studied within the salmonids. Here, I investigate the time course of β-NHE short-circuiting by paCA in rainbow trout blood following in vitro stimulation by isoproterenol, a synthetic catecholamine, as well as natural catecholamines in vivo to determine how long after adrenergic stimulation this system may remain operational. A significant increase in plasma ∆PO2 due to β-NHE short-circuiting by paCA was found in rainbow trout blood stimulated in vitro up to one hour after removal of isoproterenol; this was reduced to less than 30 min under natural catecholamines. A significant ∆PO2 of approximately 5 Torr was determined even 6 h after stimulation, as well as in pharmaceutically blocked blood, suggesting that enhanced O2 unloading may be operational in resting fish. The system was also investigated in two teleost species distantly related to the salmonids, cobia (Rachycentron canadum) and mahi-mahi (Coryphaena hippurus).  In cobia and mahi-mahi, evidence of a RBC β-NHE was found, and it is predicted that cobia may exhibit up to a 61% increase in enhanced O2 unloading with no change in blood flow through short-circuiting of RBC β-NHE, a value consistent with salmonids. This second phylogenetically distant group provides support that this system may be functional in teleosts in general. iv  Lay Summary Bony fishes are the most diverse and successful group of any vertebrate. One recent proposition suggests that their success may be related to a unique system used to oxygenate their tissues. Specifically, these fishes are thought to be able to greatly enhance the amount of oxygen available to the tissues, more so than any other vertebrate. However, the major effects of this system for enhanced oxygen unloading have only been studied under the context of high stress. In addition, despite its extrapolation to bony fishes as a whole, encompassing almost 30,000 species, this system has only been studied in salmon and trout. In my thesis, I show that the system has a significant effect even at rest, greatly expanding the situations in which enhanced oxygen unloading may be used, and show evidence for the system in a distantly related bony fish, providing support for its presence throughout the bony fishes.   v  Preface  This thesis is the original unpublished work of the author, J. Shu, with editorial input from Colin Brauner and Till Harter.  Dr Brauner and I conceived the experiments in Chapter 2, with input from Dr Harter. I carried out all experiments and analyses.  Experiments in Chapter 3 were designed with input from Dr Brauner. All experiments and analyses in Series I were conducted by me. Series II sampling was performed by Dr Rachael Heuer at the University of Miami, and oxygen equilibrium curves were produced and analysed with the help of Anne Kim at the University of British Columbia.  Animal husbandry and all experiments were conducted according to the guidelines of the Canadian Council on Animal Care and approved by the UBC Animal Care Committee (Protocol no. A15-0266).vi  Table of Contents Abstract ............................................................................................................................................ ii Lay Summary .................................................................................................................................. iv Preface ............................................................................................................................................. v Table of Contents ........................................................................................................................... vi List of Tables ................................................................................................................................. vii List of Figures ................................................................................................................................. ix List of Symbols and Abbreviations .................................................................................................. x Acknowledgements ........................................................................................................................ xi Chapter 1: General Introduction .................................................................................................... 1 1.1 The teleost oxygen transport system ......................................................................................................... 1 1.2 Time course of enhanced O2 unloading by β-NHE short-circuiting .................................................. 4 1.3 Enhanced O2 unloading in a marine teleost ............................................................................................ 5 1.4 Investigating enhanced O2 unloading ....................................................................................................... 7 Chapter 2: Time course of β-NHE short-circuiting following catecholamine removal ................ 8 2.1 Introduction .................................................................................................................................................. 8 2.2 Materials and Methods .............................................................................................................................. 11 2.2.1 Experimental animals ........................................................................................................................ 11 2.2.2 Series I: In vitro experiments ............................................................................................................. 11 2.2.3 Series II: In vivo experiments ............................................................................................................ 13 2.2.4 Closed-system ∆PO2 assay ............................................................................................................... 14 2.2.5 Hematological analyses ..................................................................................................................... 15 2.2.6 Data analysis ....................................................................................................................................... 16 2.3 Results .......................................................................................................................................................... 17 2.3.1 Series I: In vitro experiments ............................................................................................................. 17 2.3.2 Series II: In vivo experiments ............................................................................................................ 17 2.4 Discussion ................................................................................................................................................... 24 2.4.1 Series I: In vitro experiments ............................................................................................................. 24 2.4.2 Series II: In vivo experiments ............................................................................................................ 27 vii  2.4.3 Conclusions ........................................................................................................................................ 30 Chapter 3: Enhanced oxygen unloading in a marine teleost ....................................................... 32 3.1 Introduction ................................................................................................................................................ 32 3.2 Materials and Methods .............................................................................................................................. 35 3.2.1 Experimental animals ........................................................................................................................ 35 3.2.2 Series I: β-adrenergic cell swelling response .................................................................................. 35 3.2.3 Series II: Oxygen equilibrium curves and modelling of enhanced oxygen unloading ............ 36 3.3 Results .......................................................................................................................................................... 39 3.3.1 Series I: β-adrenergic cell swelling response .................................................................................. 39 3.3.2 Series II: Oxygen equilibrium curves and modelling of enhanced oxygen unloading ............ 42 3.4 Discussion ................................................................................................................................................... 45 3.4.1 Series I: β-adrenergic cell swelling response .................................................................................. 45 3.4.2 Series II: Oxygen equilibrium curves and modelling of enhanced oxygen unloading ............ 47 3.4.3 Conclusions ........................................................................................................................................ 49 Chapter 4: General Discussion ..................................................................................................... 51 4.1 Implications ................................................................................................................................................. 52 4.2 Limitations and future directions ............................................................................................................ 53 4.2.1 Rainbow trout as a model organism ............................................................................................... 53 4.2.2 OEC modelling of enhanced O2 unloading .................................................................................. 54 4.2.3 Further in vivo studies ........................................................................................................................ 55 4.2.4 Other species of teleosts ................................................................................................................... 56 4.3 Conclusion .................................................................................................................................................. 57 References ...................................................................................................................................... 58 Appendix ........................................................................................................................................ 62  viii  List of Tables Table 1. Blood parameters in rainbow trout blood ............................................................................................ 23 Table 2. Hematological parameters in caudally sampled (Rachycentron canadum) and mahi-mahi (Coryphaena hippurus) blood. ................................................................................................................................................ 41 Table 3. Hematological parameters for cobia (Rachycentron canadum) whole blood. ....................................... 43    ix  List of Figures Figure 1. Representative trace of the closed-system ∆PO2 assay for rainbow trout whole blood equilibrated with 10% O2 and 0.5% CO2, stimulated with isoproterenol (ISO). ................................. 19 Figure 2. The influence of time following isoproterenol removal on the magnitude of the change in partial pressure of oxygen in closed-system blood (∆PO2, standardized to hemoglobin concentration), following acidification and addition of carbonic anhydrase. ....................................... 20 Figure 3. Closed-system change in the partial pressure of oxygen (∆PO2) after carbonic anhydrase (CA) addition in the in vivo (Series II) and in vitro (Series I) trials. ..................................................................... 21 Figure 4. Adrenaline and noradrenaline concentrations measured in plasma sampled from cannulated rainbow trout. .................................................................................................................................................. 22 Figure 5. The β-adrenergic cell swelling response in caudally sampled blood from cobia (Rachycentron canadum) and mahi-mahi (Coryphaena hippurus). ........................................................................................... 40 Figure 6. Oxygen equilibrium curves of cobia (Rachycentron canadum) whole blood. ...................................... 44     x  List of Symbols and Abbreviations atm atmospheres of pressure, where 1 atm is 760 Torr or 101.33 kPa A absorbance AE anion exchanger ANOVA analysis of variance β-NHE β-adrenergically stimulated sodium/proton exchanger CA carbonic anhydrase Cl- chloride ion CO2 carbon dioxide ∆ delta; change (e.g. ∆PO2) H+ proton Hb hemoglobin Hct hematocrit HCO3- bicarbonate ISO isoproterenol kPa kilopascals, where 1 kPa is 0.0099atm or 7.50 Torr MCHC mean corpuscular hemoglobin concentration MS-222 tricaine methanesulphonate Na+ sodium ion nH Hill coefficient O2 oxygen ºC degrees Celsius OEC oxygen equilibrium curve PO2 partial pressure of oxygen P50 PO2 at which 50% of the hemoglobin is saturated with oxygen paCA plasma-accessible carbonic anhydrase ∆Pa-vO2 arterial-venous difference in partial pressure of O2 PCO2 partial pressure of CO2 ∆pHa-v arterial-venous difference in blood pH pHe extracellular, plasma pH pHi intracellular, red blood cell pH RBC red blood cell S.E.M. standard error of the mean S saturation of blood with oxygen    xi  Acknowledgements First and foremost, I’d like to acknowledge my supervisor, Dr Colin Brauner. Colin, you’ve been such an inspiration to me and have helped me grow so much, both as a scientist and a person. Thank you for your unwavering support and encouragement, and for sticking with me through all these years that I’m sure you didn’t think you were signing up for when I first sat in your office as a nervous undergrad 4 years ago. The positivity and grace with which you handle pretty much everything has been so refreshing and always much appreciated and admired. Thank you for everything. My immense gratitude is due also to Till Harter, who co-supervised me with much patience, wisdom, and gentle amusement during both my Honours and Master’s theses. Till, thank you for your invaluable input in designing experiments, your sharp editorial eye, and for teaching me everything. This project would not be anywhere close to what it is if it weren’t for you.  Thanks are due to the rest of the Brauner lab, Comparative Physiology group, and Zoology Department for the good times and the laughs in between the long lab days. You guys made it enjoyable to come in every day, even when the science wasn’t going too hot. Thank you especially to Anne Kim, pH-OEC queen and fellow lover of food for the help with this project and for eating with me.  Thank you to the members of my supervisory committee, Rick Taylor and Jeff Richards, for the guidance and advice on this project, and for encouraging me to think about the biochemical and ecological worlds outside of my physiology bubble. Acknowledgements are also due to the Grosell lab at the University of Miami for hosting me for a month. Thanks for letting me use your precious fish and zoom around in your beloved golf cart, and for the warmth and hospitality with which you welcomed us. Thank you especially to Dr Rachael Heuer, for dealing with all the blood samples and timing and shipping. To my friends and in particular my lifegroup, thank you for the constant prayers, good food, and laughs throughout this whole time. xii  Thanks of course to my parents, for supporting me all this time and for always bringing food.  To my new labmates of the Mank lab, thank you for being patient with me as I finish this degree. It’s been great so far and I have high hopes that this next chapter will be good times as well. Thanks everybody, it’s been fun. 1  Chapter 1: General Introduction 1.1 The teleost oxygen transport system Teleost fishes are one of the most successful and diverse groups of vertebrates, making up almost half of all vertebrate species and inhabiting virtually every aquatic habitat on the planet (Nelson et al., 2016). Multiple hypotheses have been proposed for the reasons behind the great success of teleosts, such as the importance of a whole genome duplication, or the evolution of key innovations like the swimbladder. Among the many hypotheses is one that takes into consideration the era and environment during which teleosts first began to radiate and diverge. Just prior to the teleost adaptive radiation, atmospheric O2 levels had plummeted as a result of volcanic activity and fires in the Permian crisis, wiping out most marine fishes and leaving many aquatic habitats and niches unoccupied (Helfman et al., 2009). This was followed by approximately 100 million years of low global O2 levels (Clack, 2007), creating an opportunity that could be exploited by marine animals that could perform well under low O2 conditions. It is during this period that the teleosts underwent what is likely the most extensive adaptive radiation in all of vertebrate history (Helfman et al., 2009). It has been proposed that during this time of chronic low O2 levels, the unique teleost Hb-O2 transport system played an important role in the radiation of teleosts. Through this system, teleosts are able to fill the swimbladder at depth, increase O2 supply to the eye, and potentially enhance O2 unloading to their tissues in general, processes that may have helped to firmly establish teleosts as the widely diverse group we see today (Randall et al., 2014; Rummer and Brauner, 2015; Scholander and Van Dam, 1954; Wittenberg and Wittenberg, 1974).  The teleost O2 transport system has been subjected to over 200 million years of evolution, with numerous adaptations at every level, from vasodilation to ventilation. However, common to almost all teleosts despite their vast diversity is an extremely pH-sensitive hemoglobin (Hb). All vertebrates possess Hb that is sensitive to changes in pH, where a decrease in pH will result in a decrease in Hb-O2 affinity and a right-shift of the oxygen equilibrium curve (OEC) in a phenomenon known as the Bohr effect (Bohr et al., 1904). Unique to the teleosts, however, is Hb that exhibits the Root effect, where a decrease 2  in pH results not only in a reduction in Hb-O2 affinity (Bohr effect), but in O2 carrying capacity as well (Root effect). In some cases, the Root effect is so pronounced that at low pH, Hb cannot be completely saturated even in the presence of 100 atm of pure O2 (Scholander and Van Dam, 1954).  The Root effect has long been attributed the crucial role of unloading O2 to specialized tissues: one such tissue is the swimbladder (Scholander and Van Dam, 1954). At a depth of several thousand metres, hydrostatic pressure compresses gas within the swimbladder to several hundred times that of atmospheric pressure, resulting in a PO2 that a typical blood O2 gradient would otherwise be unable to overcome. Yet, teleosts are able to inflate their swimbladder and maintain buoyancy against these high PO2 gradients, at depths of up to 7000 m, through the use of Root effect Hb (Nielsen and Munk, 1964). At these specialized tissues, the Root effect is induced via a large arterial-venous change in pH (∆pHa-v) produced via glycolysis and the pentose-phosphate shunt. A counter-current exchanger in a network of capillaries called a rete, present at both the swimbladder and the avascular retina in the eye, localizes the ∆pHa-v and acts as an O2 multiplication system. This localized acidosis greatly reduces Hb-O2 binding and drives O2 from Hb into solution via the Root effect. The resulting blood PO2 is sufficient to cause O2 diffusion into these specialized tissues, even against the very high PO2 tensions within (Baeyens et al., 1971; Pelster, 2004).  Under unstressed conditions, acidoses are kept localized to these specialized tissues, allowing Hb-O2 loading at the gills and unloading at tissues to occur as normal. However, stressors such as exercise or hypoxia may result in a generalized metabolic or respiratory acidosis, where pH decreases throughout the body, including the gills. In this case, such pH-sensitive Root effect Hb can become a liability, compromising O2 loading at the gills. At a time when O2 is in high demand or low supply, this could be greatly detrimental. Thus, to protect O2 loading at the gills, most teleosts with Hb exhibiting the Root effect also possess a red blood cell (RBC) β-adrenergic Na+/H+ exchanger (β-NHE) to regulate RBC intracellular pH (pHi). During stressful conditions, catecholamines such as noradrenaline and adrenaline are released into the blood. One result of this hormone release is activation of the β-NHE on 3  the RBC membrane, which works to maintain pHi by exporting H+ from the RBC in exchange for Na+. In the plasma, H+ combine with plasma HCO3- to form CO2 that will diffuse back into the cells. However, in the absence of carbonic anhydrase (CA) at the teleost gill, this reaction is slow and negligible (reviewed in Harter and Brauner, 2017). Thus, as H+ are extruded, pHi is maintained, and a H+ disequilibrium is produced across the RBC membrane. Despite the extracellular acidosis, the β-NHE works to keep the RBC relatively alkaline, preventing compromised Hb-O2 binding in Root effect Hb. As a result, O2 uptake at the gills is maintained. It is important to note that full oxygenation of Hb during a generalized acidosis is only possible if RBC pHi is protected as a result of a H+ disequilibrium that is maintained across the RBC membrane. This is the case in the absence of plasma CA activity, where continued β-NHE activity effectively extrudes H+. However, any CA activity accessible to the plasma would rapidly catalyze the dehydration of H+ and plasma HCO3- back into CO2, which would freely diffuse back into the RBC and short-circuit the H+ disequilibrium. In most other vertebrates, including mammals and Chondrichthyes, plasma-accessible CA (paCA) is abundant at the gas exchange surfaces, allowing for rapid interconversion of CO2 and HCO3- for CO2 excretion (Henry et al., 1997; Henry and Swenson, 2000). In teleosts, however, paCA is thought to be absent at the gills (reviewed in Harter and Brauner, 2017), allowing the H+ disequilibrium produced by the β-NHE to be maintained, ultimately protecting Hb-O2 loading and securing O2 uptake at the gills despite a generalized acidosis. Until recently, it was thought that due to β-NHE activity and RBC pHi regulation, the benefit of the Root effect was limited to specialized tissues where a large ∆pHa-v could be induced, namely at the swimbladder and eye where retia are present. However, recent studies have proposed a mechanism whereby paCA at the tissues short-circuits the H+ disequilibrium produced by the β-NHE, acidifying the RBC and producing a similar ∆pHa-v in the absence of a rete (Rummer and Brauner, 2011). As a result, Hb-O2 saturation decreases dramatically in Root effect Hb, enhancing the amount of O2 that can be unloaded to the tissue (Rummer and Brauner, 2011). Thus, by this mechanism, increased O2 unloading 4  by Root effect Hb appears not to be limited only to specialized tissue, but may occur in general tissue as well, as long as paCA is present.  In general, it is our current understanding of this system for enhanced O2 unloading that three basic components are required: 1) pH-sensitive Hb; 2) rapid and active regulation of RBC pHi during a generalized acidosis, via a RBC β-NHE; and 3) paCA at the tissues, but not at the gills, to permit localized short-circuiting of the pHi protection generated by the β-NHE, thus inducing the Root effect and enhancing O2 unloading at the tissues. Of these, pH-sensitive Hb has been widely observed in most teleosts investigated to date (Berenbrink et al., 2005). These pH-sensitive Hb are usually accompanied by the presence of RBC β-NHE activity, both of which have been demonstrated in salmonids such as rainbow trout (Oncorhynchus mykiss), coho salmon (O. kisutch), and Atlantic salmon (Salmo salar; Shu et al., 2017). Further research is required to confirm the distribution of paCA in the teleost vasculature, but based on the few species that have been studied, the absence of paCA at the gills and presence in the tissues is thought to be a general trait among most teleosts (reviewed in Harter and Brauner, 2017). 1.2 Time course of enhanced O2 unloading by β-NHE short-circuiting Despite the proposed breadth and significance of the teleost system for enhanced O2 unloading, our understanding of the mechanism of this system and its effects is still sparse. From previous studies, we know that enhanced O2 unloading helps to reduce cardiac work by 30% in swimming Atlantic salmon, and can increase tissue PO2 by 30 Torr (Harter, 2018; Rummer et al., 2013). In addition, the time course of RBC short-circuiting and recovery within the vasculature has been shown to fall within RBC transit times of the circulatory system (Harter et al., 2018). These studies point to the likely role that enhanced O2 unloading plays in vivo, but beyond this, little has been done to further investigate how this system may apply within a fish and under what conditions this can occur. Our current understanding of the system, based on empirical evidence, requires stress-induced high levels of circulating catecholamines to stimulate β-NHE activity that can then be short-circuited by paCA at the tissues. However, previous 5  studies have found evidence for enhanced O2 unloading even in Atlantic salmon at rest (Harter, 2018) and in rainbow trout displaying low levels of catecholamines (Rummer et al., 2013). Thus, our understanding of the conditions under which enhanced O2 unloading may occur is incomplete, and maximally stressed conditions with high levels of catecholamines may not be a requirement in order for enhanced O2 unloading to occur.  One possibility for enhanced O2 unloading in unstressed conditions is an initial activation of the β-NHE that continues past catecholamine removal, allowing for enhanced O2 unloading even after most of the catecholamines have been removed or metabolized. Catecholamines in vivo are relatively short-lived, with the rapid removal and metabolism of the majority of catecholamines occurring within minutes following release (Nekvasil and Olson, 1986b; Tang and Boutilier, 1988). Thus, a mechanism in which the effects of enhanced O2 unloading are prolonged could be advantageous in increasing the amount of O2 available to the tissues, unconstrained by the short time period during which catecholamines are high. Having enhanced O2 unloading available to the tissues past the duration of a stressor could play a significant role in assisting recovery from stress. Indeed, excess post-exercise oxygen consumption (EPOC), as well as excess post-hypoxic oxygen consumption (EPHOC), have been observed following exercise and hypoxic stress in salmonids (Lee et al., 2003; Scarabello et al., 1992; Svendsen et al., 2012). This suggests that, post-stress, extra O2 is required in order to recover and regain routine function, a process that could be greatly assisted by the prolonged effect of enhanced O2 unloading following a stressor. Thus, for the first goal of my thesis, I set out to determine the time course of enhanced O2 unloading and its duration and magnitude following a stressor and the rapid removal of catecholamines.  1.3 Enhanced O2 unloading in a marine teleost Previous studies on this mechanism for enhanced O2 unloading in teleosts have all expressed the great significance this system could have, not only on teleost physiology, but also on our understanding of the evolutionary history of these fish. Shown to increase muscle PO2 by up to 30 Torr, as well as decrease cardiac work by up to 30%, this system likely plays an important physiological role in modern-6  day teleosts such as rainbow trout and Atlantic salmon (Harter, 2018; Rummer and Brauner, 2015). Evolutionarily, enhanced O2 unloading to the tissues during the hypoxic Permian and Triassic eras would likely have played an important role in allowing for the initial success of teleosts (Randall et al., 2014), possibly contributing to what has ultimately become the vastly diverse group we see today. However, despite its proposed importance both in teleost physiology as well as their evolutionary history, the number and diversity of species in which this system has been studied is notably limited, with studies to date having been conducted almost entirely within the salmonids: namely rainbow trout, coho salmon, and Atlantic salmon (Rummer et al., 2013; Rummer and Brauner, 2015; Shu et al., 2017). Salmonids are common, relatively large species with active lifestyles, are easily raised in captivity, and have a long history of research in fish physiology. Consequently, these fish serve well as a starting point in which to study this system. However, if we are to continue to extrapolate our findings to all teleosts and make general assumptions about the evolutionary history of this group as a whole, a foray into the approximate 30,000 other species of teleosts is required (Nelson et al., 2016). Thus, as a small step to address this knowledge gap, I investigated whether this mechanism of enhanced O2 unloading might exist in a distantly related teleost species to the salmonids. For my study, I chose to investigate cobia (Rachycentron canadum) and mahi-mahi (Coryphaena hippurus). These fish are sister taxa within the percomorphs, possibly the most diverse and speciose group within the teleosts (Betancur-R. et al., 2013). Percormorphs also diverged from the salmonids early in the teleost lineage, resulting in over 200 million years of independent evolution to the present day. Despite this divergence, cobia and mahi-mahi are similar to the salmonids in their athletic ability. Salmonids are well known for their long oceanic and upstream spawning migrations, often travelling thousands of kilometers in order to reproduce (Cooke et al., 2006; Quinn, 1991). Similarly, cobia and mahi-mahi are pelagic, long-distance swimmers with extensive seasonal migrations (Dippold et al., 2017). Associated with this active, aerobically demanding lifestyle is the clear benefit that a system for enhanced O2 unloading to the tissues would provide. Thus, cobia and mahi-mahi are fish that are distantly related to 7  salmonids in which enhanced O2 unloading would likely be advantageous. Given their phylogenetic position and potential positive selection for this system, studying these fish would provide us with an additional point of reference with which to frame our understanding of enhanced O2 unloading and its evolution among the teleosts. Of course, more extensive research into further teleost species, as well as more ancestral actinopterygians, is required in order to truly get a sense of the evolutionary history of this system. However, as a teleost group that diverged from salmonids over 200 mya, cobia and mahi-mahi provide an informative first step into the investigation of this system in teleosts as a whole. 1.4 Investigating enhanced O2 unloading  By studying the system for enhanced O2 unloading, I aim to further our knowledge of this unique phenomenon that likely played a significant role not only in the initial diversification and success of all teleosts, but also in the physiology of modern teleosts today. Among the teleosts, there is exceptional diversity in morphology, physiology, and lifestyle. The possibility that such different animals all share this unifying characteristic of enhanced O2 unloading is a fascinating one; the idea that this characteristic may have provided a basis for which evolution could act upon and produce such diversity is even more intriguing. With my thesis, I hope to shed some light on how and in whom this system for enhanced O2 unloading works: thus, the overarching goals of my thesis are to investigate the physiological significance of this system by determining the time course that short-circuiting of the RBC β-NHE remains operational following catecholamine release, and to expand our knowledge of this system to a different teleost species. I hypothesize that the teleost system for enhanced O2 unloading to the tissues lasts beyond catecholamine removal and can be applied to teleosts outside of the salmonids. As a system that likely has a profound impact on the physiology and evolutionary history of teleosts, my investigation into the mechanism for enhanced O2 unloading, as well as its scope, may have implications not only for our understanding of the origins of this group, but also how nearly 30,000 teleost species may function today. 8  Chapter 2: Time course of β-NHE short-circuiting following catecholamine removal 2.1 Introduction Since the first proposal and proof-of-principle of the teleost system for enhanced O2 unloading conducted by Rummer and Brauner (2011) in rainbow trout, a number of studies have investigated the mechanism and in vivo relevance of enhanced O2 unloading by paCA (Alderman et al., 2016; Harter et al., 2018; Rummer et al., 2013; Rummer and Brauner, 2011, 2015; Shu et al., 2017). However, our understanding of this system is still lacking in many areas. For example, our current model of this mechanism is based on empirical evidence where catecholamines are required to trigger β-NHE activity, which in turn produces a H+ disequilibrium that can then be short-circuited by paCA for enhanced O2 unloading to the tissue. However, noradrenaline, the main catecholamine associated with β-NHE activity (Cossins and Kilbey, 1989; Tetens et al., 1988), is relatively short-lived in vivo (Gamperl et al., 1994, from Tang and Boutilier, 1988). Within 5 min following noradrenaline release, only approximately 10% of the original concentration remains active and in circulation, with the rest either metabolized or removed by the gills (Nekvasil and Olson, 1986a, 1986b). With this rapid decrease in active catecholamines in the blood, it is uncertain whether the system for enhanced O2 unloading continues to function, and if so, for how long. Thus, the goal of this study was to determine the time course of potential enhanced O2 unloading via β-NHE short-circuiting by paCA following the introduction and rapid removal of catecholamines. To investigate this time course, a closed-system ∆PO2 assay was used, as has previously been conducted on rainbow trout blood as a proof-of-principle of this system (Rummer and Brauner, 2011). In this assay, cannulated blood is drawn from the animal, equilibrated to gas tensions, then placed in a closed system where blood PO2 can be monitored in real time. The blood is β-adrenergically stimulated, acidified via HCl injection mimicking an in vivo acidosis, then CA is introduced to short-circuit any β-NHE activity. Throughout, blood PO2 is recorded using a fiber-optic PO2 optode. As pHi decreases, 9  pH-sensitive teleost Hb will unload O2 to the blood, resulting in a measurable increase in blood PO2; any increase in blood PO2 following CA addition can then be interpreted as enhanced O2 unloading via β-NHE short-circuiting by paCA. This ∆PO2 following CA may be comparable to what would be observed in vivo as RBCs encounter paCA at the tissues, and provides a way of determining the potential for enhanced O2 unloading. By conducting this assay at different time points following catecholamine release and removal, I hoped to determine the time course of enhanced O2 unloading. For my investigation of this time course, I used a combination of in vitro and in vivo experiments, in conjunction with the ∆PO2 assay. I first used an in vitro approach, stimulating blood using saturating concentrations of isoproterenol (ISO; a synthetic noradrenaline analogue) that I then rinsed off before determining the ∆PO2 following CA in the ∆PO2 assay (from here on, this will be referred to simply as ∆PO2). By using a stable catecholamine at the same concentration each time under controlled lab conditions, I hoped to account for potential variability due to different in vivo levels of catecholamines and possibly obtain a cleaner response. However, while this in vitro approach provides us with an appropriate situation in which to study the β-NHE time course, it may not accurately depict what occurs in vivo. As was previously mentioned, ISO is a synthetic noradrenaline analogue, stable and well-suited for laboratory use, but is slightly more potent than natural catecholamines (Cossins and Kilbey, 1989; Salama, 1993; Tetens et al., 1988). In addition, at the time of in vitro experimentation, tonometry was the most efficient method of equilibrating blood to gas tensions. Due to the non-linear nature of Hb-O2 binding, the same O2 saturation of approximately 75% was targeted in each run by equilibrating blood to custom-mixed gases using tonometry. However, tonometry is limited in the speed of equilibration in the larger volumes of blood used (≥2 mL) and relatively small surface area. Later, Harter et al. (2018) developed a continuous flow apparatus, or “oxygenator”, that allowed for a more rapid equilibration of blood using counter-current gas flow and a high surface area. Thus, in addition to an in vitro stress, I also conducted a ∆PO2 assay on blood from fish stressed in vivo, and attempted to account for possible limitations of the in vitro 10  experiment. Studying enhanced O2 unloading following an in vivo stress allowed for investigation into effects of the system after stimulation by and removal of natural catecholamines. Instead of using tonometry to equilibrate blood to gases over 30 min, blood was passed counter-currently to gases through the oxygenator and equilibrated within 5 min. This decreased the delay between sampling and measuring ∆PO2, allowing for measurements at earlier time points in the time course. By using this two-pronged in vitro and in vivo approach in studying the time course of enhanced O2 unloading via paCA, I hoped to develop a more thorough understanding of the time course of the system and its mechanisms. Thus, the objectives of this chapter were to determine the time course over which rainbow trout RBC β-NHE short-circuiting could occur after the removal of catecholamines: 1) in vitro, by stimulating RBCs with isoproterenol (ISO); and 2) in vivo, via the release of natural catecholamines by inducing a stress response in rainbow trout. Based on previous evidence and the potential benefit of enhanced O2 unloading past catecholamine removal, I predicted that a significant ∆PO2 would continue even in the absence of catecholamines, both in vitro and in vivo. If this is the case, we may be able to expand our current knowledge of enhanced O2 unloading and apply it to many more conditions than just during short-lived stress. As a system that has much potential to affect aerobic metabolism, this would greatly increase the number of situations during which enhanced O2 unloading could be used.    11  2.2 Materials and Methods 2.2.1 Experimental animals Female rainbow trout (Oncorhynchus mykiss, 640.9 ±156 g) were obtained from a commercial hatchery, Miracle Springs Inc. (Mission, BC), and kept at the UBC Department of Zoology Aquatics Facility in a 4000 L tank supplied with flow-through dechlorinated Vancouver tap water under 24 h light. All fish were kept at approximately 12ºC and fed every other day with commercial trout pellets (Skretting, Orient 4-0, Vancouver, BC), with feeding suspended a day before surgeries. Animal husbandry and all experiments were conducted according to the guidelines of the Canadian Council on Animal Care and approved by the UBC Animal Care Committee (AUP #A15-0266).  2.2.2 Series I: In vitro experiments 2.2.2.1 Sampling protocol Fish were anesthetized in tricaine methanesulphonate (MS-222, 0.1 g/L) buffered with NaHCO3 (0.2 g/L) dissolved in Vancouver dechlorinated tap water, then transferred to a surgery table where the gills were irrigated with 0.05 g/L MS-222 buffered with NaHCO3 (0.1 g/L). The dorsal aorta was cannulated according to Soivio (1975). Following surgery, fish were placed individually in black acrylic boxes supplied with flow-through water at 12°C and allowed to recover for at least 24 h before sampling. Cannulae were flushed at least twice a day with heparinized (Sigma-Aldrich, H3393, 50 i.u./mL) Cortland’s saline (in mM: 124.1 NaCl; 5.1 KCl; 1.6 CaCl2; 0.9 MgS04; 11.9 NaHCO3; 3.0 NaH2PO4, at pH 7.4; Wolf 1963). Following the recovery period, up to 10% of the total blood volume (assuming blood volume is 5% of total body mass) was sampled from the cannula into heparinized syringes. Sampling was stopped at the first sign of struggle to ensure a low plasma catecholamine concentration. Hematocrit (originally approximately 15-25%) was standardized to 25% by removing plasma or adding Cortland’s saline, and blood was transferred to Eschweiler tonometers and equilibrated to hypoxic, hypercapnic conditions (10 kPa O2, 0.5 kPa CO2 balanced with N2) for at least one hour. These 12  gas tensions were chosen to produce ~75% Hb-O2 saturation, and have been shown previously to produce conditions where a β-NHE response can be elicited (Harter, 2018). Following equilibration with gases, 150 µL subsamples were taken for analysis of extracellular pH (see below). 2.2.2.2 Stimulation of RBCs with isoproterenol To investigate whether RBC β-NHE could be short-circuited following removal of catecholamines, gas-equilibrated blood was stimulated with ISO, then rinsed 3x before being returned to the tonometer for a given time interval.  For stimulation of RBC β-NHE, ISO (Sigma-Aldrich I5627) was prepared in Cortland’s saline. ISO is a synthetic noradrenaline analogue and in rainbow trout has been shown to be slightly more potent than its naturally occurring counterpart, noradrenaline (EC50 of 9x10-9 M vs 1.30x10-8 M; Tetens et al., 1988). However, it is a stable and reliable β-adrenergic agonist that has been used in multiple studies to stimulate the RBC β-NHE response and investigate β-NHE short-circuiting in the context of enhanced O2 unloading (Caldwell et al., 2006; Motais et al., 1989; Rummer and Brauner, 2011; Shu et al., 2017). In this study, ISO was added to macrocentrifuge tubes with 3 mL blood to a final concentration of 10-5 M. This concentration was chosen as it elicits the maximal β-NHE response in rainbow trout, and has been used previously to study β-NHE short-circuiting (Rummer and Brauner, 2011; Shu et al., 2017). Following the addition of ISO, the macrocentrifuge tubes were inverted every minute for 5 min to ensure mixing, then centrifuged to separate plasma and RBCs. Plasma was removed and replaced with a mix of approximately 75% fresh plasma (obtained from plasma pooled during hematocrit standardization, or from a donor fish) and 25% heparinized Cortland’s to prevent blood clotting. The stimulated RBCs were rinsed a total of three times in Cortland’s saline to remove residual ISO. The whole process of stimulating and rinsing took approximately 25 min, after which the blood was returned to the tonometer for re-equilibration with gases for 0.5, 1, 2, 4, or 6 h. For data at 0 min, ISO was left in the blood and inverted to mimic rinsing and handling, then re-equilibrated to gas tensions for 0.5 h. Following re-equilibration, a 100 µL subsample was removed for analysis of hemoglobin concentration 13  ([Hb]), and a 2 mL sample was removed and subjected to the closed-system ∆PO2 assay as described below.  To further examine the mechanisms behind the ∆PO2 values observed, multiple controls were conducted. To demonstrate the involvement of NHE activity in the observed ∆PO2 after CA addition, a trial was run in the presence of the NHE inhibitor 5-(N-Ethyl-N-isopropyl)amiloride (EIPA, dissolved in DMSO and Cortland’s; final concentration of 10-4 M in blood). In addition, to show the specific effect of β-NHE activation on ∆PO2, blood was stimulated and rinsed in the presence of propranolol (final concentration of 2x10-5 M), a competitive inhibitor of the RBC β-adrenergic membrane receptor. To show that the ∆PO2 was due to CA and not the introduction of external O2 injected into the system, saline was injected into the closed system instead of CA. A trial was conducted in which blood was rinsed six times instead of three times to ensure that a prolonged ∆PO2 was not due to residual ISO. An unstimulated control was also conducted, where blood was subjected to the same methodology as ISO stimulated blood as described above, but in the absence of ISO. 2.2.3 Series II: In vivo experiments Simulating a stress in blood in vitro produces more controlled lab conditions under which to study the ∆PO2 response, but also has limitations in the extent to which this data can be extrapolated to situations in vivo. In particular, using synthetic catecholamines may not result in an identical β-NHE response as natural catecholamines; in addition, the tonometry used to equilibrate blood in Series I limited the earliest time point at which the ∆PO2 could be assayed. To address these limitations and obtain a better understanding of the time course over which the β-NHE could be short-circuited following catecholamine elevation in vivo, the ∆PO2 assay was repeated on blood drawn from a fish exposed to net handling and air exposure stress. Fish were anesthetized and the dorsal aorta was cannulated as in Series I. Following cannulation and 24 h of recovery, fish were net handled and air exposed for 90 s, then returned to boxes and allowed to recover. An air exposure of 90 s was chosen as a moderately high stress to elevate catecholamine levels (Arends et al., 1999; Deitch et al., 2006), but still 14  allow for recovery after sampling. Blood samples of approximately 5 mL were drawn at rest or at 0, 15, or 30 min after air exposure. The first 1 mL in each blood sample was spun down immediately in a Labnet Spectrafuge Mini at 6000 rpm for analysis of plasma catecholamines. Plasma was pipetted in 400 µL subsamples into a 0.5 mL microcentrifuge tube with 40 µL of 0.2 M each of EDTA and glutathione, then rapidly frozen in liquid nitrogen for determination of catecholamine concentrations using an ELISA kit.  The remaining 4 mL of blood sampled from the fish was rapidly equilibrated to 10% O2 and 0.5% CO2 prior to conducting the ∆PO2 assay described below. Gas equilibration was achieved using a peristaltic pump and an oxygenator, a continuous-flow apparatus consisting of an acrylic tube containing gas-permeable polymethyl-pentene fibers (Oxyplus, Membrana GmbH, Wuppertal, Germany). Custom-mixed gases were perfused through the oxygenator fibers while blood was pumped through the tube outside of the fibers in a counter-current direction, allowing for a large surface area and rapid equilibration; for more details, see Harter et al. (2018). This method equilibrates the blood with the respective gas composition within 5 min, compared to the minimum 30 min required in Series I tonometry, minimizing the delay between blood sampling and the ∆PO2 assay. To compare the results of the ∆PO2 assay using the continuous-flow oxygenator (Series II) and tonometry (Series I), blood was also sampled from resting fish and stimulated with ISO prior to gas equilibration with the continuous-flow apparatus. Following all equilibrations, a 100 µL subsample was taken for analysis of pH, Hct, and [Hb]. For the sake of time, blood Hct was measured, but not standardized as in Series I; instead, ∆PO2 values were standardized to [Hb] to account for differences in Hb content. Immediately after gas equilibration, 2 mL of blood were analyzed in the closed-system ∆PO2 assay as described below.  2.2.4 Closed-system ∆PO2 assay For both Series I and Series II, the magnitude of β-NHE short-circuiting was determined via a modified protocol of Rummer and Brauner (2011). Briefly, 2 mL of the gas-equilibrated blood was placed in a glass vial sealed with a septum, carefully excluding any air. The vial was held in a water bath at 15  12°C while a magnetic stir bar ensured thorough mixing of the blood. Blood PO2 was measured in real time using a fiberoptic O2 microsensor (Loligo, Viborg, Denmark; PreSens MicroTX3 meter, Regensburg, Germany) pierced through the septum sealing the glass vial. Blood was allowed to equilibrate for 2 min in the closed system. Thereafter, 20 µL of 0.2 M HCl prepared in Cortland’s saline was injected into the blood to obtain a final concentration of 2x10-3 M (Rummer and Brauner, 2011). This acidified the blood, as would occur during a generalized acidosis in an animal. After approximately 5 min, when the system had fully equilibrated, CA (Sigma-Aldrich C3934) prepared in Cortland’s saline was injected to achieve a final concentration of 10-6 M, with the goal of short-circuiting any red blood cell β-NHE activity as indicated by an increase in blood PO2 (∆PO2) described in more detail below. 2.2.5 Hematological analyses Blood pH was measured using a microelectrode (16-705 and 16-702; Microelectrodes Inc., Bedford, NH). The Hct was determined by centrifuging blood in 15 µL microcapillary tubes in triplicate at 11,500 rpm for 3 min (IEC Micro Capillary Centrifuge Model MB, Chattanooga, TN). [Hb] was analyzed in triplicate using the cyanomethemoglobin method, by lysing rinsed RBCs in Drabkin’s solution (Sigma-Aldrich, D5941) and measuring absorbance at 540 nm with a Shimadzu UV-160 spectrophotometer (Kyoto, Japan). An extinction coefficient of 10.99 was used to calculate [Hb] (van Assendelft and Zijlstra, 1975). [Hb] (in mM) was divided by Hct (as a decimal) to determine mean cell hemoglobin concentrations (MCHC) for each sample. All hematological analyses were performed in triplicate.  Catecholamine concentrations were measured using an ELISA kit (Abnova KA1877, Taipei City, Taiwan) following the manufacturer’s instructions. A four-parameter logistic model was fitted to standard curves of adrenaline and noradrenaline and used to determine catecholamine concentrations. For adrenaline, five samples at 0 min were outside the range of the standard curve, and thus concentrations were calculated using a single parameter logistic equation modelled using the three 16  highest standards instead. Samples with undetectable catecholamine concentrations (<0.09x10-9 M for adrenaline, <0.55x10-9 M for noradrenaline) were assumed to be 0.  2.2.6 Data analysis The effect of β-NHE short-circuiting due to CA addition in the closed-system preparation was calculated from the continuous PO2 recordings. The ∆PO2 following CA addition was determined by taking the difference between the mean of the last 10 points prior to CA injection and the first 10 points after CA injection to account for noise. This ∆PO2 was then divided by the [Hb] to standardise the ∆PO2 to the amount of Hb present in the vial. These results were plotted against time since removal of ISO (Series I), or time since in vivo stress (Series II). Several non-linear models were fitted to the data and the goodness of fit was assessed using the Akaike Information Criterion (AIC; Akaike, 1974), where the Hill equation resulted in the lowest AIC and best fit for the data. Based on this result, all data were analyzed using a Hill model. Series II data at 0 min was found to have a non-normal distribution, so these data were log-transformed to satisfy the assumptions of parametric tests before further analysis. A two-way ANOVA and Tukey’s HSD was used to compare the effects of time, treatment, and their interaction. Statistical analyses were conducted in R 3.2.2, using the stats and nlme packages (Pinheiro et al., 2018), with α = 0.05. Data are presented as means ±S.E.M.   17  2.3 Results 2.3.1 Series I: In vitro experiments The time course of β-NHE short-circuiting after catecholamine removal was studied by measuring changes in blood PO2 in a closed system in vitro, where HCl and CA were injected to acidify and short-circuit the blood, respectively. A representative PO2 trace (Figure 1) shows the changes in PO2 that occurred after each injection. Following addition of the blood to the closed system, the mean PO2 was 62.7 ±2.8 Torr. Injection of HCl caused PO2 to increase significantly (p <0.0001) to 112.6 ±5.2 Torr before CA was injected (Figure 1). The ∆PO2 following CA injection was plotted as a function of time after catecholamine removal (Figure 2). At 0, 30, and 60 min following catecholamine removal, ∆PO2 was significantly higher compared to EIPA-treated control samples (p <0.001). The decrease in ∆PO2 over time followed a sigmoidal pattern and was best described by a Hill equation, according to ∆PO2 =-0.857t2.1158.782+t2.11. From this, the half-time of this response was estimated to be 59 min. After 120 min, the ∆PO2 response was not found to be significantly different from the EIPA control (p >0.05), but was still significantly different from 0 (p <0.01). A second stimulation of blood with ISO after 6 h resulted in a ∆PO2 that was not significantly different from the initial ∆PO2 response at 0 min (Figure 2). In addition, rinsing the blood six times instead of three times had no significant effect on the resulting ∆PO2 at 60 min after ISO removal. There was no interaction effect of treatment and time (p >0.3). 2.3.2 Series II: In vivo experiments To investigate whether the same time course of β-NHE short-circuiting would occur after an in vivo stress, blood samples were taken from fish at rest or at 0, 15, or 30 min after net handling and air exposure stress. Blood was rapidly equilibrated to custom-mixed gases and then subjected to the closed-system ∆PO2 assay as before. The resulting ∆PO2 following CA addition was then compared between the in vitro ISO stimulated samples and the in vivo stressed samples at 0 and 30 min, along with the different controls (Figure 3). 18  At 0 min, ∆PO2 following CA in in vivo stressed samples ranged from 5 to 37 Torr, with data points falling into lower and higher subpopulations (see Figure A1). The mean ∆PO2 of these points was approximately 20 Torr, and was not significantly different from ISO stimulated blood from the in vitro (Series I) or in vivo (Series II) experiments (Figure 3). Additionally, regardless of whether the samples were equilibrated by tonometry (Series I) or using an oxygenator (Series II), no significant differences in ∆PO2 after CA addition was observed in ISO stimulated samples.  At 30 min, the ∆PO2 in blood from in vivo stressed fish had decreased significantly (p <0.05) to 6.6 ±1.0 Torr, and was no longer different from the propranolol and unstimulated control samples. However, this ∆PO2 was still higher than the EIPA and saline controls (p <0.01). All controls, with the exception of the saline control, resulted in a ∆PO2 that was significantly different from 0 (p <0.01). For Series II fish, plasma was taken immediately after sampling for analysis of catecholamine concentrations (Figure 4). Both adrenaline and noradrenaline concentrations were highest immediately following blood sampling and decreased significantly (p <0.001) by 15 and 30 min; these latter time points were not significantly different from one another or from fish at rest (p >0.05). Blood pH, Hct, [Hb], and MCHC were recorded in blood samples from both series after gas equilibration (Table 1). Blood pH was lower in Series I, but not significantly different between time points throughout Series II. The Hct was standardized to approximately 25% in Series I, but differed between time points in Series II, where Hct was not standardized. There was no significant difference in [Hb] or MCHC throughout these experiments.     19    Figure 1. Representative trace of the closed-system ∆PO2 assay for rainbow trout whole blood equilibrated with 10% O2 and 0.5% CO2, stimulated with isoproterenol. A indicates injection of HCl to acidify the blood samples, which increased PO2 by an average of 53.0 ±2.7 Torr, and B indicates injection of carbonic anhydrase (CA) into blood. The ∆PO2 following CA addition represents the β-NHE short-circuiting, and when standardized for hemoglobin concentration, corresponds with the y-axis of Figure 2. A B ∆PO2 20   Figure 2. The influence of time following isoproterenol removal on the magnitude of the change in partial pressure of oxygen in closed-system blood (∆PO2, standardized to hemoglobin concentration), following acidification and addition of carbonic anhydrase. Blood was previously stimulated for 5 mins with isoproterenol (ISO) and then rinsed three times (ISO+rinsed) or six times with saline (6x rinsed). In one treatment, stimulated and rinsed blood was re-stimulated at 6 h prior to acidification and CA addition (restimulated). Control sample was treated with EIPA instead of ISO (EIPA ctrl). Each point represents the mean ±S.E.M. of blood samples from different individuals. Curve was modelled using the Hill equation ∆PO2 = -0.857t2.1158.782+t2.11, yielding a halftime of 59 min. Different letters indicate significant differences between means, tested with a two-way ANOVA (p <0.05); numbers in parentheses indicate sample size.        21   Figure 3. Closed-system change in the partial pressure of oxygen (∆PO2) after carbonic anhydrase (CA) addition in the in vivo (Series II) and in vitro (Series I) trials. ∆PO2 values (standardized to hemoglobin concentration) were measured following addition of CA to blood adrenergically stimulated by the addition of isoproterenol (ISO) or by natural catecholamines in vivo (nat cats), at 0 or 30 min following the stressor (Series II) or removal of ISO (Series I). Controls consist of unstimulated (unstim.) blood, blood treated with 5-(N-Ethyl-N-isopropyl)amiloride (EIPA), propranolol-blocked blood (prop.), and blood injected with saline instead of CA. Letters that differ indicate statistically significant differences, while numbers in parentheses indicate sample size. A (4) 22     Figure 4. Adrenaline and noradrenaline concentrations measured in plasma sampled from cannulated rainbow trout. Samples were taken at rest or 0, 15, or 30 min after air exposure and net handling stress (Series II). Different letters indicate statistically significant differences between means at each time point, tested with a one-way ANOVA (p <0.05); n = 7 for all time points.   23  Table 1. Blood parameters in rainbow trout blood at rest and at 0, 15, or 30 min after stimulation of red blood cells, with isoproterenol in vitro (Series I) or with natural catecholamines in vivo (Series II).  Series Sample time pH Hct (%) [Hb] (mmol/L) MCHC (mmol/L) I rest (all) 7.65 ±0.03b 25.3 ±0.2a 1.07 ±0.04a 4.23 ±0.16a II rest 7.95 ±0.06a 16.0 ±2.1c 0.86 ±0.21a 4.83 ±0.18a II 0 min 7.84 ±0.05a 22.4 ±1.2b 0.94 ±0.06a 3.83 ±0.24a II 15 min 7.87 ±0.03a 22.5 ±1.8ab 1.02 ±0.12a 4.13 ±0.41a II 30 min 7.84 ±0.04a 21.1 ±1.7b 0.90 ±0.06a 4.33 ±0.14a Letters that differ within the same column denote statistically significant differences. Hct, hematocrit; [Hb], hemoglobin concentration; MCHC, mean cell hemoglobin concentration   24  2.4 Discussion In this chapter, my objectives were to determine the time course over which rainbow trout RBC β-NHE short-circuiting could occur following an in vitro stress with RBCs stimulated with ISO, and an in vivo stress via natural catecholamines as a result of net handling and air exposure. To address this, I measured ∆PO2 at different time intervals after stress. In general, I found a significant non-zero ∆PO2 values following both an in vitro and in vivo stress even after catecholamine removal, but with differing results depending on the nature of the stressor. Specifically, I determined that 1) an in vitro stress using ISO results in a significant elevated ∆PO2 for at least an hour after the removal of catecholamines, 2) a baseline ∆PO2 of approximately 5 Torr remains even up to 6 h after stimulation, as well as in unstimulated and β-blocked blood, and 3) stimulation of RBCs with natural catecholamines in vivo can elicit the same initial magnitude of ∆PO2, but the effect diminishes significantly faster. From this, we can infer that enhanced O2 unloading is likely able to occur to some extent even in the absence of or following removal of catecholamines, and this response may vary depending on the catecholamine used. 2.4.1 Series I: In vitro experiments To determine the time course of enhanced O2 unloading in vitro, I stimulated rainbow trout whole blood with ISO, then removed remaining ISO by rinsing the blood with fresh plasma and saline. After a given time interval, I measured blood PO2 in a closed system during which I acidified the blood with HCl, then added paCA to short-circuit any remaining β-NHE activity. The ∆PO2 following paCA was interpreted as the magnitude of enhanced O2 unloading, comparable to the amount of additional O2 that could be available to the tissues under this system in vivo. Initial ∆PO2 values following ISO and removal by rinsing were similar to those found by Rummer and Brauner (2011) in blood stimulated maximally with ISO (approximately 22 Torr in the current study, vs 25 Torr in Rummer and Brauner), suggesting that enhanced O2 unloading can continue following catecholamine removal. Even 30 min after the removal of catecholamines, the addition of CA to previously stimulated RBCs resulted in a significant ∆PO2 that was not different from initial values 25  after stimulation (0 min). These findings indicate that both β-NHE activity as well as the potential for enhanced O2 unloading extend beyond catecholamine removal, and remain significant for at least 1 h. Only at 120 min did the ∆PO2 fall to levels that were not significantly different from the EIPA blocked controls. Furthermore, 6 h after rinsing, the initial ∆PO2 could be recovered by renewed stimulation of the RBCs with ISO. This not only indicates that the cells remained viable throughout the entire experimental protocol, but also suggests that RBCs can undergo repeated cycles of stimulation and recovery; thus, multiple repeated stressors may not diminish the magnitude of enhanced O2 unloading. Several control experiments were carried out to validate these findings. Because ISO concentrations were not directly measured in the rinsed samples, the possibility of small amounts of residual ISO cannot be definitively ruled out. However, rinsing blood six times compared to three times resulted in no significant difference in the resulting ∆PO2. Therefore, a confounding effect of residual catecholamines in the plasma cannot explain the prolonged duration in elevated β-NHE activity. Interestingly, even in the unstimulated, propranolol, and EIPA controls, ∆PO2 was significantly different from 0, suggesting some baseline H+ disequilibrium across the RBC membrane that was short-circuited by the addition of CA, independent of β-adrenergic stimulation of the RBCs or NHE activity. It is possible that the unstimulated control, taken from a cannulated fish, could still contain low concentrations of catecholamines that resulted in β-NHE activity that translated to a non-zero ∆PO2. However, as propranolol blocks the β-adrenoceptor, the non-zero ∆PO2 suggests that β-adrenergic stimulation is not entirely necessary to elicit a response. When treated with EIPA, the resulting ∆PO2 was numerically (but not statistically) lower than the propranolol control, perhaps pointing towards some “housekeeping” NHE activity that could function to regulate RBC pHi at a low background level, independent of β-adrenergic activation. This idea has been previously proposed by Rummer and Brauner (2011), who found a significant ∆PO2 in unstimulated and propranolol-treated blood, but not in EIPA-treated blood. Interestingly, in this study, even EIPA-treated blood showed a significant non-zero ∆PO2, suggesting an additional, NHE-independent mechanism causing a H+ disequilibrium that can be short-26  circuited by paCA. One such mechanism has been proposed by Randall et al. (2014), where an excess of plasma HCO3- formed at the gills is quickly converted to CO2 by paCA at the tissues, resulting in RBC acidification and a similar enhanced O2 unloading. However, empirical evidence for this mechanism has not yet been shown. Further studies investigating the mechanisms behind this low level of enhanced O2 unloading in unstimulated and pharmaceutically inhibited blood are required, perhaps in blood from fish lacking β-NHE activity such as the sablefish, catfish, or basal actinopterygians (Berenbrink et al., 2005; Rummer et al., 2010). At 120, 240, and 360 min following ISO and rinse, stimulated RBCs still showed a significant ∆PO2 of approximately 5 Torr, similar to that in unstimulated and pharmaceutically blocked blood. Possible explanations for this baseline ∆PO2 may include residual ISO, a “housekeeping” NHE, low background levels of β-NHE activity, or a blood HCO3- disequilibrium, as was previously discussed. It is also possible that the initial β-NHE activity simply continued past the removal of catecholamines: the β-NHE is activated via a signal transduction pathway, with multiple components where prolonged activity could be possible. Stimulation begins with binding of catecholamines to the β-adrenoceptor, which activates adenylate cyclase in hydrolyzing ATP into cyclic adenosine monophosphate (cAMP); this increase in cAMP concentration then leads to the activation of the β-NHE (reviewed in Nikinmaa, 1992). Thus, at any of these steps, activity could be prolonged independent of whether the β-adrenoceptor remains bound to an agonist. Some early work has been conducted investigating the control of the β-NHE (Baroin et al., 1984; Garcia-Romeu et al., 1988); however, these were all conducted in the presence of high concentrations of ISO. Further research is required to determine the mechanisms causing this baseline ∆PO2, and whether it is a result of prolonged initial β-NHE activity, or due to other factors.  Regardless of the mechanism, however, this 5 Torr difference at minimal catecholamine levels may play a significant role in enhanced O2 unloading even in fish not under stress. Indeed, Harter (2018) found that inhibiting paCA in Atlantic salmon in vivo caused a significant increase in cardiac output to 27  compensate for a reduction in tissue O2 extraction, even at rest. In addition, compared to other vertebrates, such as in humans, where a PO2 shift of 2 Torr is sufficient for O2 unloading at the tissues (Rummer and Brauner, 2015), the baseline 5 Torr difference found here may still be a significant enhancement of O2 unloading for teleosts. Especially when this 5 Torr difference is considered not just at one single site, but throughout the body, the cumulative effect may have a significant impact on the amount of O2 available to the tissues. Again, further research is required to determine the role and significance of this baseline ∆PO2 during routine activity. Studying the β-NHE response in controlled in vitro conditions with consistent, saturating concentrations of a synthetic catecholamine has its benefits, but also has its limitations. As a stable and reliable β-adrenergic agonist, ISO has been used in multiple studies to stimulate the RBC β-NHE response (Borgese et al., 1987a, 1987b; Cossins and Kilbey, 1989; Motais et al., 1989; Nikinmaa et al., 1987) and investigate β-NHE short-circuiting in the context of enhanced O2 unloading (Rummer and Brauner, 2011; Shu et al., 2017). However, ISO is a more potent catecholamine than noradrenaline (EC50 of 9x10-9 M vs 1.30x10-8 M), resulting in comparable, but slightly higher cAMP accumulation, H+ efflux, and ∆pHi (Cossins and Kilbey, 1989; Salama, 1993; Tetens et al., 1988). Additionally, in order to remove plasma ISO, the blood was rinsed and re-suspended in fresh plasma, which may reduce β-adrenoceptor density and potentially reduce the β-adrenergic effect (Reid et al., 1991). Another limitation of the in vitro experiment is the equilibration technique of the blood to gas tensions: here, I used tonometry, an effective and widely used gas equilibration method, but one that requires upwards of half an hour for larger volumes of blood to fully equilibrate. In a study investigating the effect of time, this limits the level of resolution that can be attained, especially during early time points. Thus, these limitations of the in vitro experiment were accounted for in the in vivo portion of this work by using natural catecholamines and their in vivo removal and degradation, and by equilibrating blood using a continuous flow oxygenator that allows for analysis of closed-system ∆PO2 at earlier time points. 2.4.2 Series II: In vivo experiments 28  To corroborate my in vitro findings in a more physiologically relevant condition, I conducted an in vivo experiment, where I stressed fish by net handling and air exposure, then waited a pre-determined amount of time for catecholamines to be removed and metabolized by the gills in vivo before obtaining a blood sample. By using natural catecholamines as a result of an in vivo stress, this series provided a more physiologically relevant approach to compare with the in vitro time course. When RBCs were stimulated in vivo with natural catecholamines, the initial (0 min) ∆PO2 was not different compared to that observed during in vitro experiments, despite the difference in catecholamine source (ISO vs natural catecholamines) and equilibration technique (tonometry vs oxygenator). This finding indicates that in vivo levels of β-NHE stimulation with natural catecholamines result in a similar ∆PO2 value relative to stimulation by saturating levels of ISO in vitro. Given that ISO is more potent than noradrenaline and adrenaline (Cossins and Kilbey, 1989; Tetens et al., 1988), and the concentration of ISO used is known to maximally stimulate β-NHE activity (Caldwell et al., 2006), this is an interesting and surprising finding. This shows that, although it is a more potent catecholamine, ISO may provide a comparable portrayal of in vivo stress conditions, at least with regard to the magnitude of enhanced O2 unloading. One possible explanation for this may be due to in vivo β-NHE stimulation by adrenaline in addition to noradrenaline: despite its lower potency for β-NHE stimulation (Cossins and Kilbey, 1989; Tetens et al., 1988), high levels of adrenaline may have played a role in further stimulating the β-adrenoceptor. Similarly, cortisol is also known to increase β-NHE sensitivity (Reid and Perry, 1991), and may have resulted in a higher in vivo response. Because of the similarities between the ∆PO2 elicited by both natural and synthetic catecholamines, we may be able to more confidently infer in vivo effects from previous in vitro studies using ISO, at least regarding the magnitude of ∆PO2 values in this system immediately following a stressor. Even though the initial ∆PO2 was similar between the in vivo and in vitro experiments, differences were observed at later time points. At both 15 (data not shown) and 30 min, ∆PO2 was significantly lower in in vivo stressed blood compared to those observed in vitro. At 30 min, the in vivo ∆PO2 had 29  dropped to approximately the same baseline ∆PO2 seen in the later in vitro time points, as well as in the unstimulated and propranolol controls. However, the 30 min ∆PO2 was still significantly higher than the EIPA control, suggesting some degree of continued NHE activity. These results indicate that, in vivo, the effect of enhanced Hb-O2 unloading may not last as long as the in vitro experiments would suggest. The mechanisms behind the prolonged ∆PO2 in vitro compared to in vivo have not been investigated, and require further research. However, possibilities may include differences between ISO and noradrenaline, such as a higher binding affinity for the β-adrenoceptor: weaker binding would result in more catecholamine in solution, where it is more susceptible to degradation. In addition, it is possible that metabolism and removal of catecholamines by the gills in vivo are simply more effective than rinsing with plasma. Natural catecholamines are known to degrade over time (Tetens et al., 1988), possibly further reducing the amount of β-NHE stimulation. Salama (1993) found that stimulating RBCs with ISO vs. noradrenaline and adrenaline at the same concentration resulted in a higher concentration of RBC cAMP, suggesting that the potency of ISO is observed beyond the β-adrenoceptor to adenylate cyclase. However, the noradrenaline and adrenaline levels observed in the in vivo study are orders of magnitude lower than the ISO concentrations used here (approx. 7x10-8 M noradrenaline, 1.5x10-7 M adrenaline, vs 1x10-5 M ISO). Thus, some other mechanism unaccounted for in this study is likely responsible for resulting in the same initial ∆PO2 value between in vitro and in vivo stimulated blood, but a less prolonged response in vivo. Between the in vivo and in vitro experiments, I have shown that stimulating RBCs results in a prolonged β-NHE response even past catecholamine removal. In some cases (such as ∆PO2 magnitude immediately following CA), ISO and natural catecholamines may have a very similar effect, and using a synthetic catecholamine may be representative of an in vivo stress. For other factors, such as in duration of effect, ISO is longer lasting than natural catecholamines, and thus using natural catecholamines instead of ISO may provide a more in vivo relevant time response. 30  In the in vivo experiment, I also measured catecholamine concentrations before and after stress. Catecholamine concentrations were, as expected, highest immediately following the stressor. Literature values for noradrenaline vary depending on the stressor, but in general range between 16x10-9 M (following a 2 min tail grab, or acute hypoxia; Fiévet et al., 1990; Nakano and Tomlinson, 1967) to 85x10-9 M (after 30 min acute hypoxia or repeated burst swimming; Perry and Reid, 1992; Butler et al., 1986; reviewed in Gamperl et al., 1994), with one study reporting upwards of 445x10-9 M (following 6 min exhaustive exercise; van Dijk and Wood, 1988). Noradrenaline in the present study was found to be approximately 73x10-9 M, suggesting a moderate- to high-level stress. Similarly, adrenaline concentrations were found to be approx. 150x10-9 M immediately following the stressor, falling in the mid-range of previously reported adrenaline levels that typically range from 8 to 300x10-9 M (reviewed by Gamperl et al., 1994). After 15 min, catecholamines had decreased substantially to levels that resembled those prior to the stressor. This is in line with previous observations of blood catecholamine levels declining drastically within minutes following release (Nekvasil and Olson, 1986b; Tang and Boutilier, 1988), and suggests that whatever residual β-NHE activity seen at the 30 min mark was likely not due to additional stimulation by circulating catecholamines. 2.4.3 Conclusions Here, I first showed that enhanced O2 unloading in ISO-stimulated rainbow trout blood can continue to occur for at least an hour after catecholamine removal. I also showed that, although this effect is prolonged when stimulated by ISO in vitro, this does not last as long for blood stimulated by natural catecholamines in vivo, with ∆PO2 returning to baseline within 30 min following an in vivo stressor. However, the initial ∆PO2 in both in vivo and in vitro experiments was similar, suggesting that the degree of β-NHE activity and enhanced O2 unloading between blood stimulated by saturating concentrations of ISO and in vivo levels of natural catecholamines is comparable. In the context of enhanced O2 unloading immediately following a stressor, this may mean that previous studies using ISO may be applicable to in 31  vivo conditions. However, following prolonged periods of time, extrapolations from in vitro stressors to in vivo conditions must be made with caution.  I also found that, even in blood left for 6 h after catecholamine stimulation and removal, as well as pharmaceutically blocked blood, paCA still resulted in a significant ∆PO2 of approximately 5 Torr, suggesting some level of baseline enhanced O2 unloading even in unstimulated RBCs. The mechanisms behind this effect remain uncertain, but may suggest a “housekeeping” NHE or an NHE-independent disequilibrium that is short-circuited by paCA. Thus, this unique system for enhanced O2 unloading is likely not limited only to maximally stressed periods, but may also help to increase the O2 available to tissues in the time following stress or even at rest. As there is significant EPOC and EPHOC in rainbow trout following exercise or hypoxia, having a prolonged system for enhanced O2 unloading likely alleviates the metabolic O2 demand during recovery. In addition to possibly contributing to the early success of teleosts, this system may also play an important role in allowing modern teleosts to achieve the remarkable physiological feats we observe today.    32  Chapter 3: Enhanced oxygen unloading in a marine teleost 3.1 Introduction Since the first study by Rummer and Brauner (2011) investigating the system for enhanced O2 unloading via Root effect Hb, β-NHE activity, and short-circuiting by paCA, further research has confirmed its role in vivo, including its physiological significance and time course for pHi recovery within the circulatory system (Harter, 2018; Harter et al., 2018; Rummer et al., 2013). These studies have allowed us to better appreciate the mechanism and significance of this system, but our understanding of it remains far from complete. In particular, a major limitation of previous studies is the narrow range of species that have been investigated in the context of enhanced O2 unloading. Despite this, enhanced O2 unloading has often been extrapolated to teleosts as a whole, a group containing enormous diversity in morphology, physiology and lifestyles, and encompassing nearly half of all vertebrate species. However, enhanced O2 unloading has only really been studied within the salmonids, and in particular rainbow trout, coho salmon, and Atlantic salmon (Harter, 2018; Harter et al., 2018; Rummer et al., 2013; Rummer and Brauner, 2011; Shu et al., 2017). Salmonids are well suited as a study organism for this system given their strong selective pressures for sustained activity: many are semelparous, with a single breeding opportunity dependent upon the completion of what is often a long and strenuous migration. In addition, there is a wealth of knowledge among the salmonids from the extensive other studies on their physiology, in particular on rainbow trout. However, as only one small group within the vastly diverse and numerous teleost species, salmonids provide us with an informative starting point, but one that must be expanded upon in order to more confidently generalize this system to the whole teleost clade. As distantly related teleosts to the rainbow trout, the percomorph cobia (Rachycentron canadum) and its colourful cousin, mahi-mahi (dolphinfish; Coryphaena hippurus) provide us with additional interesting model species in which to study enhanced O2 unloading. Similar to salmonids, both cobia and mahi-mahi are very active swimmers, and may also benefit from a system for enhanced O2 unloading. 33  Cobia and mahi-mahi are known to travel long distances, making annual migrations along the Atlantic coast of North America as well as the Gulf of Mexico (Dippold et al., 2017). In addition, these recreational game fish are popular for catch-and-release, a practice that can result in exhaustive exercise, stress, and a generalized acidosis (Tufts et al., 1991), conditions that are known to promote enhanced O2 unloading via β-NHE short-circuiting. Unlike salmonids, however, cobia and mahi-mahi are warm-water marine fish, and this environmental difference may be reflected in their physiology. Mahi-mahi, in addition to three other species of percomorphs, have been shown previously to possess β-NHE activity (Berenbrink et al., 2005), while nothing is currently known about cobia. Interestingly, another species of percomorph, the sablefish (Anoplopoma fimbria), has been shown to lack β-NHE activity (Rummer et al., 2010). As two species of percomorphs that would likely benefit from enhanced O2 unloading, cobia and mahi-mahi could provide additional information of this system in percomorphs in general, as well as allow us to make inferences about the most recent common ancestor of percormorphs and salmonids. This, in conjunction with their active, warm-water, marine, and pelagic lifestyle, provide us with an interesting model in which to study this system. Previous studies of the mechanism for enhanced O2 unloading have shown that β-NHE activity is an important component for short-circuiting by paCA (Rummer et al., 2013). Thus, as a first step, I endeavoured to confirm the presence or absence of β-NHE activity in cobia and mahi-mahi by measuring β-adrenergic cell swelling as a proxy, as has been previously conducted in salmonids (Caldwell et al., 2006; Rummer and Brauner, 2011; Shu et al., 2017). As a result of β-NHE activity, H+ are exported in a 1:1 ratio in exchange for Na+. In addition, the anion exchanger exports HCO3- for Cl-. As Na+ and Cl- enter the cell, water follows by osmosis, resulting in an increase in cell volume that can be quantified via an increase in hematocrit (Hct) and a decrease in mean cell hemoglobin concentration (MCHC). Mahi-mahi had previously been shown to possess β-NHE activity in an overview of teleost species by Berenbrink et al. (2005), but nothing was known about cobia; to produce comparable results both between these species as well as with salmonids from previous studies, a β-adrenergic cell swelling assay 34  was conducted on both cobia and mahi-mahi blood to determine β-NHE activity. Determining β-NHE activity in both cobia and mahi-mahi could also provide additional support for the hypothesis that the percomorph ancestor possessed β-NHE activity (Berenbrink et al., 2005).  Prior to this study, basic blood-O2 transport characteristics were unknown in cobia and mahi-mahi. OECs and blood parameters such as P50 were lacking in these species, and are important parameters to consider when designing any experiment where a particular Hb-O2 saturation is desired, as O2 binding is non-linear and often differs between species. Currently, with the exception of some basic hematology (Chen et al., 2005), little to nothing was known about the O2 transport system in cobia or mahi-mahi. The closest species in which OECs had been analyzed was in tuna, which had diverged from cobia approximately 125 mya (Betancur-R. et al., 2013). As cobia are becoming increasingly prevalent in aquaculture, basic information on their physiology will likely be valuable in raising them under optimal conditions. Thus, I also determined representative OECs for cobia at two different pH levels (a similar study on mahi-mahi is planned, but has not yet been conducted due to time and animal constraints). Through these OECs, I not only provide, to my knowledge, the first investigation into the basic Hb-O2 transport characteristics of these fish, but also produce a powerful tool by which we can determine the potential for enhanced O2 unloading. Given a set arterial-venous change in pH (∆pHa-v) and PO2 (∆Pa-vO2), we can use these OECs to model the change in Hb-O2 saturation as the blood moves through the tissues and is short-circuited by paCA, as has been done previously in salmonids (Rummer and Brauner, 2015; Shu et al., 2017).  Given this, the objectives of this chapter were to: 1) determine β-NHE activity in cobia and mahi-mahi by measuring cell swelling as a proxy; 2) characterize the Hb-O2 transport system in cobia by determining OECs and Hb-O2 parameters such as P50, Bohr effect, and Hill coefficient; and 3) use these OECs to model the potential for enhanced O2 unloading under β-NHE short-circuiting by paCA.  In addition to being species that would be interesting to compare with salmonids in an evolutionary context, cobia and mahi-mahi may also be of interest to study commercially. These fish are 35  popular recreational and game fishing species, as well as fish that are quickly gaining popularity in aquaculture. Thus, the physiology of these fish, and in particular their respiratory physiology and how they react to stress and high activity, are of interest and importance to study. 3.2 Materials and Methods 3.2.1 Experimental animals Cobia (Rachycentron canadum, 916.4 ±373 g) and mahi-mahi (Coryphaena hippurus, 408 ±114 g) were kept in cylindrical tanks supplied with flow-through seawater at the University of Miami Experimental Hatchery on a natural photoperiod, at approximately 29°C. Fish were fed daily with a combination of chopped squid and sardines, as well as pelletized commercial marine fish feed (Skretting, Toole UT).  3.2.2 Series I: β-adrenergic cell swelling response 3.2.2.1 Sampling protocol Fish were anesthetized in tricaine methanesulphonate (MS-222, 0.2 g/L) buffered with NaHCO3 (0.4 g/L) dissolved in filtered seawater until unresponsive, but still ventilating. Approximately 6 mL of blood was sampled from each fish via caudal venipuncture into heparinized syringes, rinsed and re-suspended in marine teleost saline (in mM: 150 NaCl, 5.4 KCl, 1.5 MgCl2, 3.2 CaCl2, 10 glucose, and 10 HEPES, at pH 7.7; Brette et al., 2014), then left at 4°C overnight to allow catecholamines to degrade (Caldwell et al., 2006). 3.2.2.2 Experimental protocol Blood from each fish was divided into two 2 mL aliquots and placed in Eschweiler tonometers. Samples were allowed to equilibrate at 29ºC to a humidified custom-mixed gas (5% O2 and 1% CO2, balanced with N2) for at least 30 min before Hct was standardized to 25% by adding or removing marine teleost saline after centrifuging the blood at approximately 6000 rpm. Following Hct standardization, 1 mL of the adjusted blood was returned to each duplicate tonometer to equilibrate for at least an additional 30 min before starting the experiment. 36  After equilibration, a subsample (200 µL) was taken for the analysis of initial (0 min) pHe, Hct, and [Hb]. Thereafter, ISO (Sigma-Aldrich, I5627) was added to one duplicate tonometer per fish to a final concentration of 10-5 M, a concentration known to maximally stimulate the β-NHE in rainbow trout (Rummer and Brauner, 2011); the remaining tonometer was left unstimulated as a paired control. Subsamples of 150 µL were taken for measurements of Hct and [Hb] at 5, 15, 30, and 60 min. Changes in Hct were measured as a proxy for β-NHE activity and [Hb] was measured to ensure there were no changes throughout the experiment, such as may result from dehydration of the samples or cell lysis. As an additional control, the experiment was repeated with propranolol (a β-blocker) that was added 5 min before ISO to confirm β-adrenergic activity. 3.2.2.3 Data analysis Measures of pHe, Hct, [Hb], and MCHC were calculated as described in Chapter 2. To determine β-NHE activity, the effect of time on Hct in the presence and absence of ISO was analysed using a repeated measures ANOVA, followed by a post-hoc Dunnett’s test. The total change in MCHC (∆MCHC) was calculated between 0 min and 60 min in the presence and absence of ISO.  3.2.3 Series II: Oxygen equilibrium curves and modelling of enhanced oxygen unloading 3.2.3.1 Sampling protocol To characterize blood Hb-O2 transport in cobia, OECs were generated. Fish were anesthetized in MS-222 (0.2 g/L) buffered with NaHCO3 (0.4 g/L) dissolved in filtered seawater until unresponsive, but still ventilating. Blood samples were collected into EDTA-coated syringes via caudal venipuncture and then shipped overnight on ice in EDTA-coated 3 mL BD Vacutainer tubes to UBC. Upon arrival, blood from each fish was pooled in heparinized 10 mL Vacutainer tubes and kept on ice. Over the next 72 h, blood samples were stored at 4°C and OECs were generated using a parallel assay of oxygen equilibria as described below (Lilly et al., 2013; Shu et al., 2017). This duration is known to have no effect on erythrocyte organic phosphate levels (Caldwell et al., 2006). 37  3.2.3.2 Spectrophotometric analysis Six subsamples (3 µL) of rinsed RBCs from each fish were prepared for the analysis of Hb-O2 binding properties via spectrophotometry, as was done in Shu et al. (2017). A parallel assay of OECs was conducted as described by Lilly et al. (2013), using a SpectraMax 190 microplate reader (Molecular Devices, Sunnyvale, CA). Briefly, samples were loaded between two sheets of polyethylene film (a thin, O2-permeable membrane) secured in place with two O-rings on a microcuvette, then placed in a multi-cuvette tonometer that positioned the samples for spectrophotometric analysis in the microplate reader. A Wösthoff gas mixing pump (Bochum, Germany) was used to produce, humidify, and pump mixtures of N2, O2, and CO2 over the microcuvettes while absorbance of blood samples was measured in parallel at 430 nm (the approximate peak absorption for deoxygenated Hb; Iuchi, 1973), and 390 nm (an isosbestic point; Hoxter, 1979). The OECs were determined for each species at 0.25 and 1 kPa CO2 by measuring absorbance after blood had equilibrated with stepwise increases of O2 (1, 2, 3, 4, 5, 6, 10, 15, and 21 kPa at the respective CO2 tensions, and 25 kPa O2 with no CO2, balanced with N2), as was done for salmonids in Shu et al. (2017). All OEC analyses were conducted at 29ºC.   3.2.3.3 Gas equilibration by tonometry Because there was no means of measuring blood pH within the microcuvettes, pH values for parallel blood samples at each PCO2 were determined via tonometry, as was done in Shu et al. (2017). 400 µL blood subsamples from each fish were placed in tonometers and allowed to equilibrate for at least 50 min at 29ºC with custom-mixed gases from a Wösthoff gas mixing pump before pHe was measured using a pH microelectrode (InLab Micro, Mettler Toledo, Columbus OH). PO2 levels of 1, 3, 5, and 21 kPa were used for incubation at each of 0.25 and 1 kPa CO2, balanced with N2. Hct and [Hb] were also measured prior to tonometry. 3.2.3.4 Calculations of hematological parameters Fractional hemoglobin saturation (S) at a specific PO2 was calculated as in Shu et al. (2017), then plotted against incubation PO2. A curve was fitted to the points using a three-parameter logistic 38  equation. Using this model, OECs were generated and P50 values were calculated for each fish at each PCO2. A second-degree polynomial was used to correlate S with pH measurements, and pH at P50 (pH50) was interpolated based on the estimated parameters. Representative OECs were reconstructed using the Hill equation from mean P50 and nH values found at each PCO2. The Bohr factor was calculated by taking the slope of a regression line fitted to all points via a linear mixed-effects model for each PCO2 level (Weber et al., 1976). pH, P50, and Hill coefficients were compared between PCO2 levels using one-way ANOVAs, with α = 0.05. 3.2.3.5 Modelling enhanced O2 unloading With evidence of an active β-NHE, the potential for enhanced O2 unloading was modelled using the cobia OECs generated here, given a fixed ∆Pa-vO2 at two pH levels, following Shu et al. (2017) and Rummer and Brauner (2015). A PaO2 of 13.5 kPa (a in Figure 6) and a PvO2 of 4 kPa (v in Figure 6) were used, corresponding with in vivo arterial and venous PO2, respectively, measured in routinely swimming rainbow trout (Brauner et al., 2000). In the absence of a pH shift, O2 unloading (∆S) was calculated as the difference in S as a result of a ∆Pa-vO2 on the OEC determined at 0.25 kPa CO2 (Figure 6, change from a to v). With a ∆pHa-v, such as one that would accompany an acidosis and β-NHE short-circuiting, an additional O2 load would be available to tissues at the same ∆Pa-vO2; this was modelled under a -0.2 ∆pH by calculating the change in S that would result from the right-shift, assuming a constant PO2 of unloading (Figure 6, change from v to v’). 39  3.3 Results 3.3.1 Series I: β-adrenergic cell swelling response In general, the addition of ISO resulted in a significant (p <0.001) increase in Hct in both cobia and mahi-mahi, a response that was abolished by the addition of propranolol (Figure 5). In cobia, Hct increased significantly 5 min following stimulation with ISO (25.2 ±0.1% at 0 min to 26.2 ±0.3% at 5 min; p <0.0001; Figure 5). An increase in Hct continued over the course of the experiment to a final Hct of 27.8 ±0.4% at 60 min. Similarly, mahi-mahi Hct also increased significantly by 5 min (25.1 ±0.3% at 0 min to 26.5 ±0.4% at 5 min; p <0.001; Figure 5), and reached a final Hct of 27.8 ±0.5% at 60 min. The Hct for both control (unstimulated) and propranolol-treated samples did not differ significantly from 0 min at any time point for either species. At 60 min, the final Hct for both ISO-stimulated cobia and mahi-mahi blood were significantly different from the unstimulated and propranolol controls (p <0.0001; Figure 5). Between 0 and 60 min, cobia MCHC decreased significantly in ISO treated samples, but not in control or propranolol samples (Table 2). Similarly, mahi-mahi MCHC had also decreased significantly at 60 min in ISO treated samples but not controls. No significant changes in [Hb] were detected within each run, and thus values were averaged over all time points within each species and treatment (Table 2). In addition, no significant differences were detected in pHe between treatments or species.    40   Figure 5. The β-adrenergic cell swelling response in caudally sampled blood from cobia (Rachycentron canadum) and mahi-mahi (Coryphaena hippurus). Blood was stimulated with isoproterenol (ISO), left unstimulated (ctrl), or blocked with propranolol before stimulating with ISO (prop), and hematocrit monitored over 60 min. Asterisks (*) indicate statistically significant differences from 0 min, different letters indicate differences in hematocrit at 60 min between treatments and controls.   41  Table 2. Hematological parameters in caudally sampled (Rachycentron canadum) and mahi-mahi (Coryphaena hippurus) blood. Blood was rinsed and re-suspended, then stimulated with isoproterenol (ISO), left unstimulated (ctrl), or blocked with propranolol before stimulating (prop). ∆MCHC was calculated between 0 and 60 min; [Hb] was not found to differ between time points and was averaged within each treatment; pHe was measured after gas equilibration and averaged for each species.  Species Treatment ∆MCHC (mmol/L) [Hb] (mmol/L) pHe cobia ISO (12) -0.40 ±0.07a 1.08 ±0.01ab 7.16 ±0.02a ctrl (6) 0.19 ±0.09b 1.04 ±0.02a prop (6) 0.01 ±0.06b 1.12 ±0.01b mahi-mahi ISO (6) -0.34 ±0.06a 0.92 ±0.01c 7.08 ±0.04a ctrl (6) 0.01 ±0.06b 0.92 ±0.01c Numbers in parentheses indicate sample size, while letters that differ within the same column denote statistically significant differences. ∆MCHC, change in mean cell hemoglobin concentration; [Hb], hemoglobin concentration;     42  3.3.2 Series II: Oxygen equilibrium curves and modelling of enhanced oxygen unloading To determine Hb-O2 binding characteristics for cobia, OECs were constructed at two PCO2 levels with measurements of corresponding pHe values. From these, mean P50 and Hill coefficients (nH) were calculated (Table 3). In general, cobia showed pH-sensitive Root effect Hb, with a significant right-shift and down-shift at lower pH (Figure 6). When blood was equilibrated at 1 kPa PCO2, pHe decreased by approximately 0.2 pH units compared to samples measured at 0.25 kPa PCO2, resulting in a right-shifted curve (Figure 6) and corresponding to a significantly higher P50 (p <0.01; Table 3). In addition, the curve was down-shifted as well, as is characteristic of fish species exhibiting a Root effect. The Bohr factor was calculated to be -0.6 (see Figure A2 for Bohr plot). The OECs produced were used to model enhanced O2 unloading as was described by Rummer and Brauner (2015) and previously conducted on Atlantic salmon, coho salmon, and rainbow trout (Shu et al., 2017; Figure 6). Without the presence of an acidosis, the change in Hb-O2 saturation (∆S) at a PaO2 of 13.5 kPa and a PvO2 of 4.1 kPa was calculated to be 36.5% (Figure 6, black arrow). With a -0.2 ∆pH, such as occurred at 1 kPa CO2, the ∆S at the same ∆Pa-vO2 was calculated to be 58.8% (Figure 6, orange arrow), corresponding to a 61% increase in O2 unloading relative to the control condition without a ∆pHa-v.   43  Table 3. Hematological parameters for cobia (Rachycentron canadum) whole blood. Blood was sampled caudally and used to produce oxygen equilibrium curves at two PCO2 levels.  Parameter 0.25 kPa CO2 1 kPa CO2 pHe at P50 7.72 ±0.07a 7.52 ±0.03b P50 (Torr) 25.72 ±1.99a 43.27 ±2.43b Hill coeff. (nH) 2.41 ±0.41a 1.42 ±0.13a Bohr factor -0.6 ±0.21 Hct (%) 39.7 ±3.09 [Hb] (mmol/L) 1.47 ±0.04 MCHC (mmol/L) 3.83 ±0.33 Letters that differ within each row denote statistically significant differences; n = 6. Hct, hematocrit; [Hb], hemoglobin concentration; MCHC, mean cell hemoglobin concentration Hct, [Hb], and MCHC represent mean ±S.E.M. from all individuals at both PCO2 levels.    44   Figure 6. Oxygen equilibrium curves of cobia (Rachycentron canadum) whole blood. Curves were calculated from mean P50 and nH values, representing Hb-O2 binding at resting pH and at a ∆pH of -0.2, determined at PCO2 levels of 0.25 and 1 kPa. a indicates the Hb-O2 saturation at an arterial partial pressure of oxygen (PaO2) of 13.5 kPa, while v and v’ demarcate the Hb-O2 saturation at a venous partial pressure of oxygen (PvO2) of 4.1 kPa at each pH. These PaO2 and PvO2 values correspond with in vivo arterial and venous PO2 found in routinely swimming rainbow trout (Brauner et al., 2000). Arrows indicate the amount of O2 unloaded at each pH.     45  3.4 Discussion In this chapter, my objectives were to investigate the possibility for enhanced O2 unloading in cobia and mahi-mahi, two teleost species distantly related to the salmonids. I did this by first determining that both cobia and mahi-mahi display an increase in Hct in response to β-adrenergic stimulation of RBCs, providing evidence for β-NHE activity, an important component in enhanced O2 unloading. I also characterized the Hb-O2 transport system of cobia by generating OECs on whole blood and determining Hb-O2 binding parameters including P50, Hill coefficient, and Bohr factor. I then used these data to model the potential for enhanced O2 unloading that would result from β-NHE short-circuiting in this species. My results indicate that, with a -0.2 ∆pHa-v that could result from β-NHE short-circuiting, Hb-O2 unloading could increase by 61% when compared to conditions without this pH shift. 3.4.1 Series I: β-adrenergic cell swelling response In general, the cell swelling responses of cobia and mahi-mahi were quite similar to coho salmon and rainbow trout, with no significant difference in final Hct (see Figure A3). Hct in both cobia and mahi-mahi increased quickly, showing a significant response within 5 min, which was similar to coho salmon, but faster than in rainbow trout (Shu et al., 2017). Furthermore, cobia and mahi-mahi showed no change in [Hb], resulting in a decrease in MCHC, providing strong evidence for an increase in cell volume. Based on these data, I conclude that both cobia and mahi-mahi do, in fact, possess β-NHE activity. Following stimulation of the RBC β-adrenoceptor, an increase in RBC osmolarity causes an increase in RBC volume and Hct, providing us with an easily measured, but indirect way of determining β-NHE activity. Thus, while monitoring Hct and cell volume provides evidence for β-NHE activity, it does not directly provide mechanistic insight into β-NHE transport rates or the level of activity. Cell volume regulation is a ubiquitous process in RBCs and mediated by membrane transporters that include the Na+/K+ ATPase, Na+/K+/Cl- cotransporters, and K+/Cl- cotransporters, among others (reviewed by Nikinmaa, 1992). From the cell swelling response, it is clear that β-NHE activity is present in cobia 46  and mahi-mahi, but the magnitude and rate of cell swelling may be a result of a myriad of interacting regulatory volume mechanisms, in addition to β-NHE activity. From my study, I can conclude that cobia and mahi-mahi possess a β-NHE response that is comparable to that of the salmonids at each animal’s in vivo relevant temperature, but whether there are differences in levels of activity or cell volume regulation is not yet certain. Possible future directions for investigating species-specific differences in β-adrenergic activity include measuring Na+ fluxes, H+ movement, or cAMP accumulation. Despite the possibility of other factors at play, however, this assay shows that β-adrenergic stimulation results in an increase in cell volume that can be blocked by propranolol, providing evidence for β-NHE activity in cobia and mahi-mahi.  The assays on cobia and mahi-mahi blood were conducted at 29°C to resemble in vivo conditions of these warm-water fish, while previous studies on salmonids were done at 12°C (Rummer and Brauner, 2011; Shu et al., 2017). Temperature has been shown to have a large effect on rainbow trout RBC β-NHE activity, with a Q10 effect of 5.37 and H+ efflux increasing approximately 4-fold from 12 to 22°C (Cossins and Kilbey, 1990). From this, one might expect greatly increased β-NHE activity in warm-water fish; however, cell swelling was quite similar between cobia, mahi-mahi, rainbow trout, and coho salmon despite temperature differences, with no difference in magnitude and only a slightly slower rate in rainbow trout (see Figure A3; Shu et al., 2017). Although this similarity at different temperatures may be due to other mechanisms regulating cell volume, a possible and interesting explanation could be that some sort of physiological optimum of β-NHE activity and cell swelling has been retained among even distantly related species, regardless of external temperature. If this is the case, further studies into the similarities and differences between β-NHE activity and enhanced O2 unloading in distantly related groups of teleosts at different temperatures could be worth exploring. In this study, I confirmed β-NHE activity via cell swelling in cobia, a previously uncharacterized species, as well as mahi-mahi, in which β-NHE activity has previously been shown (Berenbrink et al., 2005). As the most diverse and phylogenetically derived teleost group, the percormorphs encompass a 47  wide array of different fishes (Betancur-R. et al., 2013). Within this clade, a small number of fish have been studied with regard to β-NHE activity: namely, the cobia and mahi-mahi investigated here, as well as the European perch (Perca fluviatilis), striped bass (Morone saxitalis), and sablefish (Berenbrink et al., 2005; Rummer et al., 2010), with β-NHE activity ranging from high within the cobia and mahi-mahi to low in the bass, to none in sablefish. Given the vast diversity of this group, this range in β-NHE activity may not be altogether surprising. By adding another group, the cobia, I provide further evidence for the proposed hypothesis by Berenbrink et al. (2005), in which the common ancestor of all percomorphs possessed β-NHE activity. However, as is the case with the rest of the teleosts, the vast majority of the percomorphs remain unstudied. Some percomorphs that may be of interest for future studies regarding β-NHE activity and enhanced O2 unloading could include gobies and toadfish, which are remarkably hypoxia tolerant, as well as other highly active fishes such as tunas and swordfish. 3.4.2 Series II: Oxygen equilibrium curves and modelling of enhanced oxygen unloading In addition to determining β-NHE activity via cell swelling in cobia and mahi-mahi, I also characterized the Hb-O2 transport system in cobia, providing some insight into the previously unstudied respiratory physiology of this tropical marine pelagic fish. Notably, Hct and [Hb] in cobia are substantially higher than in salmonids. In addition, at the same PCO2 levels of 0.25 and 1 kPa, cobia pH decreased by approximately 0.2 units, compared to 0.35 in salmonids (Shu et al., 2017). This may be a result of the higher [Hb], a major blood buffer, but may also be due to differences in the non-bicarbonate buffer capacity of the plasma. In general, however, the remaining O2 transport parameters, including P50 values, Hill coefficients, and Bohr factors between salmonids and cobia were not significantly different at the PCO2 levels tested here (see Table A1), despite the phylogenetic and environmental differences between these fishes. Whether this is a result of convergent evolution or retention of ancestral traits is uncertain, and requires further investigation into more teleost species. 48  Based on the present data, it appears that cobia largely meet the mechanistic requirements to enhance Hb-O2 unloading via β-NHE short-circuiting. My thesis provides evidence for β-NHE activity and pH-sensitive Hb-O2 binding in cobia. Given this, and under the assumption that paCA appears to be present in the tissue capillaries of all fishes (Henry et al., 1997), I modelled the potential for enhanced O2 unloading in cobia. This was calculated as the change in Hb-O2 saturation, or ∆S, that results from a ΔpHa-v at the tissues, using the modelling method that has been used previously for rainbow trout, coho salmon, and Atlantic salmon in Rummer and Brauner (2015) and Shu et al. (2017). When blood pH was modelled to decrease by 0.2 pH units during capillary transit, I found that the arterial-venous ∆S increased by 61% over that without a ∆pH (from 37% to 59%). This is comparable to what has been found in salmonids previously (27%, 76%, and 62% increase in Atlantic salmon, coho salmon, and rainbow trout, respectively; Shu et al., 2017), and suggests that, over a broad phylogenetic spectrum, teleosts appear to have significant potential to enhance O2 unloading at their tissues by short-circuiting by paCA. It is important to note, however, that this is only one scenario under which the magnitude of enhanced O2 unloading was determined. The actual values may vary substantially in vivo depending on environmental conditions and the physiological state of the animal. In addition, vertebrates have a number of different strategies to meet a given MO2 at their tissues. For my modelling approach, I assumed no change in tissue perfusion, a 0.2 decrease in pHi, and a fixed ∆Pa-vO2. With regards to this last assumption, no in vivo PO2 measurements have been made in cobia or mahi-mahi, and thus PO2 levels from routinely swimming rainbow trout were used (Brauner et al., 2000). However, as our knowledge of O2 transport grows and cobia and mahi-mahi become increasingly accessible, future research measuring in vivo blood parameters such as PO2, pH, and blood flow can be more easily conducted. By combining these values with the model presented here, we can make a more accurate prediction of what is happening within these fish. Therefore, the OECs generated in my thesis provide us with a useful tool for determining the potential for enhanced O2 unloading at the tissues of cobia under a given ∆Pa-vO2.  49  From these results, we can conclude that there is at least a large potential for enhanced O2 unloading in these fish, and that, despite phylogenetic and environmental differences between cobia and salmonids, β-NHE activity and the potential for enhanced O2 unloading by β-NHE short-circuiting by paCA appear to be comparable. Prior to this experiment, the species in which enhanced O2 unloading by paCA had been studied was limited almost exclusively to the salmonids. By investigating this system in cobia and mahi-mahi, species that diverged from the salmonids more than 200 mya (Betancur-R. et al., 2013), my thesis further extends the spectrum of our knowledge of teleosts that may use enhanced O2 unloading. From previous studies, β-NHE short-circuiting appears to be a fundamental aspect of the O2 transport system, with evidence that it reduces cardiac work by 30% in Atlantic salmon (Harter, 2018), may sustain the O2 supply to the spongy myocardium in the heart of coho salmon (Alderman et al., 2016), and may enable an adequate O2 supply to the avascular teleost eye (Damsgaard et al, unpublished). Although these studies were conducted solely within the salmonids, the similarities seen in the system for enhanced O2 unloading in my work may suggest some level of conservation among these species, providing support for the hypothesis that not only the salmonids, but teleosts in general may be able to take advantage of this system. 3.4.3 Conclusions In this chapter, I showed evidence for β-NHE activity in both cobia and mahi-mahi, and characterized the Hb-O2 transport system in cobia. I found significant cell swelling following β-adrenergic stimulation in both species, providing evidence for the presence of an active RBC β-NHE in both cobia and mahi-mahi. I generated cobia OECs and determined the P50, Hill coefficient, and Bohr factor, providing a basis on which further future respiratory and cardiovascular studies on this species can be conducted. By using the generated OECs as a model for enhanced O2 unloading, I determined that there was potential for up to a 61% increase in O2 unloading under a -0.2 ∆pHa-v in the blood. Despite phylogenetic and environmental differences between cobia and the salmonids, I found few differences between their Hb-O2 transport systems, suggesting conservation of this physiological trait 50  across diverse teleost taxa. Prior to this study, enhanced O2 unloading had only been studied within the salmonids, yet often extrapolated to teleosts as a whole. With cobia and mahi-mahi as additional points of reference, we may be able to more confidently apply our current knowledge of this system, not only in cobia and mahi-mahi, but perhaps more widely to other teleosts as well.    51  Chapter 4: General Discussion In my thesis, I expanded on our knowledge of the teleost system for enhanced O2 unloading by first investigating the time course after the stimulation of RBCs and the removal of catecholamines, then by broadening our knowledge to teleosts outside of the salmonids. My hypothesis was that the teleost system for enhanced O2 unloading to the tissues lasts beyond catecholamine removal, and is also present in other teleosts. The major findings of my thesis support this hypothesis. In my first set of experiments, I determined that significant enhanced O2 unloading following stimulation and removal of ISO remains elevated for up to 60 min in vitro. I found this response to be mirrored in the magnitude of initial enhanced O2 unloading under a natural stressor in vivo, but determined a shorter time course of less than 30 min. I also found that a baseline level of enhanced O2 unloading occurred even in the absence of β-NHE stimulation. In my second set of experiments, I found evidence for pH-sensitive Hb and β-NHE activity, two key components of the system for enhanced O2 unloading, in cobia and mahi-mahi, representing an entirely different clade of teleosts. I then determined a large potential for enhanced O2 unloading due to short-circuiting by paCA in cobia. Until recently, our understanding of enhanced O2 unloading was based almost solely on an in vitro proof-of-principle of the mechanism (Rummer and Brauner, 2011) and a single study in a paralyzed trout showing a significant decrease in red muscle PO2 when paCA was inhibited (Rummer et al., 2013). These studies were pivotal in providing evidence for a system proposed to play an important role in the evolutionary history and physiology of all teleosts (Randall et al., 2014); however, evidence for its functional significance, physiological relevance, and application to teleosts outside of the salmonids was lacking. In recent years, our understanding of this system has grown greatly from a number of studies, including a validation of the time course of RBC recovery in the venous system (Harter et al., 2018) and the determination of its contribution to the cardiorespiratory system in a swimming fish (Harter, 2018). However, prior to my thesis, information on the physiological significance, and in particular the duration, of enhanced O2 unloading was still absent. In addition, despite the implications drawn to teleosts as a 52  whole, this system had never been explored outside of the salmonids. Through my thesis, I addressed these two knowledge gaps by determining the time course of enhanced O2 unloading, then by showing the potential of this system in cobia. 4.1 Implications In my first set of experiments, I found that rainbow trout blood stimulated by ISO showed a prolonged ∆PO2 following short-circuiting by paCA that could last up to an hour, but determined that this effect was not as prolonged in vivo. This suggests that ISO may be a more long-lasting β-adrenergic stimulator than noradrenaline. However, I also found that the initial ∆PO2 immediately following catecholamine release was not significantly different between blood stimulated with ISO vs. blood stimulated with natural catecholamines. This may suggest that, even though ISO has been found to be a more potent β-adrenergic stimulator, its effect on enhanced O2 unloading is similar to that of an in vivo stressor immediately after stimulation. Given the number of studies that have used ISO to stimulate blood, this may mean that many previous findings may also be applicable to enhanced O2 unloading in vivo, at least immediately after a stressor. This greatly enlarges the pool of potentially relevant knowledge that could be applied and added to our limited understanding of enhanced O2 unloading. As the research that has been done on this system is still quite sparse, such a resource could be valuable. While determining the time course of enhanced O2 unloading, I also found a consistent baseline ∆PO2 even in unstimulated blood, suggesting that enhanced O2 unloading occurs even at rest. This contradicts our initial understanding of the system, where high levels of circulating catecholamines and β-NHE activity associated with stress were thought to be required in order for enhanced O2 unloading to occur. However, theories and evidence are accumulating that this may not be entirely necessary. In the first in vivo study of this system, Rummer et al. (2013) found a significant difference in in vivo muscle PO2 even in the absence of elevated catecholamines. Similarly, Harter (2018) found that this system played a significant role in reducing cardiac work even in Atlantic salmon at rest. Randall et al. (2014) has proposed that simply a HCO3- disequilibrium produced at the gills is enough to cause significant 53  enhanced O2 unloading due to short-circuiting by paCA. To add to all of this, I found in my current study that enhanced O2 unloading could occur after catecholamine removal and even in the absence of β-NHE stimulation, producing a significant ∆PO2 of approximately 5 Torr. Thus, even in routine situations with minimal catecholamine levels, teleosts may be able to take advantage of some level of enhanced O2 unloading. This system likely still plays an especially important role during an aerobically challenging stressor, allowing teleosts to perform under extreme circumstances in stressful situations. While these situations are important and often have critical consequences, the vast majority of the life of an animal is not typically spent under a constant state of high stress. Here, I showed that enhanced O2 unloading is functional even in the absence of catecholamines and under resting conditions. Even under routine activity, short-circuiting by paCA likely plays a role in tissue oxygenation, and may almost always be active to some extent. Utilizing enhanced O2 unloading may help to alleviate the demand on other costly aspects of respiration such as cardiac output, as has been shown in resting Atlantic salmon (Harter, 2018). Compared to other vertebrates without this system, teleosts may be able to obtain the same level of tissue oxygenation at a lower cost to the rest of the respiratory system. 4.2 Limitations and future directions 4.2.1 Rainbow trout as a model organism My determination of the time course of enhanced O2 unloading was conducted on rainbow trout. Because of its rich research history in general fish physiology, as well as being the subject of most of the studies on enhanced O2 unloading to date, rainbow trout was well suited as a model organism to investigate the duration of the system. However, it is important to note that this experiment was conducted in this single species of teleost, using a farmed, all-female population of adult rainbow trout, acclimated to freshwater at 12°C. Previous studies have shown that β-NHE activity is affected by a multitude of factors, including temperature, seasonality, and social status (Cossins and Kilbey, 1989, 1990; Thomas and Gilmour, 2006); in addition, factors such as sex and maturity may have an effect as 54  well, but have not been studied in this respect. Thus, there is much potential for variation not only between species, but also within a species. To study this intraspecific variation, wild salmon may be of particular interest, given the wide range of temperatures, challenges, and lengths of spawning migrations among different populations. Indeed, cardiorespiratory differences including aerobic scope, heart size, and coronary supply have been observed among different populations of Fraser river sockeye salmon (Eliason et al., 2011); variation in the degree and significance of enhanced O2 unloading between these fish may be possible as well. However, given that much of what we know so far about enhanced O2 unloading in salmonids appears also to apply to cobia, a distantly related teleost that is marine, warm-water, and in my study was of both sexes and only recently domesticated, general overall conservation of this system within as well as between species may not be out of the question. 4.2.2 OEC modelling of enhanced O2 unloading In my study, I used OECs as a model to determine the potential for enhanced O2 unloading. The original concept of using OECs as a model for enhanced O2 unloading was based on observations made in vivo that inhibition of paCA resulted in a 30 Torr decrease in rainbow trout red muscle PO2 (Rummer and Brauner, 2015). In comparing this value with the OECs obtained for rainbow trout, it was noted that a right-shifted curve at a ∆pH of -0.2 corresponded with a ∆PO2 of 30 Torr at the same Hb-O2 saturation. Thus, this right-shifted curve was used as an estimation of Hb-O2 saturation under short-circuiting by paCA. This same principle was applied to coho salmon and Atlantic salmon (Shu et al., 2017), as well as in cobia in this study. However, it is important to note that this modelling has yet to be validated by in vivo measurements in cobia, and simply acts as an estimation of what may occur. In vivo measurements of cobia arterial and venous PO2 in the presence and absence of C18 would help us to confirm the values generated by the model presented here. At the least, this study is the first venture into investigating enhanced O2 unloading outside of the salmonids. My results suggest great potential for this system in cobia, as well as many similarities with rainbow trout in the O2 transport system and enhanced 55  O2 unloading, providing us with increased confidence in applying our current knowledge within the salmonids to the rest of the teleosts. 4.2.3 Further in vivo studies In my experiments that determined the time course of enhanced O2 unloading in rainbow trout, I used an in vivo stress in addition to an in vitro stress. Through this two-pronged approach, I was able to investigate the extent and duration of this system both in a controlled lab environment, as well as following a stressor within a fish. Investigating the time course of enhanced O2 unloading after an in vivo stress provides us with more insight into the physiological relevance of this system than an exclusively in vitro experiment would, but still may not capture the full effect of enhanced O2 unloading working in conjunction with the rest of the cardiorespiratory system. Respiration is a complex process, and the ultimate PO2 driving O2 diffusion to active tissues is a result of a myriad of interacting mechanisms. For example, catecholamines are not the only hormone released during stress. Especially during a chronic stress, cortisol levels rise, increasing the sensitivity of the RBC β-adrenergic response to catecholamines (Perry and Reid, 1993). In addition, RBCs within a fish are in a much more dynamic state than those sampled from a cannula. As the RBCs travel throughout the vasculature, they are in a constant state of flux, becoming oxygenated at the gills, deoxygenated at the tissues, and undergoing repeated cycles of short-circuiting (Harter et al., 2018). This is quite different from the single instance of gas equilibration and paCA injection in the closed-system vial used here, and these additional factors may affect the system in ways that are difficult to quantify outside of the animal. A systematic approach to investigating each of these factors in turn, in a fish in vivo, may allow us to determine the effects of other factors on enhanced O2 unloading. Studying the detailed mechanisms of this complex system is essential in understanding how it works, but does not provide a good indication of the importance of this system and its ultimate effect on the life and fitness of an animal. As previously mentioned, one study has been conducted investigating the contribution of this system in swimming Atlantic salmon (Harter, 2018), but our understanding of its 56  role in other species, as well as during other activities, is severely lacking. In particular, aerobic challenges such as high temperature, hypoxia, and exercise may pose situations where a system for enhanced O2 unloading could play an important role, both during the stress as well as after. For further insight on the full effect of this system following a stress and during recovery, a simple potential experiment could be to measure EPOC or EPHOC following an exercise or hypoxia challenge, in the presence and absence of C18, a paCA inhibitor. If enhanced O2 unloading by paCA plays a significant role during these aerobic challenges, inhibiting the system by C18 would likely result in increased EP(H)OC. In a similar manner, additional in vivo studies could be conducted by measuring hypoxia tolerance, thermal tolerance, or maximum swimming speed in the presence or absence of C18. These studies would be interesting to conduct in rainbow trout, to combine with our current knowledge of the system, but would also be of interest to compare among other teleosts, to further expand the scope of this system. 4.2.4 Other species of teleosts We now have further insight into this system in cobia in addition to salmonids, but despite being quite distant on the teleost phylogenetic tree, both are highly migratory fish for which a system that increases O2 supply would likely be selected for. In a less active, sluggish fish that may not benefit from enhanced O2 unloading to the same extent, selection pressures for such a system may vary. Thus, studying less active fish could provide valuable insight into the conservation of this mechanism among different teleosts, as well as its cost and significance outside of high sustained swimming performance. A group that falls into this category and may be of particular interest is the Esociformes, encompassing the pikes and mudminnows. Although they are the sister taxon to the Salmoniformes (which contain only the Salmonidae), pikes and mudminnows tend to be lie-in-wait ambush predators, and often spend the majority of their time stationary. In addition, pikes and mudminnows have a high proportion of anaerobic white muscle, used for the sudden fast-start to capture prey (Frith and Blake, 1995). Thus, there is likely a decreased O2 demand in the active tissues of these fishes, which could translate to different requirements on the O2 transport system compared to salmonids and cobia. This, alongside 57  their phylogenetically close relationship to the salmonids, provides a fascinating model in which to study the role of enhanced O2 unloading in a different type of teleost. 4.3 Conclusion At the start of my thesis, the teleost system for enhanced O2 unloading was thought to be an important mechanism during extreme conditions, under the high levels of circulating catecholamines associated with stress. In addition, despite the frequent extrapolations of this system to the widely diverse and numerous teleosts as a whole, studies had only been conducted within 3 of the approximate 30,000 species of teleosts, with all 3 species encompassed by a single family. From my thesis, I provided evidence that enhanced O2 unloading can still occur in the time following catecholamine release, and that high levels of catecholamines are not necessary to trigger a significant response. I also showed that there was great potential for enhanced O2 unloading in a distantly related teleost that had diverged from the salmonids over 200 mya, providing added support for our extrapolations of this system to the rest of the teleosts. Clearly, there is still much to be learned, and many potential directions for future research on the system for enhanced O2 unloading. However, through this work, I have added to the framework for such future studies by deepening and widening our understanding of this remarkable system that likely affects almost half of all vertebrates.     58  References Akaike, H. (1974). A new look at the statistical model identification. IEEE Transactions on Automatic Control, 19(6), 716–723. Alderman, S. L., Harter, T. S., Wilson, J. M., Supuran, C. T., Farrell, A. P., & Brauner, C. J. (2016). Evidence for a plasma-accessible carbonic anhydrase in the lumen of salmon heart that may enhance oxygen delivery to the myocardium. Journal of Experimental Biology, 219(5), 719–24. Arends, R. J., Mancera, J. M., Muñoz, J. L., Wendelaar Bonga, S. E., & Flik, G. (1999). The stress response of the gilthead sea bream (Sparus aurata L.) to air exposure and confinement. The Journal of Endocrinology, 163(1), 149–57. Baeyens, D. A., Hoffert, J. R., & Fromm, P. O. (1971). Aerobic glycolysis and its role in maintenance of high O2 tensions in the teleost retina. Experimental Biology and Medicine, 137(2), 740–744. Baroin, A., Garcia-Romeu, F., Lamarre, T., & Motais, R. (1984). A transient sodium-hydrogen exchange system induced by catecholamines in erythrocytes of rainbow trout, Salmo gairdneri. The Journal of Physiology, 356(1), 21–31. Berenbrink, M., Koldkjaer, P., Kepp, O., & Cossins, A. R. (2005). Evolution of oxygen secretion in fishes and the emergence of a complex physiological system. Science, 307(5716), 1752–7. Betancur-R., R., Broughton, R. E., Wiley, E. O., Carpenter, K., López, J. A., Li, C., … Ortí, G. (2013). The Tree of Life and a New Classification of Bony Fishes. PLoS Currents. Bohr, C., Hasselbalch, K., & Krogh, A. (1904). Ueber einen in biologischer Beziehung wichtigen Einfluss, den die Kohlensäurespannung des Blutes auf dessen Sauerstoffbindung übt. Skandinavisches Archiv Für Physiologie, 16(2), 402–412. Borgese, F., Garcia-Romeu, F., & Motais, R. (1987a). Ion movements and volume changes induced by catecholamines in erythrocytes of rainbow trout: effect of pH. The Journal of Physiology, 382, 145–57. Borgese, F., Garcia-Romeu, F., & Motais, R. E. N. R. (1987b). Control of cell volume and ion transport by beta-adrenergic catecholamines in erythrocytes of rainbow trout, Salmo gairdneri. The Journal of Physiology, 382, 123–44. Brauner, C. J., Thorarensen, H., Gallaugher, P., Farrell, A. P., & Randall, D. J. (2000). The interaction between O2 and CO2 exchange in rainbow trout during graded sustained exercise. Respiration Physiology, 119. Brette, F., Machado, B., Cros, C., Incardona, J. P., Scholz, N. L., & Block, B. A. (2014). Crude oil impairs cardiac excitation-contraction coupling in fish. Science (New York, N.Y.), 343(6172), 772–6. Butler, P. J., Metcalfe, J. D., & Ginley, S. A. (1986). Plasma catecholamines in the lesser spotted dogfish and rainbow trout at rest and during different levels of exercise. Journal of Experimental Biology, 123, 409–21. Caldwell, S., Rummer, J. L., & Brauner, C. J. (2006). Blood sampling techniques and storage duration: effects on the presence and magnitude of the red blood cell beta-adrenergic response in rainbow trout (Oncorhynchus mykiss). Comparative Biochemistry and Physiology. Part A, Molecular & Integrative Physiology, 144(2), 188–95. Chen, G., Zhou, H., Zhang, J. D., & Wu, Z. H. (2005). Hematological study and observation on development of blood cells in cobia, Rachycentron canadum. Acta Hydrobiologica Sinica, 05. Clack, J. A. (2007). Devonian climate change, breathing, and the origin of the tetrapod stem group. Integrative and Comparative Biology, 47(4), 510–523. Cooke, S. J., Hinch, S. G., Crossin, G. T., Patterson, D. A., English, K. K., Healey, M. C., … Farrell, A. P. (2006). Mechanistic basis of individual mortality in Pacific salmon during spawning 59  migrations. Ecology, 87(6), 1575–86. Cossins, A. R., & Kilbey, R. V. (1989). The seasonal modulation of Na+/H+ exchanger activity in trout erythrocytes. Journal of Experimental Biology, 144(1), 463–478. Cossins, A. R., & Kilbey, R. V. (1990). The temperature dependence of the adrenergic Na+/H+ exchanger of trout erythrocytes. Journal of Experimental Biology, 148, 303–312. Deitch, E. J., Fletcher, G. L., Petersen, L. H., Costa, I. A. S. F., Shears, M. A., Driedzic, W. R., & Gamperl, A. K. (2006). Cardiorespiratory modifications, and limitations, in post-smolt growth hormone transgenic Atlantic salmon Salmo salar. Journal of Experimental Biology, 209(7), 1310–1325. Dippold, D. A., Leaf, R. T., Franks, J. S., & Read Hendon, J. (2017). Growth, mortality, and movement of cobia (Rachycentron canadum). Fishery Bulletin2, 115, 460–472. Eliason, E. J., Clark, T. D., Hague, M. J., Hanson, L. M., Gallagher, Z. S., Jeffries, K. M., … Farrell, A. P. (2011). Differences in Thermal Tolerance Among Sockeye Salmon Populations. Science, 332(6025). Fiévet, B., Caroff, J., & Motais, R. (1990). Catecholamine release controlled by blood oxygen tension during deep hypoxia in trout: effect on red blood cell Na+/H+ exchanger activity. Respiration Physiology, 79(1), 81–90. Frith, H., & Blake, R. (1995). The mechanical power output and hydromechanical efficiency of northern pike (Esox lucius) fast-starts. Journal of Experimental Biology, 198(9). Gamperl, A., Vijayan, M., & Boutilier, R. (1994). Experimental control of stress hormone levels in fishes: techniques and applications. Reviews in Fish Biology and Fisheries, 4, 215–255. Garcia-Romeu, F., Motais, R., & Borgese, F. (1988). Desensitization by External Na+ of the Cyclic AMP-dependent Na+/H+ Antiporter in Trout Red Blood Cells. Journal of General Physiology, 91(4), 529–548. Harter, T. S. (2018). The functional significance of plasma-accessible carbonic anhydrase for cardiovascular oxygen transport in teleosts. University of British Columbia. Retrieved from https://open.library.ubc.ca/cIRcle/collections/ubctheses/24/items/1.0372345 Harter, T. S., & Brauner, C. J. (2017). The O2 and CO2 Transport System in Teleosts and the Specialized Mechanisms That Enhance Hb–O2 Unloading to Tissues (pp. 1–106). Harter, T. S., May, A. G., Federspiel, W. J., Supuran, C. T., & Brauner, C. J. (2018). Time course of red blood cell intracellular pH recovery following short-circuiting in relation to venous transit times in rainbow trout, Oncorhynchus mykiss. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 315(2), R397–R407. Helfman, G. S., Collette, B. B., Facey, D. E., & Bowen, B. W. (2009). The Diversity of Fishes (2nd ed.). Oxford, UK: Blackwell Publishing. Henry, R. P., Gilmour, K., Wood, C. M., & Perry, S. F. (1997). Extracellular carbonic anhydrase activity and carbonic anhydrase inhibitors in the circulatory system of fish. Physiological Zoology, 70(6), 650–9. Henry, R. P., Smatresk, N. J., & Cameron, J. N. (1988). The distribution of branchial carbonic anhydrase and the effects of gill and erythrocyte carbonic anhydrase inhibition in the channel catfish Ictalurus punctatus. Journal of Experimental Biology, 134(1), 201–18. Henry, R. P., & Swenson, E. R. (2000). The distribution and physiological significance of carbonic anhydrase in vertebrate gas exchange organs. Respiration Physiology, 121(1), 1–12. Henry, R. P., Wang, Y., & Wood, C. M. (1997). Carbonic anhydrase facilitates CO2 and NH3 transport across the sarcolemma of trout white muscle. American Journal of Physiology - Regulatory, Integrative and Comparative Physiology, 272(6). Hoxter, G. (1979). Suggested isosbestic wavelength calibration in clinical analyses. Clin. Chem., 25(1), 143–146. 60  Iuchi, I. (1973). Chemical and physiological properties of the larval and the adult hemoglobins in rainbow trout, Salmo gairdnerii irideus. Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 44(4), 1087–1101. Lee, C. G., Farrell, A. P., Lotto, A., Hinch, S. G., & Healey, M. C. (2003). Excess post-exercise oxygen consumption in adult sockeye (Oncorhynchus nerka) and coho (O. kisutch) salmon following critical speed swimming. Journal of Experimental Biology, 206(18), 3239–3251. Lilly, L. E., Blinebry, S. K., Viscardi, C. M., Perez, L., Bonaventura, J., & McMahon, T. J. (2013). Parallel assay of oxygen equilibria of hemoglobin. Analytical Biochemistry, 441(1), 63–8. Motais, R., Fievet, B., Garcia-Romeu, F., & Thomas, S. (1989). Na+/H+ exchange and pH regulation in red blood cells: role of uncatalyzed H2CO3 dehydration. The American Journal of Physiology, 256(4 Pt 1), C728-35. Nakano, T., & Tomlinson, N. (1967). Catecholamine and Carbohydrate Concentrations in Rainbow Trout (Salmo gairdneri) in Relation to Physical Disturbance. Journal of the Fisheries Research Board of Canada, 24(8), 1701–1715. Nekvasil, N. P., & Olson, K. R. (1986a). Extraction and metabolism of circulating catecholamines by the trout gill. The American Journal of Physiology, 250(3 Pt 2), R526-31. Nekvasil, N. P., & Olson, K. R. (1986b). Plasma clearance, metabolism, and tissue accumulation of 3H-labeled catecholamines in trout. American Journal of Physiology, 250(3), R519–R525. Nelson, J. S., Grande, T. C., & Wilson, M. V. H. (2016). Fishes of the World (5th ed.). Hoboken, NJ, USA: John Wiley & Sons, Inc. Nielsen, J. G., & Munk, O. (1964). A Hadal Fish (Bassogigas profundissimus) with a Functional Swimbladder. Nature, 204(4958), 594–595. Nikinmaa, M. (1992). Membrane transport and control of hemoglobin-oxygen affinity in nucleated erythrocytes. Physiol Rev, 72(2), 301–321. Nikinmaa, M., Steffensen, J. F., Tufts, B. L., & Randall, D. J. (1987). Control of red cell volume and pH in trout: Effects of isoproterenol, transport inhibitors, and extracellular pH in bicarbonate/carbon dioxide-buffered media. Journal of Experimental Zoology, 242(3), 273–281. Pelster, B. (2004). pH regulation and swimbladder function in fish. Respiratory Physiology & Neurobiology, 144(2–3), 179–190. Perry, S. F., & Reid, S. D. (1992). Relationship between blood O2 content and catecholamine levels during hypoxia in rainbow trout and American eel. The American Journal of Physiology, 263(2), R240-9. Perry, S. F., & Reid, S. D. (1993). β-adrenergic signal transduction in fish: interactive effects of catecholamines and cortisol. Fish Physiology and Biochemistry, 11(1–6), 195–203. Pinheiro, J., Bates, D., DebRoy, S., & Sarkar, D. (2018). nlme: Linear and Nonlinear Mixed Effects Models. Quinn, T. P. (1991). Models of Pacific salmon orientation and navigation on the open ocean. Journal of Theoretical Biology, 150(4), 539–545. Randall, D. J., Rummer, J. L., Wilson, J. M., Wang, S., & Brauner, C. J. (2014). A unique mode of tissue oxygenation and the adaptive radiation of teleost fishes. Journal of Experimental Biology, 217(8), 1205–14. Reid, S. D., Moon, T. W., & Perry, S. F. (1991). Characterization of β-Adrenoreceptors of Rainbow Trout (Oncorhynchus mykiss) Erythrocytes, 216, 199–216. Reid, S. D., & Perry, S. The effects and physiological consequences of raised levels of cortisol on rainbow trout (Oncorhynchus mykiss) erythrocyte beta-adrenoreceptors (1991). Rummer, J. L., & Brauner, C. J. (2011). Plasma-accessible carbonic anhydrase at the tissue of a teleost fish may greatly enhance oxygen delivery: in vitro evidence in rainbow trout, Oncorhynchus mykiss. The Journal of Experimental Biology, 214(Pt 14), 2319–28. 61  Rummer, J. L., & Brauner, C. J. (2015). Root effect haemoglobins in fish may greatly enhance general oxygen delivery relative to other vertebrates. PloS One, 10(10), e0139477. Rummer, J. L., McKenzie, D. J., Innocenti, A., Supuran, C. T., & Brauner, C. J. (2013). Root effect hemoglobin may have evolved to enhance general tissue oxygen delivery. Science, 340(6138), 1327–9. Rummer, J. L., Roshan-Moniri, M., Balfry, S. K., & Brauner, C. J. (2010). Use it or lose it? Sablefish, Anoplopoma fimbria, a species representing a fifth teleostean group where the β-NHE associated with the red blood cell adrenergic stress response has been secondarily lost. Journal of Experimental Biology, 213(Pt 9), 1503–12. Salama, A. (1993). The role of cAMP in regulating the β-adrenergic response of rainbow trout (Oncorhynchus mykiss ) red blood cells. Fish Physiol. Biochem. 1993. Vol. 10, No. 6, Pp. 485-490, 10(6), 485–490. Scarabello, M., Heigenhauser, G. J. F., & Wood, C. M. (1992). Gas exchange, metabolite status and excess post-exercise oxygen consumption after repetitive bouts of exhaustive exercise in juvenile rainbow trout. J. exp. Biol (Vol. 167). Retrieved from http://jeb.biologists.org/content/jexbio/167/1/155.full.pdf Scholander, P. F., & Van Dam, L. (1954). Secretion of gases against high pressures in the swimbladder of deep sea fishes. I. Oxygen dissociation in blood. Biological Bulletin, 107(2), 247–259. Shu, J. J., Harter, T. S., Morrison, P. R., & Brauner, C. J. (2017). Enhanced hemoglobin-oxygen unloading in migratory salmonids. Journal of Comparative Physiology B, 188, 1–11. Svendsen, J. C., Steffensen, J. F., Aarestrup, K., Frisk, M., Etzerodt, A., & Jyde, M. (2012). Excess posthypoxic oxygen consumption in rainbow trout (Oncorhynchus mykiss): recovery in normoxia and hypoxia. Canadian Journal of Zoology, 90(1), 1–11. Tang, Y., & Boutilier, R. G. (1988). Correlation between catecholamine release and degree of acidotic stress in trout. American Journal of Physiology, 255(3 Pt 2), R395-9. Tetens, V., Lykkeboe, G., & Christensen, N. J. (1988). Potency of adrenaline and noradrenaline for beta-adrenergic proton extrusion from red cells of rainbow trout, Salmo gairdneri. Journal of Experimental Biology, 134(1), 267–280. Thomas, J. B., & Gilmour, K. M. (2006). The impact of social status on the erythrocyte β-adrenergic response in rainbow trout, Oncorhynchus mykiss. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 143(2), 162–172. Tufts, B. L., Tang, Y., Tufts, K., & Boutilier, R. G. (1991). Exhaustive exercise in “wild” Atlantic salmon (Salmo salar): acid-base regulation and blood gas transport. Canadian Journal of Fisheries and Aquatic Sciences, 48, 868–874. van Assendelft, O. W., & Zijlstra, W. G. (1975). Extinction coefficients for use in equations for the spectrophotometric analysis of haemoglobin mixtures. Analytical Biochemistry, 69(1), 43–48. van Dijk, P. L. M., & Wood, C. M. (1988). The effect of beta-adrenergic blockade on the recovery process after strenuous exercise in the rainbow trout, Salmo gairdneri Richardson. Journal of Fish Biology, 32(4), 557–570. Weber, R. E., Wood, S. C., & Lomholt, J. P. (1976). Temperature acclimation and oxygen-binding properties of blood and multiple haemoglobins of rainbow trout. Journal of Experimental Biology, 65, 333–345. Wittenberg, J. B., & Wittenberg, B. A. (1974). The choroid rete mirabile of the fish eye. I. Oxygen secretion and structure: comparison with the swimbladder rete mirabile. Biological Bulletin, 146(1), 116–36. Wolf, K. (1963). Physiological salines for freshwater teleosts. Progressive Fish-Culturist, 25(3), 135–140.  62  Appendix  Figure A1. Distribution of in vivo samples at 0 min. ∆PO2 values from blood from in vivo stressed fish were grouped into a statistically significant lower population (triangles) and higher population (circles). Total mean (diamond) at 0 min represents data displayed in Figure 3. EIPA treated (solid circle) and rest + ISO (squares) samples as in Figure 3. Data represent individual samples with means ±S.E.M.   0102030405060-2 0 2∆PO2/mmol Hb (Torr/mmol)Time since catecholamine removal (min)nat cats highnat cats lownat cats avgrest + ISOEIPA (ctrl)63   Figure A2. Bohr plot for cobia whole blood, sampled caudally. Different colours indicate different individuals, with dashed lines representing linear mixed models for each individual. Average mixed model for all individuals is shown as a solid line, with y = -0.6x + 6.0896.   1.21.31.41.51.61.71.87.3 7.4 7.5 7.6 7.7 7.8 7.9 8.0 8.1log(p50)pH at p5064    Figure A3. Changes in hematocrit following stimulation of blood with ISO in cobia and mahi-mahi, compared with coho salmon, Atlantic salmon, and rainbow trout from Shu et al. (2017). Dashed lines represent controls (ctrl) or propranolol-blocked blood (prop). No significant differences were detected at 60 min (endpoint) Hct among ISO treated blood between species.   2425262728293031320 10 20 30 40 50 60Atlantic ISO Atlantic ctrlCoho ISO Coho ctrlRainbow ISO Rainbow ctrlcobia ISO mahi ISOcobia ctrl mahi ctrlcobia propTime since stimulation (min) Hematocrit (%) 65  Table A1. Blood characteristics in cobia, compared with Atlantic salmon and coho salmon (Shu et al., 2017), and rainbow trout rinsed RBCs (adapted from Rummer and Brauner, 2015; Morrison and Brauner, unpublished data). Values for Bohr factors, Hct, [Hb], and MCHC are representative of each species at all PCO2 levels.   cobia1 Atlantic salmon2 coho salmon2 rainbow trout3 PCO2 (%) 0.25 1 0.25 1 0.25 1 0.25 1 pHe at P50 7.72 ±0.07ab 7.52 ±0.03bc 7.91 ±0.01a 7.54 ±0.03bc 7.76 ±0.06a 7.44 ±0.04c 8.01 ±0.00a 7.81 ±0.04b P50 (Torr) 25.72 ±1.99cd 43.27 ±2.43ab 22.56 ±2.94d 33.87 ±1.44bc 23.73 ±1.87d 45.04 ±1.86a 24.83 ±0.30a 40.575 ±2.18b Hill coeff. (nH) 2.41 ±0.41a 1.42 ±0.13b 1.9 ±0.1ab 2.4±0.2a 2.0 ±0.1ab 1.7 ±0.1ab 1.3 ±0.1a 1.4 ±0.0a Bohr factor -0.6 ±0.21 -0.35 -0.64 -0.444 Hct (%) 39.7 ±3.09a 25.0 ±1.5b 27.3 +3.2b 23.7* [Hb] (mmol/L) 1.47 ±0.04a 1.00 ±0.10b 0.88 ±0.06b 0.93* MCHC (mmol/L) 3.83 ±0.33ab 4.2 ±0.4a 3.3 ±0.2b 4.0* 1 present study 2 Shu et al., 2017 3 adapted from Rummer and Brauner, 2015 4 Morrison and Brauner, unpublished datas * averaged over 0.25, 0.5, and 1 kPa CO2 from Rummer and Brauner (2015) Letters that that differ within rows for cobia, Atlantic salmon, and coho salmon denote statistically significant differences, while letters that differ within rows for rainbow trout indicate statistically significant differences found by Rummer and Brauner (2015).    

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