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Vertebrate preferential intracellular pH regulation during severe acute hypercarbia Shartau, Ryan Brady 2017

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VERTEBRATE PREFERENTIAL INTRACELLULAR PH REGULATION DURING SEVERE ACUTE HYPERCARBIA  by   Ryan Brady Shartau  B.Sc., The University of Calgary, 2006 M.Sc., The University of Calgary, 2009   A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Zoology)   THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)    December, 2017   © Ryan Brady Shartau, 2017 ii  Abstract  Environmental CO2 tensions reach >8 kPa (ca. 79,000 µatm; hypercarbia) in some habitats and create severe acid-base challenges for vertebrates. Typically, during a hypercarbic-induced respiratory acidosis, changes in blood pH are compensated for, which returns pH to its normal value, and this is coupled to tissue pH (pHi) regulation. However, during acute environmental CO2 exposure, this process may be limited to <2 kPa PCO2. Some fishes fully protect tissue pH (pHi) (preferential pHi regulation) despite large sustained reductions of pHe (>1 pH unit) and can tolerate PCO2 >3 kPa. I hypothesized that preferential pHi regulation is used by adult fishes and embryonic amniotes during severe acute acid-base disturbances. This was investigated by examining (1) whether preferential pHi regulation is a general response to various types of acid-base disturbances, (2) surveying fishes for the presence or absence of preferential pHi regulation, and (3) whether preferential pHi regulation is used during development in reptiles. Using white sturgeon, I found that preferential pHi regulation is not a general response to both respiratory and metabolic acidoses. Despite a robust capacity for preferential pHi regulation during respiratory acidoses, not all tissues were protected during metabolic acidoses to the same degree. Preferential pHi regulation was observed to be a common pattern of acid-base regulation amongst fishes in response to severe acute hypercarbia. A total of 20 species, ranging from basal (“primitive”) to derived, were examined and 18 were observed to use preferential pHi regulation. Finally, developing amniotes (snapping turtle and American alligator) used preferential pHi regulation during severe acute respiratory acidosis, but the capacity for pHi regulation was progressively reduced throughout development. This thesis demonstrates that preferential pHi regulation is likely a common strategy of acid-base regulation occurring in response to severe acute hypercarbia in adult fishes and possibly amniotes. I propose that preferential pHi regulation is an embryonic vertebrate strategy, that has been retained or lost in adults depending on the environmental acid-base challenges they face.   iii  Lay Summary Acid-base homeostasis in vertebrates can be disrupted by high environmental CO2 (hypercarbia), which creates severe acid-base disturbances. Some vertebrates are exceptionally hypercarbic, likely due to their ability to tightly protect tissue pH (pHi) despite a reduction in extracellular pH (termed preferential pHi regulation). My thesis explores preferential pHi regulation in vertebrates across phylogenies and during development in response to severe acute hypercarbia. A survey of 20 fish species showed that preferential pHi regulation is used by 18 of these species; it is also used during severe acute hypercarbia in reptilian embryos. These findings suggest preferential pHi regulation is a common vertebrate pattern of pH regulation, possibly arising in embryos and retained or lost in adult vertebrates depending on their environment; this may have been important for major evolutionary transition in vertebrates, including the evolution of air breathing and the transition from life in water to life on land.  iv  Preface A version of Chapter 2 has been published. Shartau, R. B., Baker, D. W., and Brauner, C. J. (2017). White sturgeon (Acipenser transmontanus) use different strategies for pH regulation depending on the type of acid-base disturbance. Journal of Comparative Physiology B. 187:985-994. I designed the experiments, collected and analyzed the data, and wrote the manuscript with assistance from Daniel Baker and under supervision from Colin Brauner.  Chapter 3 was a collaborative project with Baker, D. W., Harter, T. S., Aboagye, D. L., Allen, P. J., Val, A. L., Crossley II, D. A., Kohl, Z. F., Hedrick, M. S., and Brauner, C. J. Preferential intracellular pH regulation may contribute to fish diversity in severely hypercarbic habitats. I designed the experiments with input from Colin Brauner. I setup the experiments and collected the data with assistance from Daniel Baker, Till Harter, Daniel Aboagye, Peter Allen, Adalberto Val, Dane Crossley, Zachary Kohl and Michael Hedrick. I analyzed the data and wrote the manuscript under supervision from Colin Brauner.  A version of Chapter 4 has been published. Shartau, R. B., Crossley II, D. A., Kohl, Z. F., and Brauner, C. J. (2016). Embryonic common snapping turtles (Chelydra serpentina) preferentially regulate tissue pH during acid-base challenges. Journal of Experimental Biology. 219(13): 1994-2002. I designed the experiments along with Dane Crossley and Colin Brauner. I collected the data with assistance from Dane Crossley and Zachary Kohl. I analyzed the data and wrote the manuscript under supervision from Colin Brauner.  A version of Chapter 5 has been published. Shartau, R. B., Crossley II, D. A., Kohl, Z. F., Elsey, R. M., and Brauner, C. J. (In press). American alligator (Alligator mississippiensis) embryos tightly regulate intracellular pH during a severe acidosis. Canadian Journal of Zoology. I designed the experiments along with Dane Crossley and Colin Brauner. I collected the data with assistance from Dane Crossley and Zachary Kohl. Ruth Elsey supplied the animals. I analyzed the data and wrote the draft under supervision from Colin Brauner.  v  The experiments in the thesis followed protocols that were approved by the UBC animal care committee (animal care no: A11-0235).  vi  Table of Contents  Abstract .......................................................................................................................................... ii	Lay Summary ............................................................................................................................... iii	Preface ........................................................................................................................................... iv	Table of Contents ......................................................................................................................... vi	List of Tables .................................................................................................................................. x	List of Figures ............................................................................................................................... xi	List of Symbols and Abbreviations .......................................................................................... xiii	Glossary ...................................................................................................................................... xiv	Acknowledgements ...................................................................................................................... xv	Dedication ................................................................................................................................... xvi	Chapter 1: General Introduction .................................................................................................1	1.1	 Acid-base regulation in vertebrates ................................................................................................. 2	1.1.1	 Extracellular compartment ....................................................................................................... 3	1.1.2	 Intracellular compartment ........................................................................................................ 6	1.1.3	 In vivo studies of pHi regulation .............................................................................................. 6	1.1.4	 In vitro pHi regulation .............................................................................................................. 8	1.1.5	 Cellular mechanisms of pHi regulation .................................................................................... 9	1.1.6	 pHe and pHi regulation beyond the bicarbonate concentration threshold .............................. 10	1.2	 Preferential pHi regulation ............................................................................................................ 11	1.2.1	 Mechanisms of preferential pHi regulation ............................................................................ 12	1.3	 Hypercarbia and acid-base regulation – a role for preferential pHi regulation? ........................... 13	1.4	 Preferential pHi regulation: a basal euteleostom strategy or embryonic strategy? ........................ 15	1.5	 Thesis objective and organization ................................................................................................. 17	1.5.1	 Is preferential pHi regulation a general strategy of acid-base regulation in white sturgeon subjected to a range of acid-base disturbances? ................................................................................. 18	1.5.2	 Is preferential pHi regulation a common strategy of acid-base regulation among a diverse range of fish species? ......................................................................................................................... 19	1.5.3	 How does the strategy of acid-base regulation shift throughout development? .................... 19	1.5.4	 Is preferential pHi regulation an embryonic strategy in amniote embryos? .......................... 21	vii  Chapter 2: White Sturgeon (Acipenser transmontanus) Acid-Base Regulation Differs in Response to Different Types of Acidoses ...................................................................................27	2.1	 Introduction ................................................................................................................................... 27	2.2	 Methods ......................................................................................................................................... 29	2.2.1	 Animal acquisition and holding ............................................................................................. 29	2.2.2	 Experimental protocol ............................................................................................................ 30	2.2.3	 Respiratory acidosis ............................................................................................................... 30	2.2.4	 Metabolic acidosis ................................................................................................................. 31	2.2.5	 Blood sampling, tissue sampling and ions ............................................................................. 31	2.2.6	 Calculations and statistical analysis ....................................................................................... 32	2.3	 Results ........................................................................................................................................... 33	2.3.1	 Extracellular acid-base status ................................................................................................. 33	2.3.2	 Intracellular acid-base status .................................................................................................. 33	2.3.3	 Hematocrit, plasma [Cl-] and [lactate] ................................................................................... 34	2.4	 Discussion ..................................................................................................................................... 34	2.4.1	 White sturgeon preferentially regulate pHi during respiratory acidoses ................................ 34	2.4.2	 Tissue pHi is differentially protected following metabolic acidoses ..................................... 36	2.4.3	 Preferential pHi regulation may be a general strategy of acid-base regulation in A. transmontanus – but not all tissues are protected all the time ............................................................ 38	2.4.4	 Conclusions ............................................................................................................................ 38	Chapter 3: Preferential Intracellular pH Regulation May Represent a Common Strategy of Acid-Base Regulation Amongst CO2 Tolerant Fishes ..............................................................46	3.1	 Introduction ................................................................................................................................... 46	3.2	 Methodology ................................................................................................................................. 49	3.2.1	 Animal acquisition and holding ............................................................................................. 49	3.2.2	 Series I: CO2 tolerance assay ................................................................................................. 51	3.2.3	 Series II: Strategy of acid-base balance during severe acute hypercarbia ............................. 51	3.2.4	 Series III: CO2 tolerance to infer pattern of pH regulation .................................................... 53	3.2.5	 Calculations and statistical analysis ....................................................................................... 53	3.3	 Results ........................................................................................................................................... 54	3.3.1	 Series I: Development of a CO2 tolerance assay .................................................................... 54	3.3.2	 Series II: Survey of pHi regulation ......................................................................................... 54	3.3.3	 Series III: CO2 tolerance ........................................................................................................ 55	viii  3.4	 Discussion ..................................................................................................................................... 55	3.4.1	 Use of CO2 assay for tolerance to acute hypercarbia ............................................................. 56	3.4.2	 Acid-base regulation during hypercarbia ............................................................................... 57	3.4.3	 Preferential pHi regulation in fishes ....................................................................................... 58	3.4.4	 Preferential pHi regulation: a strategy for expansion into hypercarbic environments? ......... 60	Chapter 4: Embryonic Common Snapping Turtles (Chelydra serpentina) Preferentially Regulate Intracellular Tissue pH during Acid-Base Challenges .............................................71	4.1	 Introduction ................................................................................................................................... 71	4.2	 Methods ......................................................................................................................................... 73	4.2.1	 Turtle embryo acquisition and incubation ............................................................................. 73	4.2.2	 Experimental protocols .......................................................................................................... 74	4.2.3	 Experimental treatments ........................................................................................................ 76	4.2.4	 Blood sampling, animal euthanasia, tissue sampling and ions .............................................. 77	4.2.5	 Calculations and statistical analyses ...................................................................................... 78	4.3	 Results ........................................................................................................................................... 79	4.3.1	 Series 1: Acid-base status during development in normocarbic normoxia following exposure to severe acute hypercarbic hypoxia .................................................................................................. 79	4.3.2	 Series 2: Response to an acute respiratory acidosis or alkalosis at 90% of incubation in embryos reared under constant hypercarbia ....................................................................................... 80	4.4	 Discussion ..................................................................................................................................... 81	4.4.1	 Capacity for preferential pHi regulation shifts during development ...................................... 81	4.4.2	 Rearing condition alters blood and tissue acid-base status .................................................... 82	4.4.3	 Acid-base regulation during development ............................................................................. 83	4.4.4	 Conclusions and perspectives ................................................................................................ 85	Chapter 5: American Alligator Embryos Tightly Regulate Intracellular pH During a Severe Acidosis .............................................................................................................................95	5.1	 Introduction ................................................................................................................................... 95	5.2	 Methods ......................................................................................................................................... 97	5.2.1	 Subjects of study .................................................................................................................... 97	5.2.2	 Surgical procedures ................................................................................................................ 97	5.2.3	 Experimental treatment and physiological measurements ..................................................... 98	5.2.4	 Calculations and statistical analyses ...................................................................................... 99	5.3	 Results and discussion ................................................................................................................. 100	ix  Chapter 6: General Discussion and Conclusions ....................................................................106	6.1	 Thesis overview and major contributions ................................................................................... 106	6.2	 Preferential pHi regulation: A common and distinct pattern of acid-base regulation ................. 108	6.2.1	 Inter- and intra-specific variation of preferential pHi regulation ......................................... 112	6.3	 Preferential pHi regulation: A potential developmental and evolutionary strategy to cope with acute acid–base disturbances ................................................................................................................ 113	6.3.1	 Preferential pHi regulation: An exaptation for vertebrate evolution? .................................. 114	6.4	 Future research directions ........................................................................................................... 117	6.4.1	 Survey of fish species .......................................................................................................... 117	6.4.2	 Acid-base regulation during development ........................................................................... 118	6.4.3	 Chronic acid-base disturbances ............................................................................................ 119	6.4.4	 Role of the environment ....................................................................................................... 119	6.4.5	 Mechanism(s) of preferential pHi regulation ....................................................................... 120	6.5	 Summary and final thoughts ........................................................................................................ 121	References ...................................................................................................................................127	Appendix: A note on the methodology of pH measurements ................................................146	   x  List of Tables  Table 3.1: Plasma Cl- and osmolarity, and hematocrit of fishes subjected to hypercarbia exposure. ........ 68	Table 3.2: CO2 tolerance assay in various fish species. CO2 tension was increased at a rate of 2 kPa per hour, starting at normocarbia (~0.04 kPa PCO2) until fish reached loss of equilibrium (LOE). ................ 69	Table 4.1: Plasma ion concentrations at 90% of incubation in Chelydra serpentina embryos reared in NC and HC3.5 ................................................................................................................................................... 94	Table 6.1: Fish species investigated in this dissertation. .......................................................................... 125	 xi  List of Figures  Figure 1.1: Representation of the typical response of vertebrates utilizing coupled pH regulation during acute sustained hypercapnia.. ...................................................................................................................... 22	Figure 1.2: Representation of the typical response of vertebrates utilizing preferential intracellular pH (pHi) regulation during acute sustained hypercapnia. ................................................................................. 23	Figure 1.3: A theoretical representation of the typical extracellular pH (pHe) response to short-term (<5 days) hypercarbia in fish. ............................................................................................................................ 24	Figure 1.4: Phylogeny showing distribution of preferential intracellular pH (pHi) regulation and coupled pH regulation amongst vertebrates when exposed to acute >2 kPa PCO2 prior to dissertation research. .. 25	Figure 2.1: Effect of a hyperoxia-induced respiratory acidosis in Acipenser transmontanus white sturgeon on blood acid-base status. ........................................................................................................................... 40	Figure 2.2: Effect of metabolic acidoses in Acipenser transmontanus (white sturgeon) on blood acid-base status. ........................................................................................................................................................... 41	Figure 2.3: Effect of a hyperoxia-induced respiratory acidosis in Acipenser transmontanus (white sturgeon) on intracellular pH (pHi) of red blood cells (RBC), heart, liver, brain and white muscle (WM). ..................................................................................................................................................................... 42	Figure 2.4: Effect of metabolic acidoses in Acipenser transmontanus (white sturgeon) on intracellular pH (pHi) of red blood cells (RBC), heart, liver, brain and white muscle (WM). ............................................. 43	Figure 2.5: Effect of a hypercarbic-induced respiratory acidosis in Acipenser transmontanus (white sturgeon) on blood and tissue acid-base status following a 6 h exposure to 1.5 kPa PCO2. ...................... 44	Figure 3.1 Bar plot of CO2 tensions at loss of equilibrium in Oncorhynchus mykiss and Acipenser transmontanus when subjected to a progressive increase in PCO2 at 1, 2 or 4 kPa h-1. ............................. 62	Figure 3.2: Effect of 3 h exposure to elevated CO2 on blood and tissue acid-base status in 10 different fish species. ........................................................................................................................................................ 65	Figure 3.3: Evolution of preferential pHi regulation and coupled pHe/pHi regulation amongst adult fishes exposed to an acute (<48 h) respiratory acidosis of >1 kPa blood PCO2. .................................................. 66	xii  Figure 4.1: Effect of exposure to an acute respiratory acidosis in common snapping turtle (Chelydra serpentina) embryos (at 70 or 90% of incubation) or yearlings in Series 1 on blood and tissue acid-base status. ........................................................................................................................................................... 86	Figure 4.2: Difference in tissue [H+] from control following 1h exposure to hypercarbia hypoxia (13 kPa PCO2, 9 kPa PO2; HC13) relative to normocarbic (0.03 kPa PCO2, 21 kPa PO2; NC) reared common snapping turtles (Chelydra serpentina) of Series 1. .................................................................................... 88	Figure 4.3: Changes in blood and tissue acid-base status in common snapping turtles (Chelydra serpentina) embryos at 90% of incubation continuously reared in either normocarbia or hypercarbia. .... 89	Figure 4.4: Effect of exposure to an acute respiratory acidosis in snapping turtle embryos (Chelydra serpentina) at 90% of incubation in Series 2 reared continuously and sampled in hypercarbia (3.5 kPa PCO2, 21 kPa PO2; HC3.5) or following 1 h exposure to hypercarbic hypoxia (13 kPa PCO2, 9 kPa PO2; HC13). ......................................................................................................................................................... 90	Figure 4.5: Effect of exposure to normocarbia in snapping turtle embryos (Chelydra serpentina) at 90% of incubation in Series 2 reared continuously and sampled in hypercarbia (3.5 kPa PCO2, 21 kPa PO2; HC3.5) or following 1 h exposure to normocarbia (0.03 kPa PCO2, 21 kPa PO2; NC) for either 3 or 24 h. ..................................................................................................................................................................... 91	Figure 4.6: Difference in blood and tissue pH of turtles during development following exposure to hypercarbia relative to normocarbia in common snapping turtles (Chelydra serpentina) (70 and 90% of incubation and yearlings; this study) and adult western painted turtles (Chrysemys picta bellii). ............. 93	Figure 5.1: Effect of exposure to an acute respiratory acidosis in Alligator mississippiensis embryos on blood and tissue acid-base status. ............................................................................................................. 104	Figure 5.2: Difference in blood pH (pHe) and tissue pH (pHi) during development following a respiratory acidosis in embryonic Alligator mississippiensis (American alligator; 70% to hatch) and Chelydra serpentina (snapping turtle; 70 and 90% to hatch), and in post-hatch C. serpentina, Chrysemys picta bellii (western painted turtle), Anolis equestris (knight anole) and Dipsosaurus dorsalis (desert iguana). ...... 105	Figure 6.1: Phylogeny showing the distribution of preferential intracellular pH (pHi) regulation and coupled pH regulation amongst vertebrates when exposed to acute >2 kPa PCO2 following completion of dissertation research. ................................................................................................................................. 122	Figure 6.2: Difference in tissue pH during development in turtles. .......................................................... 124	 xiii  List of Symbols and Abbreviations  βNHE  β-adrenergic Na+/H+ exchanger AE  Anion exchanger ENaC  Epithelial Na+ channel Hb  Hemoglobin HC13  Hypercarbic hypoxia (13kPa PCO2, 9kPa PO2) HC3.5  Hypercarbic (3.5kPa PCO2, 21kPa PO2) condition Hct  Hematocrit MCT  Monocarboxylate transporter kPa  kilo Pascal, a unit of pressure mM  millimolar MRC  Mitochondrion rich cell (ionocyte) NBC  Na+/HCO3- co-transporter NC  Normocarbic (0.03kPa PCO2, 21kPa PO2) condition NHE  Na+/H+ exchanger PCO2  Partial pressure of CO2 pH -log[H+] pHe  Blood (extracellular) pH pHi  Tissue (intracellular) pH pK’ Apparent negative log of dissociation constant (dependent on temperature and ionic strength) PO2  Partial pressure of O2 RBC  Red Blood Cell s.e.m.  Standard error of the mean VHA  V-type H+-ATPase   xiv  Glossary  Coupled pH regulation  pHi changes in a qualitatively similar fashion as pHe Exaptation An adaptation that has been co-opted for another, unrelated use Hypercapnia   Elevated internal CO2 Hypercarbia   Elevated environmental CO2 Hypochloremia  Reduced level of chloride ions in the blood Metabolic acidosis Reduced pH because of a reduction in HCO3- at a constant PCO2 pH compensation The process of pH recovery involving one or more mechanisms pH recovery A return of pH to its normal value following an acid-base disturbance Preferential pHi regulation pHi is regulated independently of pHe Respiratory acidosis Reduced pH because of increased blood CO2 from an environmental or internal source  xv  Acknowledgements  The work undertaken in this thesis would not have been possible without the help, support and guidance of many people. Firstly, I want to thank my supervisor, Colin Brauner, for his excellent mentorship. He has provided the opportunities to pursue numerous interesting avenues of research, writing and travel throughout my degree that have been instrumental in shaping my growth as a scientist. I am also greatly appreciative of the guidance of my committee members, Bill Milsom, Jeff Richards, and Rick Taylor, who provided valuable insight and feedback on my work.  The various members of the Brauner lab made it a great place to be, both in the lab and during various adventures abroad. In particular, thanks to Dan Baker for showing me the ropes when I first started. Till Harter, Phil Morrison and Mike Sackville were often with me while traveling to exotic locales, conducting interesting research, or chatting about science and life, typically while enjoying a cold brew (or perhaps a warm one watered down with ice). The Zoology Department is a fantastic place, and I had the pleasure of interacting with many members of its administrative staff, technicians and the numerous students, post-docs, and faculty. I was fortunate to start my time in the department with a great cohort of fellow graduate students, including Georgie Cox, Yvonne Dzal, Taylor Gibbons, and Gigi Lau. Over the course of my degree, I have had the good fortune to collaborate with many individuals, which has greatly contributed to this dissertation. In particular, I would like to thank Dane Crossley at the University of North Texas, Peter Allen at Mississippi State University, and Adalberto Val at the National Institute for Amazonian Research (INPA). Next, I want to thank my Mom and Dad. You were my first teachers, and you encouraged and supported me throughout all my years of education. As well, thanks to the rest of my family for your support and taking an interest in my work. And, to my four-legged buddy, Liam, who thinks thesis stuff is pointless and would rather chase squirrels, thanks for providing a useful and fun distraction. Finally, Sabrina – my wife and best friend. Thank you for your support, patience and love over the years. You have played an important role over the course of this thesis and many of the ideas are influenced from discussions with you. xvi  Dedication      For Sabrina, thank you for your love and support.   1 Chapter 1: General Introduction   The overall goal of my thesis was to examine the strategies of vertebrate acid-base regulation in response to severe acute acid-base disturbances, predominantly induced by exposure to elevated environmental CO2 (hypercarbia). Specifically, this thesis explores preferential intracellular pH regulation, a pattern of acid-base regulation that is markedly different from what has previously been considered the typical vertebrate pattern (Brauner and Baker, 2009; Shartau et al., 2016a). Typically, vertebrates exposed to severe hypercarbia experience a large rapid reduction in both extracellular pH (pHe) and intracellular pH (pHi), and compensation of pHi is dependent on partial compensation of pHe (Fig. 1.1); this is referred to as coupled pH regulation (Shartau et al., 2016a). In contrast, a few vertebrates are able to preferentially regulate pHi despite large uncompensated extracellular acidoses during exposure to severe hypercarbia (Fig. 1.2) (Shartau et al., 2016a), a phenomenon that is poorly understood and had only been observed in three fishes and one aquatic tetrapod prior to this dissertation. Using a diverse selection of species ranging from basal to derived fishes, and amniotes, this thesis explores preferential pHi regulation in vertebrates across phylogenies and during ontogeny. My overall hypothesis for this thesis is that preferential pHi regulation is a widely used strategy amongst vertebrates to maintain pH homeostasis during severe acute acid-base disturbances. Based on this hypothesis, it is predicted that preferential pHi regulation will: (1) confer protection against different types of severe acute pH disturbances, (2) be a widely used pattern of pH regulation amongst vertebrates, and (3) confer pHi protection in animals unable to acutely utilize coupled pH regulation. This General Introduction will review what is presently known about acid-base regulation in vertebrates in relation to coupled pH regulation and preferential pHi regulation. The challenges associated with acid-base regulation during hypercarbia will be explored, the putative origins of preferential pHi regulation are discussed and finally, the objectives and organization of the subsequent data chapters are provided.   2 1.1 Acid-base regulation in vertebrates  It is well known that absolute physiological pH values differ between species, differ between body compartments within species and are affected by temperature (Rahn, 1974); however, within a given system, pH values are regulated within a relatively narrow range (Cameron, 1989a; Heisler, 1984). Deviations from normal physiological pH values can affect molecular charge, altering the structure and function of proteins, lipids, carbohydrates and nucleic acids, and, ultimately, reducing whole-animal performance (e.g. reduce heart and skeletal muscle contractility, alter metabolic pathways, and disrupt cellular signalling and processes such as volume regulation) (Occhipinti and Boron, 2015; Putnam and Roos, 1997). The degree to which a pH change affects function depends on the system in question. Disturbances to acid-base homeostasis may arise from respiratory or metabolic sources. Respiratory acidoses occur due to an increase in blood CO2, either from the environment (hypercarbia) or by retention of metabolically produced CO2 (hypercapnia). Typical arterial PCO2 values for adult water and bimodally breathing fishes, reptiles and mammals are 0.1–0.5 (Ultsch, 1996), 0.5–3.5 (Shartau and Brauner, 2014), 1.8–4.3 (Ultsch, 1996) and 4.5–5.6 kPa PCO2 (Arieff et al., 1976; Malan et al., 1985; Wood and Schaefer, 1978; Yaksh and Anderson, 1987), respectively. Any increase in arterial PCO2 beyond those values shifts the equilibrium of the CO2 hydration reaction (CO2 + H2O n H+ + HCO3−), promoting the formation of H+ and HCO3−, thus lowering pH and resulting in acidosis. Metabolic acidoses occur due to the production of metabolically generated acid, which lowers HCO3- at relatively constant PCO2 (Occhipinti and Boron, 2015); often metabolic acidoses occur alongside respiratory acidoses (mixed acidosis) (Kieffer et al., 1994; Wang et al., 1994). In fishes, compensation of a metabolic acidosis is primarily dependent on net exchange of acid-base relevant ions at the gills (Evans et al., 2005; Hwang et al., 2011), and to a lesser degree, excretion of acidic equivalent in the form of titratable acidity and ammonium ions through renal pathways (Kwong et al., 2014). During an acute respiratory acidosis, reductions in pHe are associated with qualitatively similar reductions in pHi. Following the onset of a respiratory acidosis, the  3 compensation of pHi is often more rapid than that of pHe, but complete correction of pHi generally requires pHe compensation of >50% (Shartau et al., 2016a). This is referred to as ‘coupled pH regulation’ whereby changes in pHi are coupled to changes in pHe. Vertebrates relying on pHe regulation are considered to use coupled pH regulation to maintain acid-base homeostasis during an acute persistent acidosis; this is the most widely observed response both in vivo and in vitro that had been observed prior to this thesis (Shartau et al., 2016a).  1.1.1 Extracellular compartment Acid-base disturbances in vertebrates can be minimized or compensated by either (i) direct transfer of acid-base relevant ions between the cell and blood, and/or the blood and the environment, (ii) buffering with bicarbonate and non-bicarbonate buffers, or (iii) altering ventilation rate to modify blood PCO2 and, thus, pH via the CO2-HCO3- buffer system (Brauner and Baker, 2009; Evans et al., 2005; Heisler, 1984). The primary mechanism of short-term acid-base compensation in terrestrial air breathers consists of the latter because blood PCO2 is high relative to environmental levels (e.g. ~5 kPa vs. <0.1 kPa PCO2, respectively), so considerable adjustment of pHe can be accomplished through changes in ventilation; thus the buffering power of the CO2-HCO3- system is large in these animals (Occhipinti and Boron, 2015). In water breathers this mechanism is much less effective due to the low blood PCO2 levels (~0.3-0.7 kPa vs. <0.1 kPa PCO2, respectively) and similarity to environmental levels (Heisler, 1984). Thus, water breathing fishes rely on buffering to minimise acid-base changes, and direct transfer of acid-base relevant ions to compensate acid-base disturbances (Brauner and Baker, 2009; Perry and Gilmour, 2006). Studies investigating compensation for an acute respiratory acidosis in fishes have been conducted on a relatively limited number of species including a few elasmobranchs [e.g. big skate Raja ocellata (Wood et al., 1990), dogfish Scyliorhinus stellaris (Heisler et al., 1988), starspotted dogfish Mustelus manazo (Hayashi et al., 2004)] or several teleosts [e.g. rainbow trout Oncorhynchus mykiss (Larsen and Jensen, 1997; Wood and LeMoigne, 1991), carp Cyprinus carpio (Claiborne and Heisler, 1984), European eel Anguilla anguilla (McKenzie et al., 2002), Conger eel Conger conger (Toews et al.,  4 1983), brown bullhead Ictalurus nebulosus (Goss et al., 1992), Japanese founder Paralichthys olivaceus (Hayashi et al., 2004), and yellowtail Seriola quinqueradiata (Hayashi et al., 2004)]. In general, when these fishes experience an increase in blood PCO2, there is a corresponding rapid reduction in pHe, which is then compensated over the following 24-96 h. The degree of pHe reduction depends on the severity of acidosis and the buffer capacity of the blood. Bicarbonate and non-bicarbonate buffers help minimize the magnitude of pH disturbance, with the former being the CO2-HCO3- system and the latter including phosphate buffers and haemoglobin (Hb) (due to the presence of histidine and their associated imidazole side chains that buffer H+ at physiological pH) (Shartau and Brauner, 2014). Fishes, in general, have lower blood and tissue buffer values than other vertebrates (Cameron, 1989a; Heisler, 1984). Within the blood, however, buffer values vary among fishes, with the more basal groups (chondrichthyans, basal actinopterygians) having higher blood buffer values than teleosts (Berenbrink et al., 2005). When the capacity of the blood to buffer against acid-base disturbances is exceeded, pH changes occur and compensation typically occurs by net transport of acid-base equivalents between the fish and environment, with the gills, kidney and intestine all involved; the gills are believed to account for >90% of the net acid-base relevant ion transport during pH compensation (Brauner and Baker, 2009; Heisler, 1984). In a few fish species, models of the cellular mechanism(s) underlying compensation of the extracellular compartment in response to an acidosis have been developed. Within the gill epithelium of O. mykiss, mitochondrion rich cells (MRCs) (or ionocytes) are believed to be the primary site of extracellular acid-base regulation. Two populations of MRCs exist, those with peanut lectin agglutinin (PNA) binding sites on their apical membranes (PNA+ MRCs) and those lacking such sites (PNA- MRCs). PNA- MRC are proposed to be responsible for acid excretion where it is believed that H+ elimination occurs via an apical NHE, or a VHA coupled to an apical epithelial Na+ channel (ENaC). The result is hyperpolarization of the plasma membrane by transporting H+ via VHA across the membrane, resulting in a favorable electrochemical gradient for diffusion of Na+ via ENaC (Hwang et al., 2011). Net acid excretion is then achieved by the combined actions of apical H+ efflux and basolateral HCO3- influx. Exchange of HCO3- is believed to occur via a HCO3-/Cl- exchanger, such as those found in the SLC4  5 or SLC26 family and by the Na+/HCO3- co-transporter (NBC – also found in the SLC4 family) (Evans et al., 2005; Hwang et al., 2011; Parks et al., 2009; Perry et al., 2009). The PNA+ MRC is proposed to be responsible for base excretion in which the apical membrane HCO3-/Cl- exchanger links Cl- uptake to HCO3- excretion. Apical membrane HCO3- efflux along with basolateral H+ efflux, via a VHA, would result in net transepithelial base excretion (Gilmour and Perry, 2009; Hwang et al., 2011). Using these membrane transporters, net acid-base equivalents can be transported from the blood to the environment to ensure pH homeostasis. In other freshwater fishes, the proposed mechanisms are similar to O. mykiss; for example, freshwater zebrafish Danio rerio (Gilmour and Perry, 2009), tilapia Oreochromis mossambicus (Hwang et al., 2011) and medaka Oryzias latipes (Hsu et al., 2014) use an apical NHE to remove H+ catalyzed from CO2, while HCO3- is moved to the blood via basolateral Na+/HCO3- (NBC) or Cl-/HCO3- (AE). Regardless of the specific cellular mechanism(s) employed, in most fishes studied to date, compensation of pHe during hypercarbia exposure is associated with a net increase in plasma [HCO3-] that is matched by an equimolar reduction in plasma [Cl-] (Brauner and Baker, 2009; Heisler, 1984). The extent of this HCO3− elevation, however, appears to be limited, in that plasma [HCO3−] rarely exceeds 27–33 mM during exposure to acute hypercapnia, which is referred to as the “apparent bicarbonate concentration threshold” (Heisler, 1984). This threshold is associated with an absence of complete pHe compensation in most fishes during acute exposure to CO2 tensions beyond 2–2.5 kPa PCO2 (Baker et al., 2009a; Brauner and Baker, 2009) (Fig. 1.3). Although the basis of this threshold is unknown, recent work has supported the hypothesis that pHe compensation during acute hypercarbia may be limited by the relative decrease in plasma Cl− levels to avoid hypochloremia (Baker et al., 2015). Teleosts typically have plasma [Cl-] of 125 – 168 mM (Edwards and Marshall, 2013), of which approximately 17-20% can be exchanged with HCO3- before the bicarbonate concentration threshold is reached at approximately 27-33 mM HCO3-. In fish with higher plasma [Cl-], a similar pattern is observed; for example, the osmo- and iono-conforming Pacific hagfish Eptatretus stoutii has a plasma [Cl−] of ~458 mM and, perhaps as a result, hagfish are able to increase plasma [HCO3−] to >80 mM, driving pHe and pHi recovery during exposure to severe  6 hypercarbia (PCO2 of ~6.5 kPa) (Baker et al., 2015). Compensation of pHe during acute hypercarbia is affected by the physicochemical characteristics of the surrounding water, such as the levels of acid–base relevant counter-ions (Larsen and Jensen, 1997). In contrast to acute hypercarbia, chronic CO2 exposure allows some teleosts to elevate [HCO3-] well beyond this threshold, aiding pHe compensation. O. mykiss subjected to increasing hypercarbia over three days to reach 3.5 kPa PCO2, and maintained at this level for an additional three days, had a blood [HCO3-] of 66 mM (Dimberg, 1988). Similarly, A. anguilla gradually exposed to and maintained at 6 kPa PCO2 for six weeks had plasma [HCO3-] of 73 mM (McKenzie et al., 2003). Differences between acute and chronic compensation indicate that different mechanisms may underlie compensation to long-term hypercarbia exposures, a possibility that remains relatively unexplored.  1.1.2 Intracellular compartment Although all cells have the capacity for some degree of pHi regulation (Boron, 2004; Occhipinti and Boron, 2015; Putnam and Roos, 1997; Vaughan-Jones et al., 2009), in animals that employ coupled pH regulation, cells cannot fully compensate pHi during a large sustained reduction in pHe; this has been thoroughly examined in vivo and in vitro in a number of species. In fishes that exhibit coupled pH regulation, compensation of pHe and pHi occurs over the initial 24–96 h during sustained hypercarbia exposure (Fig. 1.1), with pHi compensation usually occurring more rapidly that of pHe, partly because intracellular fluids typically display a lower pH than the extracellular blood environment, which places the pK’ of the CO2–HCO3− reaction (pK’ = 6.1) closer to pHi (typically 6.3 – 7.0). Thus, relatively less HCO3− is required to compensate pHi compared to pHe. This recovery is further aided by the greater buffering capacity of intracellular fluids, which moderates the initial pH disturbance (Brauner et al., 2004; Occhipinti and Boron, 2015; Ultsch, 1996).  1.1.3 In vivo studies of pHi regulation Findings from in vivo studies conducted on a relatively small selection of fishes, amphibians, reptiles and mammals have established that pHi regulation is coupled to pHe regulation. In fishes, respiratory and metabolic acidoses typically lead to reductions in  7 pHi and pHe. For example, E. stoutii exposed to hypercarbia exhibited reduced pHe and pHi of heart, brain, liver and muscle (Baker et al., 2015); R. ocellata exposed to hypercarbia had reduced pHe and pHi of heart, brain and muscle; lemon sole Parophrys vetulus exposed to hypercarbia had reduced pHe and pHi heart, brain and muscle (Wright et al., 1988), cod Gadus morhua exposed to hypercarbia had reduced pHe and pHi of heart, liver and muscle (Larsen et al., 1997), and O. mykiss experiencing hypercapnia had reduced pHe and pHi of brain and muscle (Wood and LeMoigne, 1991) (see Shartau et al., 2016a for an overview). Similarly, metabolic acidoses also reduced pHi of liver and muscle in starry flounder Platichthys stellatus (Milligan and Wood, 1987a; Milligan and Wood, 1987b), and heart and muscle pHi in sea raven Hemitripterus americanus (Milligan and Farrell, 1986). In adult tetrapods, coupled pH regulation is observed in all taxa where pHe and pHi have been measured during an acute respiratory acidosis. Exposure of the cane toad Bufo marinus (Snyder and Nestler, 1991; Toews and Heisler, 1982), knight anole Anolis equestris and desert iguana Dipsosaurus dorsalis (Snyder et al., 1995) to 5 kPa PCO2 for 1 h resulted in a respiratory acidosis with severe reductions in pHe and pHi; reductions in pHe and pHi were observed in western painted turtles Chrysemys picta bellii up to 6 h during a severe acute respiratory acidosis associated with diving (Wasser et al., 1991). Similarly, Rana catesbeiana tadpoles exposed to 5 kPa PCO2 resulted in reduced pHe and pHi of tail muscle and liver (Busk et al., 1997). Simultaneous reductions in pHe and pHi also occur during an acute metabolic acidosis following exhaustive exercise in the salt-water crocodile Crocodylus porosus (Baldwin et al., 1995). In mammals subjected to an acute respiratory acidosis, similar responses are observed. In adult dogs (Arieff et al., 1976) and cats (Yaksh and Anderson, 1987) pHe and pHi were reduced following exposure to ≥8 kPa PCO2 for 3 h and 10 min, respectively. In guinea pigs exposed to 15 kPa PCO2 there was an uncompensated reduction in pHe and pHi of lung, kidney, heart and muscle between 2 and 8 h of exposure, but at 7 days, pHe and pHi exhibited compensation of 68% and 80–106%, respectively; a response indicative of coupled pH regulation (Wood and Schaefer, 1978). This pattern has been corroborated in rats (Gonzalez and Clancy, 1986b) and hamsters (Malan et al., 1985) during acute hypercarbia exposure. Thus, in all adult amniotes  8 investigated to date in vivo (dog, cat, rat, hamster, guinea pig, western painted turtle, knight anole and desert iguana), an acute respiratory acidosis results in reduced pHe and pHi, and compensation occurs through coupled pH regulation, as has been the case for a relatively small number of fish species that have been examined. Although vertebrates are the focus of this thesis, it is worth noting that limited studies on invertebrates subjected to respiratory acidoses demonstrate coupled reductions in pHe and pHi, similar to that of vertebrates. In the few studies where both pHe and pHi have been measured during acute severe hypercarbia, reductions in pHe and pHi in a land snail Otala lactea (Barnhart and McMahon, 1988), deep sea bivalve Acesta excavata (Hammer et al., 2011), cuttlefish Sepia officinalis (Gutowska et al., 2010) and peanut worm Sipunculus nudus (Portner et al., 1998) have been observed.  1.1.4 In vitro pHi regulation In the many cell culture studies that have examined pHi regulation following transitory reductions in pHe (Bouyer et al., 2004; Filosa et al., 2002; Furimsky et al., 1999; Goldstein et al., 2000; Huynh et al., 2011b; Liu et al., 1990; Nottingham et al., 2001; Ritucci, 2005; Salameh et al., 2014), only a few have been conducted in the presence of sustained and elevated CO2. For example, isolated trout hepatocytes were unable to recover pHi in the presence of hypercarbia (Huynh et al., 2011b); this dependency of pHi on pHe is consistent with previous studies in trout and carp during chemically-induced anoxia (Krumschnabel et al., 2001). Other studies using metabolic acid–base challenges are informative about the relationship between pHi and pHe. The general pattern shown in these studies in vertebrates is that (1) reducing pHe causes pHi to be reduced and (2) complete recovery does not occur until the starting pHe is re-established (Occhipinti and Boron, 2015; Putnam and Roos, 1997; Vaughan-Jones et al., 2009). For example, when a metabolic acidosis was induced in a variety of mouse cell types (hippocampal neurons, astrocytes, medullary raphe neurons, colon cancer cells, skeletal muscle cells, macrophages, dendritic cells, melanocytes and keratinocytes) by lowering the external fluid [HCO3-] to reduce pHe, the pHi of all cell types was reduced and only fully recovered following the return of pHe to control values (Salameh et al., 2014). Generally, in vitro studies indicate  9 that acute changes in external or environmental pH will rapidly affect pHi. Although pHi recovers in all cells once the source of the external acidosis is removed, most vertebrate cells are unable to avoid an acute reduction in pHi when pHe is reduced in a cell culture environment, characteristic of coupled pH regulation (Fig. 1.1). This relationship between pHe and pHi is also observed during early development. When mouse (Siyanov and Baltz, 2013; Zhao and Baltz, 1996), hamster (Lane, 1999), bovine (Lane and Bavister, 1999) and human (Phillips et al., 2000) preimplantation embryos are subjected to an acidosis or alkalosis, they exhibit a decrease or increase in pHi, respectively. These embryonic cells can typically compensate pHi once environmental pH is returned to control values, although mammalian oocytes may not possess the capacity for pHi regulation initially, instead relying on surrounding granulosa cells to correct ooplasmic pH (FitzHarris and Baltz, 2009). However, in sea urchins, larvae are able to fully compensate pHi of primary mesenchyme cells during a hypercarbic-induced acidosis, and are able to accomplish this in the absence of pHe compensation in the body cavity (Stumpp et al., 2012).   1.1.5 Cellular mechanisms of pHi regulation The above studies indicate that pHi compensation during acid-base disturbances is almost always associated with pHe compensation and thus pHi is dependent on some degree of extracellular control of pH. Due to the importance of pHi regulation, numerous studies have investigated the transporters involved in a variety of cell types in various species. The transporters involved in pHi regulation generally include isoforms of acid-transporting Na+/H+ exchanger (NHE), V-type H+-ATPase (VHA), and base-transporting HCO3-/Cl- [anion exchangers (AE) also referred to as Cl-/HCO3- exchanger (CBE)] and Na+/HCO3- co-transporter (NBC) families. In general, during an acidosis, cells extrude H+ to the extracellular space via extrusion of intracellular H+ or uptake of extracellular HCO3-; the net effect being an increase in pHi. The response to an alkalosis is the opposite, with the cell seeking to increase net H+ concentration. In addition to those classic acid-base transporters, a lactate-H+ cotransporter (monocarboxylate transporter; MCT) has been found to function during hypoxia (where metabolic acidosis typically occurs) to remove intracellular lactate and H+. The transport of H+ via MCT is facilitated  10 by carbonic anhydrase (CA) which catalyzes the CO2 + H2O n H+ + HCO3- reaction to provide the MCT with H+ (Parks and Pouysségur, 2017; Vaughan-Jones et al., 2009). These mechanisms have been investigated in various vertebrate cell culture studies, including teleost hepatocytes (Ahmed, 2006; Furimsky et al., 2000; Huynh et al., 2011b), mammalian preimplantation embryos (FitzHarris and Baltz, 2009; Siyanov and Baltz, 2013), cardiac cells in avian embryos (Liu et al., 1990) and mammalian cardiac cells (Vaughan-Jones et al., 2009). Additionally, owing to the putative role of pH changes in cancer tumors, there is interest in understanding the mechanism of pH regulation in tumor cells. These cells are very resistant to chronic external acidosis, which is made possible via efficient pH regulatory systems. The tumor environment can reach nearly pH 6, yet tumor cells commonly demonstrate an alkaline pHi despite this chronic metabolic acidosis, which is favorable for cellular metabolism and proliferation (Parks and Pouysségur, 2017; Reshkin et al., 2014); pHi regulation in tumor cells may represent the extreme capacity for pHi regulation in vertebrates, despite their maladaptive nature to the organism.  1.1.6 pHe and pHi regulation beyond the bicarbonate concentration threshold The above studies indicate there is an intimate relationship between the regulation of pHe and pHi, and the inability to regulate the former limits the regulation of the latter. While vertebrates may tolerate extracellular acidoses of prolonged periods, they are not typically tolerant of severe changes in pHi, even if pHe recovers. In marine fishes, intracellular acidosis of heart was suggested to be the cause of mortality even though pHe was compensated over the following 8 h in response to severe hypercarbia (Hayashi et al., 2004); similarly, reduction in muscle pHi, not pHe, was hypothesized to be the ultimate cause of mortality in trout following exhaustive exercise (Wood et al., 1983). In fishes, the dependence of pHi regulation on pHe is particularly problematic when considering the limit of pHe compensation during CO2 exposure imposed by the bicarbonate concentration threshold (Fig. 1.3). As previously indicated, typically fishes cannot compensate pHe beyond ca. 2 kPa PCO2, thus regulation of pHi is also hindered in these fishes and represents a major limitation of coupled pHe/pHi regulation. Despite the putative limits restricting pHe, and thus pHi, compensation to approximately 2 kPa PCO2,  11 many fishes appear to tolerate these conditions without the expected morbidity or mortality. Acid-base regulation in a few fishes exposed to >4 kPa PCO2 has revealed the use of a novel strategy of acid-base regulation in which pHe is reduced, and remains uncompensated, but pHi is remains tightly regulated (Baker et al., 2009a; Brauner et al., 2004; Heisler, 1982); this strategy is referred to as preferential pHi regulation and is discussed below.  1.2 Preferential pHi regulation  Preferential pHi regulation is defined as ΔpHi/ΔpHe ≤ 0 immediately following onset of an acid-base disturbance. This is associated with complete pHi regulation of heart, brain, liver and muscle despite large reductions in pHe that may approach 1 pH unit, and has been proposed to confer exceptional hypercarbia tolerance (Brauner and Baker, 2009; Shartau et al., 2016a). Preferential pHi regulation was first documented during forced air breathing (induced by aquatic hypoxia) of Synbranchus marmoratus, the marbled swamp eel, an Amazonian air breathing teleost. The treatment resulted in an increase in blood PCO2 to 3.5 kPa after 96 h causing a reduction in pHe but no change in heart or muscle pHi (Heisler, 1982). Two decades following Heisler’s work, Brauner et al. (2004) observed limited pHe compensation but a remarkable ability for regulation of pHi during short-term environmental hypercarbia in Pterygoplichthys pardalis armoured catfish, a tropical air breather found in the Amazon River. Pterygoplichthys pardalis preferentially regulated pHi of heart, liver and white muscle during 24 and 72 h exposure to 1.9 and 4.3 kPa PCO2, respectively; during these exposures, pHe was reduced and was not compensated for. The strategy of preferential pHi regulation was considered to be rare amongst vertebrates by Brauner et al. (2004) and possibly associated with air breathing or living in ion-poor waters, such as those of the Amazon River basin. More recently, Baker et al. (2009a) observed the first example of preferential pHi regulation in a non-air breathing fish, Acipenser transmontanus, the white sturgeon. This is a basal euteleostom fish, which tightly protected pHi despite severe pHe reduction of ca. 0.8 pH units during 48 h exposure to 6 kPa PCO2. Similar to S. marmoratus and P. pardalis, A. transmontanus experience a large uncompensated reduction in pHe during  12 hypercarbia exposure but fully protect pH of heart, brain, liver and white muscle. pHi is exceptionally well protected, such that pHi of heart and brain experience an increase in pH, becoming slightly alkalotic relative to their normocarbic pH (Baker et al., 2009a); exposure to 12 kPa PCO2 for 6 h reduced pHe by ~1.0 pH unit, while liver pHi increased by 0.2 pH units (Baker and Brauner, 2012). Protection of pHi during exposure to hypercarbia in A. transmontanus appears to be nearly instantaneous. When heart pHi was measured in real time at 2-minute intervals using magnetic resonance imaging (MRI), there was no evidence for heart muscle pHi ever decreasing (Baker, 2010). This use of preferential pHi regulation during severe acute hypercarbia does not appear to be metabolically costly as whole animal metabolic rate during exposure to 6 and 12 kPa PCO2 corresponded to 30 and 60% reduction in MO2, respectively (Baker and Brauner, 2012). That preferential pHi regulation appears to confer exceptional CO2 tolerance may be due to the protection of intracellular pH of critical tissues (e.g. heart and brain). While some marine fishes demonstrate tremendous capacity for pHe compensation during severe acute hypercarbia exposure, mortality typically occurs despite recovery of pHe; this is postulated to be due to changes to cardiac performance (e.g. cardiac output, contractility) which leads to a reduction in organismal oxygen supply (Hayashi et al., 2004). In contrast, cardiac performance is also fully protected during exposures up to 5 and 6 kPa PCO2 in P. pardalis (Hanson et al., 2009) and A. transmontanus (Baker et al., 2011), respectively, which is believed to be associated with complete protection of heart pHi. Reduction in brain pH is associated with loss of equilibrium in Cyprinus carpio (common carp) (Yoshikawa et al., 1994) and reduced muscle pH was hypothesized to be the cause of post-exercise mortality in O. mykiss (Wood et al., 1983).  1.2.1 Mechanisms of preferential pHi regulation The mechanism(s) of preferential pHi regulation are unknown (Brauner and Baker, 2009), but likely involve transporters identified for pHi regulation; however, the specific mechanism(s) could be most effectively be investigated using cell culture. Acipenser transmontanus primary liver cells were exposed to 6 kPa PCO2 for 19-50 h experienced an initial pHi reduction which was compensated despite a sustained  13 extracellular acidosis (Huynh et al., 2011a); this is in contrast to a similar study using trout hepatocytes exposed to hypercarbia as these cells never recovered pHi while hypercarbia was maintained (Huynh et al., 2011b). The response in vitro differs from in vivo as liver pHi in sturgeon is not reduced during hypercarbia (Baker and Brauner, 2012; Baker et al., 2009a), which suggests that liver pHi regulation during hypercarbia is influenced by extrinsic factors (Huynh et al., 2011a). While preferential pHi regulation is due to active transport of acid-base equivalents (Baker et al., 2009a), the relatively high buffer capacity of the intracellular compartments compared to the blood and extracellular fluid is likely beneficial in ensuring there are no initial pHi changes at the onset of the acid-base disturbance.    1.3 Hypercarbia and acid-base regulation – a role for preferential pHi regulation?  Due to the prevalence of hypercarbia in various environments, preferential pHi regulation may be important for conferring CO2 tolerance in these habitats. High environmental CO2 is common in many environments, particularly in aquatic ecosystems (Marcé et al., 2015; McNeil and Sasse, 2016; Raymond et al., 2013; Reum et al., 2014), and may arise due to a number of factors, including high aquatic biomass, thermo-stratification and poor water mixing, surface vegetation, and anaerobic metabolism of microorganisms (Brauner and Baker, 2009; Ultsch, 1996). Since fishes comprise over half of all vertebrate species (~32,000) (Nelson, 2006), hypercarbia may be an important abiotic variable affecting life history and evolution of fishes (Hasler et al., 2016; Ultsch, 1987). Hypercarbia in tropical freshwater may reach >8% PCO2 (Furch and Junk, 1997; Heisler, 1984; Li et al., 2013) and even temperate waters can experience naturally elevated PCO2 (Atilla et al., 2011; Butman and Raymond, 2011; Weyhenmeyer et al., 2012); for example, the lower Columbia River can reach 870 µatm (ca. 0.9 kPa) PCO2 (Park et al., 1969). These values are far in excess of current atmospheric CO2 levels [400 µatm (ca. 0.04 kPa)] and still much greater than projected end of century CO2 increases that have been predicted due to climate change [~1000 µatm (ca. 0.1 kPa)] (McNeil and Sasse, 2016).  14 Although coupled pHe/pHi regulation may be generally limited to PCO2  <2 kPa, many fishes inhabit environments that may experience hypercarbia in excess of >8 kPa PCO2 (Furch and Junk, 1997; Heisler, 1984), which is beyond their ability to compensate pHe. These hypercarbic-prone environments contain a disproportionate percentage of the world’s freshwater fishes, particularly within the Amazon and Mekong river basins, as 56% of watersheds with high fish species diversity are located in the tropics (Val et al., 2005). In the marine environment, there are natural CO2 seeps at some locations which create localized hypercarbia that may reach as high as 7 kPa PCO2 (Basso et al., 2015; Melzner et al., 2009). In addition to aquatic hypercarbia, some terrestrial environments experience dramatically higher PCO2 than ambient atmospheric levels. Some burrows of subterranean rodents may have PCO2 ranging from 0.22-6.1 kPa (Burda et al., 2007; Shams et al., 2005). Typically, burrow gas composition remains constant, thus hypercarbia is chronic, however, depending on the soil type, some burrows may experience large changes in PCO2 over the course of hours to days, and thus expose rodents to severe acute hypercarbia; under laboratory conditions, subterranean mole rat Spalax sp. can survive 15 kPa PCO2 and 3 kPa PO2 for at least 8 h without physiological or behavioural changes (Shams et al., 2005). In cave environments, PCO2 may reach 6 kPa; in one cave system, numerous species inhabit high CO2 regions, including troglobites and bats (Howarth and Stone, 1990). Nests of birds and reptiles naturally experience changes in CO2 levels due to biotic (i.e. metabolic activity of embryos and microorganisms) and abiotic (i.e. diffusion gradients/barriers and nest composition) factors, often resulting in hypercarbic rearing environments for embryos; consequently, nest CO2 can reach 5-8 kPa PCO2 (Booth, 1998; Grigg et al., 2010; Lutz and Dunbar-Cooper, 1984; Seymour et al., 1986). The acid-base response in terrestrial animals exposed to these severely hypercarbic conditions has not been well investigated. While animals exposed to chronic hypercarbia in caves and burrows likely adjusted their blood-gas composition to account for those differences, it is not known what happens during acute exposure. Amongst embryonic amniotes only the extracellular response of chicken embryos to severe acute hypercarbia has been investigated (Everaert et al., 2011). Those studies demonstrate that  15 chicken embryos exposed to severe acute hypercarbia experience a typical respiratory acidosis, but there is minimal pHe compensation when hypercarbia is maintained for up to 24 h (Andrewartha et al., 2014; Burggren et al., 2012); it is unknown how pHi responds. Due to the severe effects of hypercarbia, and especially given the limitations of coupled pH regulation, preferential pHi regulation may play an important role as an acid-base regulatory strategy in a number of these species that regularly enter and experience hypercarbic conditions. At the start of this dissertation only four species were known to use preferential pHi regulation (Fig. 1.4) but it remains uncertain how other species tolerate the severe acid-base disturbances they presumably experience during those conditions.   1.4 Preferential pHi regulation: a basal euteleostom strategy or embryonic strategy?  Preferential pHi regulation has been previously hypothesized to (1) confer exceptional CO2 tolerance and (2) that it evolved in the basal actinopterygians, as it has not been observed in hagfish or elasmobranchs (Brauner and Baker, 2009). However, direct (Siren lacertina greater siren (Heisler et al., 1982)) and indirect (Lepidosiren paradoxa South American lungfish (Sanchez et al., 2005)) evidence for preferential pHi regulation amongst the sarcopterygii suggests that it may have been used in basal Euteleostomi (Sarcopterygii + Actinopterygii) as a strategy of acid-base regulation in adults. Loss of preferential pHi regulation may have occurred due to changes in physiology requiring coupled pH regulation and where environmental conditions were favorable to permit pHe compensation. In teleosts, for example, the evolution of the Root effect likely necessitated regulation of the extracellular compartment during acidoses. This is because of the pH-sensitivity of Root effect hemoglobins, which exhibit a reduction in Hb-O2 affinity as pH is reduced. Although hemoglobin is confined to the red cells, and teleosts possessing the Root effect regulate RBC pH over the short-term via beta-adrenergic Na+/H+ exchanger (βNHE) to safeguard O2 transport, this mechanism  16 remains dependent on eventual pHe recovery (Berenbrink et al., 2005; Shartau and Brauner, 2014). As the species where preferential pHi regulation has been identified do not possess a Root effect, nor do they have RBC βNHEs (Berenbrink et al., 2005), RBC pHi is not regulated (Baker et al., 2009a; Brauner et al., 2004) and thus H+ exchange is largely passive across the RBC membrane. However, because Hb-O2 affinity is not affected to a large degree by changes in pH in these species, an uncompensated pHe acidosis may not be detrimental to O2 transport. Based on these data, preferential pHi regulation was hypothesized to have evolved in the basal euteleostomi and retention or loss in extant euteleostomi groups was driven by either environmental (e.g. hypercarbia) and/or physiological constraints (e.g. Root effect). Alternatively, preferential pHi regulation in adults may represent the retention of the embryonic capacity for intracellular pH regulation. Although few studies have characterized pHi regulation during vertebrate development, there is evidence that early-stage embryos are capable of pH regulation just after fertilization, and mammalian oocytes and embryos are able to recover from an intracellular acid-base disturbance of almost 1 pH unit (FitzHarris and Baltz, 2009; Lane, 1999). Work on fish has also shown this pattern as early-stage zebrafish embryos exposed to 3.3 kPa PCO2 for 2 h in vitro display a respiratory acidosis, but are still able to restore pHi to pre-hypercarbic values (Molich and Heisler, 2005). Similarly, sea urchin larvae are able to fully compensate pHi of primary mesenchyme cells during a hypercarbic-induced acidosis in the absence of pHe compensation in the body cavity (Stumpp et al., 2012), suggesting that preferential pHi regulation may not be limited to vertebrates; no evidence presently exists for preferential pHi regulation in adult invertebrates (Shartau et al., 2016a). These studies indicate that during an acute acidosis cells have the capacity for pHi compensation at the earliest developmental time points. It is unknown for how long embryos retain this capacity for pHi regulation, as it may be reduced or enhanced following the appearance of the extracellular space and the growth of organs involved in regulating pHe; additionally, the above findings are from in vitro studies and, as indicated previously, these may or may not be representative of in vivo responses during an acid–base disturbance. Beyond the earliest developmental stages, acid–base regulation has been poorly studied in embryonic vertebrates; however, several studies to date have examined the response of  17 late-stage chicken (Gallus gallus) embryos to respiratory and metabolic acidoses (Everaert et al., 2011). Exposing chicken embryos in vivo to severe acute respiratory or metabolic acidosis for up to 24 h results in large reductions in pHe that are not fully compensated yet the embryos survive (Burggren et al., 2012; Mueller et al., 2014); while pHi was not measured, the changes in pHe are consistent with pHe changes observed in fish that preferentially regulate pHi (Brauner and Baker, 2009). Adult amniotes use coupled pH regulation (Cameron, 1989b; Shartau et al., 2016a), but during embryonic development in which high CO2 exposure may occur, they may utilize preferential pHi regulation to cope with the acid-base disturbance. Based on this limited embryonic data, I hypothesized that preferential pHi regulation is an embryonic strategy of acid-base regulation that is retained or lost throughout development. Insufficient information existed to support or reject these ideas pertaining to preferential pHi regulation as either a basal euteleostom or embryonic strategy of acid-base regulation; this was focus of investigation for my thesis.   1.5 Thesis objective and organization  Preferential pHi regulation may provide a way to avoid the putative limits of coupled pHe/pHi regulation, and thus allow those species to tolerate and survive challenging levels of CO2 exposure. The pHe and pHi responses of species using preferential pHi regulation is wholly different from those using coupled pH regulation (Brauner and Baker, 2009; Shartau et al., 2016a); however, little about preferential pHi regulation is known. The objective of this thesis is to investigate the usage, distribution/prevalence, and origin of preferential pHi regulation as a strategy of acid-base regulation in vertebrates. In this thesis, the general objective is addressed in the subsequent four chapters: 1) Is preferential pHi regulation a general strategy of acid-base regulation in white sturgeon subjected to a range of acid-base disturbances? 2) Is preferential pHi regulation a common strategy of acid-base regulation among a diverse range of fish species? 3 and 4) How does the strategy of acid-base regulation shift throughout development in amniotes known to use coupled pH regulation as an adult?  18  1.5.1 Is preferential pHi regulation a general strategy of acid-base regulation in white sturgeon subjected to a range of acid-base disturbances? Studies observing preferential pHi regulation have all been conducted during hypercarbic conditions (Shartau et al., 2016a) and it is uncertain whether preferential pHi regulation also confers protection against non-hypercarbic induced acidoses. In P. pardalis and A. transmontanus, it is clear that preferential pHi regulation allows tissues to be fully protected against a range of hypercarbic exposures. However, fishes are often exposed to various other conditions that may pose challenges for acid-base regulation (e.g. hypoxia and exercise) and it is unknown if preferential pHi regulating species are able to confer the same degree of protection during these other types of acid-base disturbances. As the origins of respiratory and metabolic acidoses are different, it is uncertain whether preferential pHi regulation functions in the latter, or if it does, if it confers the same degree of pHi protection; more specifically, is preferential pHi regulation a general strategy of acid-base regulation? Chapter 2 seeks to address this question using A. transmontanus as their capacity for preferential pHi regulation has been investigated numerous times during hypercarbia (Baker and Brauner, 2012; Baker et al., 2011; Baker et al., 2009a; Shaughnessy et al., 2015). I hypothesized that the tremendous capacity for preferential pHi regulation in A. transmontanus during hypercarbia reflects the use of preferential pHi regulation as a general strategy of acid-base regulation. This was investigated by subjecting A. transmontanus to conditions creating severe metabolic (exhaustive exercise, anoxia, and air exposure) and non-hypercarbic respiratory acidoses (hyperoxia). Following exposure to various treatments, fishes were sampled for pHe and pHi of heart, brain, liver, and white muscle to determine their acid-base regulatory response. This chapter will also inform on whether non-hypercarbic acidoses can be used in future studies to assess the presence or absence of preferential pHi regulation, and if existing literature can be used to infer on the strategy of acid-base regulation.   19 1.5.2 Is preferential pHi regulation a common strategy of acid-base regulation among a diverse range of fish species? At the start of this dissertation research, only three fishes had been identified to use preferential pHi regulation, suggesting limited use of preferential pHi regulation in fishes (Fig. 1.4). However, as few studies have measured pHe and pHi concurrently, and only amongst a relatively limited number of species, it is uncertain as to whether preferential pHi regulation is truly a rare strategy as suggested by Brauner et al. (2004). As many fishes inhabit environments prone to severe hypercarbia, with PCO2 reaching well beyond the putative capacity for pHe regulation (Brauner and Baker, 2009; Heisler, 1984), it is likely that preferential pHi regulation is more widely used; especially given that two of the three species are both tropical air breathers, it seems likely that other tropical/Amazonian fishes would possess this ability for pH regulation. Aside from those three preferential pHi-regulating fishes, the general strategy for acid-base regulation amongst fishes (and vertebrates) is coupled pHe/pHi regulation. But, is preferential pHi regulation a unique strategy in a world characterized by coupled pH regulation, or is the former more common than current data suggest? Chapter 3 seeks to address the question of whether preferential pHi regulation is a common strategy of acid-base regulation in response to severe acute hypercarbia. I hypothesize that preferential pHi regulation is a widespread strategy amongst fishes used during severe acute hypercarbia. This was investigated by conducting a survey that included 20 fish species from groups ranging from basal to derived, including lamprey, lungfish, elasmobranchs, basal actinopterygians, and various teleosts to assess the presence or absence of preferential pHi regulation. Using a CO2 tolerance assay developed for this purpose, the acute CO2 tolerance of fishes was first determined, then presence or absence of preferential pHi regulation was determined directly via pHe/pHi measurements following hypercarbia exposure, or indirectly via CO2 tolerance. This chapter will provide a better understanding of the distribution of preferential pHi regulation, and how fishes tolerate and survive in hypercarbic environments.  1.5.3 How does the strategy of acid-base regulation shift throughout development? Acid-base regulation is a critical physiological process and regulation is present  20 early in development, starting with egg and zygote (FitzHarris and Baltz, 2009; Johnson and Epel, 1981; Lane, 1999; Molich and Heisler, 2005). While these early developmental stages do not appear to completely avoid pHi changes when subjected to such conditions designed to induce an acid-base disturbance, they are capable of adjusting intracellular pH (Johnson and Epel, 1981; Molich and Heisler, 2005). Experiments at these early developmental stages demonstrate cells have robust capacity for pHi regulation; however, early in development the tissues have not formed, nor is the extracellular space developed, which makes comparisons with adult acid-base strategies inherently difficult. Following the development of tissues and the extracellular space, there may be changes in acid-base regulatory physiology as well, however, this has not been investigated. Embryonic invertebrate (Shartau et al., 2010), fish (Ciuhandu et al., 2007) and amniote (Booth, 1998; Grigg et al., 2010) species may experience challenging environmental conditions (e.g. hypoxia) that are more severe than they would experience as adults. These differences between embryo and adult may lead to different degrees of acid-base disturbances for which different strategies may be employed, such as preferential pHi regulation or coupled pH regulation. Little is known about acid-base regulation during embryonic development in most animals; however, due in part to the large size of amniote embryos, some work has looked at bird and reptile embryos. Adult amniotes do not typically experience severe hypercarbia naturally, but when they are subjected to hypercarbia they utilize coupled pH regulation; this response in embryos has not been well investigated (Everaert et al., 2011), which is perhaps surprising given the severity of hypercarbia some embryonic reptiles and birds may experience (Booth, 1998; Grigg et al., 2010; Lutz and Dunbar-Cooper, 1984; Seymour et al., 1986). Embryonic amniotes do not have the similar physiological tools of adults to respond to respiratory acidoses via adjustment to ventilation and they may be constrained by nest and eggshell diffusion (Erasmus et al., 1971), thus, their acid-base regulatory response may differ. Studies on embryonic chickens suggest they are resilient to very high CO2 and can tolerate prolonged periods of an uncompensated reduction in pHe, suggesting amniote embryos may have a greater capacity for pHi protection than adults. Chapter 4 investigates the strategy of acid-base regulation during development of a hypercarbic tolerant amniote, Chelydra serpentina (common snapping turtle); adult  21 turtles are known to use coupled pH regulation (Wasser et al., 1991). I hypothesized that embryonic C. serpentina would preferentially regulate pHi in response to acid-base disturbances. This was investigated by exposing C. serpentina embryos at 70 and 90% to hatch, and yearlings to severe acute hypercarbic hypoxia exposure and measuring pHe and pHi of various tissues. This chapter provides the first insight into how amniote embryos regulate both pHe and pHi during severe acute acid-base disturbances, and it will inform on how acid-base regulation changes throughout development.  1.5.4 Is preferential pHi regulation an embryonic strategy in amniote embryos? Indirect evidence suggests chicken embryos use preferential pHi regulation during severe acid-base challenges (Andrewartha et al., 2014; Burggren et al., 2012) and results from Chapter 4 indicates C. serpentina embryos preferentially regulate pHi (Shartau et al., 2016b); thus other embryonic amniotes subjected to severe hypercarbia may employ a similar strategy. Another amniote that is known to experience and tolerate severe hypercarbia during development are crocodylians (Eme and Crossley, 2015), which lay eggs in nests where PCO2 can reach 2-8 kPa (Grigg et al., 2010; Lutz and Dunbar-Cooper, 1984). Little is known about acid-base regulation in oviparous amniote embryos (Everaert et al., 2011). Chapter 5 investigates the strategy of acid-base regulation in another embryonic amniote. Preferential pHi regulation is likely to be used by embryonic amniotes that are subjected to severe hypercarbic conditions in nest environments. I hypothesized that Alligator mississippiensis (American alligator) embryos preferentially regulate pHi during severe acid-base disturbances. This was investigated by exposing A. mississippiensis embryos at 70% to hatch to severe hypercarbic hypoxia and measuring pHe and pHi; the same developmental time was used at which C. serpentina displayed the most robust pHi regulation. This chapter provides insight into crocodylian acid-base regulation during development and, along with C. serpentina, contribute to understanding how pH regulation in amniotes, and thus, vertebrates.  22    Figure 1.1: Representation of the typical response of vertebrates utilizing coupled pH regulation during acute sustained hypercapnia. In species utilizing coupled pH regulation, a hypercarbia-induced respiratory acidosis (initiated at t=0) leads to a rapid reduction in both pHe and pHi, with maximal pH depression occurring typically by 3 h or less. Recovery of pH then occurs by 24 h, but the rate of compensation depends on the severity of the pH depression and the ability for net acid excretion to the environment. pHi is often compensated more rapidly than pHe but complete pHi compensation generally requires >50% of complete pHe recovery.   23     Figure 1.2: Representation of the typical response of vertebrates utilizing preferential intracellular pH (pHi) regulation during acute sustained hypercapnia. In species utilizing preferential pHi regulation, a hypercarbia-induced respiratory acidosis leads to a rapid reduction in pHe but no change (or even a slight increase) in pHi. During hypercarbia, pHi remains independent of the sustained, uncompensated reduction of pHe for periods >24 h.  24  Figure 1.3: A theoretical representation of the typical extracellular pH (pHe) response to short-term (<5 days) hypercarbia in fish. Transfer from normocarbia to hypercarbia results in extracellular pH (pHe) falling along the blood non-bicarbonate buffer line, which is indicated by the black open arrowhead. pHe then recovers along a given PCO2 isopleth via a net increase in HCO3- in exchange for Cl- as indicated by black filled arrowheads. Black filled circles represent final pHe values that would be achieved based upon limits to net HCO3- accumulation within 24-96 h exposure to hypercarbia. Shaded bar indicates the greatest pH compensation able to occur at each CO2 tension, which is believed to be limited by constraints on HCO3- accumulation and is termed the ‘bicarbonate concentration threshold’. Most fishes studied to date cannot increase plasma HCO3- beyond 27-35 mmol l-1 (Baker et al., 2015; Brauner and Baker, 2009; Heisler, 1984). Consequently, compensation for an acute respiratory acidosis (<96 h) during exposure to hypercarbia is incomplete above a PCO2 of ca. 2 kPa in most fishes. Figure modified from Brauner and Baker (2009).  25  Figure 1.4: Phylogeny showing distribution of preferential intracellular pH (pHi) regulation and coupled pH regulation amongst vertebrates when exposed to acute >2 kPa PCO2 prior to dissertation research. Species using preferential pHi regulation during severe acute hypercarbia are indicated in pink, while those using coupled pH regulation are indicated in blue. Empty branches indicate species that will be examined in this dissertation – see Fig. 6.1 for the filled in phylogeny. References are indicated by numbers following species name. 1(Baker et al., 2015), 2(Wood et al., 1990), 3(Snyder and Nestler, 1991), 4(Heisler et al., 1982), 5(Wasser et al., 1991), 6(Snyder et al., 1995), 7(Snyder et al., 1995), 8(Malan et al., 1985), 9(Wood and Schaefer, 1978), 10(Gonzalez and Clancy, 1986a), 11(Yaksh and Anderson, 1987), 12(Arieff et al., 1976), 13(Baker et al., 2009a), 14(Brauner et al., 2004), 15(Wood and LeMoigne, 1991), 16(Larsen et al., 1997), 17(Wright et al., 1988), 18(Heisler, 1982). Phylogenetic relationships are based on (2009) and branch lengths are taken  26 from various references utilizing fossil and molecular estimates of divergence times (Aschliman et al., 2012; Betancur-R et al., 2013; Betancur-R et al., 2015; Blair, 2005; Macqueen and Johnston, 2014; Meredith et al., 2011; Zhang et al., 2013); the phylogenetic tree was created using Mesquite (Maddison and Maddison, 2017).  27 Chapter 2: White Sturgeon (Acipenser transmontanus) Acid-Base Regulation Differs in Response to Different Types of Acidoses   2.1 Introduction  Acid-base regulation is one of the most important physiological processes in vertebrates due to the effect of pH on proteins, as changes in pH typically alter protein charge, which can change protein function, and ultimately reduce whole animal performance (e.g. reduce heart and skeletal muscle contractility, and alter metabolic pathways) (Occhipinti and Boron, 2015; Putnam and Roos, 1997). Challenges to acid-base homeostasis may have respiratory or metabolic origins, or a combination of the two. A respiratory acidosis results in a reduction in blood pH (extracellular pH; pHe) and intracellular pH (pHi) due to an increase in blood PCO2. In fishes, compensation of pHe primarily occurs at the gills via net exchange of acid-base relevant ions, whereby an increase in plasma [HCO3-] occurs and is generally associated with an equimolar decrease in plasma [Cl-], which gradually compensates pHe at an elevated blood PCO2 (Brauner and Baker, 2009; Heisler, 1984; Perry and Gilmour, 2006; Shartau et al., 2016a). Similarly, metabolic acidoses reduce pHe and pHi, but the acidosis often originates from the cells as a consequence of increased H+ and lactate production associated with anaerobic glycolysis (Robergs et al., 2004). Compensation of a metabolic acidosis is primarily dependent on net exchange of acid-base relevant ions at the gills (Evans et al., 2005; Hwang et al., 2011) and to a lesser degree, excretion of acidic equivalents in the form of titratable acidity and ammonium ions through renal pathways (Kwong et al., 2014). Typically, some degree of pHe compensation is required for pHi compensation as the two are coupled (referred to as ‘coupled pH regulation’, see Shartau et al., 2016a). Some fishes, such as the white sturgeon Acipenser transmontanus, however, do not follow this pattern and completely regulate pHi, often at the expense of pHe regulation  28 and in the face of severe maintained reductions in pHe, termed preferential pHi regulation (Brauner and Baker, 2009; Shartau et al., 2016a). Acipenser transmontanus are basal actinopterygians, belonging to the Acipenseriformes and are found along the Pacific coast of North America in the Fraser, Columbia, Sacramento and San Joaquin river systems (Hildebrand et al., 2016). They are one of the most CO2 tolerant fishes, able to tolerate a hypercarbic-induced respiratory acidosis of at least 12 kPa PCO2 for 6 h, which reduces pHe by nearly 1 pH unit and the extracellular acidosis remains uncompensated over this time period (Baker and Brauner, 2012). This ability is attributed to preferential pHi regulation of heart, brain, liver and muscle during these exposures (Brauner and Baker, 2009; Shartau et al., 2016a); protection of muscle pHi has been observed for up to 10 days at 6 kPa PCO2 (Shaughnessy et al., 2015). Hypercarbia in A. transmontanus has been well studied (Baker and Brauner, 2012; Baker et al., 2011; Baker et al., 2009a; Cech and Crocker, 2002; Crocker and Cech, 1998; Shartau et al., 2017b), and while severe hypercarbia (> 3 kPa PCO2) is unlikely to be widely encountered in natural settings, it may occur in aquaculture settings (Crocker and Cech, 1996). Mild hypercarbia (< 1 kPa PCO2), however, may be frequently experienced by A. transmontanus in their environment and in hatchery settings (Crocker and Cech, 1998). Hypercarbia tolerance is believed to be attributed to the capacity for preferential pHi regulation as other species (e.g. marbled swamp eel Synbranchus marmoratus, armoured catfish Pterygoplichthys pardalis, striped catfish Pangasianodon hypophthalmus, spotted gar Lepisosteus oculatus and alligator gar Atractosteus spatula) exhibiting this degree of hypercarbia tolerance also preferentially regulate pHi (see Shartau and Brauner, 2014; Shartau et al., 2016a). Recently, it was observed that preferential pHi regulation confers protection against acid-base disturbances in addition to those induced by hypercarbia in the tropical air breathing P. pardalis (Harter et al., 2014). The authors concluded that preferential pHi regulation may represent a general strategy of acid-base regulation in this species (Harter et al., 2014). Given the tremendous capacity A. transmontanus have for preferential pHi regulation during hypercarbia, we hypothesized that A. transmontanus, similar to P. pardalis, utilize preferential pHi regulation as a general strategy of acid-base regulation, irrespective of the origin of the disturbance.  29 To test this hypothesis, A. transmontanus were subjected to either respiratory or metabolic acidoses and their acid-base response was measured. Determining the presence or absence of preferential pHi regulation is contingent on inducing a sufficiently severe pHe reduction to influence pHi; therefore, the treatments to impose acidoses and the sampling times were chosen to ensure a severe pHe reduction. For a respiratory acidosis, hyperoxia was used as it has not been previously examined in A. transmontanus, unlike hypercarbia (see above references). The origin of the respiratory acidosis differs between hypercarbia and hyperoxia, whereby the former induces an acidosis due to increased external CO2. The latter induces an acidosis arising from reduced ventilatory rate due to high environmental oxygen; thus, reducing CO2 excretion and leading to an increase in metabolically produced CO2 (Wood and LeMoigne, 1991), and unlike hypercarbia, hyperoxia does not reduce water pH, which may impair pHe regulation in A. transmontanus (Shartau et al., 2017b). Metabolic acidoses were induced via exhaustive exercise, anoxia or air exposure, where the acidosis is generated intracellularly (via anaerobiosis), with the associated acid exported to the extracellular space. Treatments imposing metabolic acidoses often produce a mixed acid-base metabolic and respiratory acidosis (Kieffer et al., 1994; Wang et al., 1994). The response of A. transmontanus to these metabolic acidoses may also inform, to some degree, on the acid-base relevant effects of challenges such as catch and release fishing (McLean et al., 2016) and swimming/migration (Cocherell et al., 2011; Erickson et al., 2002; Geist et al., 2005).   2.2 Methods  2.2.1 Animal acquisition and holding All experiments were performed at the International Centre for Sturgeon Studies (ICSS) at Vancouver Island University (VIU) using A. transmontanus (656 ± 181 g). All white sturgeon were maintained in large indoor flow-through tanks in dechlorinated City of Nanaimo tap water [61 µmol l-1 Na+, 69 µmol l-1 Cl- (City of Nanaimo, 2015), pH ~6.6-6.8 (Mojazi Amiri et al., 2009)] at ~15 oC under a simulated natural photoperiod and were fed daily to satiation. Food was withheld 48 h prior to experiments. All experiments  30 were approved both by the University of British Columbia and Vancouver Island University Animal Care Committees (animal care no: A11-0235; Animal Usage Protocol: 2014-02-R).   2.2.2 Experimental protocol  For all treatments, eight white sturgeon were randomly selected from the holding tank and placed in individual black plexi-glass boxes (24 L) with aeration in a re-circulating system (flow rate ~3 L min-1 per box, 15 oC; total water volume of system ~320 L) overnight prior to experiments. Confinement in darkened boxes has been suggested to not stress A. oxyrinchus Atlantic or A. brevirostrum shortnose sturgeon (Baker et al., 2005a); similarly, juvenile Scaphirhynchus albus pallid and hybrid S. albus Î S. platorynchus shovelnose sturgeon have low physiological responses to severe confinement (Barton et al., 2000). Control fish were sampled following overnight holding; experimental manipulations are described below for each individual treatment. Sampling was staggered to ensure adequate time to euthanize, sample each animal and take blood measurements.  2.2.3 Respiratory acidosis Hyperoxia induces a respiratory acidosis via the retention of metabolically produced CO2. In this treatment, fish were held overnight, then aeration was stopped to the recirculating system, while aeration to individual tanks was maintained. All boxes were isolated from the recirculating system and rapid O2 bubbling was initiated in the main header tank to increase O2 tension to ~80 kPa PO2 (~15 min). Once achieved, the boxes were re-connected to the recirculating system in a staggered fashion (~20 min), and PwO2 increased to the target tension of ~80 kPa PCO2 within 15 min. Fish were then exposed to ~80 kPa PwO2 for 180 min to achieve a hyperoxic-induced respiratory acidosis of similar magnitude and duration as previous hypercarbic-induced respiratory acidoses (Baker et al., 2009a; Baker et al., 2015; Brauner et al., 2004).   31 2.2.4 Metabolic acidosis Metabolic acidoses were induced via exhaustive exercise, anoxia or air exposure. White sturgeon subjected to exhaustive exercise were removed from tanks and subjected to a repeated exhaustive exercise protocol similar to the one used on armoured catfish (Harter et al., 2014). Fish were chased with a plastic stick for 5 min or until the fish was completely exhausted, allowed to rest for 15 min, then chased again until complete exhaustion, which occurred within 15 min. In the anoxia treatment, after overnight holding, aeration was stopped to the recirculating system, while aeration to the individual tanks was maintained. All boxes were then isolated from the recirculating system and rapid N2 bubbling was initiated in the main header tank to reduce O2 tension to <1 kPa PwO2 (~15 min). Once achieved, the boxes were re-connected to the recirculating system in a staggered fashion (~20 min), and PwO2 decreased to the target tension of <1 kPa PwO2 within 15 min. Fish were continuously exposed to <1 kPa PwO2 for 5 min; following the anoxia exposure, the box was disconnected and aerated, returning PwO2 to >90% saturation within 2 min and fish were then allowed to recover until sampling (see below). Acipenser transmontanus were exposed to air following overnight holding in individual tanks. In a staggered fashion, water was drained from the respective tank, then a damp cloth was placed over the fish to minimize desiccation and stress; air temperature was ~15 oC. After 45 min, the tank was filled with aerated water and the fish were allowed to recover until sampling. Fish subjected to exhaustive exercise or anoxia were sampled at 15 or 120 min after the challenge, different time points were used to allow for any redistribution of acidoses and to assess those changes on pH. Due to limited fish numbers, fish subjected to air exposure were only sampled at 15 min post-exposure.  2.2.5 Blood sampling, tissue sampling and ions At the time of sampling, the box in which the fish was held was isolated from the re-circulating system and anesthetic was added to the water (MS-222 0.3 g L-1 buffered with NaHCO3) under vigorous aeration to avoid hypoxemia due to reduced ventilation. Once ventilation ceased (<3 min), each fish was turned ventral side up, while gills  32 remained submerged in aerated water and blood (3 mL) was drawn caudally via a lithium-heparin (1 g L-1)- rinsed syringe (5 mL syringe, 23 G1¼ needle) and placed on ice. Following this procedure, fish were killed via cephalic concussion and the following tissues were removed within 2-3 min, placed in aluminum foil and immediately placed in liquid N2 in the following order: heart (gently squeezed and patted dry to remove any blood), liver, dorsal white muscle (left side, just posterior of the dorsal fin; skin and red muscle removed), and brain; tissues were stored longer term at -80 oC. Blood was divided into two aliquots. Blood pH and hematocrit (Hct) were measured from one aliquot; the other aliquot was centrifuged (3 min at 10,000 rpm) and plasma was removed for measurement of total CO2 (TCO2), [Cl-] and [lactate]. Blood pH was measured using a Radiometer PHM 84 (Copenhagen, Denmark) connected to a Radiometer Analytical SAS pH electrode (GK2401C, Cedex, France) thermostated at 15 oC. RBC pHi was measured using the freeze-thaw method as described by Zeidler and Kim (1977). Tissue pHi was measured using the metabolic inhibitor tissue homogenate method (MITH; see Appendix for detailed description of this method) (Baker et al., 2009b; Portner et al., 1990). Plasma TCO2 was measured using a total CO2 analyzer (Corning model 965 Analyzer); the remaining plasma was used to measure [Cl-] ions (HBI model 4425000; digital chloridometer). For determination of plasma [lactate], 200 µL 8% perchloric acid was added to 200 µL plasma and immediately frozen in LN2 and stored at -80 oC until assayed for lactate via the method described by Bergmeyer (1983).   2.2.6 Calculations and statistical analysis  Plasma [HCO3-] and PCO2 were calculated using TCO2 and pH values described by Brauner et al. (2004). CO2 solubility coefficient and the logarithmic acid dissociation constant (pK’) for plasma were determined from Boutilier et al. (1984).  All values are expressed as mean ± s.e.m. throughout; N=8 for all treatments. Data were compared by Welch’s t-test or where multiple treatments were evaluated, data were analyzed by an analysis of variance (ANOVA), followed by Tukey’s or Dunnett’s post hoc tests or if the data did not meet normality (Shapiro-Wilk normality test) or equal variance (Bartlett’s test) assumptions a Kruskal-Wallis test followed by Dunn’s multiple  33 comparison test was used (P<0.05). GraphPad Prism (v.5) was used for all statistical analyses and for preparation of figures.   2.3 Results  2.3.1 Extracellular acid-base status  The objective of all treatments was to induce a reduction in pHe to examine pH changes in the tissues. Here, all treatments were successful in reducing pHe, although the severity of pHe reduction varied amongst acidoses (Fig. 2.1 and 2.2). A respiratory acidosis induced by 180 min hyperoxia exposure increased blood PCO2 to 1 kPa PCO2. This increase in blood PCO2 led to a reduction in pHe and an increase in plasma HCO3- immediately following this exposure (Fig. 2.1). Acipenser transmontanus exercised to exhaustion experienced a large pHe reduction (0.30 units) at 15 min post-exercise along with an increase in PCO2, but no change in plasma HCO3- was observed. Compared to 15 min post-exercise, at 120 min, pHe was unchanged, but PCO2 and plasma HCO3- were lower (Fig. 2.2a). Exposure to anoxia reduced pHe by only 0.1 units by 15 min post-anoxia and increased PCO2 and plasma HCO3-; pHe, PCO2 and plasma HCO3- did not change by 120 min post-anoxia (Fig. 2.2b). Following 45 min air exposure, both blood PCO2 and plasma [HCO3-] increased, while pHe decreased by 0.35 units (Fig. 2.2c).  2.3.2 Intracellular acid-base status A hyperoxia-induced respiratory acidosis reduced RBC and muscle pHi immediately following 180 min exposure; heart, liver and brain pHi did not change (Fig. 2.3). Exhaustive exercise reduced RBC, liver and muscle pHi at 15 and 120 min post-exercise, while heart pHi was only reduced at 120 min post-exercise; brain pHi did not significantly change (Fig. 2.4a). Anoxia exposure reduced pHi of RBC, liver and brain at 15 and 120 min post-exposure; muscle pHi was reduced at 120 min post-exposure (Fig.  34 2.4b). Air exposure resulted in a reduction in RBC, liver and muscle pHi at 15 min post-exposure (Fig. 2.4c).  2.3.3 Hematocrit, plasma [Cl-] and [lactate]  Exhaustive exercise induced the greatest change in these parameters relative to control values, where plasma [Cl-], [lactate] and hematocrit all increased following exercise. Hematocrit also increased in the hyperoxia exposure, but not in anoxia or air exposure. Plasma [lactate] was elevated compared to controls after 15 min post-acidosis in anoxia, hyperoxia and air exposure. Plasma [Cl-] did not change following anoxia, hyperoxia or air exposure (Table 2.1).   2.4 Discussion  The goal of this study was to investigate whether preferential pHi regulation in white sturgeon is a general strategy of acid-base regulation, irrespective of the origin of the acid-base disturbance. Our results indicate that A. transmontanus preferentially regulate pHi against acidoses of respiratory origin [hyperoxia (this study) and hypercarbia (Baker et al., 2009a)]; however, during the metabolic acidosis treatments (exhaustive exercise, anoxia and air exposure), which created mixed metabolic/respiratory acidoses, preferential pHi regulation did not occur uniformly amongst the tissues. These results only partially support the hypothesis and indicate preferential pHi regulation of tissues may occur selectively amongst various acidoses; this differs from P. pardalis, where preferential pHi regulation was observed to be a general strategy of acid-base regulation (Brauner et al., 2004; Harter et al., 2014). Finally, this study demonstrates that responses to various acidoses may invoke different physiological responses; consequently, caution should be taken when extrapolating results amongst different types of acidoses.  2.4.1 White sturgeon preferentially regulate pHi during respiratory acidoses Preferential pHi regulation appears to be a general strategy of acid-base regulation during respiratory acidoses in white sturgeon. During hyperoxia, blood PCO2 increased  35 to approximately 1 kPa and reduced pHe by 0.15 units at 180 min; this was slightly less severe than previous studies using hypercarbia in A. transmontanus where exposure to 1.5 kPa PCO2 reduced pHe by 0.2 units at 6 h (Fig. 2.5a) (Baker et al., 2009a). The pHe reduction in Figure 5a is similar to other studies exposing different sized A. transmontanus to 1.5 kPa PCO2 for 3 h (Baker and Brauner, 2012; Baker et al., 2011; Shartau et al., 2017b), suggesting there is minimal pH difference between these time points and that fish size is unlikely to affect the magnitude of pH change. Similar to pHe, RBC pHi was reduced by 0.11 units, indicating that the hyperoxia-induced acidosis was sufficiently severe to reduce pH in a highly buffered tissue lacking capacity for pH regulation and is consistent with the response during hypercarbia (Brauner et al., 2004; Harter et al., 2014). Unlike the blood or RBC, pHi of heart, brain and liver did not change, indicating they were protected during hyperoxia (Fig. 2.3). The degree of pHi regulation appears to be less than during hypercarbia as heart, brain and liver exhibit an increase in pHi following 6 h exposure to 1.5 kPa PCO2 (Fig. 2.5b) (Baker et al., 2009a). Interestingly, muscle pHi was reduced during hyperoxia (Fig. 2.3) but not during hypercarbia (Fig. 2.5b). The reduction in muscle pHi may be associated with tissue anaerobiosis as suggested by the increase in plasma lactate during hyperoxia (Table 2.1). The reason for the increase in plasma lactate during hyperoxia is unknown but muscle lactate increased during 24 h exposure to 2 kPa PCO2 (Baker and Brauner, 2012). Few studies have measured pHe and pHi concurrently during hyperoxia. Hyperoxia induces an increase in blood PCO2 arising from the retention of metabolically produced CO2 of ~1 kPa PCO2. When Oncorhynchus mykiss (rainbow trout) were exposed to 72 h hyperoxia, Hobe et al. (1984) found that pHe, and white muscle and whole body pHi were reduced; similarly Wood and LeMoigne (1991) observed that pHe and pHi of brain and muscle were reduced. Not surprisingly, hypercarbia and hyperoxia induce a similar increase in internal blood PCO2, and also induce similar changes in pHe (Gilmour and Perry, 1994) and pHi (Shartau, unpublished observation) in rainbow trout. Respiratory acidoses induced by hyperoxia and hypercarbia appear to result in similar pH changes in both O. mykiss and A. transmontanus, which exhibit coupled pH regulation and preferential pHi regulation, respectively; as the acidoses have similar origins, they may affect pH similarly in these fishes.  36  2.4.2 Tissue pHi is differentially protected following metabolic acidoses Acipenser transmontanus do not uniformly protect pHi following the development of a metabolic acidosis induced by exhaustive exercise, anoxia or air exposure. As expected RBC pHi was reduced in all treatments, however, there were also reductions in liver and muscle pHi. In contrast, heart pHi remained unchanged in all treatments at 15 min post-exposure and brain pHi was protected following exhaustive exercise and air exposure (Fig. 2.4). The response of A. transmontanus to the metabolic acidoses indicates that their pattern of response differs from that of P. pardalis, which completely protect brain, heart and liver against exhaustive exercise and anoxia (Harter et al., 2014). Compared to respiratory acidoses, A. transmontanus exhibit greater variability with respect to pH regulation amongst metabolic acidoses; this may be due to differences in oxygen availability and demand during conditions leading to metabolic acidoses. Acid production during metabolic acidoses originates from the tissue due to increased anaerobic metabolism following reduced oxygen supply and/or increased oxygen demand. Consequently, anaerobic metabolism will be recruited to different degrees as oxygen supply and demand may change differentially amongst tissues; the observed pHi reduction is greatest in white muscle, while heart pHi remains well protected. Acipenser transmontanus tightly regulate heart pHi during metabolic acidoses (exhaustive exercise, anoxia and air exposure), to a degree similar to that accomplished during respiratory acidoses (hyperoxia and hypercarbia). Heart pHi may be more tightly regulated given a) the importance of the heart in O2 transport and b) that changes in pHi may lead to electrical disturbances disrupting cardiac function (Vaughan-Jones et al., 2009). Preferential regulation of heart pHi may thus be important to maintain metabolic activity and avoid issues related to cardiac function, particularly during acute acidoses. During hypercarbia (Baker et al., 2011; Hanson et al., 2009) and combined hypercarbia/hypoxia (Shartau et al., 2016b), cardiac performance is maintained along with protection of heart pHi. During exercise, heart pHi is maintained during exercise of Hemitripterus americanus (sea raven) (Milligan and Farrell, 1986) and Parophrys vetulus (lemon sole) (Wright et al., 1988). Interestingly, A. transmontanus exhibited a small reduction in heart pHi 120 min post-exercise; this reduction may have been due to the  37 redistribution of the acidosis from other tissues and/or the persistent elevation of plasma lactate which can influence pHi (Vaughan-Jones et al., 2009). Brain pHi was protected following exhaustive exercise and air exposure but not anoxia, where it was reduced by ~0.07 units (Fig. 2.4). Tight regulation of brain pHi likely ensures proper metabolic function when sufficient energy supplies exist; however, the brain is one of the most metabolically active tissues and highly sensitive to perturbation of energy supply (Soengas and Aldegunde, 2002). In low O2 conditions, anaerobic metabolism is insufficient to maintain brain ATP level; thus, in hypoxic/anoxic tolerant animals, the fall in ATP production is partially compensated for by anaerobic ATP production and supplemented by suppression of ATP use (metabolic depression) which together assist brain survival during hypoxia/anoxia (Hochachka, 1986; Nilsson, 2001; Soengas and Aldegunde, 2002). Consequently, reduction in brain pHi during anoxia may represent a survival mechanism, where in fact, maintaining pHi at normoxic levels would be maladaptive due to the energy required, possibly leading to reduced hypoxia/anoxia tolerance and survival. The difference in O2 availability for aerobic metabolism may explain in part why the response in anoxia differs from exercise and air exposure. As exercise requires increased white muscle activity, it would be expected to be the site of the greatest rate of anaerobiosis due to the higher energy demand required during exercise. This in turn could result in high rates of ATP hydrolysis, outpacing ATP production, and consequently creating an excess of protons (Robergs et al., 2004). Consequently, muscle tissue overall would be expected to exhibit the greatest reduction in pHi during exhaustive exercise. In this study, muscle pHi was reduced by 0.44 units following exhaustive exercise, the largest reduction in pHi of any tissue measured; it was also associated with a large increase in plasma lactate (Table 2.1). Reduced muscle pHi following anoxia and air exposure may have been a result of a more general reduction in O2 availability, prompting an increase in the rate of anaerobic metabolism despite reduced activity. The reduction in liver pHi across all metabolic acidoses could indicate that the liver is less capable of pHi regulation than other tissues, but this response may also be associated with handling of metabolic waste products (e.g. lactate) (Richards, 2011). For  38 example, in Carassius auratus (goldfish), both the liver and white muscle act as a store of glycogen to support whole animal metabolism during anaerobic metabolism (Jibb and Richards, 2008). Indeed, following exercise and anoxia, C. auratus reduce muscle pHi, possibly due to the accumulation metabolic wastes such as H+ and lactate arising from the depletion of glycogen stores (Mandic et al., 2008). During hypoxia, goldfish liver and muscle tissues have reduced pHi and increased lactate, with pHi reduction attributed to regulation of protein synthesis and metabolic reduction (Jibb and Richards, 2008). Reduction in liver and muscle pH are also observed in Squalus acanthias (dogfish) during recovery from severe hypoxia (Zimmer and Wood, 2014); these changes parallel those observed in A. transmontanus here (Fig. 2.4, Table 2.1).  2.4.3 Preferential pHi regulation may be a general strategy of acid-base regulation in A. transmontanus – but not all tissues are protected all the time Taken together, these finding illustrate that A. transmontanus have different responses to acid-base regulation depending on the source and tissue which the acidosis originates. During respiratory acidoses, pHi is preferentially regulated in critical tissues, but following metabolic acidoses induced by exhaustive exercise, anoxia and air exposure, only some tissues are protected (e.g. heart). This pattern differs from P. pardalis, which preferentially regulate pHi following respiratory (hypercarbia) (Brauner et al., 2004) and metabolic acidoses (exhaustive exercise, anoxia) (Harter et al., 2014); although hyperoxia and air exposure were not been examined in that study. The reason for differences in pHi regulation between these two species is unclear. However, it may be due to differences in species specific capacity for pHi regulation or it could indicate that acid-base strategies differ between environments (i.e. tropical versus temperate rivers) and that in general, tropical fishes, may have a greater capacity for pHi regulation to deal with differences of their environment; uncovering these differences is worthy of further investigation.  2.4.4 Conclusions Sturgeon demonstrate remarkable resilience to a variety of stressors including severe hypercarbia (Baker et al., 2009a), hyperoxia (Bagherzadeh Lakani et al., 2013;  39 Shartau et al., 2017b), hypoxia (Baker et al., 2005a; Crocker and Cech, 1997; Maxime et al., 1995), exercise (Baker et al., 2005b; Cocherell et al., 2011; Kieffer et al., 2001), aerial exposure (Brauner and Berenbrink, 2007), salinity (Allen et al., 2014; McEnroe and Cech, 1985; Mojazi Amiri et al., 2009; Shaughnessy et al., 2015) and fisheries related stressors (McLean et al., 2016). This study may provide insight into sturgeon acid-base regulation during these stressors, as even though the treatments imposed in this study were severe, they may occasionally be encountered naturally. For example, migrating A. transmontanus may be subjected to acute periods where they exercise to near exhaustion (Cocherell et al., 2011; Geist et al., 2005), and catch and release fishing causes them to exercise to exhaustion while enduring some degree of air exposure (angling events may last between 30 seconds to over 2 hours) (McLean et al., 2016). Additionally, sturgeon are often reared for aquaculture and in these settings, hyperoxia may be encountered and exceed >40 kPa PO2 (Bagherzadeh Lakani et al., 2013; Espmark and Baeverfjord, 2009), while hypoxia as low as 4 – 10 kPa PO2 may be experienced by Atlantic (Gunderson, 1998) and white (Crocker and Cech, 1997) sturgeon in estuaries. While tolerance and survival of these stressors requires a multifaceted physiological response, these results indicate that in A. transmontanus during the acidoses examined, the robust capacity for pHi regulation may contribute to their overall tolerance during these acid-base challenges. The molecular and cellular mechanism(s) that underlie preferential pHi regulation are unknown and currently under investigation (Shartau et al., 2017b). Further research into this area should provide insight into the differential response of pHi regulation during acidoses in sturgeon, and possibly inter-specific differences in acid-base regulatory strategy.   40  Figure 2.1: Effect of a hyperoxia-induced respiratory acidosis in Acipenser transmontanus white sturgeon on blood acid-base status. Blood pH (pHe) and plasma [HCO3-] are presented on a pH-HCO3- plot. A. transmontanus were exposed to 80 kPa PO2 for 180 min and sampled either prior to (control; ™ ) or at the end of the exposure (l). Values are presented as means ± s.e.m.; N=8. Significant differences (P<0.05) are indicated by different uppercase letters (pHe), lowercase letters (blood PCO2) and Greek letters (plasma HCO3-). Dashed line indicates the blood non-bicarbonate buffer line (-11.9 mM HCO3- pH unit-1) for A. transmontanus as determined by Baker et al. (2009a).  41    Figure 2.2: Effect of metabolic acidoses in Acipenser transmontanus (white sturgeon) on blood acid-base status. Blood pH (pHe) and plasma [HCO3-] are presented on a pH-HCO3- plot. White sturgeon were subjected to either exhaustive exercise (a), anoxia (5 min exposure; b) or air exposure (45 min; c). Fish were sampled either prior to exposure (control; ™), or following exposure after a 15 (l) or 120 minutes (n; except air exposure which was only sampled at 15 min) recovery period. Values are presented as means ± s.e.m.; N=8. Significant differences (P<0.05) are indicated by different uppercase letters (pHe), lowercase letters (blood PCO2) and Greek letters (plasma HCO3-). Dashed line indicates the blood non-bicarbonate buffer line (-11.9 mM HCO3- pH unit-1) for white sturgeon as determined by Baker et al. (2009a).  42  Figure 2.3: Effect of a hyperoxia-induced respiratory acidosis in Acipenser transmontanus (white sturgeon) on intracellular pH (pHi) of red blood cells (RBC), heart, liver, brain and white muscle (WM). Acipenser transmontanus were exposed to 80 kPa PO2 for 180 min and sampled either prior to (control; open bar) or at the end of the exposure (shaded bar). Values are presented at means ± s.e.m. Asterisk indicate significant differences from control (P<0.05).  43   Figure 2.4: Effect of metabolic acidoses in Acipenser transmontanus (white sturgeon) on intracellular pH (pHi) of red blood cells (RBC), heart, liver, brain and white muscle (WM). Acipenser transmontanus were subjected to either exhaustive exercise (a), anoxia (5 min exposure; b) or air exposure (45 min; c). Fish were sampled either prior to exposure (control; open bar), or following exposure after a 15 (shaded bar) or 120 minutes (closed bar; except air exposure) recovery period. Values are presented at means ± s.e.m. Significant differences are indicated by different letters, except for air exposure where an asterisk denotes difference from control (P<0.05).  44  Figure 2.5: Effect of a hypercarbic-induced respiratory acidosis in Acipenser transmontanus (white sturgeon) on blood and tissue acid-base status following a 6 h exposure to 1.5 kPa PCO2. Blood pH (pHe) and plasma [HCO3-] are presented on a pH-HCO3- plot; fish were sampled either prior to (control; ™) or at the end of the exposure (l) (a). Dashed line indicates the blood non-bicarbonate buffer line (-11.9 mM HCO3- pH unit-1) for A. transmontanus as determined by Baker et al. (2009a). Intracellular pH (pHi) of red blood cells (RBC), heart, liver, brain and white muscle (WM) when sampled either prior to (control; open bar) or at the end of the exposure (shaded bar) (b). Data is re-plotted from Baker et al. (2009a). Significant differences (P<0.05) for pHe, plasma HCO3- and pHi, as determined by Baker et al. (2009a), are indicated by uppercase letters, Greek letters and asterisks, respectively.   45 Table 2.1: Effect of various treatments inducing an acidosis on hematocrit, plasma [Cl-] and [lactate] in Acipenser transmontanus (white sturgeon). Treatment Post-exposure time (min) Hematocrit (%) Plasma [Cl-] (mM) Plasma [lactate] (mM) Control  27±0.5 125±4 2.9±0.4 Hyperoxia 0 29±1* 129±1 4.6±0.5* Exercise 15 35±1* 140±5* 6.2±0.5*  120 33±1* 130±1 6.2±0.5* Anoxia 15 29±1 133±5 4.1±0.2*  120 29±1 125±2 3.8±0.3 Air exposure 15 26±2 129±3 5.0±0.3* Values are indicated as means ± s.e.m., N=8; significant differences from control are indicated by an asterisk (P<0.05).   46 Chapter 3: Preferential Intracellular pH Regulation May Represent a Common Strategy of Acid-Base Regulation Amongst CO2 Tolerant Fishes   3.1 Introduction  Large transient increases in CO2 (hypercarbia) are common in many aquatic environments and pose challenges for acid-base regulation in fishes (Brauner and Baker, 2009; Hasler et al., 2016; McNeil and Sasse, 2016; Shartau and Brauner, 2014). When subjected to acute hypercarbia, fishes will experience an increase in blood PCO2 as CO2 diffuses and equilibrates across the gills, which leads to a reduction in blood and extracellular pH (pHe), referred to as a respiratory acidosis. The most common response observed in fishes [e.g. Eptatretus stoutii (Pacific hagfish) (Baker et al., 2015), Scyliorhinus stellaris (dogfish) (Heisler et al., 1988), Gadus morhua (Atlantic cod) (Larsen et al., 1997), Oncorhynchus mykiss (rainbow trout) (Hobe et al., 1984), Conger conger (conger eel) (Toews et al., 1983)] is that pHe is compensated by a net increase in plasma [HCO3-] in exchange for Cl-, with the gills playing the primary role in compensation (Brauner and Baker, 2009). Depending on the severity of the acidoses and the ionic composition of the water (Larsen and Jensen, 1997), complete pHe compensation typically occurs within 24-72 h. Putative limits on the elevation in plasma [HCO3-] appear to prevent complete pHe compensation during exposure to acute PCO2 >2 kPa (Baker et al., 2015; Brauner and Baker, 2009; Heisler, 1984). Changes in pHe are often associated with qualitatively similar changes in intracellular pH (pHi) as the two are typically coupled, referred to as ‘coupled pH regulation’; thus, recovery of pHe is important for pHi recovery (Shartau et al., 2016a). Failure to maintain acid-base homeostasis will negatively impact fitness in environments subject to severe acute hypercarbia as deviations from normal physiological pH values can affect molecular charge, altering the structure and function of biological macromolecules, and, ultimately, reducing whole-animal performance (e.g. reduce heart and skeletal muscle contractility,  47 alter metabolic pathways, and disrupt cellular signalling and processes such as volume regulation) (Boron, 2004; Occhipinti and Boron, 2015; Putnam and Roos, 1997). Many of the world’s freshwater fishes inhabit hypercarbia-prone environments. Globally, the average PCO2 of stream and river systems is ~0.3 kPa (ca. 3100 µatm), about 8-fold above current atmospheric levels (Raymond et al., 2013); some tropical freshwater lakes and rivers may experience a range of CO2 tensions up to ~2 kPa (20,249 µatm) (Cole et al., 1994) and ~5.5 kPa (54,270 µatm) (de Fátima F L Rasera et al., 2013), respectively. In many aquatic systems, there may be localized point sources for high PCO2 which may further increase PCO2 due to vegetative covering and respiration of aquatic life (Hasler et al., 2016); in the Amazon River basin PCO2 may reach 8 kPa (Furch and Junk, 1997; Heisler, 1984) and the presence of CO2 vents could produce PCO2 >50 kPa (Sorey et al., 2000). Within manmade aquatic systems, PCO2 can easily rise beyond 2-4 kPa PCO2 within recirculating aquaculture systems and aquaculture ponds (Crocker and Cech, 1996; Damsgaard et al., 2015). Additionally, modifications to river systems via creation of dams have the potential to create environments prone to experiencing hypercarbia (de Faria et al., 2015). There is also interest in controlling movement of invasive fish species using CO2 as a barrier by increasing regional PCO2 levels to as high as ~5-11 kPa (100-200 mg/L) (Dennis et al., 2016; Kates et al., 2012; Tierney, 2016). In the marine environment, there are natural CO2 seeps at some locations which create localized hypercarbia (Basso et al., 2015; Melzner et al., 2009), thus affecting marine life (Brauner and Baker, 2009; Hawkins, 2004; Lackner, 2003). There is a great variation in water PCO2 worldwide that species have adapted to, or have to deal with due to anthropogenic influences, and often PCO2 exceeds the putative limits for pHe regulation. However, relatively little is known about how fish tolerate and compensate for these high CO2 levels. Some groups of fishes may be especially well adapted to severe hypercarbia as evident in tropical freshwater environments which have a high diversity of species. Tropical environments contain 56% of watersheds with high fish species diversity (Val et al., 2005), and this is exemplified by the species rich Rio Negro, an acidic ion-poor Amazon River tributary, that contains over 1000 species (Gonzalez et al., 2017). A number of basal euteleostom fishes also reside in hypercarbic-prone habitats, with the  48 majority being bimodal breathers, which may increase the likelihood of experiencing a respiratory acidosis (Shartau and Brauner, 2014). This suggests many fishes are well adapted to responding to hypercarbia; indeed, Regan et al. (2016) observed that brain function in Pangasianodon hypophthalmus (striped catfish), a tropical bimodal breather, is adapted for the hypercarbic waters of the Mekong River and is disrupted during exposure to normocapnia. Coupled pHe/pHi regulation may be constrained in high CO2 environments, yet little is known about how fishes tolerate and survive severe acute hypercarbia. In the few fishes studied to date that tolerate hypercarbia well above 2 kPa PCO2, it appears tolerance is associated with the ability to completely regulate pHi of heart, brain, liver and muscle, despite a large, often uncompensated, reduction in pHe (preferential pHi regulation) (Shartau et al., 2016a). Preferential pHi regulation has been demonstrated in Synbranchus marmoratus (marbled swamp eel) (Heisler, 1982), Pterygoplichthys pardalis (armoured catfish) (Brauner et al., 2004) and Acipenser transmontanus (white sturgeon) (Baker et al., 2009a) exposed to PCO2 ranging from 3-6 kPa. This strategy of acid-base regulation, at least in A. transmontanus, appears to provide near instantaneous pHi regulation [(Baker, 2010); reviewed in (Shartau et al., 2016a)] and does not exert a whole animal metabolic cost (Baker and Brauner, 2012). In P. pardalis, preferential pHi regulation is a general strategy of pH regulation, used during both respiratory and metabolic acid-base challenges (Harter et al., 2014); however, in A. transmontanus, pH protection may be tissue specific during metabolic acidoses (Shartau et al., 2017a). It has been previously hypothesized that preferential pHi regulation confers exceptional CO2 tolerance in fishes, as it may be the strategy by which they are able to cope with a severe acute respiratory acidosis. Protection of tissue pH during acidoses is important as large pHi reduction in O. mykiss muscle following exhaustive exercise is believed to be responsible for increased mortality following exercise (Wood et al., 1983). Lower heart pHi is associated with reduced heart contractibility, and thus, cardiac performance may be reduced leading to diminished O2 delivery (Vaughan-Jones et al., 2009). A reduction in brain pHi due to hypercarbia has an anesthetic affect, causing a loss of equilibrium which may lead to mortality (Yoshikawa et al., 1994). By preferentially regulating pHi during a hypercarbic-induced respiratory acidosis, the above fishes may  49 avoid these damaging affects and thus, inhabit and/or travel through environments experiencing short-term severe hypercarbia. As few fish species have been shown to preferentially regulate pHi (Shartau et al., 2016a), it remains unclear if this strategy is widely used, or is confined to a select few basal actinopterygian and air breathing fishes (Shartau and Brauner, 2014); that few species are known to use this pattern of acid-base regulation is likely a result of few studies exposing fishes to severe hypercarbia while simultaneously measuring pHe and pHi. We hypothesize that CO2 tolerant fishes utilize preferential pHi regulation and that fishes from a number of different families and orders use this strategy of acid-base regulation. The main objective of this study was to conduct a survey of preferential pHi regulation and CO2 tolerance in a group of phylogenically diverse fishes that included 20 fishes originating from three continents (North America, South America and Africa), representing 11 orders, and range from basal vertebrates (e.g. lamprey) to derived actinopterygians (e.g. tilapia). We first devised a CO2 tolerance assay to assess acute CO2 tolerance (Series I). Next, acid-base response of various fish species were examined using terminal pHe/pHi sampling following severe acute hypercarbia ranging from 1.5-6 kPa PCO2, depending on their CO2 tolerance (Series II). Finally, Series III used the CO2 tolerance assay to indirectly determine preferential pHi regulation in a number of other fish species to gain a broader understanding of the phylogenetic distribution of preferential pHi regulation. Together, these objectives provide the most comprehensive examination of acid-base regulation during acute hypercarbia in fishes conducted to date.   3.2 Methodology  3.2.1 Animal acquisition and holding In this study, 20 species of fish were used and experiments were conducted as follows. From September 2012 to November 2013 measurements were made on the following species at the University of British Columbia, Vancouver, BC, Canada): Oncorhynchus mykiss (rainbow trout) (250-400g) from Miracle Springs Inc. (Mission, BC, Canada); Entosphenus tridentatus (Pacific lamprey) (~200g) caught Fall 2013 on the  50 Nechako river near Prince George, BC, Canada; Oncorhynchus kisutch (coho salmon) (~100g) UBC aquaculture facility (Vancouver, BC, Canada); Oreochromis niloticus X mossambicus X hornorum (tilapia hybrid) (~300-400g) from Redfish Ranch (Courtenay, BC, Canada). Experiments with Acipenser transmontanus (white sturgeon) (~200g) were conducted at the International Centre for Sturgeon Studies, Vancouver Island University, Nanaimo, BC, Canada using fish reared in their facility. Experiments were conducted at the Instituto Nacional de Pesquisas da Amazônia/INPA (Manaus, AM, Brazil) with fish caught in the Rio Negro near Manaus and transferred to a holding facility at INPA in 2008: Hoplosternum littorale (tamoata), Brycon amazonicus (matrinxa), Colossoma macropomum (tambaqui) and Astronotus ocellatus (oscar). In 2013 the following species were caught from the wild and investigated at INPA: Potamotrygon sp. (freshwater ray) (~50-80g), Lepidosiren paradoxa (South American lungfish) ~ 300-1500g, Synbranchus marmoratus (marbled swamp eel) ~ 50-150g, Electrophorus electricus (electric eel). The following fish were obtained from local fish farms and transferred to facilities at INPA, Arapaima gigas ~ 75-100g, C. macropomum, Astronotus ocellatus (oscar), and Pterygoplichthys pardalis (armoured catfish) ~100g. Experiments with the following farm reared species were conducted at the South Farm Aquaculture research facility at Mississippi State University (Starkville, MS, USA) in March 2013: Polyodon spathula (American paddlefish) ~ 150-400g, Atractosteus spatula (alligator gar) ~ 400-1000g, Ictalurus punctatus (channel catfish) ~ 100 – 200g, Ictalurus punctatus X I. furcatus (channel X blue catfish) ~ 100 – 200g. Experiments with the following species were conducted at the University of North Texas (Denton, TX, USA) with fish caught from nearby lakes/rivers in November 2012: Lepisosteus oculatus (spotted gar) ~ 300-700g, and I. punctatus ~ 50-200g. Typically, animals were kept for at least 2 weeks in appropriate tanks under standard conditions of food, temperature and natural photoperiod before experiments; however, some fish were wild caught and only held for 72 h due to constraints on animal housing. Fish were not fed at least 48 h before experiments.   51 3.2.2 Series I: CO2 tolerance assay  To determine the optimal rate of CO2 increase for assessing CO2 tolerance, fish species that are known to use different acid-base regulatory strategies in response to hypercarbia were used, O. mykiss (coupled pH regulation) and A. transmontanus (preferential pHi regulation) (Shartau et al., 2016a). Fish were randomly selected from the holding tank and placed in individual black plexi-glass boxes (24 L) with aeration in a re-circulating system (flow rate ~3 L min-1 per box, 15 oC; total water volume of system ~320 L) overnight prior to experiments. Fish were then exposed to progressively increasing levels of hypercarbia at a rate of 1, 2 or 4 kPa PCO2 h-1. Water PCO2 was monitored to ensure PCO2 increased at the desired rate for the duration of exposure using a thermostated (15 oC) Radiometer PCO2 electrode (E5036) (output, Radiometer PHM 73). Fish were continuously observed for loss of equilibrium (LOE), which was used as the end point to indicate their CO2 tolerance. LOE was defined as the inability to maintain dorsoventral orientation. Once LOE was reached, fish were immediately removed from the box and placed in a normocarbic, normoxic recovery tank; fish were monitored for at least 48 h after hypercarbia exposure and there were no mortalities.   3.2.3 Series II: Strategy of acid-base balance during severe acute hypercarbia  The strategy of acid-base regulation used during severe acute hypercarbia in fishes was determined by first subjecting them to the CO2 tolerance assay at a rate of 2 kPa PCO2 h-1, as was previously determined to be a suitable rate in Series I. The assay was targeted to ensure fish would tolerate one of three desired PCO2 test exposures (1.5, 3 or 6 kPa). The highest CO2 tension fish could tolerate was used in order to observe pHi during maximal pHe depression; as pHi only changes by approximately 1/3 of pHe in coupled pH regulators, (due to a lower starting pHi value and greater tissue buffer value) larger reductions in pHe allow for a more accurate determination of whether fishes preferentially regulate pHi. Once the we determined the CO2 tension that fish species of interest could tolerate, we performed the following experiment to assess whether they utilize either coupled pH regulation or preferential pHi regulation.  Fish were acclimated individually for 24 h in black plexi-glass boxes in the system described above; this period is sufficient to allow recovery from handling stress in  52 sturgeon (Baker et al., 2005b; Barton et al., 2000). Normocarbic fish were terminally sampled immediately (see below) following this acclimation period (control group). Other fish were then exposed to 3 h hypercarbia at either 1.5, 3 or 6 kPa PCO2, depending on the fish species. We also examined the response of an extremely hypercarbia tolerant species (based on series I), C. macropomum, to more severe hypercarbia of 20 kPa PCO2 as we were interested if CO2 tolerance at this high CO2 tensions is associated with preferential pHi regulation as is the case at lower, but still severe hypercarbia levels. The 3 h time point was chosen to sample fish in Series II because at this time pHi is typically maximally reduced and pHi compensation is more rapid than pHe compensation. Furthermore, this provides sufficient time for CO2 to increase in all tissues resulting in pHi depression if fish are coupled pH regulators or no change in pHi if they are preferential pHi regulators (Shartau et al., 2016a). Hypercarbia was achieved by bubbling a mixing tank with preset rates of air and 100% CO2 using Sierra Instruments mass flow controllers. Water PCO2 was measured with a PCO2 electrode to confirm target CO2 tensions; water O2 levels remained >80% saturation. At the time of sampling, each box was isolated from the recirculation system and anesthetic was added to the water (MS-222 0.3 g/L buffered with NaHCO3) while hypercarbic gas bubbling was maintained to minimize changes in blood PCO2 and avoid hypoxemia due to reduced ventilation. Once ventilation ceased (<3 min), each fish was turned ventral side up, while gills remained submerged in aerated water and blood (~2-3 mL) was drawn from the caudal vein into a lithium-heparin (1 g L-1) rinsed syringe (3 mL syringe, 23 G1¼ needle) and placed on ice. Following this procedure, fish were killed via cephalic concussion and cervical dislocation and tissues (0.5-1.0 g) were removed within 2-3 min, wrapped in aluminum foil and immediately flash frozen in liquid N2. Tissues were sampled in the following order: heart (gently squeezed and patted dry to remove any excess blood), liver, dorsal white muscle (left side, just posterior of the dorsal fin; skin and red muscle removed), and brain; tissues were stored longer term at -80 oC. Blood was divided into two aliquots. Blood pH and hematocrit (Hct) were measured from one aliquot; the other aliquot was centrifuged (3 min at 10,000 rpm) and plasma was removed for measurement of total CO2 (TCO2) and [Cl-].  53 Blood pH was measured using a Radiometer PHM 84 (Copenhagen, Denmark) connected to a thermostated Radiometer Analytical SAS pH electrode (GK2401C, Cedex, France). RBC pHi was measured using the freeze-thaw method as described by Zeidler and Kim (Zeidler and Kim, 1977). Tissue pHi was measured using the metabolic inhibitor tissue homogenate method (MITH; see Appendix for detailed description of this method) (Portner et al., 1990) and validated for use in fish by Baker et al. (2009b). Plasma TCO2 was measured using a total CO2 analyzer (Corning model 965 Analyzer); the remaining plasma was used to measure [Cl-] ions (HBI model 4425000; digital chloridometer). Plasma [HCO3-] and PCO2 were calculated using TCO2 and pH values described by Brauner et al. (2004). CO2 solubility coefficient and the logarithmic acid dissociation constant (pK’) for plasma were determined from Boutilier et al. (1984).  3.2.4 Series III: CO2 tolerance to infer pattern of pH regulation To conduct a more rapid and non-lethal assessment of pH regulation in fishes, we used the CO2 tolerance assay to determine CO2 tolerance in various species, as determined by point of LOE. Fish were placed individually in boxes, allowed to acclimate 24 h and then exposed to a target rate of increase of 2 kPa PCO2 h-1 until LOE was reached or in the case of E. tridentatus, Potamotrygon sp., P. pardalis and S. marmoratus when these fishes became unresponsive to gentle prodding with a plastic stick. As in Series I, once LOE was reached, fish were removed and allowed to recover. Fishes reaching LOE >8 kPa PCO2 are considered to be preferential pHi regulators as O. mykiss did not tolerate PCO2 beyond this tension at any other rates of increase (Fig. 3.1), and thus suggests fish tolerant to CO2 tensions greater than this are preferential pHi regulators.  3.2.5 Calculations and statistical analysis  All values are expressed as means ± s.e.m. throughout. Data were compared by Welch’s t-test or where multiple treatments were evaluated, data were analyzed by analysis of variance (ANOVA), followed by Tukey’s post hoc test. If the data did not meet the assumptions of normality (Shapiro-Wilk normality test) or equal variance (Bartlett’s test), a Kruskal-Walis test followed by Dunn’s multiple comparison test was  54 used (P<0.05). GraphPad Prism (v.5) was used for statistical analyses and preparation of figures.   3.3 Results  3.3.1 Series I: Development of a CO2 tolerance assay To determine a rate of CO2 increase to assess acute CO2 tolerance, O. mykiss and A. transmontanus were exposed to progressively increasing levels of hypercarbia at a rate of 1, 2 or 4 kPa PCO2 h-1. The mean PCO2 at which LOE occurred in rainbow trout was 5.5 ± 0.3, 4.8 ± 0.3 and 2.7 ± 0.2 kPa PCO2 at rates of 1, 2 and 4 kPa PCO2 h-1, respectively, while that in A. transmontanus was 22.1 ± 2.2, 14.6 ± 2.3 and 6.3 ± 1.8 kPa PCO2, respectively (Fig. 3.1). Within species, the PCO2 LOE at 4 kPa PCO2 h-1 was lower relative to the other rates (P<0.01); there was no difference between 1 and 2 kPa PCO2 h-1. Comparison between species at the different rates of PCO2 increase indicated that A. transmontanus had a higher PCO2 LOE at 1 and 2, but not 4 kPa PCO2 h-1 (P<0.01). No mortalities occurred in the 72 h following LOE in fish allowed to recover in normocarbia.  3.3.2 Series II: Survey of pHi regulation Acute hypercarbia exposure to 1.5 kPa PCO2 in P. spathula, 4 kPa PCO2 in H. littorale, B. amazonicus, C. macropomum and A. ocellatus, and 6 kPa PCO2 in L. oculatus, A. spatula, I. punctatus, and Oreochromis sp. reduced both pHe and RBC pHi as expected; the sole exception was A. ocellatus RBC where there was limited sample size (n=2). In contrast, pHi increased in the heart of L. oculatus, H. littorale, B. amazonicus, C. macropomum and A. ocellatus (P<0.05), A. spatula liver, I. punctatus brain and Oreochromis sp. white muscle (Fig. 3.2); no other statistically significant changes were observed in other tissues. Similarly, exposure of C. macropomum to 20 kPa PCO2 severely reduced pHe from 7.729 ± 0.03 to 6.896 ± 0.024 pH units. Red blood cell (RBC) and white muscle pHi was reduced but there are no statistically significant changes in  55 heart, liver or brain pHi (Fig. 3.2G). Results in all of these fish species are consistent with the capacity for preferential pHi regulation (Shartau et al., 2016a). Exposure of O. mykiss to 3 kPa PCO2 also resulted in the expected reduction in pHe and RBC pHi. In line with those changes, pHi was reduced in heart, liver, brain, and white muscle (Fig. 3.2F); these results are in agreement with previous studies and consistent with use of coupled pH regulation (Shartau et al., 2016a). Where measured, there were no significant changes in plasma Cl-, or osmolarity in any fishes. Only O. mykiss experienced an increase in hematocrit during exposure to 1.5 and 3 kPa PCO2 while P. spathula exhibited a reduction (Table 3.1).  3.3.3 Series III: CO2 tolerance Using the Series I CO2 tolerance assay at a rate of 2 kPa PCO2 h-1, the mean PCO2 at which LOE occurred were determined for the following species: E. tridentatus - 14.6 ± 0.4 kPa, Potamotrygon sp. 11.1 ± 0.2 kPa, P. spathula 2.7 ± 0.5 kPa, A. transmontanus 14.5 ± 2.3 kPa, A. gigas 24.4 ± 1.0 kPa, I. punctatus 8.4 ± 0.1 kPa, I. punctatus X I. furcatus 7.4 ± 0.2 kPa, P. pardalis 14.0 ± 0.9 kPa, O. mykiss 4.8 ± 0.3 kPa, O. kisutch 5.5 ± 0.7 kPa, A. ocellatus 13.9 ± 0.6 kPa, and Oreochromis sp.12.6 ± 0.5 kPa (Table 3.2). Several fish species were highly CO2 tolerant and their tolerance exceeded our ability to measure CO2 which was limited to 26.7 kPa PCO2 (200 torr PCO2); these fishes did not reach LOE at a rate of 2 kPa PCO2 h-1: S. marmoratus, L. paradoxa, C. macropomum, E. electricus (n=2). Immediately following LOE, fish were transferred to normocarbic waters and no mortalities were observed in the subsequent 48 h of recovery.   3.4 Discussion  Our objective was to investigate the prevalence of preferential pHi regulation in phylogenically diverse fishes to understand how they maintain acid-base homeostasis during severe acute hypercarbia. We show that preferential pHi regulation is used by fishes tolerant of severe acute hypercarbia, and that it is present in species from numerous phylogenetic orders; thus, likely representing a general strategy of acid-base regulation  56 amongst fishes (Fig. 3.3). These results support our hypothesis that CO2 tolerant fishes use preferential pHi regulation. However, we also show that preferential pHi regulation is not sufficient to confer CO2 tolerance (Fig. 3.3; Table 3.2) as was hypothesized by Brauner and Baker (2009). This study demonstrates that preferential pHi regulation may be an important and widespread trait allowing fishes to tolerate, and thus survive hypercarbia in diverse aquatic environments.  3.4.1 Use of CO2 assay for tolerance to acute hypercarbia Previous studies examining CO2 tolerance have generally exposed fishes to a certain PCO2 and recorded the time at which behaviour changes or LOE is achieved (Hasler et al., 2017; Hayashi et al., 2004; Kates et al., 2012); consequently, these methodologies do not provide an estimate as to the maximal acute PCO2 fish can tolerate, nor do they suggest an appropriate rate of CO2 increase to investigate hypercarbia tolerance. This study shows that 1 and 2 kPa PCO2 h-1 may provide the best estimate of acute CO2 tolerance as there was no difference in the PCO2 at which LOE were reached, whereas at 4 kPa PCO2 h-1, the LOE PCO2 was significantly lower (Fig. 3.1). It is uncertain why the faster rate of PCO2 increase resulted in a lower LOE PCO2 but could be a consequence of the rapid CO2 induced acidification outpacing the cellular defenses to mitigate the acidosis. Additionally, the lack of difference between O. mykiss and A. transmontanus LOE PCO2 at 4 kPa PCO2 h-1 prevents differentiating between strategies of acid-base regulation. Where multiple runs of this assay were conducted using different individuals (L. oculatus, P. spathula, I. punctatus; limited fish numbers precluded us from repeating the CO2 tolerance assay in all species), we observed that the PCO2 at which LOE occurs is consistent (Table 1). Recently, Hasler et al. (2017) demonstrated that hypercarbia tolerance is a repeatable, and likely a heritable, trait within individuals of Micropterus salmoides (largemouth bass). Using this CO2 tolerance assay as an indicator of acute tolerance may not accurately reflect natural environmental exposures, nor is it likely to indicate the maximal capacity of fishes to compensate for gradual, chronically induced hypercarbia. However, the rapid acute CO2 exposure in this assay does provide an approximation of fishes’ ability to protect critical tissues against rapid acidification, which is thought to be the  57 cause of LOE (Yoshikawa et al., 1994), and we propose indicates the presence of preferential pHi regulation which in the sturgeon heart has been shown to be virtually instantaneous during CO2 exposure [Baker, 2010, reviewed in (Shartau et al., 2016a)]. In fishes where pHi was measured along with CO2 tolerance, it was observed that in those that were more tolerant of hypercarbia than O. mykiss (which use coupled pH regulation), are also preferential pHi regulators. In addition to A. transmontanus, A. spatula, L. oculatus, I. punctatus, P. pardalis, A. ocellatus, Oreochromis sp., S. marmoratus, C. macropomum are CO2 tolerant as indicated by the CO2 tolerance assay developed here and direct pH measurements have confirmed their ability for preferential pHi regulation during severe acute hypercarbia. Therefore, CO2 tolerance, as demonstrated in this assay, may provide an assessment regarding the capacity for fishes to protect pHi, and thus, use preferential pHi regulation. The association between high CO2 tolerance and preferential pHi regulation was corroborated with pH measurements in a number of species; however, the relationship between low CO2 tolerance and coupled pH regulation only occurred in O. mykiss, and thus should be investigated more thoroughly among other coupled pH regulators.  3.4.2 Acid-base regulation during hypercarbia Studies measuring acid-base status in fishes exposed to acute hypercarbia have typically observed concurrent pHe and pHi reductions (Brauner and Baker, 2009; Shartau et al., 2016a). However, most of these studies have investigated fish exposed <2 kPa PCO2, and those subjecting fishes to more severe hypercarbia have not typically investigated how they maintain acid-base homeostasis (Shartau et al., 2016a). Fishes dependent on coupled pHe/pHi regulation appear to be limited to compensating pHe at PCO2 <2 kPa due to putative limits on plasma HCO3- [the so-called “HCO3- concentration threshold” (Heisler, 1984)], which may be associated with preventing hypochloremia, as pHe compensation is associated with a net increase in plasma HCO3- in equimolar exchange for plasma Cl- (Baker et al., 2015; Brauner and Baker, 2009; Heisler, 1984). As many fishes inhabit environments where CO2 may greatly exceed 2 kPa PCO2 and likely experience large, frequent oscillations in CO2 (Furch and Junk, 1997; Gonzalez et al., 2017; Heisler, 1984; Val et al., 2005), the use of coupled pH regulation is likely  58 insufficient for survival in these habitats, particularly in species rich regions such as the Amazon and Mekong river basins. In contrast preferential pHi regulation appears to offer an advantageous strategy of acid-base regulation during acute CO2 exposure, allowing fishes to protect pHi against at least PCO2 >15 kPa (Fig. 3.2G) in at least one species, which is quite remarkable. Survival during severe acute acidoses may depend on protecting pHi, not pHe as mortality in marine fishes following exposure to severe acute hypercarbia is believed to be due reduced heart pHi (Hayashi et al., 2004). In the latter, pHi was not measured, but it was suggested O2 supply was impaired as cardiac output dropped due to reduced cardiac contractility stemming from reduced cardiac pH (Vandenberg et al., 1994). Similarly, reduced brain pH may be responsible for the anesthetic effect in common carp Cyprinus carpio causing them to lose equilibrium (Yoshikawa et al., 1994). During an exercise-induced metabolic acidosis, reduced muscle pH was hypothesized to be the cause of post-exercise mortality in O. mykiss (Wood et al., 1983). The capacity for pHi regulation in those fishes may be insufficient to protect against acidoses, thus leading to deleterious changes in pHi that ultimately affect whole animal performance (see introduction). Irrespective of their ability to compensate pHe, the cellular dysfunction accompanied by pHi reduction renders these fishes sensitive to severe acid-base challenges. In contrast, species maintaining pHi during these acid-base challenges, particularly in critical tissues such as the heart, appear to be resilient to a range of respiratory and metabolic acidoses. For example, P. pardalis (Harter et al., 2014) and A. transmontanus (Shartau et al., 2017a) tolerate a range of acidoses which may be largely due to their capacity for preferential pHi regulation. Tolerance of severe acute hypercarbia in this study is likely due to the exceptional capacity for pHi regulation and is best exemplified in this study by pHi protection of heart and brain in C. macropomum during exposure to 15 kPa PCO2 (Fig. 3.2G).  3.4.3 Preferential pHi regulation in fishes Measurements of pHe and pHi in Series II reveals several species preferentially regulate pHi during acute severe hypercarbia and the CO2 assay suggests several other species also may have this ability. Use of the CO2 tolerance assay without pH  59 measurements may not consistently infer the strategy of acid-base regulation if fish are sensitive to CO2 as preferential pHi regulation alone does not appear sufficient to confer CO2 tolerance; this is demonstrated in P. spathula which fully protected pHi despite having a relatively low CO2 tolerance (2.7±0.5 kPa PCO2) (Fig. 3.2A; Table 3.2). The basis for this low tolerance in P. spathula is uncertain, although it may be due, in part, to their high P50 and high MO2 (Aboagye and Allen, 2014; Aboagye and Allen, 2017); thus, reductions in Hb-O2 affinity due to Bohr/Root effects may hinder O2 uptake and lead paddlefish to experience hypoxemia despite complete water O2 saturation. A greater degree of certainty is possible regarding the strategy of pH regulation when fishes are tolerant to high CO2 as the [HCO3-] threshold putatively confers a physiological limit to pHe regulation; consequently, fishes more tolerant than O. mykiss, where this limit has been demonstrated numerous times, are most likely to use preferential pHi regulation. The association between high CO2 tolerance and preferential pHi regulation is reinforced by the now numerous examples of highly CO2 tolerant fishes using preferential pHi regulation. Use of preferential pHi regulation is made all the more likely by the putative ionoregulatory disturbances that would occur if pHe was compensated during severe hypercarbia. For example, complete pHe compensation in C. macropomum with blood PCO2 of 15 kPa would require a plasma [HCO3-] of ca. 250 mM; thus, complete pHe compensation is not possible due to the ensuing changes in plasma osmolarity and ion balance that would occur. Even if compensation was desirable, typical freshwater teleost osmolarity is approximately 262-274 mM and plasma [Cl-] is 125-132 mM (Table 3.1) (Edwards and Marshall, 2013); consequently, there is insufficient Cl- to exchange for HCO3-, and even partial compensation would require a near total change in plasma ionic composition. Use of preferential pHi regulation does not preclude pHe compensation during hypercarbia exposure in all fishes as H. littorale, B. Amazonicus, C. macropomum, and A. ocellatus, all exhibited complete or partial pHe compensation following 24 h hypercarbia exposure (data not shown). This is different than the response observed in P. pardalis which do not compensate pHe following 96 h exposure to 4 kPa PCO2 (Brauner et al., 2004), yet is similar to the P. hypophthalmus in the Mekong which compensate pHe by 48 h at ca. 4 kPa PCO2 (Damsgaard et al., 2015) while preferentially regulating pHi (R.B.S., M. Sackville, C. Damsgaard, L.M. Phuong,  60 M. Hvas, T. Wang, M. Bayley, D.T.T. Huong, N.T. Phuong, and C.J.B., unpublished observations). Compensation of pHe by these fishes generally conforms to the limits of the putative [HCO3-] threshold and this may indicate a preference to preserve whole animal acid-base homeostasis when possible. Previously, preferential pHi regulation had only been identified in three fishes (Baker et al., 2009a; Brauner et al., 2004; Heisler, 1982); we now show, via direct and indirect measurements, that another 15 species use preferential pHi regulation during severe acute hypercarbia (Fig. 3.3). That preferential pHi regulation is a rare pattern of acid-base regulation among vertebrates (Brauner et al., 2004) is unlikely, but rather it may be an ubiquitous strategy given the putative limits to pHe regulation and the species richness in hypercarbic habitats. When previous studies (Baker et al., 2009a; Brauner et al., 2004; Heisler, 1982), unpublished observations [Amia calva, reviewed in (Brauner and Baker, 2009) and P. hypophthalmus, reviewed in (Shartau et al., 2016a)] and this study, are considered, there are 16 fish species likely to use preferential pHi regulation, representing 9 euteleostomi fish orders, as well as an elasmobranch and agnathans (Fig. 3.3); suggesting preferential pHi regulation is both a widely distributed and widely used strategy of acid-base regulation.  3.4.4 Preferential pHi regulation: a strategy for expansion into hypercarbic environments? The effect of hypercarbia in the context of anthropogenic climate change on fishes has been well studied; however, these current and future PCO2 increases are greatly surpassed by existing natural CO2 variations in many aquatic systems. The severe hypercarbic conditions in many environments pose challenges to acid-base regulation, however, this still remains an area ripe for investigation. Preferential pHi regulation likely represents a key adaptation for survival in high CO2 environments as an inability to protect pHi is likely the proximate cause of mortality during, and following severe acidoses (Hayashi et al., 2004; Shartau et al., 2017a; Wood et al., 1983; Yoshikawa et al., 1994). Hypercarbia tolerance may have been an important selective pressure for niche expansion in aquatic habitats, particularly in the tropics, which may partially explain the tremendous species richness seen in these regions (e.g. Amazon and Mekong rivers);  61 additionally, hypercarbia, along with aquatic hypoxia, may have been a selective pressure for the evolution of air breathing (Ultsch, 1987; Ultsch, 1996). Aerial respiration in fishes typically increases blood PCO2 as (1) hypoxic waters are often simultaneously hypercarbic, and (2) CO2 release still largely occurs at the gills, and gill ventilation is typically reduced during air breathing (Shartau and Brauner, 2014); thus, CO2 tolerance conferred by preferential pHi regulation may have been instrumental for the evolution of air breathing (Brauner and Baker, 2009; Shartau and Brauner, 2014). In summary, this study is the most comprehensive investigation to date examining how fishes respond to severe acute respiratory acidoses. Here, 20 fishes originating from three continents (North America, South America and Africa), representing 11 orders, which include 17 families and 20 genera (Betancur-R et al., 2013), are investigated for their response to hypercarbia; these species range from basal vertebrates (e.g. lamprey) to derived actinopterygians (e.g. tilapia). This study demonstrates that preferential pHi regulation is a widely used strategy to survive and tolerate CO2 tensions ranging from 3-20 kPa PCO2. As acid-base regulation is intimately associated with proper physiological functioning and ultimately survival, understanding how fishes (and vertebrates) co-opted preferential pHi regulation to thrive in challenging environments may provide insight into key evolutionary transitions in vertebrates, such as the evolution of air breathing and the transition from water to land.  62  Figure 3.1 Bar plot of CO2 tensions at loss of equilibrium in Oncorhynchus mykiss (open bars) and Acipenser transmontanus (grey bars) when subjected to a progressive increase in PCO2 at 1, 2 or 4 kPa h-1. Mean ± s.em. Significant differences due to rate of CO2 increase within species are indicated by letters that differ (uppercase – O. mykiss; lowercase – A. transmontanus) (P<0.05). Differences between O. mykiss and A. transmontanus, which use coupled pHe/pHi and preferential pHi regulation, respectively, at each rate of CO2 increase are indicated by an asterisk (P<0.05).  63   64   65  Figure 3.2: Effect of 3 h exposure to elevated CO2 on blood and tissue acid-base status in 10 different fish species. The relationship between extracellular pH (pHe) and intracellular tissue pH (pHi) for each species is shown on two panels. Red blood cell (RBC) pHi is plotted separately in the first panel for each species (i) as it is expected to be reduced during hypercarbia as RBCs generally appear to lack the capacity for pHi regulation but possess high intracellular buffer capacity; thus, RBC pHi demonstrate that the acidosis is sufficiently severe to reduce pHi in a tissue unable to regulate pHi and acts an internal control for the presence of an intracellular acidosis. In the second panel (ii) for each species, pHi of heart (squares), liver (triangles), brain (inverted triangles) and white muscle (WM; diamonds) is plotted; sampling occurred at 0 (closed symbols) and 3 h (open symbols). Fish were subjected to hypercarbia for 3 h depending on their CO2 tolerance as follows: Polyodon spathula (1.5 kPa PCO2; A), Lepisosteus oculatus (6 kPa PCO2; B), Atractosteus spatula (6 kPa PCO2; C), Ictalurus punctatus (6 kPa PCO2; D), Hoplosternum littorale (4 kPa PCO2; E), Brycon amazonicus (4 kPa PCO2, F), Colossoma macropomum (4 and 20 kPa PCO2, G), Oncorhynchus mykiss (3 kPa PCO2; H), Astronotus ocellatus (4 kPa PCO2, I), Oreochromis sp. (6 kPa PCO2; J). Significant differences between time points are indicated for each tissue by an asterisk (two-way t-test, P<0.05). In all species, pHe was significantly reduced at 3 h (P<0.05) and pHi of heart, brain, liver and white muscle were not reduced at any point except in O. mykiss; this is indicative of preferential pHi regulation in all fishes except O. mykiss which exhibited coupled pH regulation. 66  Figure 3.3: Evolution of preferential pHi regulation and coupled pHe/pHi regulation amongst adult fishes exposed to an acute (<48 h) respiratory acidosis of >1 kPa blood PCO2. Pattern of acid-base regulation [preferential pHi regulation (black branches) or coupled pH regulation (white branches)] was determined directly via pH measurements or indirectly via CO2 tolerance, which are indicted by superscript # or $, respectively. Adjacent to species names it is indicated whether they are water or air breather, and the habitat of their primary geographical zone is listed (temperate, sub-tropical or tropical). Ancestral states for preferential pHi regulation were reconstructed by likelihood using Mesquite (Maddison and maddison). Unless specified, all species were examined in this chapter; references are indicated below and correspond to superscript numbers: 1(Baker et al., 2015), 2(Wood et al., 1990), 3(Chapter 3; Baker et al., 2009a), 4(Chapter 3; Brauner et al., 2004), 5(R.B.S., M. Sackville, C. Damsgaard, L.M. Phuong, M. Hvas, T. Wang, M. Bayley, D.T.T. Huong, N.T. Phuong,  67 and C.J.B., unpublished observations; reviewed in Shartau et al., 2016a), 6(Chapter 3; Wood and LeMoigne, 1991), 7(Larsen et al., 1997), 8(Chapter 3; Heisler, 1982), 9(Wright et al., 1988)]. Phylogenetic relationships are based on (2009) and branch lengths are taken from various references utilizing fossil and molecular estimates of divergence times (Aschliman et al., 2012; Betancur-R et al., 2013; Betancur-R et al., 2015; Blair, 2005; Macqueen and Johnston, 2014; Meredith et al., 2011; Zhang et al., 2013); the phylogenetic tree was created using Mesquite (Maddison and Maddison, 2017).   68 Table 3.1: Plasma Cl- and osmolarity, and hematocrit of fishes subjected to hypercarbia exposure. Species PCO2 (kPa) Exposure time (h) Cl- (mM) Osmolarity (mM) Hematocrit (%) Control CO2 Control CO2 Control CO2 Polyodon spathula 1.5 3 111 ±4 113 ±3 248 ±6 252 ±5 27 ±2 18 ±2* Acipenser transmontanus1,2 1.5 6 121 ±1 114 ±2* 265 ±3 266 ±5*   6 6 119 ±3 95 ±3*   31 ±3 33 ±3 12 6 119 ±3 88 ±2*   31 ±3 24 ±2* Atractosteus spatula 6 3 128 ±5 127 ±3 286 ±9 305 ±12 33 ±3 27 ±2 Lepisosteus oculatus 6 3 107 ±9 108 ±1 266 ±14 283 ±7 33 ±8 38 ±8 Colossoma macropomum 4 3 147 ±4 138 ±6   28 ±1 31 ±2 20 3     22 ±1 24 ±2 Brycon amazonicus 4 3 119 ±4 121 ±6   42 ±1 35 ±4 Hoplosternum littorale 4 3 120 ±4 128 ±4   31 ±1 33 ±3 Pterygoplichthys pardalis3 4.3 6 111 ±5 108 ±3 247 ±7 246 ±5 40 ±2 40 ±2 Ictalurus punctatus 6 3 120 ±4 112 ±2 276 ±7 276 ±6 23 ±2 28 ±4 Oncorhynchus mykiss 1.5 3     27 ±2 35 ±2* 3 3     27 ±2 51 ±4* Oreochromis niloticus 6 3     21 ±2 28 ±8 Astronotus ocellatus 4 3 147 ±9 138 ±5     Freshwater teleost4   125-132  262-274    Significant differences between control and CO2 exposures are indicated by asterisk (P<0.05). Typical freshwater teleost Cl- and osmolarity values are shown for reference at the bottom of the table. 1(Baker et al., 2009a), 2(Baker and Brauner, 2012), 3(Brauner et al., 2004), 4(Edwards and Marshall, 2013).   69 Table 3.2: CO2 tolerance assay in various fish species. CO2 tension was increased at a rate of 2 kPa per hour, starting at normocarbia (~0.04 kPa PCO2) until fish reached loss of equilibrium (LOE). Where LOE was reached, the CO2 tension and time at which LOE was first recorded is indicated, and the max PCO2 exposure fish were able to tolerate (within range of our equipment) is noted, and finally, the median and mean CO2 tension that LOE occurred are indicated. The exposure was repeated in a couple of species due to sufficient number of animals. There were a couple of species where CO2 exposure ended once the first animal reached LOE as that endpoint was deemed sufficient to determine the presence of preferential pHi regulation (e.g. Lepisosteus oculatus). CO2 tolerance in a few species was high as they tolerated CO2 tensions higher than our equipment could measure (26.6 kPa PCO2) and thus we used that CO2 tension as an endpoint instead of LOE (e.g. Colossoma macropomum). Species n # fish reaching LOE Time to first LOE (h) PCO2 range of LOE (PCO2 at 1st LOE – PCO2 at last LOE) Median PCO2 at LOE Mean PCO2 at LOE Entosphenus tridentatus 10 10 6.6 13.2 – 17.1 14.7 14.6±0.4 Potamotrygon spp. 10 10 5.0 10.1 – 11.9 11.0 11.1±0.2 Polyodon spathula 7 7 1.0 1.9 – 5.9 2.0 2.7±0.5 8 8 0.6 1.1 – 1.9 1.4 1.4±0.1 Acipenser transmontanus 9 9 2.7 5.3 – 26 11.5 14.6±2.3 Lepisosteus oculatus 6 1 6.1 12.1 – n/a n/a n/a 6 1 6.0 11.9 – n/a n/a n/a Atractosteus spatula 8 1 6.0 12 – n/a n/a n/a Arapaima gigas 10 8 9.7 19.3 – 26.7 26.7 24.4±1.0 Colossoma macropomum 10 0 >13.4 n/a (>26.7) n/a n/a Electrophorus electricus 2 0 >13.4 n/a (>26.7) n/a n/a Ictalurus punctatus 9 1 4.3 8.5 – n/a n/a n/a 10 10 4.1 8.1 – 8.7 8.5 8.4±0.1    70 Species n # fish reaching LOE Time to first LOE (h) PCO2 range of LOE (PCO2 at 1st LOE – PCO2 at last LOE) Median PCO2 at LOE Mean PCO2 at LOE Ictalurus punctatus X Ictalurus furcatus 10 10 3.2 6.3 – 8.4 7.5 7.4±0.2 Pterygoplichthys pardalis 10 10 5.4 10.7 – 18 13.2 14.0±0.9 Oncorhynchus mykiss 9 9 1.8 3.5 – 5.7 5.1 4.8±0.3 Oncorhynchus kisutch 10 10 1.4 2.8 – 8.8 5.2 5.5±0.7 Synbranchus marmoratus 10 0 >13.4 n/a (>26.7) n/a n/a Astronotus ocellatus 8 8 5.8 11.5 – 16.5 13.9 13.9±0.6 Oreochromis niloticus X mossambicus X hornorum 10 10 5.1 10.1 – 15.1 12.5 12.6±0.5 Lepidosiren paradoxa 8 0 >13.4 n/a (>26.7) n/a n/a   71 Chapter 4: Embryonic Common Snapping Turtles (Chelydra serpentina) Preferentially Regulate Intracellular Tissue pH During Acid-Base Challenges   4.1 Introduction  The nests of many reptiles naturally experience changes in carbon dioxide (CO2) levels, often resulting in an elevated CO2 (hypercarbia) rearing environment for the embryos. These conditions arise due to a number of biotic and abiotic factors including nest saturation from precipitation, metabolic activity of microorganisms, and from changes in embryonic metabolism (Ackerman, 1977; Grigg et al., 2010; Lutz and Dunbar-Cooper, 1984). In nests of the broad-shelled river turtle (Chelodina expansa), green turtle (Chelonia mydas), and loggerhead turtle (Caretta caretta) CO2 values can reach up to 5-8 kPa PCO2 (Booth, 1998; Prange and Ackerman, 1974); similar PCO2 tensions have been recorded in crocodilian nests (Grigg et al., 2010; Lutz and Dunbar-Cooper, 1984). The degree of disturbance and recovery from an acute hypercarbic-induced respiratory acidosis has been well described in adult amniotes, and initially it is typically characterized by reductions in both blood [extracellular pH (pHe)] and tissue pH [intracellular pH (pHi)] that change in a qualitatively similar manner. Compensation of pHi is usually more rapid that of pHe, but compensation in both compartments is coupled (Busk et al., 1997; Nestler, 1990; Siesjö et al., 1972; Wasser et al., 1991), which we define here as coupled pH regulation. This pattern of coupled pHi and pHe compensation following a respiratory acidosis is thought to be representative of vertebrates in general. However, in CO2 tolerant fishes, it is becoming increasingly clear that pHi in a number of species is tightly regulated in the complete absence of pHe regulation (Baker et al., 2009a; Brauner and Baker, 2009; Brauner et al., 2004; Harter et al., 2014; Heisler, 1982; Shartau and Brauner, 2014), termed preferential pHi regulation. Preferential pHi regulation confers exceptional CO2 tolerance by allowing animals to withstand severe  72 challenges to acid-base regulation (Brauner and Baker, 2009; Shartau and Brauner, 2014). Chicken embryos between 60 and 90% of incubation subjected to hypercarbia (5 kPa PCO2) for 24 h experienced a reduction in pHe that was largely uncompensated (Burggren et al., 2012). Embryonic chickens are exceptionally hypercarbic tolerant as they can survive 1 h exposure to PCO2 of 10 kPa where pHe is reduced by ~0.8 pH units (Andrewartha et al., 2014), a degree of pHe depression typically observed in animals that preferentially regulate pHi (Shartau and Brauner, 2014). Amniotic embryos are enclosed within structures (e.g. eggshell, chorioallantoic membrane) that create diffusion barriers and limit or eliminate the ability for net acid excretion with the environment necessary for pH compensation. Thus, tolerance of a respiratory acidosis may be associated with preferential pHi regulation, a phenomenon that has not been investigated previously in embryonic amniotes. Embryonic turtles can survive chronic high CO2 in both nest (see above) and incubation environments (Wearing et al., 2014), suggesting a high degree of CO2 tolerance for chronic, and likely acute, CO2 exposure. We were interested in how turtles respond to severe acute respiratory acid-base disturbances as the ability to tolerate high CO2 could be associated with the capacity for preferential pHi regulation, as observed in a number of fishes and a salamander during acute hypercarbia (Brauner and Baker, 2009; Shartau and Brauner, 2014), but never in amniotes. We hypothesized that embryonic turtles preferentially regulate pHi allowing them to tolerate severe acute acid-base challenges. To test this hypothesis, we conducted two series of experiments. Series 1 investigated the pattern of acid-base regulation in normocarbia/normoxia-reared animals subjected to an acute respiratory acidosis at three developmental stages (70 and 90% of incubation, and yearlings) to assess the pattern of acid-base regulation during development. Next, in Series 2, we were interested if the pattern of acid-base regulation differed in embryos (at 90% of incubation) that had been reared under constant hypercarbia (representative of typical CO2 tensions in a natural nest environment) and then exposed to a more severe acute respiratory acidosis or to an acute respiratory alkalosis. The acid-base status of turtles was assessed in the blood compartment by measuring pHe, and in the tissues by measuring pHi of heart, brain, liver, white muscle,  73 kidney, and lung. The results of this study indicate that embryonic turtles preferentially regulate pHi, while the capacity for preferential pHi regulation is reduced in yearlings as the transition to coupled pH regulation occurs.   4.2 Methods  4.2.1 Turtle embryo acquisition and incubation Common snapping turtle eggs (Chelydra serpentina (Linnaeus, 1758)) were collected in north-western Minnesota, USA and transported by automobile to the laboratory at the University of North Texas (Minnesota Department of Natural Resources Permit No. 19772 to DAC). Eggs were staged to determine approximate age of each clutch (53-55 d total incubation period at 30°C (Yntema, 1968)); a clutch being embryos from the same nest. Eggs were incubated at 30°C in a walk-in, constant temperature room on a 14h:10h light:dark photoperiod. All embryos were incubated in plastic containers, placed in a bed of moist vermiculite mixed in a 1:1 ratio of vermiculite:water. Water content of the vermiculite was maintained by weighing the box twice weekly and adding water as needed to keep the mass constant. Embryos from each clutch were divided into two groups, and reared in normocarbic/normoxic (0.03 kPa PCO2, 21 kPa PO2; “NC”) or hypercarbic/normoxic (3.5 kPa PCO2, 21 kPa PO2; “HC3.5”) conditions from that point onward. Exposure began at ~18-22% of incubation (10-12 days post-laying, where 100% of incubation would correspond with hatch), determined by dissection of at least two representative embryos from each clutch as described previously (Crossley and Altimiras, 2005; Eme et al., 2011). For NC incubation, embryos were sealed inside large Ziplock bags, with two holes in the bag that allowed parallel inflow and outflow of gas in normoxic/normocapnic conditions in a walk-in Percival® incubator (Percival Scientific, Perry, IA). HC3.5 embryos were incubated in separate 0.3 m3 Percival incubators (model I30NLX, Percival Scientific, Perry, IA) fitted with IntellusUltra™ controllers and an IntellusUltra™ Web Server that allowed CO2 to be regulated ±0.2% and for O2 and CO2 levels to be monitored remotely. The target gas tensions (3.5 kPa PCO2, 21 kPa PO2) were achieved  74 using rotameters and Intellus™ solenoid controllers, which controlled the upstream supply of compressed O2 and CO2, respectively. Incoming O2 and CO2 levels were monitored with analyzers (S-A/I and CD-3A, respectively; Ametek Applied Electrochemistry, IL, USA) connected to a PowerLab® with LabChart Pro® software (v 7 ADInstruments, CO, USA).  Yearlings from the previous clutch year (2013) were kept in 70 l tanks at 28oC with sufficient water for voluntary submergence and access to room air. They were fed 3 times weekly, and animals were fasted for 5 days prior to experimentation. Measurements were made in embryos at 70% (N=8) and 90% (N=8) of incubation, which reflected developmental stages 22/23 and 25/26, respectively, or in yearlings (N=6) that were approximately one year old. This study used embryos from 13 clutches; each experimental exposure used typically one embryo, and occasionally two, per clutch. Three clutches of yearlings were used, two animals per clutch for each experimental exposure. All studies were approved by UNT IACUC #11-007.  4.2.2 Experimental protocols Embryos: Surgical procedures and experimental set-up  Embryos were removed from their respective incubation chambers and candled to identify a tertiary chorioallantoic membrane (CAM) artery. Embryos were placed in a temperature-controlled surgical chamber (30oC) under normocarbic/normoxic (NC) conditions and ~1 cm2 of the eggshell was removed under a dissection microscope (Leica MZ6 or MZ3; Leica Microsystems, Waukegan, IL, USA). A tertiary CAM artery was isolated for arterial pressure monitoring and blood sampling in the experimental series described below. An occlusive catheter was inserted into a tertiary CAM using heat-pulled, heparinized, and saline-filled PE-50 tubing, as previously described (Crossley and Altimiras, 2005; Crossley and Altimiras, 2000). The surgical preparations were minimally invasive and no anesthesia/analgesia was used; the entire surgical procedure took 7-10 min. Following catheterization, the catheter was fixed to the shell with cyanoacrylic glue and embryos were placed in a water jacketed multi-chamber experimental unit (~700 cm3 per chamber, one embryo per chamber, placed on cotton)  75 and allowed to acclimate for at least 60 min prior to experimentation (described below) at incubation gas tensions. Temperature in the chambers was maintained at 30oC by recirculating water from a constant temperature circulator (VWR International, LLC, West Chester, PA, USA). Each chamber consisted of a container fitted with a lid with three ports that allowed the catheter and airlines to enter the chamber. To prevent changes in chamber temperature due to incoming gas flow, all incoming gas traversed a 1 m copper line submerged within the constant temperature circulator’s water bath. Gas was forced into each chamber at a flow rate of 200 ml min-1. Cardiovascular measurements of blood pressure and heart rate were obtained by connecting the arterial catheter with saline-filled PE50 tubing to a pressure transducer held 1-3 cm above the egg, connected to an amplifier, and the pressure signal acquired at 40 Hz using PowerLab data recording system (ADInstruments, CO, USA) connected to a computer running Chartpro software (v 7.4 ADInstruments). Pressure transducers were calibrated prior to each measurement period with a vertical column of saline, and heart rate was determined with a software tachograph that integrated the arterial pressure trace. Cardiovascular measurements were made to verify embryos were alive during these acid-base exposures and to avoid sampling unhealthy animals, as well as to quantify cardiovascular changes during acid-base challenges.  Yearlings: Experimental set-up Yearling turtles were placed in a water-jacketed, multi-chamber, stainless steel experimental apparatus (~4000 cm3 per chamber, one animal per chamber) containing ~1000 ml tap water and allowed to acclimate for at least 90 min prior to experiments (described below). Temperature in the chambers was maintained at 30oC by recirculating water within the water jacket from a constant temperature circulator (VWR International, West Chester, PA, USA). Each chamber consisted of a container fitted with a lid with small holes that allowed air lines to enter the chamber. Air or N2/O2/CO2 gas mix was bubbled into the water using an air stone to ensure sufficient gas flow.   76 4.2.3 Experimental treatments  Series 1: Acid-base status during development in normocarbic normoxia following exposure to severe hypercarbic hypoxia. The specific objective of this series was to induce a severe respiratory acidosis and investigate for the presence or absence of preferential pHi regulation rather than mimicking the natural rearing environment of the turtle. NC reared animals that had been placed in individual chambers as described above were sampled (as described below) at either 70% of incubation or 90% of incubation, or as yearlings after exposure to 1 h of NC (control) or 1 h exposure to severe hypercarbic hypoxia (13 kPa PCO2 and 9 kPa PO2; HC13). The 1 h exposure time was chosen because in fish preferential pHi regulation is observed at maximal pHe depression, which occurs within 1 h of hypercarbia exposure (Baker, 2010; Baker et al., 2009a); no comparable embryonic or reptile studies exist to provide guidance for exposure times (Everaert et al., 2011). HC13 was generated using three mass flow controllers (GFC Aalborg; Orangeburg, NY, USA) and command module (Model SDPROC, Aalborg; Orangeburg, NY, USA) supplied with compressed O2, CO2, and N2 to achieve the desired gas mix. O2 and CO2 levels were monitored with analyzers (S-A/I and CD-3A, respectively; Ametek Applied Electrochemistry, IL, USA). Gas composition in the chamber changed within 1-2 min and was maintained for the remaining hour prior to sampling.  Series 2: Response to a respiratory acidosis or respiratory alkalosis at 90% of incubation in embryos reared under constant hypercarbia levels. Embryos reared in HC3.5 at 90% of incubation were sampled to examine the effect of hypercarbic rearing on acid-base balance at CO2 tensions likely representative of the natural nest environment. Next, the effect of respiratory acidosis on HC3.5 reared embryos was examined by exposing HC3.5 embryos at 90% of incubation to HC13 for 1 h and then sampled as described below. To examine the effect of a respiratory alkalosis, HC3.5 reared embryos were exposed to normocarbic normoxia for either 3 or 24 h and then sampled as below.   77 Due to limited numbers of HC3.5 reared embryos in Series 2, only embryos at 90% of incubation were investigated. We chose this developmental stage over 70% of incubation because we felt they would be more likely to tolerate the severe acid-base challenges and increase the likelihood of Series 2 being successful. There were no turtles continuously reared to yearlings under HC3.5, thus, we could not include yearlings in Series 2.  4.2.4 Blood sampling, animal euthanasia, tissue sampling and ions - Embryos Embryonic heart rate and blood pressure were continuously recorded prior to sampling. Following a 1 h exposure period approximately 70-200 µl of blood was sampled from the cannulated CAM artery by disconnecting the cannula from the pressure transducer and allowing the blood to passively flow into a 1ml heparinized plastic syringe; blood pH (pHe) and total CO2 (TCO2) were measured immediately. pHe was measured using a thermostated capillary pH electrode (model BMS 3 MK 2; Radiometer; Copenhagen, Denmark) that was calibrated daily with buffer solutions (BDH5050, pH 7.38 and BDH5058, pH 6.86; VWR; Radnor, PA, USA). TCO2 was measured using a total CO2 analyzer (Corning model 965 Analyzer; Essex, United Kingdom) and was calibrated using freshly prepared 0, 10, and 25 mmol l-1 NaHCO3. Embryos were then euthanized with an overdose of sodium pentobarbital (100mgkg-1) injected into the CAM artery. Tissues (heart, brain, liver, white muscle, kidney, and lung) were then quickly dissected (within 5 min), placed in micro-centrifuge tubes, frozen in liquid nitrogen and stored at -80oC for later measurements of pHi. Tissue was later ground under liquid nitrogen and pHi was measured using the metabolic inhibitor tissue homogenate method (MITH: see Appendix for detailed description of this method); this technique has been validated (Baker et al., 2009b; Portner et al., 1990) and used in fish (Baker and Brauner, 2012; Baker et al., 2015; Brauner et al., 2004; Regan et al., 2016) and non-fish (Busk et al., 1997; Galli and Richards, 2012) studies. Plasma Na+, K+, Cl-, and Ca2+ were measured in embryos at 90% of incubation at each rearing condition using Nova Biomedical Stat profile prime (Waltham, MA, USA).    78 – Yearlings  To sample blood and tissues in yearlings, turtles were removed from the chamber, euthanized with an overdose of isoflurane and the plastron removed and the heart exposed. Blood was sampled (~200-300 µl) from the right aorta using a 1 ml syringe with a 30 gauge heparinized needle. Tissues (heart, brain, liver, white muscle, kidney, and lung) were immediately dissected out (within 6-7 min) and frozen for later analysis as described above. Due to the greater blood volume collected in yearlings, blood PCO2 was measured at the same time as pHe using a PCO2 electrode (E201/E5037; Loligo Systems; Denmark) thermostated at 30oC in a Radiometer BMS 3 MK 2 (Copenhagen, Denmark) calibrated daily with humidified pre-mixed gases. All measurements of pHi and pHe, and TCO2 were measured as described above.   4.2.5 Calculations and statistical analyses Plasma [HCO3-] and PCO2 were calculated using measured TCO2 and pH values as described by Brauner et al. (2004). The CO2 solubility coefficient and pKa were calculated using equations from Heisler (1984) which were adapted, and experimentally validated, for use with reptile blood (Stabenau and Heming, 1993). To determine how a 1 h HC13 exposure changes [H+] relative to NC (control) [H+], pHi values were converted to [H+] ([H+]=10-pH) and HC13 [H+] was subtracted from NC [H+] to calculate the net [H+] difference. This was done for each tissue at each developmental age and was plotted as mean ± s.e.m. All data was analyzed using R version 3.1.0 (The R Foundation for Statistical Computing). Homogeneity of variances was tested with the Levene’s test (P<0.05) and normality of distributions was tested with the Shapiro-Wilkinson test (P<0.05). Differences between control and treatment group means of individual measurements were compared using a Welch two-sample t-test (P<0.05). Comparisons of means across treatments, tissues and/or developmental age were conducted using either a one-way or two-way ANOVA (Tukey post hoc, P<0.05) as appropriate. Data that did not meet the assumption of normality for a one-way ANOVA were analyzed using the Kruskal-Wallis test (P<0.05). Absolute blood pressure was corrected for the pressure transducer’s distance above the egg. Mean arterial pressure (kPa) and mean heart rate (beats min-1)  79 were calculated from the individual mean values for embryos in each exposure group. Mean arterial pressure and mean heart rate for individual embryos were based on stable period at 10 min intervals over the exposure time period. Mean arterial pressure and mean heart rate during exposure were compared to unexposed measurements using a one-way ANOVA, followed by a Tukey post hoc (P<0.05). All values are presented as mean±s.e.m; sample size for NC embryos are N=8, NC yearlings are N=6, and HC3.5 embryos are N=6. All figures were created using GraphPad Prism v5.0 (GraphPad Software Inc., 2007).   4.3 Results  4.3.1 Series 1: Acid-base status during development in normocarbic normoxia following exposure to severe acute hypercarbic hypoxia Animals reared at NC and transferred to HC13 for 1 h exhibited a significant reduction in pHe and a significant increase in blood PCO2 at all three developmental ages (Welch 2-sample t-test, P<0.05) (Fig. 4.1A) as expected a priori. Blood [HCO3-] did not change significantly (Fig. 4.1A). The pattern of changes in pHi, however, differed between ages. At 70% of incubation, hypercarbia was associated with a significant increase in pHi of the brain, white muscle, and lung but no statistically significant change was observed in heart, liver, or kidney (Fig. 4.1B); at 90% of incubation only heart pHi significantly increased while no changes in liver, brain, white muscle, lung, or kidney were observed (Fig. 4.1C). In yearlings there were no significant changes in pHi of any tissues (Welch 2-sample t-test, P>0.05), however, there was a trend toward a reduction in pHi in most tissues (Fig. 4.1D). To assess the effect of development and tissue type on acid-base changes following acute hypercarbia, [H+] was calculated from pHi, then tissue [H+] following 1 h hypercarbia was subtracted from the respective NC (control) tissue [H+] for each tissue type and at each developmental age. There was a significant effect of developmental age on the difference in tissue [H+] from control, where a progressive statistically significant increase in tissue [H+] was observed with an increase in developmental age (two-way  80 ANOVA, Tukey’s post hoc; P<0.01) indicating a progressive reduction in the ability to preferentially regulate pHi. Additionally, the various tissues respond differently as development proceeds as the interaction of developmental age and tissue significantly affected the net change in tissue [H+] (i.e. the changes between treatment and control [H+] between tissue differ significantly when developmental age is considered) (two-way ANOVA, P<0.05) (Fig. 4.2). Cardiovascular measurements indicated that embryos at 70% of incubation reared in NC and exposed to HC13 exhibited no significant changes in blood pressure (0.50 ± 0.08 kPa) or heart rate (48.3 ± 9.1 beats min-1) from controls (one-way ANOVA, P>0.05). In embryos at 90% of incubation, blood pressure and heart rate were reduced during HC13 exposure from 1.14 ± 0.09 kPa to 0.82 ± 0.06 kPa and 53.2 ± 4.6 beats min-1 to 36.7 ± 2.7 beats min-1, respectively (one-way ANOVA, Tukey’s post hoc, P<0.001).  4.3.2 Series 2: Response to an acute respiratory acidosis or alkalosis at 90% of incubation in embryos reared under constant hypercarbia Embryos at 90% of incubation reared at HC3.5 had increased pHe, blood PCO2 and [HCO3-] compared to those reared in NC (Fig. 4.3A-C). pHi was also significantly elevated in all tissues, except liver (Fig. 4.3D-I). Exposure of HC3.5 reared embryos at 90% of incubation to HC13 for 1 h resulted in a significant reduction in pHe and a significant increase in blood PCO2 but no change in blood [HCO3-] (Welch 2-sample t-test, P<0.001) (Fig. 4.4A). Heart pHi was significantly reduced; other tissues did not change (Welch 2 sample t-test, P<0.05) (Fig. 4.4B). Plasma ions (Na+, K+, Cl-, and Ca2+) were measured in untreated embryos at 90% of incubation to assess for differences due to rearing conditions that may affect acid-base status between the groups. The HC3.5 reared embryos had a greater [K+] compared to the NC reared embryos (t-test, P<0.05). There were no differences in other ion concentrations (Table 4.1). Embryos at 90% of incubation reared in HC3.5 and transferred to NC for 3 or 24 h exhibited a significant increase in pHe (one-way ANOVA, P<0.0001) and reduction in blood PCO2 (one-way ANOVA, Tukey’s post hoc, P<0.001) (Fig. 4.5A). There was a significant reduction in [HCO3-] following 24 h NC exposure (one-way ANOVA, Tukey’s post hoc, P<0.01) (Fig. 4.5A). Tissue pHi was unchanged at 3 h but at 24 h, heart  81 and brain pHi were significantly reduced (one-way ANOVA, Tukey’s post hoc, P<0.05) (Fig. 4.5B,C). Cardiovascular measurements showed that embryos at 90% of incubation reared at HC3.5 had reductions in blood pressure and heart rate during HC13 exposure from 0.96 ± 0.05 kPa to 0.67 ± 0.04 kPa and 58.1 ± 1.3 beats min-1 to 39.6 ± 1.5 beats min-1, respectively (one-way ANOVA, Tukey’s post hoc, P<0.001).   4.4 Discussion  Preferential pHi regulation has been documented in a number of fishes, and in an aquatic salamander, but never before in amniotes (Cameron, 1989a; Everaert et al., 2011; Shartau and Brauner, 2014). We hypothesized that embryonic turtles preferentially regulate pHi during a severe acute acidosis, which is supported by our findings here on snapping turtles; this is the first time this pattern of pH regulation has been identified in an amniote. These results suggest that coupled pH regulation is not the strategy used during embryonic development of snapping turtles and demonstrates that preferential pHi regulation is likely important for tolerating acute respiratory acid-base disturbances in this amniote species at this development stage.  4.4.1 Capacity for preferential pHi regulation shifts during development Exposure of NC reared turtles to HC13 greatly increased blood PCO2 (Fig. 4.1A); the difference between blood and environmental PCO2 of 13 kPa likely represents non-equilibrium between the animals and the environment due to the short exposure time. Despite the lack of complete CO2 equilibration, turtles experience large reductions in pHe (which was the objective of the treatment) but there was no reduction in pHi (Fig. 4.1) consistent with preferential pHi regulation. However, there appears to be a reduction in the capacity for pHi regulation between the younger embryos and yearlings. During 1 h HC13 exposure, three tissues exhibited a significant increase in pHi in embryos at 70% of incubation, while this was observed in only one tissue in 90% of incubation embryos and none in yearlings (Fig. 4.1B-D), suggesting younger embryos possess a greater capacity for preferential pHi regulation. When contrasted to adult western painted turtles, the lack  82 of pHi change during hypercarbia in embryos is impressive as adult western painted turtles (the only known study to measure pHe and pHi in adult turtles exposed to hypercapnia) (Wasser et al., 1991) experiencing 1 h of hypercapnia exhibited severe reductions in pHe, and pHi of heart, liver, brain, and skeletal muscle. The difference between pH of hypercapnic exposed and control animals is plotted for blood and tissues (Wasser et al., 1991) in Figure 4.6, along with relevant results from this study to highlight the large pHi reductions in adult turtles compared to embryos. The differences in the pattern of acid-base regulation between snapping turtle embryos and yearlings, and western painted turtle adults is likely due to changes in the capacity for preferential pHi regulation and buffering capacity. An increase in pHi from control values during an acidosis (or decrease during an alkalosis) is due to preferential pHi regulation and not buffer capacity, as the latter can only delay or minimize the reductions in pH during an acidosis (or increases during an alkalosis). Turtles appear to transition from preferentially regulating pHi to having coupled pH regulation.  4.4.2 Rearing condition alters blood and tissue acid-base status Rearing condition appears to affect blood and tissue acid-base status. Embryos at 90% of incubation reared at HC3.5 had a blood PCO2 of 3.6 kPa PCO2 (Fig. 4.3B), which was slightly higher than incubation PCO2 of 3.5 kPa PCO2. This indicates that these embryos were in equilibrium with environmental PCO2, as would be expected, and the slightly higher blood PCO2 would permit the release of metabolically produced CO2 to their environment. Additionally, these embryos experienced a higher pHe and blood [HCO3-] compared to NC reared embryos (Fig. 4.3A,C) suggesting these embryos have compensated pHe in chronic hypercarbia; pHi was also elevated in all tissues, except liver (Fig. 4.3D-I). The increase in blood HCO3- (Fig. 4.3C) and plasma K+ (Table 4.1) may indicate that these embryos compensate pHe similar to chicken embryos during chronic elevations in CO2, as the latter control pHe by a combination of HCO3- uptake from the shell and excretion of H+ into albumen in exchange for K+ (Bruggeman et al., 2007; Crooks and Simkiss, 1974; Rowlett and Simkiss, 1989). The increase in blood HCO3- may facilitate pHi regulation in turtle embryos by providing a greater HCO3- gradient of HCO3-/Cl- exchange.  83  4.4.3 Acid-base regulation during development Changes in the pattern of pHi regulation during development are expected as a single cell develops into a complex organism. In the earliest developmental stages, cells cannot rely on extracellular pH regulation as the extracellular compartment does not yet exist; appropriately, in vitro studies measuring pHi of post-fertilization single celled oocytes of mammals have shown that they are capable of regulating and defending pHi against external acid-base challenges (Erdogan et al., 2005; FitzHarris and Baltz, 2009; Lane, 1999; Squirrell et al., 2001). Similarly, Molich and Heisler (Molich and Heisler, 2005) found that early stage embryonic cells of zebrafish (Danio rerio) regulate pHi when exposed to changes in ambient PCO2. Aside from studies on pHe regulation in chicken embryos, which show incomplete pHe regulation and are suggestive of preferential pHi regulation, there are no other studies, to our knowledge, investigating acid-base regulation in embryonic amniotes or vertebrates once the extracellular space and circulatory system develops (Brauner, 2008; Everaert et al., 2011). Recently, however, the authors investigated the response of American alligator embryos to severe respiratory acidosis and found that they also preferentially regulate pHi, similar to turtle embryos shown here (Shartau et al., in press). During ontogeny, the capacity for coupled pH regulation increases due to the development of the extracellular space and necessary structures (e.g. cardiovascular, respiratory, and renal systems). Preferential pHi regulation has not been identified in adult amniotes as pHi is coupled to changes in pHe during acid-base disturbances (Baldwin et al., 1995; Malan et al., 1985; Nestler, 1990; Siesjö et al., 1972; Wasser et al., 1991; Wood and Schaefer, 1978); however, this is not the case in all adult vertebrates. A number of fishes (Brauner and Baker, 2009; Shartau and Brauner, 2014), including a salamander (Heisler et al., 1982), preferentially regulate pHi when subjected to severe acute acid-base disturbances despite reductions of pHe > 1 pH units. Snapping turtle embryos and yearlings are tolerant of acute hypercarbia, similar to other species capable of preferential pHi regulation; this pattern of pH regulation appears to confer exceptional tolerance to CO2 tensions up to 12 kPa PCO2 (Baker et al., 2009a; Brauner and Baker, 2009; Shartau and Brauner, 2014). Without preferentially regulating  84 pHi, it is unlikely these animals could tolerate, and thus, be able to maintain acid-base status during high CO2 tensions due to putative limitations on pHe regulation. The “bicarbonate concentration threshold”, originally described by Heisler (Heisler, 1984; Heisler et al., 1982) limits plasma [HCO3-] uptake to approximately 27-33 mmol l-1 which limits complete pHe compensation to CO2 tensions below ~2-2.5 kPa PCO2 (Brauner and Baker, 2009). In addition to conferring exceptional tolerance to hypercarbic-induced acidosis, preferential pHi regulation appears to play a role in short-term pHi regulation during both metabolic acidoses, metabolic alkalosis (Harter et al., 2014), and respiratory alkalosis (Fig. 4.5). Similar to some fishes, including the armoured catfish (Pterygoplichthys pardalis), preferential pHi regulation acts as a general pattern of acid-base regulation in turtle development as it protects against respiratory/metabolic acidosis of HC13 exposure (Fig. 4.1). Additionally, embryos reared at 3.5 kPa PCO2, which likely mirror natural nest conditions, largely maintained pHi during both HC13 and NC exposure, which create an acidosis and alkalosis, respectively (Fig. 4.4; 4.5); this suggests that preferential pHi regulation is a pattern of acid-base regulation that is used during the course of development, conferring robust capacity to cope with acid-base challenges.  Cardiovascular function may be protected by preferential pHi regulation Preferential pHi regulation may protect cardiac function in embryos at 70% of incubation. Blood pressure and heart rate did not change during severe acute acidosis, this response is similar to what is seen in white sturgeon (Baker et al., 2011) and armoured catfish (Hanson et al., 2009) during acute hypercarbia, both preferential pHi regulators; however, cardiac function in embryos at 90% of incubation was not preserved. Difference in cardiac function between development ages may be due to the increased metabolic demand of older embryos being depressed by changes in CO2 and O2 (Erasmus et al., 1971), as in adult turtles cardiac function is reduced during lower metabolic demand (Jackson, 1987; Jackson et al., 1991).   85 4.4.4 Conclusions and perspectives Preferential pHi regulation has only been described a handful of times in fishes and amphibians (Baker et al., 2009a; Brauner and Baker, 2009; Brauner et al., 2004; Harter et al., 2014; Heisler, 1982; Heisler et al., 1982; Shartau and Brauner, 2014), but now our findings indicate that an amniote, the common snapping turtle, can also preferentially regulate pHi. It is intriguing to think that preferential pHi regulation may represent the “default” pattern of acid-base regulation used during development, starting from the single cell oocyte, and in some animals is maintained from this embryonic condition through to the adult stage. Clearly this is an area worthy of further investigation. Understanding the pattern of acid-base regulation in embryos and adults, and the transition between these different patterns of pH regulation will provide significant insight into acid-base homeostasis during development of amniotes, and vertebrates in general. In conclusion, we demonstrated the first occurrence of preferential pHi regulation in an amniote; furthermore, we also found that the capacity for preferential pHi regulation changed during development between embryo to yearling. Preferential pHi regulation in developing snapping turtles and other amniotes, such as American alligators (Shartau et al., in press), likely plays an important role in allowing embryos to successfully develop when faced with acute acid-base challenges for which typical adult mechanisms of acid-base compensation are unavailable. Future studies should investigate whether preferential pHi regulation is used during development of other amniotes, and vertebrates; it would be interesting to assess if the capacity for pHi regulation changes from embryo to adult in animals that are able to preferentially regulate pHi as adults. Additionally, investigating the cellular and molecular mechanisms of preferential pHi regulation, and how they change during development will be an important contribution to understanding acid-base physiology in vertebrates.  86  Figure 4.1: Effect of exposure to an acute respiratory acidosis in common snapping turtle (Chelydra serpentina) embryos (at 70 or 90% of incubation) or yearlings in Series 1 on blood and tissue acid-base status. Blood pH (pHe) and blood [HCO3-] are presented on a pH-HCO3- plot. Embryos at 70% of incubation (l), 90% of incubation (n), or yearlings (p) were sampled in normocarbia (0.03 kPa PCO2, 21 kPa PO2; NC) or following 1 h hypercarbic hypoxia (13 kPa PCO2, 9 kPa PO2; HC13) exposure; curved lines represent PCO2 isopleths (A). The relationship between pHe and tissue pH (pHi) in snapping turtles is indicated for 70% of incubation (B), 90% of incubation (C), and yearlings (D) following 1 h exposure to HC13. Tissues are indicated by the following symbols: heart (l, H), liver (n, L), lung (p, U), kidney (q, K), brain (u, B), white muscle (¡, WM) and red cell (n, RBC - yearlings only). Values are presented as means±s.e.m; n=8 for 70 and 90% of incubation, and n=6 for yearlings. A: symbols indicate significant differences (P<0.05) between  87 control (NC) and treatment (HC13) for pHe (*), blood PCO2 (∇), and blood HCO3- (Φ). B, C and D: *significant differences in pHi from the NC group, letter next to asterisk indicates tissue (P<0.05).  88  Figure 4.2: Difference in tissue [H+] from control following 1h exposure to hypercarbia hypoxia (13 kPa PCO2, 9 kPa PO2; HC13) relative to normocarbic (0.03 kPa PCO2, 21 kPa PO2; NC) reared common snapping turtles (Chelydra serpentina) of Series 1. Concentrations of H+ were calculated from tissue pH ([H+]=10-pH) and the mean NC [H+] was subtracted from individual HC13 [H+] values to calculate a mean difference [H+] ± s.e.m. This was done for each tissue at each developmental age. 70% of incubation (¡), 90% of incubation (n) and yearlings (p). Positive [H+] values indicate an increase in tissue [H+] and negative [H+] values indicate a reduction in tissue [H+]. Significant differences between [H+] changes across developmental ages and tissues were determined using a 2-way ANOVA, followed by Tukey’s post hoc (n=8 for 70 and 90% of incubation, and n=6 for yearlings). Uppercase letters that differ indicate significant differences between tissues in the same developmental age and lowercase letters that differ indicate significant differences between developmental age in the same tissue following separate 1-way ANOVA followed by Tukey’s post hoc (P<0.05).  89  Figure 4.3: Changes in blood and tissue acid-base status in common snapping turtles (Chelydra serpentina) embryos at 90% of incubation continuously reared in either normocarbia or hypercarbia. (A) blood pH, (B) blood PCO2 (kPa), (C) blood HCO3- (mmol l-1), (D) heart pH, (E) liver pH, (F) brain pH, (G) kidney pH, (H) lung pH, and (I) white muscle pH, where different incubation conditions are indicated as follows: normocarbia (0.03 kPa PCO2, 21 kPa PO2; NC, n) and hypercarbia (3.5 kPa PCO2, 21 kPa PO2; HC3.5, n). Data are means ± s.e.m.; n=8 for NC embryos, and n=6 for HC3.5 embryos. These data are re-plotted from figures 1 and 4. Significant differences between rearing conditions are indicated by asterisk (P<0.05).  90  Figure 4.4: Effect of exposure to an acute respiratory acidosis in snapping turtle embryos (Chelydra serpentina) at 90% of incubation in Series 2 reared continuously and sampled in hypercarbia (3.5 kPa PCO2, 21 kPa PO2; HC3.5) or following 1 h exposure to hypercarbic hypoxia (13 kPa PCO2, 9 kPa PO2; HC13). Blood pH (pHe) and blood [HCO3-] are presented on a pH-HCO3- plot. Embryos were sampled in HC3.5 or following 1 h HC13 exposure; curved lines represent PCO2 isopleths (A). The relationship between pHe and tissue pH (pHi) in snapping turtles following 1h exposure to HC13. Tissues are indicated by the following symbols: heart (l, H), liver (n, L), lung (p, U), kidney (q, K), brain (u, B), and white muscle (¡, WM). Values are presented as means ± s.e.m. (n=6). A: symbols indicate significant differences (P<0.05) between HC3.5 and HC13 for pHe (*) and blood PCO2 (∇). B: *significant differences in pHi from the NC group, letter next to asterisk indicates tissue (P<0.05).   91  Figure 4.5: Effect of exposure to normocarbia in snapping turtle embryos (Chelydra serpentina) at 90% of incubation in Series 2 reared continuously and sampled in hypercarbia (3.5 kPa PCO2, 21 kPa PO2; HC3.5) or following 1 h exposure to normocarbia (0.03 kPa PCO2, 21 kPa PO2; NC) for either 3 or 24 h. Blood pH (pHe) and blood [HCO3-] are presented on a pH-HCO3- plot. Embryos were sampled in HC3.5 or following 1 h HC13 exposure; curved lines represent PCO2 isopleths (A). The relationship between pHe and tissue pH (pHi) in snapping turtles following 1h  92 exposure to HC13 for 3 h (B) or 24 h (C). Tissues are indicated by the following symbols: heart (l, H), liver (n, L), lung (p, U), kidney (q, K), brain (u, B), and white muscle (¡, WM). A: different letters indicate significant differences (P<0.05) between control (NC) and treatment (HC13) for pHe (Uppercase letters), blood PCO2 (lowercase letters), and blood HCO3- (Greek letters). B, C and D: *significant differences in pHi from the NC group, letter next to asterisk indicates tissue (P<0.05).  93  Figure 4.6: Difference in blood and tissue pH of turtles during development following exposure to hypercarbia relative to normocarbia in common snapping turtles (Chelydra serpentina) (70 and 90% of incubation and yearlings; this study) and adult western painted turtles (Chrysemys picta bellii) (Wasser et al., 1991). Control pH values for pHe, and pHi of heart, liver, brain and muscle were subtracted from the values determined following either 1 h HC13 (13 kPa PCO2, 9 kPa PO2) exposure (from Figure 4.1) in snapping turtles or 1 h 6.5 kPa PCO2 exposure in western painted turtles. Mean control (normocarbic) pH was subtracted from individual hypercarbic values to calculate a mean; differences are shown as means only to visualize the large reductions in pHi in adult turtles compared to either embryos or yearlings in the present study. 70% of incubation (¡), 90% of incubation (n), yearlings (p) and adult western painted turtle (★).    94 Table 4.1: Plasma ion concentrations at 90% of incubation in Chelydra serpentina embryos reared in NC and HC3.5  Na+ (mmol l-1) K+ (mmol l-1) Cl- (mmol l-1) Ca2+ (mmol l-1) NC 129.0±3.7 3.5±0.1 116.4±3.4 1.4±0.1 HC3.5 135.0±3.2 3.9±0.1* 117.0±3.7 1.4±0.1  NC, normocarbia (0.03 kPa PCO2, 21 kPa PO2; N=8) and HC3.5, hypercarbia (13 kPa PCO2, 9 kPa PO2; N=6) reared in (common snapping turtles) embryos. Data are means ± s.e.m. *Significance between rearing conditions (P<0.05).    95 Chapter 5: American Alligator Embryos Tightly Regulate Intracellular pH During a Severe Acidosis   5.1 Introduction  Acid-base regulation in adult amniotes relies on net H+ exchange with the environment through ventilatory and/or renal pathways (Cameron, 1989a); however, during embryonic development of oviparous animals this is constrained as the egg shell structure limits environmental interaction (Eme and Crossley, 2015; Erasmus et al., 1971; Everaert et al., 2011). Acid-base balance is one of the most important physiological parameters and tight pH regulation is critical as small deviations can have large effects on molecular function, and ultimately reduce whole animal performance (Putnam and Roos, 1997). In adult amniotes, compensation of intracellular pH (pHi) following an acid-base disturbance is usually more rapid than that of blood pH (extracellular pH; pHe) but compensation in both compartments is coupled, termed coupled pH regulation. Complete pHi recovery during a sustained respiratory acidosis occurs only following approximately >50% pHe compensation (Shartau et al., 2016b; Shartau et al., 2016a). Thus, acid-base regulation in adult amniotes is characterized by the regulation of pHe, which ensures pHi is protected (Shartau et al., 2016a) – the response in embryonic amniotes constrained within an egg shell is poorly understood (Eme and Crossley, 2015; Everaert et al., 2011). Limited evidence from snapping turtle embryos Chelydra serpentina during severe acute respiratory metabolic acidosis suggests that the pattern of acid-base regulation in embryos may differ from adults. Exposure to an acute elevated environmental CO2 tension (hypercarbia) of C. serpentina embryos at two developmental ages resulted in a dramatic reduction in pHe; however, pHi was observed to be protected (Shartau et al., 2016b). This trait is referred to as preferential pHi regulation and has only previously been observed in a few adult anamniotes (e.g. white sturgeon [Acipenser transmontanus], armoured catfish [Pterygoplichthys pardalis] and greater siren [Siren lacertina]), but never in adult amniotes (Shartau and Brauner, 2014; Shartau et al., 2016b).  96 Vertebrates capable of preferential pHi regulation exhibit no detectable pHi reduction during a severe respiratory acidosis, and notably, pHe compensation is not required for pHi protection (Shartau et al., 2016a). Regulation of pHe for coupled pH regulation is limited in embryos due to barriers created by the eggshell and associated membranes (e.g. chorioallantoic membrane) (Erasmus et al., 1971; Everaert et al., 2011; Shartau et al., 2016a). Additionally, the absence or incomplete formation of an extracellular compartment and necessary cardiorespiratory and renal structures, limit pHe regulation (Eme and Crossley, 2015; Everaert et al., 2011; Shartau et al., 2016b); consequently, preferential pHi regulation may be a more common trait in reptilian embryos as observed in C. serpentina, particularly those that are known to be tolerant of conditions that induce an acid-base disturbance such as elevated CO2. The nests of some reptilian species naturally experience large increases in CO2 and reductions in O2 (Grigg et al., 2010; Lutz and Dunbar-Cooper, 1984); this may create challenges for acid-base regulation and be further constrained by limited nest and eggshell diffusion (Erasmus et al., 1971). It has been documented that the mound nests of crocodilians can naturally experience CO2 levels of 2-8.5 kPa (Grigg et al., 2010; Lutz and Dunbar-Cooper, 1984). Chronic high CO2 tensions may not adversely affect crocodilian embryos during rearing (Eme and Crossley, 2015), but nothing is known about their acid-base status during either chronic or acute CO2 exposure (Everaert et al., 2011). We were interested in determining whether embryonic crocodilians can protect tissue pH during acute hypercarbia hypoxia, similar to that observed in C. serpentina. I hypothesized that Alligator mississippiensis embryos would preferentially regulate pHi during a severe acute respiratory acidosis. This hypothesis was tested by exposing embryos to a severe acute respiratory metabolic acidosis to examine the impact of this exposure on blood and tissue acid-base status to gain insight into the pattern of pH regulation in embryonic crocodilians and determine whether preferential pHi regulation may be a general trait of CO2 tolerant reptilian embryos.    97 5.2 Methods  5.2.1 Subjects of study Alligator mississippiensis embryos were collected from the Rockefeller Wildlife Refuge at Grand Chenier, LA, USA and transported to the laboratory at the University of North Texas. Embryos were staged to determine approximate age of each clutch (72 d total incubation period at 30oC) and were incubated at 30oC in a walk-in incubator ensuring all embryos developed as female. All embryos were placed in plastic containers and placed in a bed of moist vermiculite mixed in a 1:1 ratio of vermiculite to water. Water content of vermiculite was maintained by weighing the box twice weekly and adding water as needed to keep the mass constant.  5.2.2 Surgical procedures Embryos were removed from the incubation chamber and candled to identify a tertiary chorioallantoic membrane (CAM) artery. They were then placed in a temperature-controlled surgical chamber (30oC) under normoxic/normocarbic conditions and ~1 cm2 of the eggshell was removed under a dissection microscope (Leica MZ6 Leica Microsystems, Waukegan, IL). A tertiary CAM artery was isolated for arterial pressure monitoring and blood sampling. An occlusive catheter was inserted into the vessel using heat-pulled, heparinized, and saline-filled PE-50 tubing, as previously described (Crossley and Altimiras, 2005). The surgical preparations were minimally invasive and no anesthesia/analgesia is required; the entire surgical procedure took 7-10 minutes. Following catheterization, the catheter was fixed to the shell with cyanoacrylic glue and the embryo was placed in a water-jacketed multi-chamber experimental unit (~700 cm3 per chamber, one embryo per chamber) and allowed to acclimate for at least 60 minutes. Temperature in the chambers was maintained at 30oC with a circulating water bath (VWR International, LLC, West Chester, PA, USA). Each chamber consisted of a container fitted with a lid with three ports that allowed the catheter and airlines to enter the chamber. To prevent changes in chamber temperature due to incoming air flow, all incoming gas traversed a 1 m copper line submerged within the constant temperature circulator’s water bath. Air was forced into each chamber at a flow of 200 ml min-1. Each  98 arterial catheter was attached to a pressure transducer 1-3 cm above the egg via saline-filled PE50 tubing, connected to an amplifier, and the pressure signal acquired at 40 Hz using PowerLab data recording system (ADInstruments, CO, USA) connected to a computer running ChartPro software (v 7.4 ADInstruments CO, USA). Pressure transducers were calibrated prior to each measurement period with a vertical column of saline, and heart rate was determined with a software tachograph that integrated the arterial pressure trace. Cardiovascular measurements were made to quantify cardiovascular changes during acid-base challenge.  5.2.3 Experimental treatment and physiological measurements Embryos reared in normocarbic/normoxic (0.03 kPa PCO2, 21 kPa PO2; air) were removed at 70% of incubation and subjected to either 1 h exposure to air or hypercarbic hypoxia (13 kPa PCO2 and 9 kPa PO2; H13). This treatment was chosen as previously it has been shown to induce a sufficiently severe acidosis (Andrewartha et al., 2014), allowing for the determination of preferential pHi regulation (Shartau et al., 2016b); the exposure time of 1 h was chosen as pHe and pHi typically reach maximal depression at that point (Baker et al., 2009a). The conditions for H13 were generated using compressed O2, CO2, and N2 regulated with mass flow controllers for nitrogen, oxygen and carbon dioxide (GFC Aalborg; Orangeburg, NY, USA) regulated with a command module (Model SDPROC, Aalborg; Orangeburg, NY, USA) to achieve the desired gas mix. O2 and CO2 levels were monitored with analyzers (S-A/I and CD-3A, respectively; Ametek Applied Electrochemistry, IL, USA). Gas composition in the chamber changed within 60-120 seconds. Following exposure two aliquots of blood, approximately 150-300 µL, were sampled from the CAM artery by disconnecting the pressure catheter with blood passively flow into a 1 mL heparinized syringe. Blood pH (pHe; model BMS 3 MK 2; Radiometer) and total CO2 (TCO2) (model 965 Analyzer; Corning) were measured immediately using the first aliquot and the second aliquot was centrifuged (3 min at 10,000 rpm), plasma removed and red cells frozen for later analysis of pHi. Embryos were then euthanized with an overdose of sodium pentobarbital (100 mg kg-1) injected into the CAM artery. Tissues (heart, brain, liver, white muscle and kidney) were then quickly dissected (within 5 min), placed in micro-centrifuge tubes, frozen in liquid  99 nitrogen and stored at -80oC for later measurements of pHi. Tissue was later ground under liquid nitrogen and pHi was measured using the metabolic inhibitor tissue homogenate method (MITH; see Appendix for detailed description of this method); this technique has been validated (Baker et al., 2009b; Portner et al., 1990) and has been previously used in reptiles (Galli and Richards, 2012; Shartau et al., 2016b). Red blood cell (RBC) pHi was measured using the freeze-thaw technique (Baker et al., 2009a).  5.2.4 Calculations and statistical analyses Plasma [HCO3-] and PCO2 were calculated using measured TCO2 and pH values as previously described by Brauner et al. (2004). The CO2 solubility coefficient and pKa were calculated using equations from Heisler (1984) which were adapted, and experimentally validated, for use with reptile blood (Stabenau and Heming, 1993). Comparison of acid-base changes between control and treatment were conducted using both pH and proton concentration ([H+]); this was done to mitigate concern regarding the perceived problem of using pH, a logarithmic value, in statistical analyses (Boutilier and Shelton, 1980). [H+] was calculated from individually measured pH values ([H+]=10-pH) and plotted as mean ± s.e.m. All data was analyzed using GraphPad Prism v5.0 (GraphPad Software Inc., 2007). Differences between the acid-base parameters of air and H13 exposed groups were compared using a two-sample t-test (P< 0.05). Mean arterial pressure (kPa) and mean heart rate (beats min-1) were calculated from the individual mean values for embryos in each exposure group and were based on stable individual mean values for 45 min during the exposure period. Absolute blood pressure was corrected for the pressure transducer’s distance above the egg. Mean blood pressure and heart rate were compared using a two-sample t-test (P< 0.05). All values are presented as mean ± s.e.m.; sample sizes are N=6-7 except for RBC where N=5.    100 5.3 Results and discussion  Alligator mississippiensis embryos preferentially regulate pHi despite a reduction in pHe during a severe acute respiratory metabolic acidosis. Following 1 h exposure to H13, blood PCO2 increased from 3.3 ± 0.5 to 8.6 ± 1.0 kPa PCO2, which was accompanied by a large reduction in pHe from 7.516 ± 0.027 to 7.010 ± 0.019; blood [HCO3-] did not differ (Fig. 5.1A). Despite pHe being reduced by 0.506 pH units, pHi of tissues was not reduced; heart and brain pHi increased (6.346 ± 0.051 to 6.572 ± 0.066 and 6.512 ± 0.046 to 6.693 ± 0.061 pH units, respectively) (Fig. 5.1B), while no change in pHi of liver, white muscle, or kidney were observed (Fig. 5.1C). As pH is a measure of [H+], the changes in [H+] of blood and tissues following this acidosis reflected those of pH. Blood [H+] increased from 30.9 ± 1.9 to 80.1 ± 3.3 nM, which was accompanied by reductions in [H+] of heart and brain (Fig. 5.1D); no change in [H+] of liver, white muscle or kidney occurred (Fig. 5.1E). Using [H+] did not yield different statistical conclusions compared to using pH and thus indicate that despite the logarithmic nature of pH, use of pH should not be an issue in these analyses. This is corroborated by Boutilier and Shelton who conclude that the use of pH is as valid of that of [H+] for statistical analysis; they suggest this conclusion is applicable to all vertebrates that have fairly precise pH (or [H+]) regulation (Boutilier and Shelton, 1980). Our calculations, along with Boutilier and Shelton (1980), provide additional reassurance regarding the acceptability of using pH in statistical analyses in this study, and others. Exposure to elevated CO2 was expected to reduce pHe, however, a pure respiratory acidosis would be associated with an increase in plasma [HCO3-] along the blood buffer line from 19.5 mM to 26.4 mM based on a blood buffer value of -16 mM HCO3- pH unit-1 from chicken embryos (Burggren et al., 2012) (Fig. 5.1A). That [HCO3-] is well below the blood buffer line is indicative of net acid excretion from the intracellular to the extracellular compartment and is a characteristic of preferential pHi regulation (Harter et al., 2014; Shartau et al., 2016a). The RBC were the only tissue to exhibit a reduction of pHi which is a common trait among fish that preferentially regulate pHi (Harter et al., 2014). While clearly the RBCs do not actively regulate pHi in the way that other tissues do, they have a tissue buffer value similar to tissues such as the heart  101 and brain (Wood and LeMoigne, 1991) and thus inform on the reduction in pHi that might be expected in tissues lacking the capacity for preferential pHi regulation (Harter et al., 2014). Differences amongst tissues for pHi regulation may reflect varying capacity for preferential pHi regulation; this may be tissue specific or reflect differential organ maturation at 70% of incubation (Shartau et al., 2016b). The capacity for preferential pHi regulation may depend on the relative importance of the respective tissue to the physiology of the embryo at this developmental stage. For example, the capacity of heart to regulate its pHi may underlie the constant blood pressure and heart rate (0.65 ± 0.05 kPa and 69.5 ± 6.3 beats min-1, respectively;) during acute H13 exposure. Preferential pHi regulation in the heart is also likely responsible for maintaining cardiac function in embryonic C. serpentina during acute hypercarbia hypoxia at 70% of incubation but at 90% of incubation cardiac function was reduced (Shartau et al., 2016b). Similarly, preferential pHi regulation is suggested to preserve cardiac performance in adult white sturgeon (Baker et al., 2011) and armoured catfish (Hanson et al., 2009) during exposure up to 3 and 5 kPa PCO2, respectively; however, beyond those CO2 tensions those fishes exhibited modest reduction in cardiac performance. The ability of A. mississippiensis embryos to protect (and elevate) pHi while preserving cardiac function at this developmental stage, suggests they possess a robust capacity to tolerate this respiratory metabolic acidosis, and that the impact on whole embryo performance, at least acutely, is minimal. In white sturgeon, PCO2 tensions greater than 6 kPa PCO2 are also associated with a reduction in metabolic rate while still preferentially regulating pHi (Baker and Brauner, 2012). Other studies have shown metabolic depression in response to hypercarbia-induced respiratory acidosis (Baker and Brauner, 2012; Michaelidis et al., 2005; Stapp et al., 2015) and that the response, including protein synthesis, differs between tissues and organisms (Stapp et al., 2015). During hypercarbia in the peanut worm (Sipunculus nudus), there is a metabolic depression which is associated with a shift to less ATP costly ion-transporters which allow for compensation of the accompanying intracellular acidosis (Portner et al., 1998; Portner et al., 2000). Given the differential response of pHi in A. mississippiensis embryos in response to hypercarbia hypoxia, it may be that tissues are affected differently by the extracellular acidosis. The protection of pHi  102 may be reflected in the relative ability to shift to the optimal acid-base ion transporters, such that the most acid sensitive tissues such as brain and heart, possess the greatest capacity for this and thus exhibit the most robust capacity for pHi regulation against reduction in pHe. Embryonic A. mississippiensis, like embryonic turtles in Chapter 4, were subjected to both respiratory and metabolic acidosis as a consequence of hypercarbia and hypoxia, respectively. As hypoxia leads to anaerobic metabolism and the increased production of lactate, this may have affected tissues differently. While lactate concentration was not measured, exposure of similar stage embryonic chickens to 1 h hypercarbia hypoxia resulted in a pHe reduction of ~0.8 pH unit and increase in blood lactate from 0.8 to 14 mM (Andrewartha et al., 2014), intracellular pH or lactate were not measured so it is not known how the tissues responded but they likely experienced large pHi reduction and lactate increase. The response of pHi to hypercarbia or hypoxia alone in amniote embryos are unknown; consequently, the possible differences between them on pHe and pHi should be considered. Preferential pHi regulation likely confers the CO2 tolerance exhibited by A. mississippiensis embryos, which may enhance embryonic survival by allowing them to tolerate acid-base disturbances in an environment that is not favorable to net acid excretion. The ability of C. serpentina and A. mississippiensis embryos to preferentially regulate pHi, species where adults use coupled pH regulation (Shartau et al., 2016a; Wasser et al., 1991), suggests the pattern of acid-base regulation is modified during reptilian development. Compared to older embryos and post-hatch animals, the capacity for pHi regulation in turtles appears to be the greatest at 70% of incubation (Fig. 5.2). The differential ability of embryos and post-hatch reptiles to regulate pHe and pHi (only pHi for brain and muscle are shown) following an acute acidosis is shown in Figure 2. Although pHe is consistently reduced, pHi is well regulated in 70% of incubation embryos, but in post-hatch turtles (C. serpentina and C. picta bellii) and lizards (A. equestris and D. dorsalis), pHi is reduced. These results show that embryonic A. mississippiensis fully protect pHi against severe reduction in pHe during hypercarbia hypoxia, corroborating results in turtle (Shartau et al., 2016b), further demonstrating that preferential pHi regulation occurs in  103 reptilian embryos; a strategy of acid-base regulation that has only been identified in a few adult anamniotes vertebrates (Shartau et al., 2016a). Although more work is needed to support the hypothesis recently proposed by Shartau et al. (2016a) that preferential pHi regulation is an embryonic strategy of acid-base regulation in vertebrates, these findings in A. mississippiensis provide the second example of this strategy in an embryonic reptile, and the first in a crocodilian. Further studies are required to assess whether A. mississippiensis embryos exhibit a similar transition from preferential pHi regulation to coupled pH regulation like that in turtles, and whether this strategy of acid-base regulation manifests in non-reptilian embryonic vertebrates.  104  Figure 5.1: Effect of exposure to an acute respiratory acidosis in Alligator mississippiensis embryos on blood and tissue acid-base status. Blood pH (pHe), blood [HCO3-] are presented on a pH-HCO3- plot. Embryos at 70% of incubation were sampled in air or following a 1 h hypercarbic hypoxia (13 kPa PCO2, 9 kPa PO2: H13) exposure; curved lines represent PCO2 isopleths and dashed line represents the non-bicarbonate blood buffer value (adapted from Burggren et al., 2012) (A). Relationship between blood pH (extracellular pH [pHe]) and tissue pH (pHi) (B, C) or relationship between in air or blood proton concentration [H+] (nM) and tissue [H+] (D, E) following 1 h exposure to H13. Brain (l), liver (p) and heart (n) (B, D), and red blood cells (RBC, q), white muscle (WM, n) and kidney (u) (C, E). Values are presented as means ± s.e.m. Symbols (*, +) indicate significant differences (P<0.05) between air (open symbols) and H13 (closed symbols) treatment for pH or [H+] (*) and blood HCO3- (+). 105  Figure 5.2: Difference in blood pH (pHe) and tissue pH (pHi) during development following a respiratory acidosis in embryonic Alligator mississippiensis (American alligator; 70% to hatch) and Chelydra serpentina (snapping turtle; 70 and 90% to hatch), and in post-hatch C. serpentina, Chrysemys picta bellii (western painted turtle), Anolis equestris (knight anole) and Dipsosaurus dorsalis (desert iguana). Control pH values for pHe (open bar), and pHi of brain (black bar) and muscle (grey bar) were subtracted from the values determined following either 1 h exposure to 13 kPa PCO2, 9 kPa PO2 in A. mississippiensis (this study) and C. serpentina (Chapter 4), or 1 h dive which increased arterial PCO2 to 6.5 kPa in C. picta bellii (Wasser et al., 1991), or 1 h exposure to 5 kPa PCO2 in A. equestris and D. dorsalis (Snyder et al., 1995). Differences are shown as means only to visualize the changes across developmental ages. Values that are ≥0 are representative of preferential pHi regulation while those that are <0 indicate a net acidosis.   106 Chapter 6: General Discussion and Conclusions   The objective of my thesis was to investigate the usage, distribution/prevalence, and origin of preferential pHi regulation as a strategy of acid-base regulation in vertebrates. This objective was addressed by investigating how adult fishes and embryonic amniotes respond to severe acute acid-base disturbances. Chapter 2 suggests that preferential pHi regulation is not used by all tissues to protect against all types of acidosis in white sturgeon. Chapter 3 indicates that preferential pHi regulation occurs in many fish species and in numerous fish phylogenetic groups in response to severe acute hypercarbia. Finally, Chapters 4 and 5 demonstrate that preferential pHi regulation occurs in reptile embryos and may be lost in adults. Together, these results demonstrate that preferential pHi regulation is a widely used strategy of acid-base regulation amongst vertebrates in response to severe acute hypercarbia. Additionally, it may be an embryonic strategy that is either retained or lost in adults, and that differences exist in the degree of pHi protection between the tissues in response to an acid-base disturbance, and differences in pHi regulation occur between different types of acute acid-base disturbances. This General Discussion will examine preferential pHi regulation as a distinct pattern of acid-base regulation compared to the more familiar/traditional strategy of coupled pHe/pHi regulation. The significance of using preferential pHi regulation will be explored, including how it may have played a critical function in a number of key transitions in vertebrate evolution.   6.1 Thesis overview and major contributions   Chapter 2 investigated whether preferential pHi regulation is a general strategy of acid-base regulation in response to different types of acidoses. The goal of this chapter was to determine if preferential pHi regulation could be assessed in fishes using various acid-base challenges such as exhaustive exercise, in addition to hypercarbia where it has  107 been observed previously (Baker et al., 2009a). If different acidoses can be used to demonstrate the presence or absence of preferential pHi regulation, this would allow for the use of acid-base exposures other than hypercarbia (e.g. exhaustive exercise) to conduct the survey of presence or absence of preferential pHi regulation in Chapter 3. Additionally, inference regarding the presence or absence of preferential pHi regulation from the literature could occur as most acid-base challenges are not conducted using hypercarbia. I hypothesized preferential pHi regulation is a general acid-base regulatory strategy in Acipenser transmontanus (white sturgeon). The results from Chapter 2 indicated that A. transmontanus, which preferentially regulate pHi during hypercarbia, do not exhibit the same capacity for pHi regulation in all tissues during metabolic acidoses induced by anoxia, exhaustive exercise and air exposure. This suggests that response between acidoses varies and thus, metabolic acidoses may not indicate whether fishes possess the capacity for preferential pHi regulation. These results help shape subsequent chapters by emphasizing the importance of consistently using the same type of acid-base disturbance (i.e. hypercarbia) to assess the presence or absence of preferential pHi regulation. Chapter 3 investigated the presence or absence of preferential pHi regulation in fishes. The objective of this chapter was to determine if preferential pHi regulation is a common and widespread strategy of acid-base regulation amongst fishes as prior to this thesis preferential pHi regulation had only been observed in three fishes (A. transmontanus, Pterygoplichthys pardalis, Synbranchus marmoratus). I hypothesized that preferential pHi regulation would occur in fishes tolerant of severe acute hypercarbia; this was supported by the results. Chapter 3 shows that an additional 15 fish species use preferential pHi regulation; these fishes include water and air breathers, as well as tropical and temperate species (Table 6.1), and span numerous phylogenetic groups (Fig. 6.1). These findings represent a major contribution to understanding the distribution of acid-base regulatory strategies in fishes as preferential pHi regulation can now be considered relatively common (or at least, not rare) and broadly used. As preferential pHi regulation was demonstrated in Chapter 3 to be closely associated with tolerance to severe hypercarbia in adult fishes, I was interested in investigating whether this strategy might be used by amniotes, some of which are highly  108 hypercarbia tolerant during embryonic development. Chapter 4 investigated the pattern of acid-base regulation during development of a hypercarbia tolerant amniote, Chelydra serpentina (common snapping turtle). I hypothesized that embryonic turtles would preferentially regulate pHi. Results from this chapter indicate that embryonic turtles preferentially regulate pHi and that capacity for pHi regulation is reduced throughout development (Fig. 6.2). These findings are highly significant as they demonstrate for the first time that preferential pHi regulation occurs in an amniote, and that the pattern of acid-base regulation changes throughout ontogeny. This suggests that preferential pHi regulation may be an embryonic pattern of acid-base regulation and that preferential pHi regulation may be retained or lost in adults. Chapter 5 investigated acid-base regulation in another hypercarbia tolerant amniote, Alligator mississippiensis (American alligator), to see if I could corroborate findings from Chapter 4; i.e. whether other amniote embryos use preferential pHi regulation. As alligator embryos are hypercarbia tolerant, I hypothesized that embryos will preferentially regulate pHi; this is supported by the results. The importance of this finding is that it demonstrates preferential pHi regulation occurs during development in another, distantly related, amniote species, which further strengthens support for the hypothesis that preferential pHi regulation is an embryonic pattern of acid-base regulation. Together, these chapters greatly expand the understanding of preferential pHi regulation as strategy of acid-base regulation that provides exceptional pHi protection during hypercarbia-induced respiratory acidoses in a large number of diverse fishes, and may represent an embryonic strategy as indicated by its use in embryonic amniotes.   6.2 Preferential pHi regulation: A common and distinct pattern of acid-base regulation  Before this dissertation, preferential pHi regulation was considered to be a novel and rare pattern of acid-base regulation (Fig. 1.4) (Fig. 1.4; Brauner and Baker, 2009; Brauner et al., 2004); however, based upon my findings in this thesis, this does not appear to be the case. The findings in Chapter 4 and 5 that preferential pHi regulation is  109 used by developing amniotes was intriguing but perhaps is expected given the putative limitations embryos face during development. While the mechanisms remain yet to be uncovered, use of preferential pHi regulation allows embryos to compensate for acid–base challenges to pHi, despite the incomplete formation of the extracellular compartment and associated structures (e.g. cardiovascular, respiratory, and renal systems) that are required for coupled pH regulation. Embryos may also experience additional challenges due to encapsulation within extra-embryonic structures (e.g. eggshells or egg capsules) (Goldberg et al., 2008; Tazawa, 1980). These structures typically permit the perfusion of O2 and CO2 but create diffusion gradients (Ciuhandu et al., 2007; Goldberg et al., 2008; Tazawa, 1980); additionally, in the aquatic, but not terrestrial environment, limited exchange of ions may occur (Alderdice, 1988; Everaert et al., 2011) depending on the permeability of the specific extra-embryonic structure. Reduced exchange of gases and acid-base ion equivalents with the external environment may putatively limit the ability of encapsulated embryos to use coupled pH regulation (Erasmus et al., 1971). The results in Chapters 4 and 5, along with the physical limitations posed by encapsulation, led me to hypothesize that preferential pHi regulation may represent the basal pattern of acid–base regulation in vertebrates as an acid–base regulatory strategy during development, with adults either retaining or losing this trait. Thus, preferential pHi regulation may in fact be ubiquitous amongst vertebrates (and possibly invertebrates, which can face many of the same challenges as vertebrates) (e.g. Kikkawa et al., 2008; Portner et al., 1998; Spicer et al., 2007; Spicer et al., 2011). Further studies should examine other embryos at various developmental stages to 1) assess how they respond to acid-base challenges, 2) determine at which developmental stage preferential pHi regulation is lost, and 3) assess the role of encapsulation on the pattern of acid-base regulation (e.g. compare encapsulated and free-swimming embryos). If preferential pHi regulation is an embryonic trait, then the occurrence of preferential pHi regulation in adult species may represent the retention of the embryonic trait; whereas, species exhibiting only coupled pH regulation would imply the loss of preferential pHi regulation and the acquisition of coupled pH regulation, which may represent a derived strategy of acid-base regulation. In comparison to the start of this  110 thesis (Fig. 1.4), the number of species identified to use preferential pHi regulation has increased greatly, which represents a fundamental shift in the quantity and phylogenetic distribution of species using preferential pHi regulation. Based upon my work (in collaboration with others) preferential pHi regulation has been identified for the first time in adult lamprey (Entosphenus tridentatus), elasmobranchs (e.g. Potamotrygon spp.), basal sarcopterygians (e.g. Lepidosiren paradoxa), and numerous additional species have been included in groups where only a single species had previously been known to use preferential pHi regulation (e.g. basal actinopterygians, Siluriformes, Perciformes). The diversity of these species includes tropical, subtropical and temperate species; additionally, there is a mix of water and bimodal breathers (Table 6.1). This suggests preferential pHi regulation is not restricted to a particular taxonomic group, geographic area or mode of respiration, and it may be that the physical characteristics of the environment are strong selectors for this pattern of acid-base regulation. As preferential pHi regulation was believed to be a rare strategy at the start of this thesis, I specifically targeted fishes that would likely live in high CO2 environments as they would be more likely to use preferential pHi regulation; this approach likely created a bias towards identifying species that preferentially regulate pHi compared to those using coupled pH regulation in Chapter 3. All the fishes sampled here primarily reside in freshwater, which is likely the more challenging environment for acid-base regulation compared to marine environments, due to the greater likelihood of severe hypercarbia (Brauner and Baker, 2009; Furch and Junk, 1997; McNeil and Sasse, 2016; Raymond et al., 2013). If marine fishes were sampled, it is likely this would include many coupled pH regulators as the marine environment is typically more stable and the availability of acid-base relevant counter ions for pHe regulation would allow for the loss of preferential pHi regulation as coupled pH regulation would be feasible. However, some marine environments may pose challenges for acid-base regulation, such as the intertidal zone (Richards, 2011) and near deep-sea CO2 vents (Ishimatsu et al., 2008); these could be environments that select for the retention of preferential pHi regulation in adults. Despite that my selection of species likely underrepresented the prevalence of coupled pH regulation, that preferential pHi regulation was identified in so many species was  111 unexpected and represent an important finding demonstrating that it is relatively common and widespread among fishes. The putative retention of preferential pHi regulation in many adult vertebrates is likely influenced by the environment. Many adult fishes using preferential pHi regulation inhabit environments characterized by challenging conditions for acid-base regulation. For example, Amazonian fishes (e.g. Colossoma macropomum) live in ion-poor waters with naturally low pH that experience large fluctuations in water PCO2 and PO2 (Pinardi et al., 2014; Val et al., 2005). Using preferential pHi regulation may be the only viable solution to the acid-base challenges associated with these environments; particularly under conditions such as severe acute hypercarbia, it is highly unlikely that compensation of pHe could occur. Conversely, the putative loss of preferential pHi regulation in some adult vertebrates may reflect the fact that they inhabit environments where pHe can be sufficiently regulated. For example, fishes such as Atlantic cod and rainbow trout typically reside in normocarbic, normoxic waters with sufficient ions for acid-base relevant ion exchange that allow them to easily compensate pHe during the acid-base challenges they may experience. Similarly, adult amniotes (e.g. turtles and alligators), which are terrestrial air breathers, are able to adjust air convection requirements to compensate for an acid-base disturbance. While the direct involvement of the environment in influencing the pattern of acid-base regulation has not been thoroughly assessed, some work on this has been conducted. Environmental influences on acid-base regulation have been implicated in O. mykiss exposed to hypercarbia in waters containing different ionic composition which resulted in large differences in the degree and speed of pHe compensation (Larsen and Jensen, 1997). In Pangasianodon hypophthalmus exposed to hypercarbia, they were observed to preferentially regulate pHi but also compensate pHe in unaltered pond water; however, when placed in hypercarbia in pond water with artificially lowered pH, they only regulated pHi and not pHe (R.B.S., M. Sackville, C. Damsgaard, L.M. Phuong, M. Hvas, T. Wang, M. Bayley, D.T.T. Huong, N.T. Phuong, and C.J.B., unpublished observations and reviewed in Shartau et al., 2016a); this is consistent with the idea of environmental influence on the pattern of acid-base regulation. This indicates that patterns of pH regulation are not fixed, and that animals can switch between coupled pH regulation and preferential pHi regulation, and that pHe regulation is  112 vulnerable to changes in environmental parameters. Whether patterns of pH regulation are fixed/determined or if animals can switch between coupled pH regulation and preferential pHi regulation is unknown, but it does appear that pHe regulation itself is vulnerable to environmental changes.  6.2.1 Inter- and intra-specific variation of preferential pHi regulation Amongst preferential pHi regulators, the capacity for pHi regulation is not uniform. While Chapter 2 demonstrated that A. transmontanus do not protect all tissues against metabolic acidoses (Shartau et al., 2017a), a similar study on P. pardalis indicated that preferential pHi regulation is a general strategy as they protected pHi following exhaustive exercise, anoxia and metabolic alkalosis (Harter et al., 2014). As both A. transmontanus and P. pardalis exhibit tremendous pHi protection during hypercarbia it is unclear as to why the former has less control over pHi during metabolic acidoses; however, it may be associated with differences in their capacity for pHe regulation. Unlike A. transmontanus, P. pardalis appear to have little to no capacity for pHe regulation, as evident from the near complete lack of pHe compensation at all PCO2 exposures over 24-96 h, including at ca. 1 kPa PCO2 (Brauner et al., 2004); A. transmontanus can fully compensate pHe while preferentially regulating pHi at 1.5 kPa PCO2 (Baker et al., 2009a). Thus, tissues in P. pardalis may possess a more robust capacity for pHi regulation to deal with the lack of any pHe compensation, which could be further underlined by the need to respond to naturally occurring severe acid-base challenges. The capacity for pHi regulation varies between tissues, and different species exhibit different degrees of variability in those tissues. In some species that use preferential pHi regulation, the degree of pHi regulation varies between tissues, for example, L. oculatus exposed to 6 kPa PCO2 exhibit a significant increase in heart pHi but no pH change in other tissues (Fig. 3.2B); however, Atractosteus spatula exposed to 6 kPa PCO2 exhibit a significant increase in liver pHi but no pH change in other tissues (Fig. 3.2C). While most tissues amongst pHi preferentially regulating species remain unchanged during hypercarbia, Figure 3.2 demonstrates the variation amongst species and tissues. The reason for this variation is not known but it could indicate that those  113 tissues which are becoming alkalotic relative to normocarbia possess a greater capacity for pHi regulation and/or that those tissues err on the side of pHi overcompensation. Conversely, that other tissues do not experience pHi change during hypercarbia could indicate that they have a greater capacity to tightly maintain intracellular pH homeostasis, which could be the optimal strategy to avoid intracellular disruption. Another possibility is that differences in pHi regulation amongst tissues reflect animals prioritizing some tissues over others, which may occur if pHi regulation becomes more challenging as blood acidity increases. In this situation, less critical tissues (e.g. muscle) may be less tightly regulated to avoid exacerbating the pHe reduction to ensure pHi of critical tissues (e.g. brain) can be well protected. These differences in pHi regulation may reflect differences in cellular mechanisms between tissues. As pHi regulation is likely nearly instantaneous, based on the A. transmontanus heart pHi response to hypercarbia (Baker, 2010; Shartau et al., 2016a), it is likely that the cellular transporters involved in acid-base regulation are present in the plasma membrane. Transporters could increase in activity as pHi deviates from the transporter’s pH set point, thus triggering pHi regulation. Indeed, in mammalian cells, increased activation of acid-base transporters such as NHEs and MCTs occurs when pH moves away from the pH set point of steady state pH levels and restoration of pHi is initiated (Demaurex, 2002; McBrian et al., 2013; Schapiro and Grinstein, 2000; Tokudome et al., 1990). The cellular and molecular mechanism of preferential pHi regulation remain unknown and thus this remains an important area of future research on this topic.   6.3 Preferential pHi regulation: A potential developmental and evolutionary strategy to cope with acute acid–base disturbances  Preferential pHi regulation may be the embryonic strategy of acid-base regulation as it allows embryos to protect their cells and tissues without relying on pH regulation of the external medium (i.e. pHe); this may be particularly important during severe acid-base disturbances. Retention or loss of preferential pHi regulation in adulthood may due to  114 environmental and physiological factors as use of preferential pHi regulation in more challenging environments is likely to enhance survival; however, many vertebrates appear to lose the capacity for preferential pHi regulation and acquire coupled pH regulation. The reason for the loss of preferential pHi regulation is not understood. Preferential pHi regulation offers exceptional tolerance to severe acute respiratory acid-base disturbances and does not appear to be metabolically costly at the whole animal level during severe hypercarbia exposure (Baker and Brauner, 2012); however, the putative advantages of preferential pHi regulation may not apply uniformly to metabolic acidoses (Shartau et al., 2017a) and it is not known how well preferential pHi regulation functions during chronic acid-base disturbances. Furthermore, although the metabolic cost of preferential pHi regulation has been determined to be low, this was only investigated A. transmontanus (Baker and Brauner, 2012); it is not known if this applies to other species. Additionally, there may be costs not yet determined or quantified with having a greater imbalance in pH between the extracellular and intracellular compartments; these could be related to disruption of membrane proteins affecting cellular function.  6.3.1 Preferential pHi regulation: An exaptation for vertebrate evolution? Vertebrates are believed to have had a marine origin (Carrete Vega and Wiens, 2012; Halstead, 1985); the marine environment is generally characterized by being relatively stable with respect to PCO2, PO2, and temperature (compared to freshwater), which may limit the occurrence of severe environmental acid-base disturbances. Additionally, marine environments are ion-rich, which may have facilitated the use of coupled pH regulation during acid-base challenges; use of this strategy is observed in a basal marine vertebrate, the Eptatretus stoutii (Pacific hagfish), an osmo- and iono-conformer, which compensates pHe and pHi during a hypercarbic-induced respiratory acidosis (Baker et al., 2015), as well as marine elasmobranches and teleosts (Brauner and Baker, 2009; Shartau et al., 2016a; Wood et al., 1990). The transition of vertebrates from marine to ion-poor fresh water likely posed a challenge for acid-base regulation due to the reduced availability of counter-ions for coupled pH regulation. Consequently, the transition to freshwater may have led to the broader retention of preferential pHi  115 regulation in adults. This transition likely occurred approximately 420-430 million years ago in the late Silurian (Halstead, 1985) by the ancestors of the basal euteleostom fishes where global average temperature and atmospheric CO2 tensions were higher than present day levels (Clack, 2007). These conditions may have resembled present day tropical systems, such as the Amazon River, which have warm, ion-poor, CO2-rich waters (Furch and Junk, 1997) and may have promoted the retention of preferential pHi regulation to maintain acid-base homeostasis; thus, protecting against acid-base disturbances that would otherwise be intolerable if relying on coupled pH regulation (Brauner and Baker, 2009; Shartau and Brauner, 2014). As fishes colonized tropical freshwater environments, preferential pHi regulation may have been beneficial when encountering severe hypercarbic conditions present in many of these habitats (Furch and Junk, 1997; Heisler, 1984; Li et al., 2013; Ultsch, 1987). Preferential pHi regulation confers exceptional CO2 tolerance in nearly all species investigated in this thesis (Chapter 3) and CO2 tolerance in these environments may be important as hypercarbia is often associated with aquatic hypoxia (Ultsch, 1987); the latter of which is believed to be the primary driver of the evolution of air breathing in fishes (Graham, 1997; Randall et al., 1981). As fishes developed the capacity for air breathing, and became bimodal breathers, the retention of preferential pHi regulation may provide a means to cope with acid–base challenges associated with air breathing (Shartau and Brauner, 2014). Bimodal breathing fishes take up O2 from water or air, but typically excrete the majority of CO2 to the water as the capacitance of water for CO2 is much greater than air (Graham, 1997; Randall et al., 1981). Consequently, in bimodal breathing fishes, air breathing leads to a rapid increase in blood PCO2, as CO2 excretion rates at the gills are reduced due to emersion or reduced gill blood flow and/or gill ventilation. Depending on the species and conditions, blood PCO2 can increase to >3 kPa PCO2 during an air-breathing episode causing a reduction in pHe (Shartau and Brauner, 2014). Air-breathing in fishes is thought to have evolved in tropical environments that likely experience both hypoxia and hypercapnia (Ultsch, 1987); thus, these fishes may have already been subjected to selection pressures to retain preferential pHi regulation, which could then serve as an exaptation for dealing with an air breathing induced respiratory acidosis (Brauner and Baker, 2009; Shartau and Brauner, 2014).  116 The transition of vertebrates from water to land posed a number of physiological challenges due to the physical differences between the aquatic and terrestrial environments; one of these is acid-base regulation. Transitioning from an aquatic water breather to a terrestrial air breather involved changes in blood acid-base status as the former have low PCO2, low plasma [HCO3-], and high pHe, while the latter have high PCO2, high plasma [HCO3-], and low pHe (Randall et al., 1981; Ultsch, 1996). Additionally, acid-base regulation between the two differs in that water breathers rely on physicochemical buffering and net transport of acid-base equivalents, while air breathers depend mainly on changes in ventilation rate to alter PCO2 and thus pH (Brauner and Baker, 2009). Exactly how early vertebrates made this transition is not known but it is hypothesized that use of preferential pHi regulation may have played an important role in minimizing the effects of respiratory acidosis (Brauner and Baker, 2009; Shartau and Brauner, 2014; Shartau et al., 2016a). Although early terrestrial vertebrates were semi-aquatic bimodal breathers, they still excreted the majority of CO2 into the water (Janis et al., 2012); therefore, preferential pHi regulation may have been important to deal with the respiratory acidosis associated with terrestrial excursion when venturing onto land, to forge, escape predation, or related to reproduction. As vertebrates became more dependent on air breathing (i.e. moved from being facultative to obligate air breathers), and terrestrial excursions became longer, the rise in blood PCO2 would have become greater, resulting in increasingly severe respiratory acidosis (Janis et al., 2012). The increase in plasma [HCO3-] in air breathers may be due to increased renal HCO3- production and retention (Gonzalez et al., 2010). Additionally, it has been postulated that highly vascularized dermal bone was involved in providing HCO3- to buffer respiratory acidosis (Janis et al., 2012), which may have been used during the terrestrialization of tetrapods as bone and shell contribute to buffering acidosis associated with anoxia in reptiles (Jackson, 2003; Jackson et al., 2000a; Jackson et al., 2000b) and amphibians (Warren, 2005), but not in fish (Harter et al., 2014). Preferential pHi regulation may have acted as an important intermediate step protecting tissues during these transitory stages. Increased blood PCO2 would have made excretion of CO2 into the air easier due to the large diffusion gradient; consequently, tight regulation of pHe would  117 be possible via control of ventilatory rate. Unlike water breathers where ventilatory rate is typically O2 dependent as O2 is often more limited in aquatic environments, terrestrial air breathers are typically not O2 limited; thus, control of ventilation is moderated by regulation of blood pH, which is adjusted by adjusting blood PCO2 by changing air convection requirement (Cameron, 1989a). This, along with the use of the kidneys as the primary organ for acid-base regulation permits tetrapods to tightly control pHe, and thus maintain pHi homeostasis. These ideas are highly speculative but presently little is known about this transition in acid-base status.   6.4 Future research directions  This thesis has made a significant advancement to the area of preferential pHi regulation; yet more work is needed to fully understand this pattern of acid-base regulation, and as such, many interesting areas remain to be examined. Some of the topics for future research I believe are worth investigating are described below.  6.4.1 Survey of fish species With an estimated 32,000+ fish species (Nelson, 2006), investigating the strategy of acid-base regulation in even a fraction of these species would be a challenging task. However, it would be highly informative if a few additional species were examined, allowing for a better understanding of the diversity (or lack thereof) of acid-base regulatory strategies amongst fishes. While Chapter 3 attempted to include a diverse sample of species, there is still considerable room for improvement in order to gain a broader appreciation for how fishes regulate pH and to avoid overgeneralizing. As previously indicated, many of the species were selected to obtain a high success rate of identifying species using preferential pHi regulation when I thought it was a relatively rare phenomenon. To obtain a more comprehensive understanding of the strategy of acid-base regulation amongst various groups it would be beneficial to include a few additional species and groups. This thesis has not investigated any marine fishes, which include a phylogenetically diverse range of fishes. Similar to freshwater fishes, it would be  118 informative to investigate basal and derived marine species, as well as air breathers. A couple of basal marine groups that should be examined in more detail include the Chondrichthyes and coelacanths. Few studies have investigated acid-base regulation in Chondrichthyes in response to severe pH disturbances and almost none have measured both pHe and pHi. The coelacanths are the only extant marine sarcopterygian fishes and thus would be fascinating based on their habitat and phylogenetic position (however, obtaining these fishes would be undoubtedly challenging and/or cost prohibitive). There are numerous marine air breathing fishes amongst the teleosts, ranging from the basal to derived including the Elopiformes (Megalops atlanticus Atlantic tarpon and M. cyprinoides ox eye herring), three families within the Salmoniformes (Umbridae, Lepidogalaxiidae, and Galaxiidae), sculpins in the order Scorpaeniformes (family Cottidae), and mudskippers in the order Perciformes (family Gobiidae) (Graham, 1997). Many of the species investigated in Chapter 3 are tropical or sub-tropical air breathers; one unique air breather that would be interesting to include in this survey would be the Arctic air breathing fish Dallia pectoralis (Alaska blackfish) (Lefevre et al., 2014). While the polar environment differs greatly from those in the tropics/sub-tropics, they are subjected to periods of hypoxia as lakes freeze and thus, may be exposed to acid-base challenges where preferential pHi regulation would be useful. These groups and species are not exhaustive in terms of which fishes might be worth investigating, but hopefully provide some idea where to continue this survey of acid-base regulation in fishes.  6.4.2 Acid-base regulation during development Similar to the survey in Chapter 3, the discovery of preferential pHi regulation in embryonic amniotes in Chapters 4 and 5 warrant further investigations into other species. The work in Chapters 4 and 5 has resulted in a new hypothesis that preferential pHi regulation is an embryonic pattern of acid-base regulation that is either retained or lost in adults; however, additional work is needed to fully support this hypothesis and investigating acid-base regulation in other embryonic vertebrates are needed, including fishes, especially those using coupled pH regulation (e.g. Oncorhynchus mykiss). Among other vertebrates, embryonic chickens would be useful to examine as they are well studied with respect to changes in pHe during hypercarbia but nothing is known about  119 how pHi is affected (Andrewartha et al., 2014; Burggren et al., 2012; Everaert et al., 2011; Mueller et al., 2014). Investigating a broad phylogenetic representation of amphibians, reptiles, birds, and mammals during development would be invaluable towards supporting (or refuting) this new hypothesis.  6.4.3 Chronic acid-base disturbances This thesis has exclusively focused on acute acid-base challenges, yet chronic acid-base disturbances are common and it is largely unknown if preferential pHi regulation also protects against chronic disturbances. One study examined Anguilla anguilla (European eel) during a six-week exposure to hypercarbia at 2, 4 or 6 kPa PCO2 and found that despite pHe being only partially compensated at 6 weeks, pHi of heart and white muscle were protected (McKenzie et al., 2003). This suggests that A. anguilla preferentially regulate pHi during chronic acid-base disturbances (the response during acute hypercarbia is not known in A. anguilla). As responses to acute and chronic acid-base disturbances can vary considerably in species using coupled pH regulation, as demonstrated by O. mykiss (Brauner and Baker, 2009; Smart et al., 1979). It would be valuable to understand if there are differences in capacity/ability for preferential pHi regulation between acute and chronic exposures by examining the acute (<48 h) and chronic (>4 weeks) acid-base disturbances.  6.4.4 Role of the environment As indicated previously, it is hypothesized that the loss or retention of preferential pHi regulation is determined by environmental and physiological factors. Results from P. hypophthalmus suggest that indeed, the environment may be associated with the ability of fishes to regulate pHe and thus require them to use preferential pHi regulation (R.B.S., M. Sackville, C. Damsgaard, L.M. Phuong, M. Hvas, T. Wang, M. Bayley, D.T.T. Huong, N.T. Phuong, and C.J.B., unpublished observations). It has been shown that rainbow trout pHe regulation is dependent on environmental ion availability (Larsen and Jensen, 1997). Studies investigating the role of the environment on the pattern of acid-base regulation would be informative about the selective pressures for the putative retention of preferential pHi regulation. Possible experiments could include examining pHe and pHi  120 changes in response to hypercarbia exposure in water with various ion composition, similar to Larsen and Jensen’s study (Larsen and Jensen, 1997), with ionic concentrations ranging from ion-poor, such as found in the Rio Negro (Brauner et al., 2004), to that of typical hard water (Lecuyer, 2014). This could also include varying water pH as low water pH inhibits pHe regulation (Lin and Randall, 1995; Shartau et al., 2017b), which could promote preferential pHi regulation. One particularly interesting study would be to see if P. pardalis are capable of pHe regulation if the optimal conditions are provided as their capacity for pHe compensation is extremely limited in their natural environment.  6.4.5 Mechanism(s) of preferential pHi regulation Lastly, one area that needs to be addressed is the molecular and cellular mechanisms of preferential pHi regulation as nothing is presently known. This may be a challenging task given that different tissues have different responses (Shartau et al., 2017a), that there are differences between developmental stages (Shartau et al., 2016b), all of which may use different mechanisms. Additionally, while Huynh et al. indicated that cell culture approach may work for investigating the mechanism of preferential pHi regulation, they also showed that the in vitro response differs from the in vivo response; possibly suggesting that extrinsic factors are involved (Huynh et al., 2011a). However, as preferential pHi regulation did occur in vitro, this may represent one possible approach. Another possible technique is to use tissue slices (e.g. liver) exposed to hypercarbia and treated with various pharmacological inhibitors to assess how pHi changes. This technique leaves cells in their extracellular matrix, allowing them to associate with each other and may be more natural than cell culture; this approach has been widely used in toxicology work in salmonids (Lemaire et al., 2011; Singh et al., 1996; Thohan et al., 2001). Regardless of the approach used, understanding the mechanisms will be important to fully understand how preferential pHi is regulated and how it functions in response to different types of acid-base disturbances and in different environments.    121 6.5 Summary and final thoughts  At the start of this dissertation, coupled pH regulation had been considered ‘the’ pattern of acid-base regulation for decades (Albers, 1970; Cameron, 1989b; Heisler, 1984; Occhipinti and Boron, 2015; Roos and Boron, 1981) and preferential pHi regulation was a novel and rare strategy of acid-base regulation limited to a mere four species: three fishes and one aquatic tetrapod. This dissertation provides evidence that preferential pHi regulation is no longer a novel, nor rare strategy of acid-base regulation and thus may represent a paradigm shift regarding vertebrate acid-base regulation. Unexpectedly, preferential pHi regulation does not confer uniform protection for tissues against all types of acid-base disturbances, at least in white sturgeon (Chapter 2). However, my findings indicate that adults of at least an additional 15 species (Chapter 3) and embryos of two species (Chapters 4 and 5), use preferential pHi regulation during severe acute hypercarbia, which is an exciting expansion in the number of species exhibiting this strategy. The most interesting and surprising finding in this dissertation is that developing amniotes use preferential pHi regulation (Chapters 4 and 5). The work in Chapters 3-5 required the original hypothesis of my thesis, that preferential pHi regulation evolved in the ancestors of the basal euteleostomi, to be modified to: preferential pHi regulation is an embryonic strategy that is either retained or lost in adults. The implication of this, which remains to be fully tested, is that all vertebrates use preferential pHi regulation at one point during their life history. The putative retention of this embryonic strategy of acid-base regulation may have been an exaptation for maintaining pH homeostasis as adults in challenging environments, including during the major evolutionary transition to air breathing and the transition from life in water to life on land.  122  Figure 6.1: Phylogeny showing the distribution of preferential intracellular pH (pHi) regulation and coupled pH regulation amongst vertebrates when exposed to acute >2 kPa PCO2 following completion of dissertation research. Species using preferential pHi regulation during severe acute hypercarbia are indicated in pink, while those using coupled pH regulation are indicated in blue. This phylogeny builds on Figure 1.4 and includes species examined prior to, and during this dissertation. All species were examined during Chapter 3 with the exception of L. osseus, O. bicirrhosum, P. hypophthalmus, and M. salmoides. Data from L. osseus19, O. bicirrhosum20, and M. salmoides22 is not included in this thesis due to limited n’s from sampling; however, based on the CO2 tolerance assay, they preferentially regulate pHi and are included in this figure to further demonstrate the prevalence of preferential pHi regulation. Results from P. hypophthalmus are part of an unpublished project 21(R.B.S., M. Sackville, C. Damsgaard, L.M. Phuong, M. Hvas, T. Wang, M. Bayley, D.T.T. Huong, N.T.  Phuong, C.J.B., unpublished). Other relevant references are indicated by numbers following  123 species name - 1(Baker et al., 2015), 2(Wood et al., 1990), 3(Snyder and Nestler, 1991), 4(Heisler et al., 1982), 5(Wasser et al., 1991), 6(Snyder et al., 1995), 7(Snyder et al., 1995), 8(Malan et al., 1985), 9(Wood and Schaefer, 1978), 10(Gonzalez and Clancy, 1986a), 11(Yaksh and Anderson, 1987), 12(Arieff et al., 1976), 13(Baker et al., 2009a), 14(Brauner et al., 2004), 15(Wood and LeMoigne, 1991), 16(Larsen et al., 1997), 17(Wright et al., 1988), 18(Heisler, 1982). Phylogenetic relationships are based on (2009) and branch lengths are taken from various references utilizing fossil and molecular estimates of divergence times (Aschliman et al., 2012; Betancur-R et al., 2013; Betancur-R et al., 2015; Blair, 2005; Macqueen and Johnston, 2014; Meredith et al., 2011; Zhang et al., 2013); the phylogenetic tree was created using Mesquite (Maddison and Maddison, 2017).  124  Figure 6.2: Difference in tissue pH during development in turtles. Difference in tissue pH (pHi) during development is shown following exposure to hypercarbia relative to normocarbia in Chelydra serpentina [common snapping turtle; at 70% and 90% to hatch and in yearlings] and adult Chrysemys picta bellii (western painted turtles). Control pH values for pHi of heart (red circle), liver (yellow square), brain (blue triangle) and muscle (grey inverse triangle) were subtracted from the values determined following either 1 h exposure to 13 kPa PCO2, 9 kPa PO2 in C. serpentina or 1 h exposure to 6.5 kPa arterial PCO2 in C. picta bellii. Values ≥ 0 in the light red portion of the figure are indicative of preferential pHi regulation while values ≤ in the light blue portion of the figure are indicative of coupled pH regulation. This figure shows that turtles preferentially regulate pHi early in development and that the capacity for pHi regulation is reduced throughout development. Significant changes in pHi from control are indicated by asterisk (P<0.05); in all developmental stages extracellular pH (pHe) was significantly reduced during hypercarbia exposure (P<0.05).   125 Table 6.1: Fish species investigated in this dissertation. Order Family Species Pattern of acid-base regulation Biogeographical realm1 Water/air breather Petromyzontiformes Petromyzontidae Entosphenus tridentatus ppHi Nearctic Water Myliobatiformes Potamotrygonidae Potamotrygon sp. ppHi Neotropic Water Ceratodontiformes Lepidosirenidae Lepidosiren paradoxa  ppHi Neotropic Air Acipenseriformes Polydontidae Polyodon spathula ppHi Nearctic Water Acipenseridae Acipenser transmontanus2,3 ppHi Nearctic Water Leisosteiformes Lepisosteidae Lepisosteus oculatus ppHi Nearctic Air Atractosteus spatula ppHi Nearctic Air Osteoglossiformes Osteoglossidae Arapaima gigas ppHi Neotropic Air Gymnotiformes Gymnotidae Electrophorus electricus ppHi Neotropic Air Characiformes Serrasalmidae Colossoma macropomum ppHi Neotropic Water Characidae Brycon amazonicus ppHi Neotropic Water Siluriformes Callichthyidae Hoplosternum littorale ppHi Neotropic Air Loricariidae Pterygoplichthys pardalis2,4 ppHi Neotropic Air Ictaluridae Ictalurus punctatus ppHi Nearctic Water I. punctatus X I. furcatus ppHi Nearctic Water Pangasiidae Pangasianodon hypophthalmus5 ppHi Indomalayan Air    126 Order Family Species Pattern of acid-base regulation Biogeographical realm1 Water/air breather Salmoniformes Salmonidae Oncorhynchus kisutch Coupled Nearctic Water Oncorhynchus mykiss2,6 Coupled Nearctic Water Synbranchiformes Synbranchidae Synbranchus marmoratus2,7 ppHi Neotropic Air Perciformes Cichlidae Oreochromis niloticus ppHi Africotropical Water Astronotus ocellatus ppHi Neotropic Water  Taxonomic information for order and family are indicated, pattern of acid-base regulation (preferential pHi regulation – ppHi, or coupled pH regulation – coupled) as determined from Chapter 3 results are shown. Biogeographical realms are indicated in order to show that the survey included fishes from various regions. A mix of water and air breathers were used, this is indicated. All fishes were primarily freshwater or freshwater-brackish inhabitants. All fishes were examined as part of Chapter 3 unless otherwise stated, footnotes indicate applicable references.1(Udvardy, 1975), 2Chapter 3, 3(Baker et al., 2009a), 4(Brauner et al., 2004), 5(R.B.S., M. Sackville, C. Damsgaard, L.M. Phuong, M. Hvas, T. Wang, M. Bayley, D.T.T. Huong, N.T.  Phuong, C.J.B., unpublished), 6(Wood and LeMoigne, 1991), 7(Heisler, 1982).  127 References  Aboagye, D. L. and Allen, P. J. (2014). Metabolic and locomotor responses of juvenile paddlefish Polyodon spathula to hypoxia and temperature. Comp. Biochem. Phys. A 169, 51–59. Aboagye, D. L. and Allen, P. J. (2017). Effects of acute and chronic hypoxia on acid–base regulation, hematology, ion, and osmoregulation of juvenile American paddlefish. J. Comp. Physiol. B. doi.org/10.1007/s00360-017-1104-7. Ackerman, R. A. (1977). The respiratory gas exchange of sea turtle nests (Chelonia, Caretta). Respir. Physiol. 31, 19–38. Ahmed, K. H. (2006). Signalling pathways involved in hypertonicity- and acidification-induced activation of Na+/H+ exchange in trout hepatocytes. J. Exp. Biol. 209, 3101–3113. Albers, C. (1970). Acid-base balance. In Fish Physiology (eds. Hoar, W. S. and Randall, D. J., pp. 173–208. New York: Academic Press. Alderdice, D. F. (1988). Osmotic and ionic regulation in teleost eggs and larvae. In Fish Physiology (eds. Hoar, W. S. and Randall, D. J.), pp. 163–251. New York: Academic Press. Allen, P. J., Mitchell, Z. A., DeVries, R. J., Aboagye, D. L., Ciaramella, M. A., Ramee, S. W., Stewart, H. A. and Shartau, R. B. (2014). Salinity effects on Atlantic sturgeon (Acipenser oxyrinchus oxyrinchus Mitchill, 1815) growth and osmoregulation. J. Appl. Ichthyol. 30, 1229–1236. Andrewartha, S. J., Tazawa, H. and Burggren, W. W. (2014). Acute regulation of hematocrit and acid-base balance in chicken embryos in response to severe intrinsic hypercapnic hypoxia. Respir. Physiol. Neurobiol. 195C, 1–10. Arieff, A. I., Kerian, A., Massry, S. G. and DeLima, J. (1976). Intracellular pH of brain: alterations in acute respiratory acidosis and alkalosis. Am. J. Physiol. 230, 804–812. Aschliman, N. C., Nishida, M., Miya, M. and Inoue, J. G. (2012). Body plan convergence in the evolution of skates and rays (Chondrichthyes: Batoidea). Mol. Phylogen. Evol. 63, 28-42. Atilla, N., McKinley, G. A., Bennington, V. and Baehr, M. (2011). Observed variability of Lake Superior pCO2. Limnol. Oceanogr. 56, 775–786. Bagherzadeh Lakani, F., Sattari, M., Sharifpour, I. and Kazemi, R. (2013). Effect of hypoxia, normoxia and hyperoxia conditions on gill histopathology in two weight groups of beluga (Huso huso). Caspian J. Env. Sci. 11, 77–84.    128 Baker, D. W. (2010). Physiological responses associated with aquatic hypercarbia in the CO2-tolerant white sturgeon, Acipenser transmontanus. PhD thesis. University of British Columbia, Vancouver, BC. Baker, D. W. and Brauner, C. J. (2012). Metabolic changes associated with acid–base regulation during hypercarbia in the CO2-tolerant chondrostean, white sturgeon (Acipenser transmontanus). Comp. Biochem. Phys. A 161, 61–68. Baker, D. W., Hanson, L. M., Farrell, A. P. and Brauner, C. J. (2011). Exceptional CO2 tolerance in white sturgeon (Acipenser transmontanus) is associated with protection of maximum cardiac performance during hypercapnia in situ. Physiol. Biochem. Zool. 84, 239–248. Baker, D. W., Matey, V., Huynh, K. T., Wilson, J. M., Morgan, J. D. and Brauner, C. J. (2009a). Complete intracellular pH protection during extracellular pH depression is associated with hypercarbia tolerance in white sturgeon, Acipenser transmontanus. Am. J. Physiol. Regul. Integr. Comp. Physiol. 296, R1868–80. Baker, D. W., May, C. and Brauner, C. J. (2009b). A validation of intracellular pH measurements in fish exposed to hypercarbia: the effect of duration of tissue storage and efficacy of the metabolic inhibitor tissue homogenate method. J. Fish Biol. 75, 268–275. Baker, D. W., Sardella, B., Rummer, J. L., Sackville, M. and Brauner, C. J. (2015). Hagfish: Champions of CO2 tolerance question the origins of vertebrate gill function. Sci. Rep. 5, 11182. Baker, D. W., Wood, A. M. and Kieffer, J. D. (2005a). Juvenile Atlantic and shortnose sturgeons (family: Acipenseridae) have different hematological responses to acute environmental hypoxia. Physiol. Biochem. Zool. 78, 916–925. Baker, D. W., Wood, A. M., Litvak, M. K. and Kieffer, J. D. (2005b). Haematology of juvenile Acipenser oxyrinchus and Acipenser brevirostrum at rest and following forced activity. J. Fish Biol. 66, 208–221. Baldwin, J., Seymour, R. S. and Webb, G. J. W. (1995). Scaling of anaerobic metabolism during exercise in the estuarine crocodile (Crocodylus porosus). Comparative Biochemistry and Physiology 112, 285–293. Barnhart, M. C. and McMahon, B. R. (1988). Depression of aerobic metabolism and intracellular pH by hypercapnia in land snails, Otala lactea. J. Exp. Biol. 138, 289–299. Barton, B. A., Bollig, H., Hauskins, B. L. and Jansen, C. R. (2000). Juvenile pallid (Scaphirhynchus albus) and hybrid pallid×shovelnose (S. albus×platorynchus) sturgeons exhibit low physiological responses to acute handling and severe confinement. Comp. Biochem. Phys. A 126, 125–134.    129 Basso, L., Hendriks, I. E. and Rodríguez-Navarro, A. B. (2015). Extreme pH conditions at a natural CO2 Vent System (Italy) affect growth, and survival of juvenile pen shells (Pinna nobilis). Estuar. Coasts 1986–1999. Berenbrink, M., Koldkjaer, P., Kepp, O. and Cossins, A. R. (2005). Evolution of oxygen secretion in fishes and the emergence of a complex physiological system. Science 307, 1752–1757. Bergmeyer, H. U. (1983). Methods of Enzymatic Analysis. New York: Academic Press. Betancur-R, R., Broughton, R. E., Wiley, E. O., Carpenter, K., Lopez, J. A., Li, C., Holcroft, N. I., Arcila, D., Sanciangco, M., Cureton, J. C., II, et al. (2013). The tree of life and a new classification of bony fishes. PLoS Curr. 5, 1-45.  Betancur-R, R., Orti, G. and Pyron, R. A. (2015). Fossil‐based comparative analyses reveal ancient marine ancestry erased by extinction in ray‐finned fishes. Ecol. Lett. 18, 441–450. Blair, J. E. (2005). Molecular Phylogeny and Divergence Times of Deuterostome Animals. Mol Biol. Evol. 22, 2275–2284. Booth, D. T. (1998). Nest temperature and respiratory gases during natural incubation in the broad-shelled river turtle, Chelodina expansa (Testudinata: Chelidae). Aust. J. Zool. 46, 183–191. Boron, W. F. (2004). Regulation of intracellular pH. AJP: Advances in Physiology Education 28, 160–179. Boron, W. F. and De Weer, P. (1976). Intracellular pH transients in squid giant axons caused by CO2, NH3, and metabolic inhibitors. J. Gen. Physiol. 67, 91–112. Boutilier, R. G. and Shelton, G. (1980). The statistical treatment of hydrogen ion concentration and pH. J. Exp. Biol. 84, 335–339. Boutilier, R. G., Heming, T. A. and Iwama, G. K. (1984). Appendix: physicochemical parameters for use in fish respiratory physiology. In Fish Physiology (eds. Hoar, W. S. and Randall, D. J.), pp. 403–430. New York: Academic. Bouyer, P., Bradley, S. R., Zhao, J., Wang, W., Richerson, G. B. and Boron, W. F. (2004). Effect of extracellular acid–base disturbances on the intracellular pH of neurones cultured from rat medullary raphe or hippocampus. J. Physiol. 559, 85–101. Brauner, C. J. (2008). Acid-base balance. In Fish Larval Physiology (eds. Finn, R. N. and Kapoor, B. G.), pp. 185–198. Enfield, NH: Science Publishers. Brauner, C. J. and Baker, D. W. (2009). Patterns of acid-base regulation during exposure to hypercarbia in fish. In Cardio-Respiratory Control in Vertebrates: Comparative and Evolutionary Aspects (eds. Glass, M. L. and Wood, S. C.), pp. 43–63. Berlin, Germany: Springer-Verlag.  130 Brauner, C. J. and Berenbrink, M. (2007). Gas transport and exchange. In Primitive Fishes, Fish Physiology (eds. McKenzie, D. J., Farrell, A. P., and Brauner, C. J.), pp. 213–282. New York: Elsevier. Brauner, C. J., Wang, T., Wang, Y., Richards, J. G., Gonzalez, R. J., Bernier, N. J., Xi, W., Patrick, M. and Val, A. L. (2004). Limited extracellular but complete intracellular acid-base regulation during short-term environmental hypercapnia in the armoured catfish, Liposarcus pardalis. J. Exp. Biol. 207, 3381–3390. Bruggeman, V., Witters, A., De Smit, L., Debonne, M., Everaert, N., Kamers, B., Onagbesan, O. M., Degraeve, P. and Decuypere, E. (2007). Acid-base balance in chicken embryos (Gallus domesticus) incubated under high CO2 concentrations during the first 10 days of incubation. Respir. Physiol. Neurobiol. 159, 147–154. Burda, H., Šumbera, R. and Begall, S. (2007). Microclimate in Burrows of Subterranean Rodents — Revisited. In Subterranean Rodents, pp. 21–33. Berlin, Heidelberg: Springer Berlin Heidelberg. Burggren, W. W., Andrewartha, S. J. and Tazawa, H. (2012). Interactions of acid–base balance and hematocrit regulation during environmental respiratory gas challenges in developing chicken embryos (Gallus gallus). Respir. Physiol. Neurobiol. 183, 135–148. Busk, M., Larsen, E. H. and Jensen, F. B. (1997). Acid-base regulation in tadpoles of Rana catesbeiana exposed to environmental hypercapnia. J. Exp. Biol. 200, 2507–2512. Butman, D. and Raymond, P. A. (2011). Significant efflux of carbon dioxide from streams and rivers in the United States. Nature Geosci 4, 839–842. Cameron, J. N. (1989a). The Respiratory Physiology of Animals. New York: Oxford University Press. Cameron, J. N. (1989b). Acid-Base Homeostasis: Past and Present Perspectives. Physiol Zool 62, 845–865. Carrete Vega, G. and Wiens, J. J. (2012). Why are there so few fish in the sea? Proc. R. Soc. B Biol. Sci. 279, 2323–2329. Cech, J. J. and Crocker, C. E. (2002). Physiology of sturgeon: effects of hypoxia and hypercapnia. J. Appl. Ichthyol. 18, 320–324. Ciuhandu, C. S., Wright, P. A., Goldberg, J. I. and Stevens, E. D. (2007). Parameters influencing the dissolved oxygen in the boundary layer of rainbow trout (Oncorhynchus mykiss) embryos and larvae. J. Exp. Biol. 210, 1435–1445. Clack, J. A. (2007). Devonian climate change, breathing, and the origin of the tetrapod stem group. Integr. Comp. Biol. 47, 510–523. Claiborne, J. B. and Heisler, N. (1984). Acid-base regulation and ion transfers in the carp  131 (Cyprinus carpio) during and after exposure to environmental hypercapnia. J. Exp. Biol. 108, 25–43. Cocherell, D. E., Kawabata, A., Kratville, D. W., Cocherell, S. A., Kaufman, R. C., Anderson, E. K., Chen, Z. Q., Bandeh, H., Rotondo, M. M., Padilla, R., et al. (2011). Passage performance and physiological stress response of adult white sturgeon ascending a laboratory fishway. J. Appl. Ichthyol. 27, 327–334. Cole, J. J., Caraco, N. F. and Kling, G. W. (1994). Carbon dioxide supersaturation in the surface waters of lakes. Science 265, 1568–1570. Crocker, C. E. and Cech, J. J., Jr (1998). Effects of hypercapnia on blood-gas and acid-base status in the white sturgeon, Acipenser transmontanus. J. Comp. Physiol. B 168, 50–60. Crocker, C. E. and Cech, J. J., Jr (1997). Effects of environmental hypoxia on oxygen consumption rate and swimming activity in juvenile white sturgeon, Acipenser transmontanus, in relation to temperature and life intervals. Environ. Biol. Fish 50, 383–389. Crocker, C. E. and Cech, J. J., Jr (1996). The effects of hypercapnia on the growth of juvenile white sturgeon, Acipenser transmontanus. Aquaculture 147, 293–299. Crooks, R. J. and Simkiss, K. (1974). Respiratory acidosis and eggshell resorption by the chick embryo. J. Exp. Biol. 61, 197–202. Crossley, D. A. and Altimiras, J. (2005). Cardiovascular development in embryos of the American alligator Alligator mississippiensis: effects of chronic and acute hypoxia. J. Exp. Biol. 208, 31–39. Crossley, D. and Altimiras, J. (2000). Ontogeny of cholinergic and adrenergic cardiovascular regulation in the domestic chicken (Gallus gallus). Am. J. Physiol. Regul. Integr. Comp. Physiol. 279, R1091–R1098. Damsgaard, C., Gam, L. T. H., Dang, D. T., Van Thinh, P., Huong, D. T. T., Wang, T. and Bayley, M. (2015). High capacity for extracellular acid-base regulation in the air-breathing fish Pangasianodon hypophthalmus. J. Exp. Biol. 218, 1290–1294. de Faria, F., Jaramillo, P. and Sawakuchi, H. O. (2015). Estimating greenhouse gas emissions from future Amazonian hydroelectric reservoirs. Environ. Res. Lett. 10, 124019. de Fátima F L Rasera, M., Krusche, A. V., Richey, J. E., Ballester, M. V. R. and Victória, R. L. (2013). Spatial and temporal variability of pCO2 and CO2 efflux in seven Amazonian Rivers. Biogeochemistry 116, 241–259. Demaurex, N. (2002). pH Homeostasis of Cellular Organelles. Physiology 17, 1–5. Dennis, C. E., Wright, A. W. and Suski, C. D. (2016). Potential for carbon dioxide to act as a non-physical barrier for invasive sea lamprey movement. J. Great Lakes Res. 42, 150–155.  132 Dimberg, K. (1988). High blood CO2 levels in rainbow trout exposed to hypercapnia in bicarbonate-rich hard fresh water - a methodological verification. J. Exp. Biol. 134, 463–466. Eddy, F. B. (1976). Acid-base balance in rainbow trout (Salmo gairdneri) subjected to acid stresses. J. Exp. Biol. 64, 159–171. Edwards, S. L. and Marshall, W. S. (2013). Principles and patterns of osmoregulation and euryhalinity in fishes. In Euryhaline Fishes: Fish Physiology (eds. McCormick, S. D., Farrell, A. P., and Brauner, C. J.), pp. 1–44. New York: Academic Press. Eme, J. and Crossley, D. A. (2015). Chronic hypercapnic incubation increases relative organ growth and reduces blood pressure of embryonic American alligator (Alligator mississippiensis). Comp. Biochem. Phys. A 182, 53–57. Eme, J., Altimiras, J., Hicks, J. W. and Crossley, D. A., II (2011). Hypoxic alligator embryos: Chronic hypoxia, catecholamine levels and autonomic responses of in ovo alligators. Comp. Biochem. Phys. A 160, 412–420. Erasmus, B. W., Howell, B. J. and Rahn, H. (1971). Ontogeny of acid-base balance in the bullfrog and chicken. Respir. Physiol. 11, 46–53. Erdogan, S., FitzHarris, G., Tartia, A. P. and Baltz, J. M. (2005). Mechanisms regulating intracellular pH are activated during growth of the mouse oocyte coincident with acquisition of meiotic competence. Dev. Biol. 286, 352–360. Erickson, D. L., North, J. A., Hightower, J. E., Weber, J. and Lauck, L. (2002). Movement and habitat use of green sturgeon Acipenser medirostris in the Rogue River, Oregon, USA. J. Appl. Ichthyol. 18, 565–569. Espmark, Å. M. and Baeverfjord, G. (2009). Effects of hyperoxia on behavioural and physiological variables in farmed Atlantic salmon (Salmo salar) parr. Aquacult. Int. 17, 341–353. Evans, D. H., Piermarini, P. M. and Choe, K. P. (2005). The multifunctional fish gill: Dominant site of gas exchange, osmoregulation, acid-base regulation, and excretion of nitrogenous waste. Physiol. Rev. 85, 97–177. Everaert, N., Willemsen, H., Willems, E., Franssens, L. and Decuypere, E. (2011). Acid-base regulation during embryonic development in amniotes, with particular reference to birds. Respir. Physiol. Neurobiol. 178, 118–128. Filosa, J. A., Dean, J. B. and Putnam, R. W. (2002). Role of intracellular and extracellular pH in the chemosensitive response of rat locus coeruleus neurones. J. Physiol. 541.2, 493–509. FitzHarris, G. and Baltz, J. M. (2009). Regulation of intracellular pH during oocyte growth and maturation in mammals. Reproduction 138, 619–627. Furch, K. and Junk, W. J. (1997). Physicochemical Conditions in the Floodplains. In The  133 Central Amazon Floodplain, pp. 69–108. Berlin, Heidelberg: Springer Berlin Heidelberg. Furimsky, M., Moon, T. W. and Perry, S. F. (1999). Intracellular pH regulation in hepatocytes isolated from three teleost species. J. Exp. Zool. 284, 361–367. Furimsky, M., Moon, T. W. and Perry, S. F. (2000). Evidence for the role of a Na+/HCO3− cotransporter in trout hepatocyte pHi regulation. J. Exp. Biol. 203, 2201–2208. Galli, G. and Richards, J. G. (2012). The effect of temperature on mitochondrial respiration in permeabilized cardiac fibres from the freshwater turtle, Trachemys scripta. J. Therm. Biol. 37, 195–200. Geist, D. R., Brown, R. S., Cullinan, V., Brink, S. R., Lepla, K., Bates, P. and Chandler, J. A. (2005). Movement, swimming speed, and oxygen consumption of juvenile white sturgeon in response to changing flow, water temperature, and light level in the Snake River, Idaho. T Am Fish Soc 134, 803–816. Gilmour, K. and Perry, S. (1994). The effects of hypoxia, hyperoxia or hypercapnia on the acid-base disequilibrium in the arterial blood of rainbow trout. J. Exp. Biol. 192, 269–284. Gilmour, K. M. and Perry, S. F. (2009). Carbonic anhydrase and acid-base regulation in fish. J. Exp. Biol. 212, 1647–1661. Goldberg, J. I., Doran, S. A., Shartau, R. B., Pon, J. R., Ali, D. W., Tam, R. and Kuang, S. (2008). Integrative biology of an embryonic respiratory behaviour in pond snails: the 'embryo stir-bar hypothesis'. J. Exp. Biol. 211, 1729–1736. Goldstein, J. I., Mok, J. M., Simon, C. M. and Leiter, J. C. (2000). Intracellular pH regulation in neurons from chemosensitive  and nonchemosensitive regions of Helix aspersa. Am. J. Physiol. Regul. Integr. Comp. Physiol. 279, R414–R423. Gonzalez, N. C. and Clancy, R. L. (1986a). Acid-base regulation in prolonged hypoxia: effect of increased PCO2. Respir. Physiol. 64, 213–227. Gonzalez, N. C. and Clancy, R. L. (1986b). Intracellular pH regulation during prolonged hypoxia in rats. Respir. Physiol. 65, 331–339. Gonzalez, R. J., Brauner, C. J., Wang, Y. X., Richards, J. G., Patrick, M. L., Xi, W., Matey, V. and Val, A. L. (2010). Impact of ontogenetic changes in branchial morphology on gill function in Arapaima gigas. Physiol. Biochem. Zool. 83, 322–332. Gonzalez, R. J., Jones, S. L. and Nguyen, T. V. (2017). Ionoregulatory Characteristics of Non–Rio Negro Characiforms and Cichlids. Physiol. Biochem. Zool. 000–000. Goss, G. G., Laurent, P. and Perry, S. F. (1992). Evidence for a morphological component in acid-base regulation during environmental hypercapnia in the brown bullhead (Ictalurus nebulosus). Cell Tissue Res. 268, 539–552.  134 Graham, J. B. (1997). Air breathing fishes: Evolution, Diversity, and Adaptation. San Diego, CA: Academic press. Grigg, G., Thompson, M., Beard, L. and Harlow, P. (2010). Oxygen levels in mound nests of Crocodylus porosus and Alligator mississippiensis are high, and gas exchange occurs primarily by diffusion, not convection. Aust. Zool. 35, 235–244. Gunderson, T. E. (1998). Effects of hypoxia and temperature on survival, growth, and respiration of juvenile Atlantic sturgeon, Acipenser oxyrinchus. Fish Bull. 96, 603–613. Gutowska, M., Melzner, F., Langenbuch, M., Bock, C., Claireaux, G. and Pörtner, H. (2010). Acid–base regulatory ability of the cephalopod (Sepia officinalis) in response to environmental hypercapnia. J. Comp. Physiol. B 180, 323–335. Halstead, L. B. (1985). The Vertebrate Invasion of Fresh Water. Philos. Trans. R. Soc. B 309, 243–258. Hammer, K. M., Kristiansen, E. and Zachariassen, K. E. (2011). Physiological effects of hypercapnia in the deep-sea bivalve Acesta excavata (Fabricius, 1779) (Bivalvia; Limidae). Mar Environ Res 72, 135–142. Hanson, L. M., Baker, D. W., Kuchel, L. J., Farrell, A. P., Val, A. L. and Brauner, C. J. (2009). Intrinsic mechanical properties of the perfused armoured catfish heart with special reference to the effects of hypercapnic acidosis on maximum cardiac performance. J. Exp. Biol. 212, 1270–1276. Harter, T. S., Shartau, R. B., Baker, D. W., Jackson, D. C., Val, A. L. and Brauner, C. J. (2014). Preferential intracellular pH regulation represents a general pattern of pH homeostasis during acid-base disturbances in the armoured catfish, Pterygoplichthys pardalis. J. Comp. Physiol. B 184, 709–718. Hasler, C. T., Butman, D., Jeffrey, J. D. and Suski, C. D. (2016). Freshwater biota and rising pCO2? Ecology Letters 19, 98–108. Hasler, C., Bouyoucos, I. A. and Suski, C. D. (2017). Tolerance to hypercarbia is repeatable and related to a component of the metabolic phenotype in a freshwater fish. Physiol. Biochem. Zool. 90, 583–587. Hawkins, D. G. (2004). No exit: thinking about leakage from geologic carbon storage sites. Energy 29, 1571–1578. Hayashi, M., Kita, J. and Ishimatsu, A. (2004). Acid-base responses to lethal aquatic hypercapnia in three marine fishes. Mar. Biol. 144, 153–160. Hedges, S. B. and Kumar, S. (2009). The Timetree of Life. Oxford University Press. Heisler, N. (1982). Intracellular and extracellular acid-base regulation in the tropical fresh-water teleost fish Synbranchus marmoratus in response to the transition from water breathing to air  135 breathing. J. Exp. Biol. 99, 9–28. Heisler, N. (1984). Acid-base regulation in fishes. In Fish Physiology (eds. Hoar, W. S. and Randall, D. J.), pp. 315–401. San Diego: Academic. Heisler, N., Forcht, G., Ultsch, G. R. and Anderson, J. F. (1982). Acid-base regulation in response to environmental hypercapnia in two aquatic salamanders, Siren lacertina and Amphiuma means. Respir. Physiol. 49, 141–158. Heisler, N., Toews, D. P. and Holeton, G. F. (1988). Regulation of ventilation and acid-base status in the elasmobranch Scyliorhinus stellaris during hyperoxia-induced hypercapnia. Respir. Physiol. 71, 227–246. Hildebrand, L. R., Drauch Schreier, A., Lepla, K., McAdam, S. O., McLellan, J., Parsley, M. J., Paragamian, V. L. and Young, S. P. (2016). Status of White Sturgeon (Acipenser transmontanus Richardson, 1863) throughout the species range, threats to survival, and prognosis for the future. J. Appl. Ichthyol. 32, 261–312. Hobe, H., Wood, C. M. and Wheatly, M. G. (1984). The mechanisms of acid-base and ionoregulation in the freshwater rainbow trout during environmental hyperoxia and subsequent normoxia. I. Extra- and intracellular acid-base status. Respir. Physiol. 55, 139–154. Hochachka, P. (1986). Defense strategies against hypoxia and hypothermia. Science 231, 234–241. Howarth, F. G. and Stone, F. D. (1990). Elevated carbon dioxide levels in Bayliss Cave, Australia: Implications for the evolution of obligate cave species. Pac. Sci. 44, 207–218. Hsu, H.-H., Lin, L.-Y., Tseng, Y.-C., Horng, J.-L. and Hwang, P.-P. (2014). A new model for fish ion regulation: identification of ionocytes in freshwater- and seawater-acclimated medaka (Oryzias latipes). Cell Tissue Res. 357, 225–243. Huynh, K. T., Baker, D. W., Harris, R., Church, J. and Brauner, C. J. (2011a). Capacity for intracellular pH compensation during hypercapnia in white sturgeon primary liver cells. J. Comp. Physiol. B 181, 893–904. Huynh, K. T., Baker, D. W., Harris, R., Church, J. and Brauner, C. J. (2011b). Effect of hypercapnia on intracellular pH regulation in a rainbow trout hepatoma cell line, RTH 149. J. Comp. Physiol. B 181, 883–892. Hwang, P.-P., Lee, T.-H. and Lin, L.-Y. (2011). Ion regulation in fish gills: recent progress in the cellular and molecular mechanisms. Am. J. Physiol. Regul. Integr. Comp. Physiol. 301, R28–R47. Ishimatsu, A., Hayashi, M. and Kikkawa, T. (2008). Fishes in high-CO2, acidified oceans. Mar. Ecol. Prog. Ser. 373, 295–302.  136 Jackson, D. C. (2003). Lactate sequestration by osteoderms of the broad-nose caiman, Caiman latirostris, following capture and forced submergence. J. Exp. Biol. 206, 3601–3606. Jackson, D. C. (1987). Cardiovascular Function in Turtles During Anoxia and Acidosis: In vivo and in vitro Studies. Amer. Zool. 27, 49–58. Jackson, D. C., Arendt, E. A., Inman, K. C., Lawler, R. G., Panol, G. and Wasser, J. S. (1991). 31P-NMR study of normoxic and anoxic perfused turtle heart during graded CO2 and lactic acidosis. Am. J. Physiol. 260, R1130–6. Jackson, D. C., Crocker, C. E. and Ultsch, G. R. (2000a). Bone and shell contribution to lactic acid buffering of submerged turtles Chrysemys picta bellii at 3oC. Am. J. Physiol. 278, R1564–R1571. Jackson, D. C., Ramsey, A. L., Paulson, J. M., Crocker, C. E. and Ultsch, G. R. (2000b). Lactic acid buffering by bone and shell in anoxic softshell and painted turtles. Physiol. Biochem. Zool. 73, 290–297. Janis, C. M., Devlin, K., Warren, D. E. and Witzmann, F. (2012). Dermal bone in early tetrapods: a palaeophysiological hypothesis of adaptation for terrestrial acidosis. Proc. R. Soc. B Biol. Sci. 279, 3035–3040. Jibb, L. A. and Richards, J. G. (2008). AMP-activated protein kinase activity during metabolic rate depression in the hypoxic goldfish, Carassius auratus. J. Exp. Biol. 211, 3111–3122. Johnson, C. H. and Epel, D. (1981). Intracellular pH of sea urchin eggs measured by the dimethyloxazolidinedione (DMO) method. J. Cell Biol. 89, 284. Kates, D., Dennis, C., Noatch, M. R., Suski, C. D. and MacLatchy, D. L. (2012). Responses of native and invasive fishes to carbon dioxide: potential for a nonphysical barrier to fish dispersal. Can. J. Fish Aquat. Sci. 69, 1748–1759. Kieffer, J. D., Wakefield, A. M. and Litvak, M. K. (2001). Juvenile sturgeon exhibit reduced physiological responses to exercise. J. Exp. Biol. 204, 4281–4289. Kieffer, J., Currie, S. and Tufts, B. (1994). Effects of environmental temperature on the metabolic and acid-base responses of rainbow trout to exhaustive exercise. J. Exp. Biol. 194, 299–317. Kikkawa, T., Watanabe, Y., Katayama, Y., Kita, J. and Ishimatsu, A. (2008). Acute CO2 tolerance limits of juveniles of three marine invertebrates, Sepia lycidas, Sepioteuthis lessoniana, and Marsupenaeus japonicus. Plankton Benthos Res. 3, 184–187. Krumschnabel, G., Manzl, C. and Schwarzbaum, P. J. (2001). Regulation of intracellular pH in anoxia-tolerant and anoxia-intolerant teleost hepatocytes. J. Exp. Biol. 204, 3943–3951. Kwong, R. W. M., Kumai, Y. and Perry, S. F. (2014). The physiology of fish at low pH: the zebrafish as a model system. J. Exp. Biol. 217, 651–662.  137 Lackner, K. S. (2003). A guide to CO2 sequestration. Science 300, 1677–1678. Lane, M. (1999). Bicarbonate/chloride exchange regulates intracellular pH of embryos but not oocytes of the hamster. Biol. Reprod. 61, 452–457. Lane, M. and Bavister, B. D. (1999). Regulation of intracellular pH in bovine oocytes and cleavage stage embryos. Mol. Reprod. Dev. 54, 396–401. Larsen, B. K. and Jensen, F. B. (1997). Influence of ionic composition on acid-base regulation in rainbow trout (Oncorhynchus mykiss) exposed to environmental hypercapnia. Fish Physiol. Biochem. 16, 157–170. Larsen, B. K., Portner, H. O. and Jensen, F. B. (1997). Extra- and intracellular acid-base balance and ionic regulation in cod (Gadus morhua) during combined and isolated exposures to hypercapnia and copper. Mar. Biol. 128, 337–346. Lecuyer, C. (2014). Water on Earth. (eds. Mariotti, A. and Pomerol, J.-C.) John Wiley and Sons. Lefevre, S., Damsgaard, C., Pascale, D. R., Nilsson, G. E. and Stecyk, J. A. W. (2014). Air breathing in the Arctic: influence of temperature, hypoxia, activity and restricted air access on respiratory physiology of the Alaska blackfish Dallia pectoralis. J. Exp. Biol. 217, 4387–4398. Lemaire, B., Beck, M., Jaspart, M., Debier, C. and Calderon, P. B. (2011). Precision-cut liver slices of Salmo salar as a tool to investigate the oxidative impact of CYP1A-mediated PCB 126 and 3-methylcholanthrene metabolism. Toxicol. In Vitro 25, 335–342. Li, S., Lu, X. X. and Bush, R. T. (2013). CO2 partial pressure and CO2 emission in the Lower Mekong River. J. Hydrol. 504, 40–56. Lin, H. and Randall, D. (1995). Proton pumps in fish gills. In Fish Physiology (eds. Wood, C. M. and Shuttleworth, T. J.), pp. 229–255. Fish Physiology. Liu, S., Piwnica-Worms, D. and Lieberman, M. (1990). Intracellular pH regulation in cultured embryonic chick heart cells. Na+-dependent Cl-/HCO3- exchange. J. Gen. Physiol. 96, 1247–1269. Lutz, P. L. and Dunbar-Cooper, A. (1984). The nest environment of the American crocodile (Crocodylus acutus). Copeia 1984, 153–161. Macqueen, D. J. and Johnston, I. A. (2014). A well-constrained estimate for the timing of the salmonid whole genome duplication reveals major decoupling from species diversification. Proc. R. Soc. B 281, 20132881. Maddison, W. P. and maddison, D. R. Mesquite: a modular system for evolutionary analysis. Malan, A., Rodeau, J. L. and Daull, F. (1985). Intracellular pH in hibernation and respiratory  138 acidosis in the European hamster. J. Comp. Physiol. B 156, 251–258. Mandic, M., Lau, G. Y., Nijjar, M. and Richards, J. G. (2008). Metabolic recovery in goldfish: A comparison of recovery from severe hypoxia exposure and exhaustive exercise. Comp. Biochem. Phys. C 148, 332–338. Marcé, R., Obrador, B., Morguí, J.-A., Lluís Riera, J., López, P. and Armengol, J. (2015). Carbonate weathering as a driver of CO2 supersaturation in lakes. Nature Geosci. 8, 107–111. Maxime, V., Nonnotte, G., Peyraud, C., Williot, P. and Truchot, J. P. (1995). Circulatory and respiratory effects of an hypoxic stress in the Siberian sturgeon. Respir. Physiol. 100, 203–212. McBrian, M. A., Behbahan, I. S., Ferrari, R., Su, T., Huang, T.-W., Li, K., Hong, C. S., Christofk, H. R., Vogelauer, M., Seligson, D. B., et al. (2013). Histone Acetylation Regulates Intracellular pH. Mol. Cell 49, 310–321. McEnroe, M. and Cech, J. J., Jr (1985). Osmoregulation in juvenile and adult white sturgeon, Acipenser transmontanus. Environ. Biol. Fish 14, 23–30. McKenzie, D. J., Piccolella, M., Valle, A. Z. D., Taylor, E. W., Bolis, C. L. and Steffensen, J. F. (2003). Tolerance of chronic hypercapnia by the European eel Anguilla anguilla. J. Exp. Biol. 206, 1717–1726. McKenzie, D. J., Taylor, E. W., Dalla Valle, A. Z. and Steffensen, J. F. (2002). Tolerance of acute hypercapnic acidosis by the European eel (Anguilla anguilla). J. Comp. Physiol. B 172, 339–346. McLean, M. F., Hanson, K. C., Cooke, S. J., Hinch, S. G., Patterson, D. A., Nettles, T. L., Litvak, M. K. and Crossin, G. T. (2016). Physiological stress response, reflex impairment and delayed mortality of white sturgeon Acipenser transmontanus exposed to simulated fisheries stressors. Conserv. Physiol. 4, cow031. McNeil, B. I. and Sasse, T. P. (2016). Future ocean hypercapnia driven by anthropogenic amplification of the natural CO2 cycle. Nature 529, 383–386. Melzner, F., Gutowska, M. A., Langenbuch, M., Dupont, S., Lucassen, M., Thorndyke, M. C., Bleich, M. and Portner, H. O. (2009). Physiological basis for high CO2 tolerance in marine ectothermic animals: pre-adaptation through lifestyle and ontogeny? Biogeosciences 6, 2313–2331. Meredith, R. W., Janecka, J. E., Gatesy, J., Ryder, O. A., Fisher, C. A., Teeling, E. C., Goodbla, A., Eizirik, E., Simao, T. L. L., Stadler, T., et al. (2011). Impacts of the Cretaceous Terrestrial Revolution and KPg Extinction on Mammal Diversification. Science 334, 521–524. Michaelidis, B., Ouzounis, C., Paleras, A. and Portner, H. O. (2005). Effects of long-term  139 moderate hypercapnia on acid-base balance and growth rate in marine mussels Mytilus galloprovincialis. Mar. Ecol. Prog. Ser. 293, 109–118. Milligan, C. L. and Farrell, T. P. (1986). Extracellular and intracellular acid-base status following strenuous activity in the sea raven (Hemitripterus americanus). J. Comp. Physiol. B 156, 583–590. Milligan, C. L. and Wood, C. M. (1987a). Muscle and liver intracellular acid-base and metabolite status after strenuous activity in the inactive, benthic starry flounder Platichthys stellatus. Physiol Zool 60, 54–68. Milligan, C. L. and Wood, C. M. (1987b). Effects of strenuous activity on intracellular and extracellular acid-base status and H+ exchange with the environment in the inactive, benthic starry flounder Platichthys stellatus. Physiol. Zool. 60, 37–53. Mojazi Amiri, B., Baker, D. W., Morgan, J. D. and Brauner, C. J. (2009). Size dependent early salinity tolerance in two sizes of juvenile white sturgeon, Acipenser transmontanus. Aquaculture 286, 121–126. Molich, A. and Heisler, N. (2005). Determination of pH by microfluorometry: intracellular and interstitial pH regulation in developing early-stage fish embryos (Danio rerio). J. Exp. Biol. 208, 4137–4149. Mueller, C., Tazawa, H. and Burggren, W. (2014). Dynamics of acid-base metabolic compensation and hematological regulation interactions in response to CO2 challenges in embryos of the chicken (Gallus gallus). J. Comp. Physiol. B 184, 641–649. Nelson, J. S. (2006). Fishes of the world. New York: John Wiley and Sons. Nestler, J. R. (1990). Intracellular pH during daily torpor in Peromyscus maniculatus. J. Comp. Physiol. B 159, 661–666. Nilsson, G. E. (2001). Surviving anoxia with the brain turned on. Physiology 16, 217–221. Nottingham, S., Leiter, J. C., Wages, P., Buhay, S. and Erlichman, J. S. (2001). Developmental changes in intracellular pH regulation in medullary neurons of the rat. Am. J. Physiol. Regul. Integr. Comp. Physiol. 281, R1940–R1951. Occhipinti, R. and Boron, W. F. (2015). Mathematical modeling of acid-base physiology. Prog. Biophys. Mol. Biol. 117, 557–573. Park, P. K., Gordon, L. I., Hager, S. W. and Cissell, M. C. (1969). Carbon Dioxide Partial Pressure in the Columbia River. Science 166, 867–868. Parks, S. K. and Pouysségur, J. (2017). Targeting pH regulating proteins for cancer therapy-Progress and limitations. Semin. Cancer Biol. 43, 66–73. Parks, S. K., Tresguerres, M. and Goss, G. G. (2009). Cellular mechanisms of Cl− transport in  140 trout gill mitochondrion-rich cells. Am. J. Physiol. Regul. Integr. Comp. Physiol. 296, R1161–R1169. Perry, S. F. and Gilmour, K. M. (2006). Acid-base balance and CO2 excretion in fish: unanswered questions and emerging models. Respir. Physiol. Neurobiol. 154, 199–215. Perry, S. F., Vulesevic, B., Grosell, M. and Bayaa, M. (2009). Evidence that SLC26 anion transporters mediate branchial chloride uptake in adult zebrafish (Danio rerio). Am. J. Physiol. Regul. Integr. Comp. Physiol. 297, R988–R997. Phillips, K. P., Léveillé, M.-C., Claman, P. and Baltz, J. M. (2000). Intracellular pH regulation in human preimplantation embryos. Hum. Reprod. 15, 896–904. Pinardi, M., Rossetto, M., Viaroli, P. and Bartoli, M. (2014). Daily and seasonal variability of CO2 saturation and evasion in a free flowing and in a dammed river reach. J. Limnol. 73, 468-481. Portner, H. O., Bock, C. and Reipschlager, A. (2000). Modulation of the cost of pHi regulation during metabolic depression: A P-31-NMR study in invertebrate (Sipunculus nudus) isolated muscle. J. Exp. Biol. 203, 2417–2428. Portner, H. O., Boutilier, R. G., Tang, Y. and Toews, D. P. (1990). Determination of intracellular pH and PCO2 values after metabolic inhibition by fluoride and nitrilotriacetic acid. Respir. Physiol. 81, 255–274. Portner, H. O., Reipschlager, A. and Heisler, N. (1998). Acid-base regulation, metabolism and energetics in Sipunculus nudus as a function of ambient carbon dioxide level. J. Exp. Biol. 201, 43–55. Prange, H. D. and Ackerman, R. A. (1974). Oxygen consumption and mechanisms of gas exchange of green turtle (Chelonia mydas) eggs and hatchlings. Copeia 1974, 758–763. Putnam, R. W. and Roos, A. (1997). Intracellular pH. In Handbook of physiology, vol 14, Cell physiology (eds. Hoffman, J. and Jamieson, J., pp. 389–440. Oxford: Oxford University Press. Rahn, H. (1974). Body temperature and acid-base regulation. Pneumonologie 151, 87–94. Randall, D. J., Burggren, W. W., Farrell, A. P. and Haswell, M. S. (1981). The evolution of air breathing in vertebrates. Cambridge: Cambridge University press. Raymond, P. A., Hartmann, J., Lauerwald, R., Sobek, S., McDonald, C., Hoover, M., Butman, D., Striegl, R., Mayorga, E., Humborg, C., et al. (2013). Global carbon dioxide emissions from inland waters. Nature 503, 355–359. Regan, M. D., Turko, A. J., Heras, J., Andersen, M. K., Lefevre, S., Wang, T., Bayley, M., Brauner, C. J., Huong, D. T. T., Phuong, N. T., et al. (2016). Ambient CO2, fish behaviour and altered GABAergic neurotransmission: exploring the mechanism of CO2- 141 altered behaviour by taking a hypercapnia dweller down to low CO2 levels. J. Exp. Biol. 219, 109–118. Reshkin, S. J., Greco, M. R. and Cardone, R. A. (2014). Role of pHi, and proton transporters in oncogene-driven neoplastic transformation. Phil. Trans. R. Soc. B 369, 20130100. Reum, J. C. P., Alin, S. R., Feely, R. A., Newton, J., Warner, M. and McElhany, P. (2014). Seasonal carbonate chemistry covariation with temperature, oxygen, and salinity in a fjord estuary: implications for the design of ocean acidification experiments. PLOS ONE 9, e89619. Richards, J. G. (2011). Physiological, behavioral and biochemical adaptations of intertidal fishes to hypoxia. J. Exp. Biol. 214, 191–199. Ritucci, N. A. (2005). Response of membrane potential and intracellular pH to hypercapnia in neurons and astrocytes from rat retrotrapezoid nucleus. Am. J. Physiol. Regul. Integr. Comp. Physiol. 289, R851–R861. Robergs, R. A., Ghiasvand, F. and Parker, D. (2004). Biochemistry of exercise-induced metabolic acidosis. Am. J. Physiol. Regul. Integr. Comp. Physiol. 287, R502–R516. Roos, A. and Boron, W. F. (1981). Intracellular pH. Physiol. Rev. 61, 296–434. Rowlett, K. and Simkiss, K. (1989). Respiratory gases and acid-base balance in shell-less avian embryos. J. Exp. Biol. 143, 529–536. Salameh, A. I., Ruffin, V. A. and Boron, W. F. (2014). Effects of metabolic acidosis on intracellular pH responses in multiple cell types. Am. J. Physiol. Regul. Integr. Comp. Physiol. 307, R1413–27. Sanchez, A. P., Giusti, H., Bassi, M. and Glass, M. L. (2005). Acid-base regulation in the South American lungfish Lepidosiren paradoxa: Effects of prolonged hypercarbia on blood gases and pulmonary ventilation. Physiol. Biochem. Zool. 78, 908–915. Schapiro, F. B. and Grinstein, S. (2000). Determinants of the pH of the Golgi Complex. J. Biol. Chem. 275, 21025–21032. Seymour, R. S., Vleck, D. and Vleck, C. M. (1986). Gas exchange in the incubation mounds of megapode birds. J. Comp. Physiol. B 156, 773–782. Shams, I., Avivi, A. and Nevo, E. (2005). Oxygen and carbon dioxide fluctuations in burrows of subterranean blind mole rats indicate tolerance to hypoxic–hypercapnic stresses. Comp. Biochem. Phys. A 142, 376–382. Shartau, R. B. and Brauner, C. J. (2014). Acid-base and ion balance in fishes with bimodal respiration. J. Fish Biol. 84, 682–704. Shartau, R. B., Baker, D. W. and Brauner, C. J. (2017a). White sturgeon (Acipenser  142 transmontanus) acid-base regulation differs in response to different types of acidoses. J. Comp. Physiol. B. 187, 985-994. Shartau, R. B., Baker, D. W., Crossley, D. A. and Brauner, C. J. (2016a). Preferential intracellular pH regulation: hypotheses and perspectives. J. Exp. Biol. 219, 2235–2244. Shartau, R. B., Brix, K. V. and Brauner, C. J. (2017b). Characterization of Na+ transport to gain insight into the mechanism of acid-base and ion regulation in white sturgeon (Acipenser transmontanus). Comp. Biochem. Phys. A 204, 197–204. Shartau, R. B., Crossley, D. A., Kohl, Z. F. and Brauner, C. J. (2016b). Embryonic common snapping turtles (Chelydra serpentina) preferentially regulate intracellular tissue pH during acid-base challenges. J. Exp. Biol. 219, 1994–2002. Shartau, R. B., Crossley, D. A., Kohl, Z. F., Elsey, R. M. and Brauner, C. J. (In press). American alligator (Alligator mississippiensis) embryos tightly regulate intracellular pH during a severe acidosis. Can. J. Zool. Shartau, R. B., Harris, S., Boychuk, E. C. and Goldberg, J. I. (2010). Rotational behaviour of encapsulated pond snail embryos in diverse natural environments. J. Exp. Biol. 213, 2086–2093. Shaughnessy, C. A., Baker, D. W., Brauner, C. J., Morgan, J. D. and Bystriansky, J. S. (2015). Interaction of osmoregulatory and acid-base compensation in white sturgeon (Acipenser transmontanus) during exposure to aquatic hypercarbia and elevated salinity. J. Exp. Biol. 218, 2712–2719. Siesjö, B. K., Folbergrová, J. and MacMillan, V. (1972). The effect of hypercapnia upon intracellular pH in the brain, evaluated by the bicarbonate-carbonic acid method and from the creatine phosphokinase equilibrium. J. Neurochem. 19, 2483–2495. Singh, Y., Cooke, J. B., Hinton, D. E. and Miller, M. G. (1996). Trout liver slices for metabolism and toxicity studies. Drug Metab. Dispos. 24, 7–14. Siyanov, V. and Baltz, J. M. (2013). NHE1 Is the Sodium-Hydrogen Exchanger Active in Acute Intracellular pH Regulation in Preimplantation Mouse Embryos. Biol. Reprod. 88, 1-11. Smart, G. R., Knox, D., Harrison, J. G., Ralph, J. A., Richards, R. H. and Cowey, C. B. (1979). Nephrocalcinosis in rainbow trout Salmo gairdneri Richardson; the effect of exposure to elevated CO2 concentrations. J. Fish Dis. 2, 279–289. Snyder, G. K. and Nestler, J. R. (1991). Intracellular pH in the toad Bufo marinus following hypercapnia. J. Exp. Biol. 161, 415–422. Snyder, G. K., Nestler, J. R., Shapiro, J. I. and Huntley, J. (1995). Intracellular pH in lizards after hypercapnia. Am. J. Physiol. 268, R889–R895.  143 Soengas, J. L. and Aldegunde, M. (2002). Energy metabolism of fish brain. Comp. Biochem. Phys. B 131, 271–296. Sorey, M. L., Werner, C., McGimsey, R. G. and Evans, W. C. (2000). Hydrothermal activity and carbon-dioxide discharge at shrub and upper Klawasi mud volcanoes, Wrangell mountains, Alaska. U. S. Geological Survey, Water-Resources Investigations Report. Spicer, J. I., Raffo, A. and Widdicombe, S. (2007). Influence of CO2-related seawater acidification on extracellular acid–base balance in the velvet swimming crab Necora puber. Mar. Biol. 151, 1117–1125. Spicer, J. I., Widdicombe, S., Needham, H. R. and Berge, J. A. (2011). Impact of CO2-acidified seawater on the extracellular acid-base balance of the northern sea urchin Strongylocentrotus dröebachiensis. J. Exp. Mar. Biol. Ecol. 407, 19–25. Squirrell, J. M., Lane, M. and Bavister, B. D. (2001). Altering intracellular pH disrupts development and cellular organization in preimplantation hamster embryos. Biol. Reprod. 64, 1845–1854. Stabenau, E. K. and Heming, T. A. (1993). Determination of the constants of the Henderson-Hasselbalch equation, (alpha)CO2 and pKa, in sea turtle plasma. J. Exp. Biol. 180, 311–314. Stapp, L. S., Kreiss, C. M., Portner, H. O. and Lannig, G. (2015). Differential impacts of elevated CO2 and acidosis on the energy budget of gill and liver cells from Atlantic cod, Gadus morhua. Comp. Biochem. Phys. A 187, 160–167. Stumpp, M., Hu, M. Y., Melzner, F., Gutowska, M. A., Dorey, N., Himmerkus, N., Holtmann, W. C., Dupont, S. T., Thorndyke, M. C. and Bleich, M. (2012). Acidified seawater impacts sea urchin larvae pH regulatory systems relevant for calcification. Proc. Natl. Acad. Sci. U.S.A. 109, 18192–18197. Tazawa, H. (1980). Oxygen and CO2 exchange and acid-base regulation in the avian embryo. Amer. Zool. 20, 395–404. Thohan, S., Zurich, M. C., Chung, H., Weiner, M., Kane, A. S. and Rosen, G. M. (2001). Tissue Slices Revisited: Evaluation and Development of a Short-Term Incubation for Integrated Drug Metabolism. Drug Metab Dispos 29, 1337–1342. Tierney, K. B. (2016). Chemical avoidance responses of fishes. Aquat. Toxicol. 174, 228–241. Toews, D. P. and Heisler, N. (1982). The effects of hypercapnia on intracellular and extracellular acid-base status in the toad Bufo marinus. J. Exp. Biol. 97, 79–86. Toews, D. P., Holeton, G. F. and Heisler, N. (1983). Regulation of the acid-base status during environmental hypercapnia in the marine teleost fish Conger conger. J. Exp. Biol. 107, 9–20. Tokudome, G., Tomonari, H., Gardner, J. P., Aladjem, M., Fine, B. P., Lasker, N., Gutkin, M., Byrd, L. H. and Aviv, A. (1990). Variations in the apparent pH set point for activation  144 of platelet Na-H antiport. Hypertension 16, 180–189. Udvardy, M. (1975). A classification of the biogeographical provinces of the world. IUCN Occassional Papers 18, 1–50. Ultsch, G. R. (1987). The potential role of hypercarbia in the transition from water-breathing to air-breathing in vertebrates. Evolution 41, 442–445. Ultsch, G. R. (1996). Gas exchange, hypercarbia and acid-base balance, paleoecology, and the evolutionary transition from water-breathing to air-breathing among vertebrates. Palaeogeogr. Palaeoclimatol. Palaeoecol. 123, 1–27. Val, A. L., De Almeida Val, V. M. F. and Randall, D. J. (2005). Tropical Environment. In The Physiology of Tropical Fishes: Fish Physiology (eds. Hoar, W. S., Randall, D. J., and Farrell, A. P.), pp. 1–45. New York: Academic Press. Vandenberg, J. I., Metcalfe, J. C. and Grace, A. A. (1994). Intracellular pH recovery during respiratory acidosis in perfused hearts. AJP: Cell Physiology 266, C489–C497. Vaughan-Jones, R. D., Spitzer, K. W. and Swietach, P. (2009). Intracellular pH regulation in heart. J. Mol. Cell Cardiol. 46, 318–331. Wang, Y., Heigenhauser, G. J. and Wood, C. M. (1994). Integrated responses to exhaustive exercise and recovery in rainbow trout white muscle: acid-base, phosphogen, carbohydrate, lipid, ammonia, fluid volume and electrolyte metabolism. J. Exp. Biol. 195, 227–258. Warren, D. E. (2005). The role of mineralized tissue in the buffering of lactic acid during anoxia and exercise in the leopard frog Rana pipiens. J. Exp. Biol. 208, 1117–1124. Wasser, J. S., Warburton, S. J. and Jackson, D. C. (1991). Extracellular and intracellular acid-base effects of submergence anoxia and nitrogen breathing in turtles. Respir. Physiol. 83, 239–252. Wearing, O., Eme, J., Kemp, A. and Crossley, D. (2014). Impact of hypercapnic incubation on hatchling common snapping turtle (Chelydra serpentina) growth and metabolism. The FASEB Journal 28 1 Supplement 1101.3. Weyhenmeyer, G. A., Kortelainen, P., Sobek, S., Müller, R. and Rantakari, M. (2012). Carbon Dioxide in Boreal Surface Waters: A Comparison of Lakes and Streams. Ecosystems 15, 1295–1307. Wood, C. M. and LeMoigne, J. (1991). Intracellular acid-base responses to environmental hyperoxia and normoxic recovery in rainbow trout. Respir. Physiol. 86, 91–113. Wood, C. M., Turner, J. D. and Graham, M. S. (1983). Why do fish die after severe exercise? J. Fish Biol. 22, 189–201. Wood, C. M., Turner, J. D., Munger, R. S. and Graham, M. S. (1990). Control of ventilation  145 in the hypercapnic skate Raja ocellata: II. Cerebrospinal fluid and intracellular pH in the brain and other tissues. Respir. Physiol. 80, 279–298. Wood, S. C. and Schaefer, K. E. (1978). Regulation of intracellular pH in lungs and other tissues during hypercapnia. J. Appl. Physiol. 45, 115–118. Wright, P. A., Randall, D. J. and Wood, C. M. (1988). The distribution of ammonia and H+ between tissue compartments in lemon sole (Parophrys vetulus) at rest, during hypercapnia and following exercise. J. Exp. Biol. 136, 149–175. Yaksh, T. L. and Anderson, R. E. (1987). In vivo studies on intracellular pH, focal flow, and vessel diameter in the cat cerebral cortex: effects of altered CO2 and electrical stimulation. J. Cereb. Blood Flow Metab. 7, 332–341. Yntema, C. L. (1968). A series of stages in the embryonic development of Chelydra serpentina. J. Morphol. 125, 219–251. Yoshikawa, H., Fumio, K. and Masao, K. (1994). The relationship between the EEG and brain pH in carp, Cyprinus carpio, subjected to environmental hypercapnia at an anesthetic level. Comparative Biochemistry and Physiology Part A: Physiology 107, 307–312. Zeidler, R. and Kim, H. D. (1977). Preferential hemolysis of postnatal calf red cells induced by internal alkanlinization. J. Gen. Physiol. 70, 385. Zhang, P., Liang, D., Mao, R.-L., Hillis, D. M., Wake, D. B. and Cannatella, D. C. (2013). Efficient Sequencing of Anuran mtDNAs and a Mitogenomic Exploration of the Phylogeny and Evolution of Frogs. Mol. Biol. Evol. 30, 1899–1915. Zhao, Y. and Baltz, J. M. (1996). Bicarbonate/chloride exchange and intracellular pH throughout preimplantation mouse embryo development. Am. J. Physiol. 271, C1512–20. Zimmer, A. M. and Wood, C. M. (2014). Exposure to acute severe hypoxia leads to increased urea loss and disruptions in acid-base and ionoregulatory balance in dogfish sharks (squalus acanthias). Physiol. Biochem. Zool. 87, 623–639.     146 Appendix: A note on the methodology of pH measurements  The majority of this thesis relies on measurements of pHe and pHi to determine the pattern of acid-base regulation in the animals examined; consequently, reliable and accurate pH measurements are critical to the work and conclusions of my thesis.  Extracellular pH measurement In Chapters 2 and 3, pHe measurements were obtained from blood taken via caudal puncture which may differ from that of dorsal aortic blood drawn from a cannulated fish as performing cannulations was not always possible. To avoid the negative effects associated with caudal puncture for blood sampling, prior to sampling I lightly anesthetized fish in their container while ensuring sufficient aeration (either air or treatment gas); fish were then sampled in the container while keeping them submerged except for the site of the caudal puncture. This ensured the fish did not struggle, nor became hypoxic during sampling. To ensure pH values obtained via caudal puncture produced pH values comparable to those taken via dorsal aorta cannulation, I compared the two techniques in rainbow trout. pHe from dorsal aorta cannulated and caudal puncture in control fish were 7.93 ± 0.03 (mean ± s.e.m.; n=12) and 7.83 ± 0.04 (n=8) pH units, respectively. In fish exposed to 1.5 kPa PCO2 for 24 h, dorsal aorta cannulated fish and caudal puncture were 7.53 ± 0.04 (n=4) and 7.41 ± 0.04 (n=7) pH units, respectively. The pHe differences between sampling technique for control and 1.5 kPa PCO2 are not significant (independent samples t-test, P>0.05) and the relative difference between techniques is similar (0.101 and 0.122 pH units, in fish sampled via cannulation and caudal puncture, respectively). Differences between techniques can be largely attributed to stress during caudal puncture sampling, and some mixing of arterial and venous blood where the latter may be slightly lower than arterial pHe in trout (Eddy, 1976). Despite caudal puncture sampling underestimating pHe, through careful technique, this difference was minimized and the relative differences between control and treatment are still maintained; thus, use of caudal puncture provides a quick and reliable method of blood sampling.     147 Intracellular pH measurement To assess pHi regulation in response to acid-base challenges, I measured pHi using the metabolic inhibitor tissue homogenate (MITH) method. The MITH method was first described and validated by Portner et al. (Portner et al., 1990) and later validated for use at high CO2 by Baker et al. (2009b). The MITH method utilizes a simple protocol, described here. First, the tissues are quickly excised from the animal, ideally in <2 minutes and placed in aluminum foil or Eppendorf tubes, and immediately placed in liquid nitrogen. Tissues can then be transferred for storage at -80oC for up to three months as validated by Baker et al. (2009b) (see below for more detail). Tissue pH is measured by grinding tissue into a fine powder under liquid nitrogen, which is then transferred to an Eppendorf tube containing metabolic inhibitor (KF [150 mmol/l], Na2NTA [6 mmol/l]; exact concentrations of KF and Na2NTA may vary depending on intracellular concentration of K and Na) at a ratio of 1:5 – 1:10 tissue:metabolic inhibitor, and gently vortexed. Finally, pHi is measured on this supernatant using a pH electrode (Radiometer Analytical SAS pH electrode; CK2401C, Cedex, France) thermostated to the temperature at which the fish had been held. The MITH method of measuring pHi has previously been found to provide accurate, repeatable measurements of pHi in several tissues from worms (Sipunculus nudas), squid (Illex illecebrosus), trout (Oncorhynchus mykiss), toads (Bufo marinus), and rats (Portner et al., 1990). This method of measuring pHi was found to provide comparable pHi values, with less variability compared to the older and commonly used dimethyloxazolidinedione (DMO) technique which is more variable and has considerable time delay to reach equilibrium in the tissue. The latter can vary from <30 s to ~ 1 h (Portner et al., 1990) while tissues can be dissected out immediately using the MITH technique. The MITH technique has been used in fish (Baker and Brauner, 2012; Baker et al., 2015; Brauner et al., 2004; Regan et al., 2016) and non-fish (Busk et al., 1997; Galli and Richards, 2012; Portner et al., 1990) studies. Use of the MITH technique has been validated for both storage duration and measurement of pHi from tissues exposed to high CO2 tensions (Baker et al. 2009b). Baker et al. (2009b) validated the use of the MITH method on sturgeon red blood cell pHi measurements following CO2 exposure of up to 10 kPa PCO2 with storage in either liquid nitrogen or -80oC for 90 days. They observed no differences in pHi of red blood cells measured immediately or following 30 days of storage at -80oC. Additionally, the pHi values obtained from the MITH  148 method were identical to those obtained using the freeze-thaw method (Zeidler and Kim, 1977), which involves repetitively freezing and thawing red blood cells and does not use any chemicals. These storage durations and procedures using the MITH procedure have been used numerous times in fish (Baker and Brauner, 2012; Baker et al., 2015; Brauner et al., 2004; Regan et al., 2016) and non-fish studies, including the freshwater turtle (Trachemys scripta; (Galli and Richards, 2012), Rana catesbeiana tadpoles (Busk et al., 1997), and as indicated above, in worms, squid, toads, and rats (Portner et al., 1990) during CO2 exposure ranging from normocarbia to 12 kPa PCO2. 

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