UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Ions before oxygen : ancestral origins of vertebrate gill function Sackville, Michael 2020

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
24-ubc_2020_november_sackville_michael.pdf [ 15.41MB ]
Metadata
JSON: 24-1.0394140.json
JSON-LD: 24-1.0394140-ld.json
RDF/XML (Pretty): 24-1.0394140-rdf.xml
RDF/JSON: 24-1.0394140-rdf.json
Turtle: 24-1.0394140-turtle.txt
N-Triples: 24-1.0394140-rdf-ntriples.txt
Original Record: 24-1.0394140-source.json
Full Text
24-1.0394140-fulltext.txt
Citation
24-1.0394140.ris

Full Text

IONS BEFORE OXYGEN: ANCESTRAL ORIGINS OF VERTEBRATE GILL FUNCTION  by Michael Sackville  B.Sc., McGill University, 2004 M.Sc., The University of British Columbia, 2010    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)  August 2020       © Michael Sackville, 2020   ii   The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Ions before oxygen: Ancestral origins of vertebrate gill function  submitted by Michael A Sackville in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Zoology  Examining Committee: Colin J Brauner, Professor, Department of Zoology, University of British Columbia Supervisor  Patricia M Schulte, Professor, Department of Zoology, University of British Columbia Supervisory Committee Member  Christopher D G Harley, Professor, Department of Zoology, University of British Columbia University Examiner Scott G Hinch, Professor, Department of Forestry, University of British Columbia University Examiner Warren W Burggren, Professor, Department of Biological Sciences, University of North Texas External Examiner  Additional Supervisory Committee Members: William K Milsom, Professor, Department of Zoology, University of British Columbia Supervisory Committee Member Eric B Taylor, Professor, Department of Zoology, University of British Columbia Supervisory Committee Member    iii Abstract  Gas exchange and ion regulation at gills play many key roles in vertebrate evolution. Current hypotheses assume gills acquired these important functions from the skin along the vertebrate stem, facilitating the evolution of larger, armoured and more active modes of life. However, this assumption lacks functional support from representatives of early vertebrates and their ancestors. To better understand how and when vertebrate gills became the primary site of gas exchange and ion regulation, I characterized gill and skin function in representatives of ancestral vertebrates (lamprey ammocoete, Entosphenus tridentatus), cephalochordates (amphioxus, Branchiostoma floridae) and hemichordates (acorn worm, Saccoglossus kowalevskii). Intraspecific comparisons within ammocoetes tested the effects of size, dermal thickness and activity on gill function, and interspecific comparisons between taxa tested ancestral origins of gill function. For ammocoetes, I measured multiple gas and ion fluxes in vivo at gills and skin of different sized animals (0.02-2.00 g). Gills accounted for ~20% of gas flux in the smallest ammocoetes in normoxia at 10oC, and contributions increased with size, hypoxia and temperature. Conversely, gills accounted for 100% of ion flux in all sizes and conditions. For acorn worms, I exploited their regenerative ability to partition animals into viable halves with and without gills for respirometry. Gills did not enhance oxygen uptake or ammonia excretion despite hypoxic or thermal challenges. Morphometry by others estimates amphioxus gills also contribute negligibly to gas exchange. However, both acorn worm and amphioxus gills displayed elevated signals for ion regulation (NKA/VHA activity; CA/NHE/AE/Foxi expression). This is the first functional support in ancestral representatives for a vertebrate origin of gas exchange at gills associated with increasing size, dermal thickness and activity. However, ion regulation at gills appears unrelated to this vertebrate transition, and results instead suggest a novel and much earlier deuterostome origin near the inception of pharyngeal arches.  iv Lay Summary  Gills play many important roles in vertebrate evolution by serving as the primary site for breathing and regulating internal ions. These functions are assumed to have first shifted to gills from the skin as early vertebrates transitioned from small, worm-like creatures to larger, more active fishes. However, this assumption lacks functional support from species that resemble vertebrate ancestors. Here, I characterized gill and skin function in representatives of ancestral vertebrates (lamprey ammocoetes), cephalochordates (lancelets) and hemichordates (acorn worms). Comparisons within ammocoetes tested the effects of size and activity on early vertebrate gill function, and comparisons between all representatives used shared traits to infer gill function in common ancestors. Results support an early vertebrate origin for breathing at gills associated with increasing body size and activity. However, ion regulation at gills appears unrelated to this vertebrate transition, and instead likely evolved long before vertebrates when gills first appeared in early deuterostomes. v Preface  I designed, performed and analyzed all experiments under the guidance of CJ Brauner at the University of British Columbia. All animal research was approved by the Canadian Council on Animal Care and conducted under the Animal Care Certificate #A15-0266. vi  Table of Contents  Abstract ......................................................................................................................................... iii	Lay Summary ............................................................................................................................... iv	Preface .............................................................................................................................................v	Table of Contents ......................................................................................................................... vi	List of Tables .............................................................................................................................. viii	List of Figures ............................................................................................................................... ix	List of Equations .......................................................................................................................... xi	List of Symbols ............................................................................................................................ xii	List of Abbreviations ................................................................................................................. xiii	Chapter 1: General Introduction .................................................................................................1	1.1	 Introduction ........................................................................................................................ 1	1.2	 Vertebrate gill structure and function ................................................................................ 3	1.2.1	 General gill structure ................................................................................................... 3	1.2.2	 Gas exchange at gills .................................................................................................. 4	1.2.3	 Ion regulation at gills .................................................................................................. 6	1.2.4	 Summary for gas exchange and ion regulation in fish gills ...................................... 10	1.3	 A vertebrate origin for gas exchange and ion regulation at gills ..................................... 11	1.3.1	 Constrained by gas exchange and ion regulation at skin .......................................... 11	1.3.2	 Freed by gas exchange and ion regulation at gills .................................................... 13	1.3.3	 More constrained by ion regulation than by gas exchange? ..................................... 14	1.3.4	 Gaps in the existing support ...................................................................................... 15	1.4	 Thesis Approach: Comparing extant representatives ...................................................... 17	1.4.1	 Study species ............................................................................................................. 18	1.4.1.1	 Vertebrate representative: Lamprey ammocoete, E. tridentatus ....................... 18	1.4.1.2	 Cephalochordate representative: Amphioxus, B. floridae ................................. 19	1.4.1.3	 Hemichordate representative: Acorn worm, S. kowalevskii .............................. 20	1.4.2	 Aim 1: How were gills recruited for gas exchange and ion regulation? ................... 20	1.4.3	 Aim 2: When were gills recruited for gas exchange and ion regulation? ................. 22	1.5	 Figures.............................................................................................................................. 23	vii  Chapter 2: The effects of body size and activity on gill recruitment for gas exchange and ion regulation in an early vertebrate representative ................................................................32	2.1	 Introduction ...................................................................................................................... 32	2.2	 Methods............................................................................................................................ 35	2.3	 Results .............................................................................................................................. 42	2.4	 Discussion ........................................................................................................................ 43	2.5	 Figures.............................................................................................................................. 48	Chapter 3: Gas exchange in a hemichordate representative ...................................................52	3.1	 Introduction ...................................................................................................................... 52	3.2	 Methods............................................................................................................................ 55	3.3	 Results .............................................................................................................................. 58	3.4	 Discussion ........................................................................................................................ 59	3.5	 Figures.............................................................................................................................. 62	Chapter 4: Ion regulation in hemichordate and cephalochordate representatives ...............64	4.1	 Introduction ...................................................................................................................... 64	4.2	 Methods............................................................................................................................ 67	4.3	 Results .............................................................................................................................. 71	4.4	 Discussion ........................................................................................................................ 72	4.5	 Figures.............................................................................................................................. 77	Chapter 5: General Discussion ...................................................................................................93	5.1	 Introduction ...................................................................................................................... 93	5.2	 A deep origin for ion regulation at gills, but for what? ................................................... 94	5.3	 Was the ancestral form of ion regulation linked to filter-feeding? .................................. 95	5.4	 Did filter-feeding limit gas exchange at gills? ................................................................. 97	5.5	 Future Directions ............................................................................................................. 99	5.6	 Figures............................................................................................................................ 102	References ...................................................................................................................................104	Appendices ..................................................................................................................................121	Appendix A -   Chapter 2 supplemental material ................................................................... 121	Appendix B -   Chapter 3 supplemental material .................................................................... 123	Appendix C -   Chapter 4 supplemental material .................................................................... 124	viii  List of Tables  Table 4.1 Percent identity between human and protovertebrate protein sequences ..................... 91	Table 4.2 Putative promoter binding sites for Foxi in B. floridae and S. kowalevskii .................. 92	Table C.1 Target genes and NCBI accession numbers ............................................................... 128	Table C.2 Primer sequences for RT-qPCR ................................................................................. 129	 ix  List of Figures  Figure 1.1 Cladogram of major deuterostome taxa ...................................................................... 23	Figure 1.2 External anatomy of study species .............................................................................. 24	Figure 1.3 General teleost fish gill structure ................................................................................. 25	Figure 1.4 Countercurrent and uniform pool arrangements for gas exchange ............................. 26	Figure 1.5 Acid and base excretion in the seawater chondrichthyan gill ..................................... 27	Figure 1.6 Salt excretion in the seawater teleost gill .................................................................... 28	Figure 1.7 Ammonia excretion in the freshwater teleost gill ........................................................ 29	Figure 1.8 Gill arch structure in hemichordates and cephalochordates ........................................ 30	Figure 1.9 Cladogram of major vertebrate taxa ............................................................................ 31	Figure 2.1 Divided chamber ......................................................................................................... 48	Figure 2.2 Percent of total gas and ion flux at gills of Entosphenus tridentatus .......................... 49	Figure 2.3 Whole body rates of gas and ion flux in Entosphenus tridentatus .............................. 50	Figure 2.4 Cutaneous anatomical diffusion factor (ADF) of Entosphenus tridentatus ................ 51	Figure 3.1 Oxygen uptake rates of whole and fragmented Saccoglossus kowalevskii ................. 62	Figure 3.2 Ammonia excretion rates of whole and fragmented Saccoglossus kowalevskii .......... 63	Figure 4.1 Skin and gill ATPase activities in B. floridae and S. kowalevskii ............................... 77	Figure 4.2 Skin and gill gene expression in B. floridae and S. kowalevskii ................................. 78	Figure 4.3 Anion exchanger phylogeny ........................................................................................ 79	Figure 4.4 Sodium-proton exchanger phylogeny .......................................................................... 80	Figure 4.5 Carbonic anhydrase phylogeny ................................................................................... 81	Figure 4.6 Ammonia transporter phylogeny ................................................................................. 82	Figure 4.7 Forkhead box phylogeny ............................................................................................. 83	Figure 4.8 Multiple sequence alignment for anion exchangers .................................................... 84	Figure 4.9 Multiple sequence alignment for sodium-proton exchangers ..................................... 86	Figure 4.10 Multiple sequence alignment for carbonic anhydrases ............................................. 88	Figure 4.11 Multiple sequence alignment for Rhesus glycoproteins ............................................ 89	Figure 4.12 Multiple sequence alignment for forkhead box I ...................................................... 90	Figure 5.1 Thesis summary ......................................................................................................... 102	Figure 5.2 Metazoans and ionocyte origins ................................................................................ 103	x  Figure A.1 Epidermal thickness of Entosphenus tridentatus ...................................................... 121	Figure A.2 AQ, Q10, RER and ventilation rate in Entosphenus tridentatus ............................... 122	Figure B.1 Gas exchange in whole and fragmented Protoglossus graveolens ........................... 123	Figure C.1 Skin and gill NKA activity in S. kowalevskii, B. floridae and E. tridentatus ........... 124	Figure C.2 All tissue ATPase activity in S. kowalevskii, B. floridae and E. tridentatus ............ 125	Figure C.3 All tissue gene expression in S. kowalevskii ............................................................. 126	Figure C.4 All tissue gene expression in B. floridae .................................................................. 127	 xi  List of Equations  Equation 1.1 V̇ = A • ∆P • D • t-1 .................................................................................................... 4	Equation 1.2 Diffusion capacity = A • D • t-1 ................................................................................. 5	Equation 2.1 O2 uptake for chamber half = ∆PO2·h-1 • αO2 • (v - mp) ........................................... 37	Equation 2.2 CO2 excretion for chamber half = ∆TCO2·h-1 • (v - mp) ......................................... 37	Equation 2.3 NH3/4+ excretion for chamber half = ∆NH3/4+ • (v - mp) • t-1 ................................... 38	Equation 2.4 Na+ uptake = Rfish • SAwater-1 • m-1 • t-1 .................................................................... 39	Equation 2.5 Ca2+ uptake = Rfish • SAwater-1 • m-1 • t-1 ................................................................... 39	Equation 2.6 t = 45.2(m)0.34 .......................................................................................................... 40	Equation 3.1 ṀO2 = ∆PO2 • αO2 • v • m-1 ....................................................................................... 57	Equation 3.2 ṀNH3/4+ =  ∆NH3/4+ • v  • m-1 • t-1 ............................................................................. 57	 xii  List of Symbols  µ = micro oC = degrees Celsius ∆ = change or difference α(gas) = solubility of specified gas P(gas) = partial pressure of specified gas Ṁ(gas) = rate of flux for mass of specified gas V̇(gas) = rate of flux for volume of specified gas Q10 = temperature effect Kir = inward-rectifier K+ channel  xiii  List of Abbreviations  ADF = anatomical diffusion factor AE = anion exchanger AMRC = ammocoete mitochondrion rich cell ATP = adenosine triphosphate AQ = ammonia quotient CA = carbonic anhydrase CFTR = cystic fibrosis transmembrane conductance regulator cpm = counts per minute dph = days post-hatch ENaC = epithelial Na+ channel FOX = forkhead box MS-222 = tricane methanesulfonate NCBI = National Centre for Biotechnology Information NHE = Na+, H+-exchanger NKA = Na+, K+-ATPase NKCC = Na+/K+/2Cl- -co-transporter NEM = N-ethylmaleimide Osm = osmole PCR = polymerase chain reaction Pd = Pendrin anion exchanger Rhag = Rhesus glycoprotein A Rhbg = Rhesus glycoprotein B Rhcg = Rhesus glycoprotein C RER = respiratory exchange ratio SA:V = surface area to volume ratio s.e.m. = standard error of the mean SLC = solute carrier family TCO2 = total CO2 VHA = vacuolar type H+-ATPase  1 Chapter 1: General Introduction  1.1 Introduction  Pharyngeal gill arches are a defining vertebrate trait. These structures and their derivatives are hypothesized to play multiple adaptive roles in vertebrate evolution, making key contributions to the broad diversity in form, function and habitat exhibited by this taxon. In early vertebrates and fishes, many of these important evolutionary roles are attributed to gills serving as the primary sites of gas exchange and ion regulation. Indeed, gill function in gas and ion homeostasis is implicated in many notable evolutionary events, including the expansion into brackish and freshwater environments (Evans, 1984), and the stem-vertebrate transition to larger, armoured and more active modes of life (Northcutt, 2005). However, despite this adaptive value, significant gaps in our understanding of when and how gills acquired their function in gas exchange and ion regulation remain. This thesis aims to address these gaps.  Gills are currently believed to have acquired their primary roles in gas exchange and ion regulation in near-identical fashion along the vertebrate stem (Figure 1.1; Northcutt, 2005; Brauner and Rombough, 2012). In these scenarios, vertebrate ancestors were initially constrained to a small, worm-like existence by their reliance on the skin to breathe and to regulate acid-base ion balance. Both processes then shifted to the filter-feeding gills, relaxing worm-like constraints and facilitating the evolution of larger, armoured and more active modes of life. These proposed origins of vertebrate gill function are based on morphological and functional data. For gas exchange, fossil and developmental work show that respiratory structures associated with breathing at gills were acquired along the vertebrate stem with larger, fish-like body forms (Morris and Caron, 2014; Green et al., 2015; Gillis and Tidswell, 2017). There is no equivalent fossil or developmental evidence for ionoregulatory structures, but the presence of specialized ion regulating cells in the gills of all extant fishes is consistent with a stem-vertebrate origin. The best functional support for both scenarios comes from larval teleosts, where gas exchange and ion regulation shift from skin to gills with increasing size, dermal thickness and activity during development (reviewed by Rombough, 2007; Brauner and Rombough, 2012).  2  Support for these hypothesized origins of vertebrate gill function is compelling but incomplete. In vertebrate ancestors, the assumed reliance on cutaneous gas exchange is based solely on morphology (Schmitz et al., 2000), and skin and gill function in ion regulation remains unknown. Furthermore, the larval teleost data linking gill function to early vertebrate size, dermal thickness and activity are confounded by development and derived aspects of teleost morphology and life history. The proposed origins of gill function thus lack functional support from representatives of early vertebrates and their ancestors.  Here, I sought to improve our understanding of when and how vertebrate gills acquired their primary roles in gas exchange and ion regulation. To do so, I characterized gill and skin function in representatives of ancestral vertebrates (lamprey ammocoete, Entosphenus tridentatus; Figure 1.2A), cephalochordates (amphioxus, Branchiostoma floridae; Figure 1.2B) and hemichordates (acorn worm, Saccoglossus kowalevskii; Figure 1.2C). With this study system, I tested two central hypotheses:  (1) Gills acquired their primary roles in gas exchange and ion regulation in the vertebrate lineage  (2) Increases to early vertebrate size, dermal thickness and activity were facilitated by gills replacing the skin as the primary site for gas exchange and ion regulation   Interspecific comparisons between taxa tested the ancestral origins of gill function (Chapters 2, 3 & 4), and intraspecific comparisons within ammocoetes tested the effects of size, dermal thickness and activity on early vertebrate gill function (Chapter 2). These comparative approaches are bolstered by (1) gill arch homology among representative species (Gillis et al., 2012; Green et al., 2015; Simakov et al., 2015) and (2) the resemblance of representative species to the presumed morphology and lifestyle of early vertebrates and their ancestors (Cameron et al., 2000; Gerhart et al., 2005; Brown et al., 2008; Swalla and Smith, 2008; Holland et al., 2015; Lowe et al., 2015).  The remainder of this introductory chapter details the foundation upon which the central hypotheses and experimental design are based. This includes a brief overview of vertebrate gill structure and function in gas exchange and ion regulation, a summary of current hypotheses and  3 support for the origins of gill function, and justification for the experimental species and approach. This introduction then concludes with specific chapter hypotheses and predictions.  1.2 Vertebrate gill structure and function  The fish gill is the dominant site of gas exchange and ion regulation. In simple terms, it is the primary epithelium across which gases and ions move between the external and internal environments. This includes the exchange of oxygen and carbon dioxide, excretion of nitrogenous waste, and movement of ions to maintain both acid-base and hydromineral balance (reviewed by Evans et al., 2005; Wilson and Laurent, 2002).  Much of the gill’s effectiveness in performing these vital homeostatic functions lies in its structural organization. This section describes general gill structure, highlighting how specific features enhance gas exchange and the various forms of ion regulation relative to the skin. Hypotheses regarding the origins of gas exchange and ion regulation in vertebrate gills are based heavily on the structures and principles of gas and ion transport outlined here.  1.2.1 General gill structure  Fish gills consist of three basic structural units: arches, filaments and lamellae (Figure 1.3). The archetypal teleost fish gills are typically arranged in four paired arches that line the lateral borders of the pharyngeal cavity (Figure 1.3A). These arches form during development as pharyngeal endoderm fuses with ectoderm, creating discrete columns separated by pharyngeal slits (Warga and Nüsslein-Volhard, 1999; Gillis and Tidswell, 2017). The arches contain a core of paraxial mesoderm and neural crest mesenchyme that gives rise to a cartilaginous endoskeleton, blood vessels, smooth muscle and nervous tissue (reviewed by Green et al., 2015). In addition to providing structural support for the pharynx, the robust cartilaginous arches give rise to numerous finer projections termed “filaments” (also known as primary lamellae; Figure 1.3B). Filaments bud laterally from the endoderm of the arches, and in turn give rise to more delicate sheet-like projections termed “lamellae” (also known as secondary lamellae; Figure 1.3C). Together, the filaments and lamellae support a vast proliferation of the blood vessels found in the arches, providing intimate contact between the internal milieu and external  4 environment. As described below, these finer projections form the primary sites of gas exchange and ion regulation.  1.2.2 Gas exchange at gills  Exploiting Fick’s law of diffusion  Vertebrate life can not exist without gas exchange. Oxygen uptake is required to fuel aerobic respiration for ATP production, and the resulting carbon dioxide waste must be excreted to maintain acid-base balance. Gases move across biological membranes largely by way of diffusion. The gill’s effectiveness as a gas exchange organ can thus be best illustrated with an equation based on Fick’s law of diffusion (Equation 1.1).  Equation 1.1                                     V̇ = A • ∆P • D • t-1  In this modified Fick equation (Equation 1.1), “V̇” is the rate of gas diffusion, “A” is epithelial area, “t” is epithelial thickness, “D” is the diffusion coefficient (determined by molecular size and solubility of a given gas) and “∆P” is the partial pressure gradient across the epithelium (P(water) - P(blood)). This iteration of the equation is commonly used to describe trans-epithelial gas flux in biological systems (Cameron, 1989).  Fish gills are effective gas exchange organs because their structure exploits the different elements of our modified Fick equation. They have a high surface area (A), thin diffusion distance (t) and maintain favourable partial pressure gradients (∆P) with counter-current blood-water flow. Relative to the skin, gills are superior in each of these ways. For example, the gills of a 10 g Atlantic salmon (Salmo salar) have a surface area of ~6000 mm2 and mean diffusion distance of ~4 µm (Wells and Pinder, 1996a). This is 20% greater than total body surface area (~5000 mm2) and 20-fold thinner than skin (-80 µm). Anatomical diffusion capacities can further illustrate how these structural differences impact gas exchange (Equation 1.2). Assuming diffusion coefficients (D) are similar between epithelia, the anatomical diffusion capacity is simply the epithelial surface area (A) divided by its mean thickness (t).    5 Equation 1.2                            Diffusion capacity = A • D • t-1  According to these numbers, the diffusive capacity for the gills of a 10 g Atlantic salmon is 24-fold higher than that of the skin, accounting for ~95% of the fish’s total capacity. Furthermore, most of this diffusive capacity occurs specifically at lamellae. Of the 6000 mm2 of total gill area, filaments and lamellae account for 1500 and 4500 mm2, respectively. Lamellar diffusion distance is also 5-fold thinner than that of filaments, at ~2 vs ~10 µm. Lamellae are thus the primary gas exchange structure within gills, possessing a diffusive capacity 15-fold higher than that of filaments and accounting for over 90% of the gill’s total capacity. Aside from a greater diffusive capacity, gills also outperform the skin for gas exchange because of their counter-current blood-water flow (Figure 1.4A). A counter-current arrangement ensures a more complete transfer of gases between blood and water by maintaining a favourable ∆P over a wider range of partial pressures. For example, the gills of many fishes are estimated to remove >80% of oxygen from inspired water, with blood PO2 nearing ~75% of atmospheric levels (Saunders, 1962). This is far more effective than the “uniform pool” arrangement present at the skin, in which PO2(water) and PO2(blood) equilibrate at an intermediate PO2 closer to 50% of atmospheric levels (Figure 1.4B).  Pillar cells enhance lamellar gas exchange The large area, thin diffusion distance and countercurrent arrangement underlying the superior gas exchange function of lamellae are facilitated in part by specialized endothelial “pillar” cells. Lamellae are sheet-like capillary beds enclosed by a thin epithelium, and are just thick enough to accommodate red blood cells (Newstead, 1967; Hughes and Morgan, 1973). In addition to a small diffusion distance, this thin, sheet-like structure allows many individual lamellae to be packed closely together on filaments, vastly increasing total lamellar area in the opercular cavity. As the name implies, pillar cells span the lamellar blood space between opposing epithelia, serving as “pillars” that preserve the integrity of this delicate sheet-like structure. They prevent lamellae from collapsing in on themselves or bursting under high blood pressure. In the absence of pillar cells, lamellae would require a more robust epithelium and/or endoskeleton, likely limiting their diffusive capacity and closely packed arrangement.  6 Pillar cells also have contractile properties that influence blood flow within the capillary spaces of lamellae (Bettex-Galland and Hughes, 1973; Sundin and Nilsson, 1998; Stenslokken et al., 1999). They therefore play an important role in counter-current exchange by directing blood flow to the appropriate lamellar channels. Furthermore, this contractile function also helps control the area of lamellar perfusion to match changes in demand for gas exchange. For example, rainbow trout (Oncorhynchus mykiss) only perfuse ~60% of total lamellar area at rest (Booth, 1978), but can likely increase this value to 100% in seconds during exercise or hypoxia (Randall and Daxboeck, 1982).  1.2.3 Ion regulation at gills   As with gas exchange, ion regulation at gills can also be affected by epithelial area, thickness and blood/water convection. However, the ways in which these factors affect ion regulation differ slightly from gas exchange. This is because ions and gases are transported across epithelia in fundamentally different ways. While gases can diffuse freely across epithelia, ions often move through transporters and channels powered by active transport. This ion transport machinery is localized to specialized epithelial cells called “ionocytes” that are distributed throughout gill filaments and lamellae (also commonly referred to as “chloride cells” and “mitochondrion-rich cells”; reviewed by Hwang et al., 2011). A brief description of ionocytes and the various forms of ion regulation is provided below. This description is then followed by an explanation of how epithelial area, thickness and blood/water convection can affect ionocyte function.  Ionocytes power ion regulation Multiple ionocyte subtypes exist across fishes, each with their own set of transport machinery specialized to their respective life histories. However, all ionocytes share certain hallmark features. These include the presence of transmembrane ATPase’s, abundant mitochondria to power the ATPase’s, and typically apical/basolateral contact with the water/blood. ATPase’s consume ATP to actively transport ions across the ionocyte cell membrane. This establishes the electrochemical gradients required to drive ions through other parts of the ionocyte machinery (termed secondary active transport). In fish gills, this driving  7 force is usually provided by Na+,K+-ATPase (NKA) and/or V-type H+-ATPase (VHA). As described below, these ATPase’s power the movement of ions through the ionocytes of fish gills to maintain acid-base balance, hydromineral balance and ammonia excretion (reviewed by Dymowska et al., 2012; Hwang and Lin, 2013).  Ionocytes maintain acid-base balance Changes in pH can affect the protonation state of proteins, which can in turn alter protein structure, function and ultimately animal performance (Hochachka and Somero, 2002). Actively regulating pH of the internal milieu therefore provides important selective benefits, allowing organisms to maintain optimal protein function despite acid-base disturbances of metabolic and/or environmental origin. In fishes, acid-base balance is maintained primarily by gill ionocytes that transport acid-base relevant ions, and the kidney and intestine are believed to play only minor roles (reviewed by Brauner et al., 2019). In the archetypal seawater chondrichthyan gill, acid and base excretion is performed by two different ionocyte subtypes (Figure 1.5; based on Wright and Wood, 2015). In acid excreting cells (Figure 1.5A), basolateral NKA actively pumps sodium into the plasma to establish an inward gradient for sodium flux. This gradient powers an apical sodium-proton exchanger (NHE), where protons are excreted to seawater in exchange for sodium. The protons for acid excretion are provided by the hydration of cytosolic carbon dioxide, which is catalyzed by carbonic anhydrase (CA). Excess bicarbonate from this hydration reaction is hypothesized to then return to the plasma down the electrochemical gradient, although a specific pathway has yet to be elucidated. The end result is net acid excretion from the animal. In base excreting cells (Figure 1.5B), basolateral VHA actively pumps protons into the plasma. As in acid excreting cells, this removal of protons from the cytosol drives further hydration of carbon dioxide within the ionocyte. Excess bicarbonate from the hydration reaction then accumulates, driving its apical excretion to seawater through a pendrin-like anion exchanger (Pd). This bicarbonate excretion is also assisted by the lower ionocyte membrane potential produced from basolateral proton extrusion by VHA. The end result is net base excretion from the animal. These ionocyte schematics are specific to seawater chondrichthyans, but similar pathways for acid-base ion regulation are proposed for gill ionocytes in all fishes (reviewed by  8 Dymowska et al., 2012; Hiroi and McCormick, 2012; Hwang and Lin, 2013). Acid-base regulation is believed to be the ancestral form of ion regulation in vertebrates (Evans, 1984). Machinery similar to that described here is believed to have been coopted for its more derived function in maintaining hydromineral balance and ammonia excretion as described below.  Ionocytes maintain hydromineral balance  Hydromineral balance refers to the concentrations of ions and water within the internal milieu. It is critical to many vital biological processes such as cell volume regulation and the maintenance of transmembrane electrochemical gradients (Hochachka and Somero, 2002). As with acid-base balance, actively regulating hydromineral balance of the internal milieu thus provides important selective benefits, allowing organisms to maintain these biological processes despite disturbances of metabolic and/or environmental origin. As an example, regulating hydromineral balance played a key role in the vertebrate expansion into brackish and freshwater environments (Evans, 1984).  Teleost plasma osmolarity is typically 300-350 mOsm. This is ~300-fold higher than freshwater (~1 mOsm) and ~1/3 that of seawater (~1000 mOsm). The resulting osmotic gradients subject teleosts to passive ion loss and water gain in freshwater, and ion gain and water loss in seawater. To combat these passive fluxes, teleosts actively move ions and water against the osmotic gradients (reviewed by Edwards and Marshall, 2012). Gill ionocytes are the primary site for active ion uptake and excretion in freshwater and seawater, respectively, while the kidney and intestine play more important roles in water flux (although the kidney is also important for divalent ion excretion in seawater). Multiple gill ionocyte subtypes have been implicated in maintaining teleost hydromineral balance (Edwards and Marshall, 2012; Hwang and Lin, 2013). Thus, in the interest of brevity, only the archetypal seawater teleost ionocyte is described here. The archetypal seawater teleost ionocyte excretes excess sodium and chloride from plasma to water (Figure 1.6). Here, basolateral NKA actively pumps sodium out of the ionocyte into the plasma, establishing a favourable gradient for inward sodium flux. Sodium from the plasma then follows this gradient back into the ionocyte through Na+,K+,2Cl--cotransporter (NKCC). As the name implies, chloride also moves into the ionocyte through NKCC by piggybacking on this favourable sodium gradient. This eventually raises intracellular chloride high enough to drive its excretion to seawater through an apical chloride channel (cystic fibrosis  9 transmembrane conductance regulator or “CFTR”). The accumulation of chloride in the apical boundary layer of ionocytes then helps establish an electrochemical gradient that draws sodium out of the plasma through paracellular junctions.   Ionocytes excrete ammonia  Ammonia is a ubiquitous waste product of protein metabolism that can serve as a lethal neurotoxin if left to accumulate (Rangroo Thrane et al., 2013). It is often referred to as a respiratory gas (NH3), but is mostly found in ionic form (NH4+) at physiological pH (teleost blood pH ~7.8, NH3 pK’ in plasma ~9.65; Cameron and Heisler, 1983). In fishes, ammonia excretion occurs primarily at gills. Many pathway specifics remain unresolved and may vary with species and environment. However, the coupling of ammonia excretion to sodium uptake and/or proton excretion in freshwater teleosts is well supported (Hwang and Lin, 2013; Wright and Wood, 2012). Ammonia excretion is thus often discussed in the context of ion regulation.  In currently accepted pathways for ammonia excretion in freshwater teleosts (Figure 1.7; redrawn from Hwang and Lin, 2013), ammonium in the plasma binds to a Rhesus glycoprotein (Rhbg) on the basolateral membrane of the ionocyte. It then deprotonates to ammonia, entering the ionocyte down its partial pressure gradient through the Rhbg channel. In the ionocyte, ammonia re-protonates to ammonium before binding Rhcg on the apical membrane. Ammonium again deprotonates to ammonia, which exits through Rhcg down its partial pressure gradient. Once in the water, ammonia is re-protonated for a final time, helping to maintain a favourable partial pressure gradient for its excretion. This “acid-trapping” by ammonium on the apical water side has been linked to sodium uptake and proton excretion in freshwater teleosts (reviewed by Dymowska et al., 2012; Wright and Wood, 2012). For example, in ammonium-dependent sodium uptake, Rhcg is believed to form a metabolon with apical NHE (Kumai and Perry, 2011; Shih et al., 2011). In this scenario, the proton cleaved from cytosolic NH4+ by Rhcg directly powers sodium uptake through NHE. Acid-trapping on the water side then helps to maintain the outward movement of NH3 and protons through this metabolon. VHA is also believed to contribute to this pathway by acidifying the boundary layer. This is proposed to enhance sodium uptake by either increasing NH3 flux through the Rhcg-NHE metabolon, or by powering sodium through separate apical ion channels (Dymowska et al., 2014). It should be noted that details of these proposed pathways are still  10 being debated and actively researched. However, the coupling of ammonia excretion with active ion transport at teleost gill ionocytes is well supported.  Ion regulation is also affected by area, thickness and convection Despite these fundamental differences from gas exchange, ion regulation at gills can be similarly affected by epithelial area, thickness and blood/water convection. For example, area can affect ion regulation by providing or limiting space for ionocytes. This is evidenced by rainbow trout acclimated to ion poor water, who maintain sufficient rates of ion uptake by doubling the gill area occupied by ionocytes through cell proliferation and expansion (Greco et al., 1995, 1996). Epithelial thickness can also affect ionocyte function. Ionocytes typically have basolateral/apical contact with blood/water, therefore requiring an epithelial thickness of one cell layer. Thus, while gas diffusion progressively declines with epithelial thickness, ion movement might abruptly terminate beyond a certain critical thickness (proposed by Brauner and Rombough, 2012). Finally, ion transport can also theoretically be affected by blood/water convection. Ventilation and perfusion of the gill epithelium could help maintain suitable ion concentrations in ionocyte boundary layers for transporter function. This has yet to be demonstrated experimentally, but perhaps because the respiratory demands placed on ventilation and perfusion in fishes may exceed the ionic demands in most situations examined.  1.2.4 Summary for gas exchange and ion regulation in fish gills  The large area, thin epithelium and convective control of blood and water make fish gills an effective organ for gas exchange and ion regulation. Gas exchange primarily occurs at lamellae, where specialized endothelial pillar cells facilitate an elevated diffusive capacity. Ion regulation occurs at both filaments and lamellae, and is driven by specialized epithelial cells called ionocytes. As described in the following sections, hypotheses regarding the origins of vertebrate gill function for gas exchange and ion regulation are based heavily on these principles and structures.    11 1.3 A vertebrate origin for gas exchange and ion regulation at gills  Gills are hypothesized to have acquired their primary roles in gas exchange and ion regulation along the vertebrate stem in near-identical fashion. This scenario has long been accepted for gas exchange (Gans and Northcutt, 1983; Northcutt, 2005), but was only more recently proposed for ion regulation (Brauner and Rombough, 2012). In this scenario, the vertebrate ancestor is widely believed to be a small, worm-like burrower that lived in the sediment on the ocean floor. It possessed ciliated gill arches for filter-feeding that lacked filaments and lamellae, and it relied on the skin for gas exchange and acid-base ion regulation. Reliance on the skin for gas exchange and ion regulation is believed to have constrained the ancestor to its worm-like existence by requiring a high surface area to volume ratio (SA:V) and thin dermis for sufficient rates of gas diffusion and ion transport. At some point along the vertebrate stem, gas exchange and ion regulation shifted from skin to the filter-feeding gills. This was facilitated in part by the appearance of a muscular pharyngeal pump, which produced a greater flow of water through the pharynx than the ciliated arches. The increased water flow is believed to have been initially selected for enhanced feeding. However, increased water flow through the pharynx also provided the gills with a greater convective potential. Filaments and lamellae then appeared on the gill arches, exploiting this improved water convection and enhancing the capacity for gas exchange and ion regulation at gills. No longer bound by the constraints of cutaneous gas exchange and ion regulation, vertebrates could abandon the high SA:V, thin outer dermis and low activity that characterized their worm-like ancestry. Shifting gas exchange and ion regulation to the gills is thus believed to have facilitated the evolution of larger, armoured and more active modes of life that characterize early vertebrate diversification. As described in the following sections, support for this scenario is compelling but incomplete.  1.3.1 Worm-like ancestors were constrained by gas exchange and ion regulation at skin  The proposed worm-like morphology, burrowing lifestyle and filter-feeding habits of the vertebrate ancestor are well supported. Fossil and biomechanical data suggest the ancestral hemichordate closely resembled burrowing, worm-like members of the extant enteropneusta  12 class (Cameron et al., 2000; Cameron, 2005; Caron et al., 2013; Nanglu et al., 2015; Vo et al., 2019), and all extant and fossil cephalochordates also appear to be worm-like burrowers (Morris and Caron, 2012; Holland et al., 2015). Extant echinoderms and urochordates are not worm-like burrowers, but genetic and fossil data suggest their pentaradial and sessile modes of life are derived traits (Sumrall and Wray, 2007; Smith, 2008; Holland, 2016; see Figure 1.1 for simplified deuterostome cladogram). The presence of rudimentary gill arches is also well supported. Pharyngeal gill arches are an unambiguous deuterostome synapomorphy (Green et al., 2015; Lowe et al., 2015). Hemichordates, cephalochordates, urochordates and vertebrates all develop pharyngeal arches from endodermal outpocketings under similar genetic control (Gillis et al., 2012; Simakov et al., 2015), and fossil evidence suggests arches were secondarily lost in echinoderms (Smith, 2005). Furthermore, arches in representatives of ancestral hemichordates and cephalochordates are organized into remarkably similar gill bars that lack filaments and lamellae (Figure 1.8). These structures are supported by skeletal rods composed of acellular collagen, and each has a simple hemolymph vessel and coelom (Gillis et al., 2012). As for filter-feeding with gills, pharyngeal arches in all of the extant invertebrate deuterostomes form a mucociliary complex for this purpose. In ancestral representatives from hemichordates and cephalochordates, cilia generate a feeding current into the pharynx through the “mouth” that directs water flow over the gill bars and out through the gill slits (Mallatt, 1981; Gonzalez and Cameron, 2009). Cilia and mucus covering the gill bars trap food particles that are ultimately passed down through the gut for digestion. Support for reliance on the skin for gas exchange is less definitive. Morphometric analyses suggest cephalochordate gills play negligible roles in gas exchange, possessing less than 5% of total body diffusive capacity (Schmitz et al., 2000). However, morphometric analyses are absent in hemichordates, and direct measurements of gas exchange at gills have not been made in either clade. Direct measurements are preferred over morphometrics when possible, as convection may alter epithelial contributions in ways that differ from calculated diffusive capacities. The case for ion regulation is even less clear. Ionocytes do not fossilize and have yet to be identified in any extant invertebrate deuterostome. In cephalochordates, positive immunoreactivity for ion transport machinery has been reported at both skin and gills, but  13 antibodies were not tested for non-specific binding (Pederzoli et al., 2014; Cuoghi et al., 2018). Classic morphological work has identified rudimentary nephridial structures associated with the gill coelom, but their function also remains unknown (Moller and Ellis, 1974). Similar nephridial structures of unknown function are also found in the proboscis of acorn worms (Balser and Ruppert, 1990), but no further exploration of ion regulation has been conducted in hemichordates. The assumption that gills of the vertebrate ancestor were not a primary site for gas exchange and ion regulation thus appears to lack support.  1.3.2 Early vertebrates were freed by gas exchange and ion regulation at gills  Morphological support Evidence from fossil and extant deuterostomes indicates that many of the structural adaptations associated with gas exchange at gills arose along the vertebrate stem with larger, fish-like body shapes. Foremost, gill filaments first appear in the fossil record alongside a muscular pharyngeal pump and more fish-like body forms in the putative stem-vertebrates Myllogkunmingia (Shu et al., 1999), Metaspriggina (Morris and Caron, 2014) and Haikouichthys (Xian-guang et al., 2002), and before the bony dermal armour of the early jawless ostracoderm fishes (Janvier, 2008). Furthermore, developmental studies reveal a common embryonic origin for gill filaments and lamellae among jawed and jawless fishes, similarly suggesting a single origin for these structures in the last common ancestor of vertebrates (Stockard, 1906; Damas, 1944; Warga and Nüsslein-Volhard, 1999; Gillis and Tidswell, 2017). Other structural innovations implicated in this scenario are derived from the neural crest, which is an embryonic cell population unique to vertebrates (Martik et al., 2019). In jawed and jawless fishes, the neural crest adds the musculature and cellular cartilage to gill arches that provides the contraction and elastic recoil, respectively, for the pharyngeal pump (Green and Bronner, 2014). The specialized pillar cells that underlie the enhanced capacity for gas exchange in gill lamellae are also derived from neural crest (Mongera et al., 2013), as are several other circulatory adaptations associated with improved blood convection (cardiac valves, pericytes, smooth muscle of the cardiovascular system, etc; Kirby et al., 1983; Kuratani and Kirby, 1991; Jiang et al., 2000; Boot et al., 2003; Green and Bronner, 2014; Tang et al., 2019).  14 As for specific ionoregulatory structures, the presence of ionocytes in the gills of all extant fishes also suggests they were present in the gills of the stem-vertebrate. However, a neural crest origin has yet to be shown, and ionocyte presence and distribution in earlier ancestors remains unknown.  Morphological data thus appear to provide good support for the proposed relationship between gas exchange at gills and early vertebrate size, dermal thickness and activity, but support for ion regulation is lacking.   Functional support There are no direct measurements of gas exchange or ion regulation at the skin or gills of extant representatives of early vertebrates or their ancestors. Due to this absence, functional support linking gas exchange and ion regulation at gills to early vertebrate size, dermal thickness and activity is mostly derived from larval teleost ontogeny (reviewed by Rombough, 2007; Brauner and Rombough, 2012). This is because larval teleost ontogeny exhibits striking parallels with early vertebrate evolution. Indeed, larval teleosts initially lack gills and thus rely on the skin for gas exchange and ion regulation. Like the presumed vertebrate ancestor, this reliance on the skin is believed to similarly constrain larval teleosts to a high SA:V, thin dermis and low activity early on. As development progresses, proliferation of gill arches, filaments, lamellae and ionocytes coincides with dramatic increases to body size, dermal thickness and activity. Direct measurements of gas exchange and ion regulation demonstrate that gills replace the skin as the primary site for these functions as these changes occur (Wells and Pinder, 1996b; Fu et al., 2010; Zimmer et al., 2014).  1.3.3 Were early vertebrates more constrained by ion regulation at the skin than by gas exchange?  The larval teleost work also suggests that early vertebrate gills might have become the primary site for ion regulation before they became the primary site for gas exchange, as the gills of every larval teleost examined exhibit this pattern (reviewed by Rombough, 2007; Brauner and Rombough, 2012). In zebrafish, gill ablation suggests gills become critical for ion regulation at 7 days post-fertilization vs 14 for gas exchange (Rombough, 2002), and in rainbow trout,  15 partitioned flux experiments show that gills replace the skin as the primary site for sodium uptake and ammonia excretion at 15 days post-hatch vs 28 for oxygen (Fu et al., 2010; Zimmer et al., 2014). Furthermore, ionocytes appear on the gills before the development of respiratory lamellae in all teleosts investigated (rainbow trout, O. mykiss, González et al., 1996 and Rombough, 1999; tilapia, Oreochromis mossambicus, Li et al., 1995; seabass, Dicentrarchus labrax, Varsamos et al., 2002; Japanese flounder, Paralichthys olivaceus, Hiroi et al., 1998; ayu, Plecoglossus altivelis, Hwang, 1990; killifish, Fundulus heteroclitus, Katoh et al., 2000; brown trout, Salmo trutta, Pisam et al., 2000; yellowfin tuna, Thunnus albacares, Kwan et al., 2019). This pattern led some to propose that early vertebrate gills also acquired a primary role in ion regulation before acquiring a primary role in gas exchange (Fu et al., 2010), and that this was because early vertebrate size, dermal thickness and activity were perhaps more constrained by cutaneous ion regulation than by cutaneous gas exchange (Brauner and Rombough, 2012). Recalling Section 1.1.3, ionocyte function typically requires simultaneous contact with blood and water, while gases can diffuse through multiple cell layers. Thus, dermal thickening could theoretically impact ion regulation more severely than gas exchange beyond a certain critical thickness. This notion is supported by work in larval teleosts, where the vast majority of cutaneous ionocytes are found on the vascularized yolk epithelium (Varsamos et al., 2005). As the yolk is absorbed and the area of vascularized epithelium declines, so too do the number of ionocytes and the capacity for cutaneous ion regulation (Rombough, 1999). Conversely, partitioned measurements of oxygen uptake in Atlantic salmon found no differences between the yolk sac epithelium and remaining outer dermis, suggesting gas exchange is less dependent on cutaneous vasculature (Wells and Pinder, 1996b). This is further supported by data from rainbow trout at yolk absorption, where the skin is responsible for 45% of total oxygen uptake and 0% of sodium (Fu et al., 2010). However, as discussed subsequently, caveats associated with larval teleost ontogeny limit the relevance of these data to early vertebrate evolution.  1.3.4 Gaps in the support for a vertebrate origin of gas exchange and ion regulation at gills  Support for this proposed scenario of when and how vertebrate gills became the primary site for gas exchange and ion regulation is compelling, but incomplete. Two main gaps exist:  16 1) Gas exchange and ion regulation in vertebrate ancestors As described above, fossil and developmental data suggest that morphology and lifestyle of the vertebrate ancestor most closely resembled those of extant representatives from cephalochordate and hemichordate taxa. However, the proposed reliance on skin rather than gills for gas exchange in the vertebrate ancestor is based largely on morphometric measurements from cephalochordates alone (Schmitz et al., 2000). These data are robust, but more convincing support would include measurements in ancestral representatives from hemichordates as well. Furthermore, direct measurements of gas exchange at skin and gills are preferred over morphometrics, as differences in convection may alter epithelial contributions in ways that differ from calculated diffusive capacities. The proposed reliance on the skin for ion regulation in the vertebrate ancestor has even less support. Ionocytes do not fossilize, and a reliable signal for ionocytes and/or ion transport machinery in the skin or gills has yet to be found in any extant representative. Without these data, the possibility remains that gills served as the primary site for gas exchange and ion regulation before the origin of vertebrates.  2) Functional data linking early vertebrate size, dermal thickness and activity to gas exchange and ion regulation at gills There are no functional data in extant representatives of early vertebrates or their ancestors that directly link gas exchange and ion regulation at gills to early vertebrate size, dermal thickness and activity. Larval teleost ontogeny has instead served as the primary functional support for this link, but this is problematic for two reasons. First, embryonic development may confound the perceived effects of size, dermal thickness and activity on larval teleost gill function. Larval teleost development is an embryonic phase where rapid and dramatic changes to many aspects of the organism’s biology occur in a highly organized and often programmed way (Finn and Kapoor, 2008). This makes it difficult to determine if gill recruitment for gas exchange and ion regulation is driven by changes to body SA:V, dermal thickness and activity, or other unrelated aspects of developmental programming. Second, larval teleosts do not resemble the presumed morphology or lifestyle of early vertebrates or their ancestors. The ancestral vertebrate is described as a marine, osmoconforming, worm-like burrower with filter-feeding gills (Brown et al., 2008; Holland et  17 al., 2015; Lowe et al., 2015). At no point during development do larval teleosts assume this morphology or lifestyle. They begin as sphere-like organisms immobilized by a large yolk sac that fuels development, and then rapidly transition to pelagic fishes never having used their gills to feed. Those teleosts referenced also live in freshwater and actively maintain a hydromineral balance very different from the external environment. The constraints and demands imposed by this derived morphology and lifestyle might therefore result in gill and skin function for gas exchange and ion regulation that differs from the ancestral state. As described previously (Section 1.2.2), compelling morphological data from ancestrally representative systems do support the link between gas exchange at gills and early vertebrate size, dermal thickness and activity. However, functional data are still preferred to account for differences in convection. Furthermore, morphological data may lack the resolution necessary to determine whether gills first replaced the skin as the primary site of gas exchange or ion regulation.  1.4 Thesis Approach: Intra- and interspecific comparisons with extant representatives  The primary goal of this thesis was to improve our understanding of when and how vertebrate gills became the primary site for gas exchange and ion regulation. The aim was to test the following central hypotheses in a way that addressed the main gaps in existing support:  (1) Gills became the primary site for gas exchange and ion regulation in the vertebrate lineage  (2) Increases to early vertebrate size, dermal thickness and activity were facilitated by gills replacing the skin as the primary site for gas exchange and ion regulation  To test these related hypotheses, gill and skin function in gas exchange and ion regulation was assessed in extant representatives of ancestral vertebrates (lamprey ammocoete, E. tridenatatus), cephalochordates (amphioxus, B. floridae; ion regulation only) and hemichordates (acorn worm, S. kowalevskii). Intraspecific comparisons within lamprey ammocoetes tested the effects of early vertebrate size, dermal thickness and activity on gill function (Aim 1; Chapter 2), and interspecific comparisons between all three taxa tested the ancestral origins of gill function  18 (Aim 2; Chapters 2, 3 & 4). These comparative approaches are bolstered by (1) gill arch homology among representative species (Gillis et al., 2012; Simakov et al., 2015) and (2) the resemblance of representative species to the presumed morphology and lifestyle of early vertebrates and their ancestors (Cameron et al., 2000; Swalla and Smith, 2008; Brown et al., 2008; Holland et al., 2015; Lowe et al., 2015). These are the first functional data in representatives of early vertebrates and their ancestors to address the ancestral origins of gas exchange and ion regulation in vertebrate gills. The following sections detail the specific hypotheses, predictions and experimental design associated with each comparative approach and how they address specific knowledge gaps. Because many readers are likely unfamiliar with these study species, brief descriptions of their general biology and rationale for inclusion are first provided below.  1.4.1 Study species  1.4.1.1 Vertebrate representative: Lamprey ammocoete, E. tridentatus  Lampreys belong to the vertebrate clade Petromyzontiformes. Along with Myxiniformes (hagfishes), these two sister taxa represent the extant jawless fishes in a group known as Cyclostomata (see Figure 1.9 for vertebrate cladogram). There are approximately 40 species of extant lampreys, most of which are broadly distributed across temperate regions in northern and southern hemispheres (Potter et al., 2015). All lampreys spawn in freshwater and begin life as cryptic juvenile “ammocoetes” (Figure 1.2A). Ammocoetes remain burrowed in the benthic substrate of their natal streams for years following embryonic development, emerging only to filter-feed on detritus nocturnally (Dawson et al., 2015). Following this ammocoete phase, lampreys undergo a metamorphosis to become adults. Many dramatic changes occur during this metamorphosis, including the loss of their burrowing, filter-feeding lifestyle, and the gain of distinct features such as eyes and a circular mouth with suctorial disc and rasping tongue (Manzon et al., 2015). Unlike the ammocoete phase, adult life histories are quite diverse (Docker and Potter, 2019). Some species spawn shortly following metamorphosis, while others adopt a carnivorous parasitic feeding mode for further growth and maturation. Of the parasitic species, many migrate to the marine environment before returning to spawn in natal streams.  19 As mentioned previously, ammocoetes were selected for study because they closely resemble the ancestral vertebrate morphology and lifestyle. In addition to being worm-like burrowers that filter feed with gills, they also possess many traits hypothesized to enhance the respiratory capacity of early vertebrate gills (reviewed by Green and Bronner, 2014; Shimeld and Donoghue, 2012). These include gill filaments and lamellae, pillar cells, a rudimentary pharyngeal pump, hemoglobin, a closed circulatory system and a muscular heart. Ammocoetes of all lamprey species are believed to possess these same general traits of interest, and E. tridentatus was chosen for study by virtue of local availability. Ammocoetes are unfortunately freshwater osmoregulators, which is not an ancestral trait. There is some concern that this derived trait may result in gill function that deviates from the ancestral condition. These concerns are partially addressed by the marine osmoconforming lifestyles of the cephalochordate and hemichordate representatives described below.  1.4.1.2 Cephalochordate representative: Amphioxus, B. floridae  Branchiostoma floridae (Figure 1.2B) is one of ~35 cephalochordate species. Also known as amphioxus or lancelets, they join urochordates and vertebrates as one of three chordate subphyla (Figure 1.1). Many excellent reviews of their general biology and suitability as a model species for studying vertebrate evolution exist (Gee, 2008; Bertrand and Escriva, 2011; Holland and Holland, 2017). They are marine organisms, found almost exclusively in shallow coastal waters of temperate and tropical regions globally. Much like ammocoetes, they are small, worm-like organisms that filter-feed from the safety of a sandy burrow. However, their pharyngeal and circulatory systems are much simpler than those of vertebrates. Water flow for feeding is directed into the pharynx by cilia rather than a muscular pharyngeal pump, and while food is still captured by mucociliary gills, they are more numerous (>100) and consist of simpler and finer collagen bars that lack filaments and lamellae. The gill bars do possess hemolymph vessels, but they lack an endothelium and are poorly defined in many regions of the body. All cephalochordates share these general characteristics in morphology and lifestyle, and all are presumed to be marine osmoconformers. B. floridae was specifically chosen for experimentation by virtue of its commercial availability and associated molecular resources (sequenced genome and transcriptome publically available).  20 1.4.1.3 Hemichordate representative: Acorn worm, S. kowalevskii  Saccoglossus kowalevskii (Figure 1.2C) is one of ~110 known species of acorn worm (Tassia et al., 2016). Acorn worms or “Enteropneusta” are one of two classes in the hemichordate phylum. Along with echinoderms and chordates, the hemichordates comprise the deuterostomes (Figure 1.1). As with amphioxus, many excellent reviews of their general biology and suitability as a model species for studying vertebrate evolution exist (Lowe et al., 2004; Gerhart et al., 2005; Rottinger and Lowe, 2012). Acorn worms are distributed globally in marine benthic environments, found anywhere between the intertidal and deep sea. Most are like S. kowalevskii in being only a few centimeters long, but some can reach over a meter. All have a tripartite body plan that consists of an anterior proboscis, collar and posterior trunk region. The muscular proboscis is used to burrow into the sediment and is covered by cilia that help direct water for feeding in through the mouth near the collar. The anterior region of the trunk is perforated by gill slits and covers pharyngeal gill bars structurally very similar to those found in cephalochordates. As in cephalochordates, hemichordate gills also serve a mucociliary function for feeding, directing water flow through the gill slits and transporting captured food posteriorly to the gut. Acorn worms are also presumed to be osmoconformers and possess a rudimentary circulatory system similar to amphioxus. However, there is no defined integument or skin lining the outer layer of the muscular trunk wall as there is in amphioxus and ammocoetes. Enteropneust worms are all believed to share these general characteristics. As with B. floridae, S. kowalevksii was specifically chosen by virtue of its commercial availability and associated molecular resources (sequenced genome and transcriptome publically available).  1.4.2 Aim 1: How were gills recruited for gas exchange and ion regulation?  This aim used ammocoetes to test how gill recruitment for gas exchange and ion regulation might be linked to early vertebrate size, dermal thickness and activity.  Primary Hypothesis 1) Increases to early vertebrate size, dermal thickness and activity were facilitated by gills replacing the skin as the primary site for gas exchange and ion regulation.  21 Secondary Hypothesis 2) Early vertebrate size, dermal thickness and activity were more constrained by cutaneous ion regulation than by cutaneous gas exchange.  Experimental design To test these hypotheses, gas exchange and ion regulation were directly measured at the skin and gills of different sizes of lamprey ammocoetes in vivo. Measurements were taken at rest and during acute hypoxic and/or thermal challenges to ensure full gill recruitment for gas exchange. This experiment was designed to test how gill function was affected by different variables of the modified Fick equation (Equation 1.1). Variation in body size tested the effects of SA:V (“A”) and dermal thickness (“t”) on gill function, while acute hypoxic and thermal challenges tested the effects of oxygen supply and demand (“∆P”).  Primary prediction 1) Ammocoete gills supplant the skin as the primary site of gas exchange and ion regulation with increasing body size and challenges to oxygen supply and demand  Secondary prediction 2) Ammocoete gills supplant the skin as the primary site of ion regulation at smaller body sizes than gas exchange in all conditions tested  Significance and justification These are the first data from an early vertebrate representative to test how gill recruitment for gas exchange and ion regulation might be linked to early vertebrate size, dermal thickness and activity. The use of ammocoetes addresses many of the caveats associated with prior work in larval teleosts, as they are post-embryonic and more closely resemble the presumed morphology and lifestyle of the ancestral vertebrate (Section 1.3.1.1). However, ammocoetes are freshwater osmoregulators. This derived life history might increase gill recruitment for ion regulation in a way that differs from the ancestral condition. This caveat is partially addressed by the representative species chosen for study in Aim 2.   22 1.4.3 Aim 2: When were gills recruited for gas exchange and ion regulation?  This aim used all three extant representatives to test the assumption that gills were not the primary site for gas exchange or ion regulation before the evolution of vertebrates.  Hypothesis 1) Gills became the primary site of gas exchange and ion regulation in the vertebrate lineage  Experimental Design To test this hypothesis, ancestral representatives from cephalochordates (amphioxus, B. floridae) and hemichordates (acorn worm, S. kowalevskii) were assessed for gill and skin function in gas exchange (Chapter 3, acorn worm only) and ion regulation (Chapter 4, acorn worm and amphioxus). Comparisons between these representatives and lamprey ammocoetes from Chapter 2 were then used to infer gill function in common ancestors (Chapter 5).  Prediction 1) Gills are the primary site for gas exchange and ion regulation in ammocoetes (vertebrates), but not amphioxus (cephalochordates) or acorn worms (hemichordates).  Significance and justification These are the first functional data to assess the relative contributions by skin and gills to gas exchange and ion regulation in extant representatives of early vertebrates and their ancestors. These data will help to determine whether or not gills were the primary site for gas exchange and ion regulation before the evolution of vertebrates. In addition to being worm-like burrowers with filter-feeding gills, B. floridae and S. kowalevskii are also marine osmoconformers (Sections 1.3.1, 1.3.2). This more closely resembles the presumed ancestral condition than the freshwater osmoregulating life history of ammocoetes, and helps to address concerns that ammocoete gill recruitment for ion regulation in Chapter 2 might be elevated by this derived aspect of their physiology     23 1.5 Figures             Figure 1.1 Cladogram of major deuterostome taxa Colours indicate key events in deuterostome gill evolution with relevance to gas exchange and ion regulation. First appearance of pharyngeal gill arches (pink), secondary loss of pharyngeal gill arches (green), gills acquire primary role in gas exchange (orange), gills acquire primary role in ion regulation (blue). Larval teleosts suggest gills acquired a primary role in ion regulation (blue) before gas exchange (orange) in the vertebrate stem. References in text.   24     Figure 1.2 External anatomy of study species The vertebrate lamprey ammocoete, Entosphenus tridentatus (A), cephalochordate amphioxus, Branchiostoma floridae (B) and hemichordate acorn worm, Saccoglossus kowalevskii (C). All representatives are aquatic burrowers that filter-feed with gills, and pharyngeal gill arches of all three representatives are homologous. Scale bars in lower right of panels. References in text, photos by Michael Sackville.   25          Figure 1.3 General teleost fish gill structure Illustration depicting location and orientation of teleost gill arches and filaments in the opercular cavity (A; redrawn from Evans et al., 2005). Scanning electron micrographs of zebrafish gills showing arches, filaments and lamellae (B, C; original unlabeled images from van der Meer et al., 2005).   26         Figure 1.4 Countercurrent and uniform pool arrangements for gas exchange Countercurrent exchange (A) results in a more complete transfer of gases between blood (dashed lines) and water (solid lines). Grey lines indicate the boundary layer in the uniform pool arrangement (B).   27               Figure 1.5 Acid and base excretion in the seawater chondrichthyan gill Presumed pathways for acid excreting (A) and base excreting (B) ionocytes in the seawater chondrichthyan gill. Carbonic anhydrase (CA), Na+/K+-ATPase (NKA), vacuolar-type H+-ATPase (VHA), Na+/H+ exchanger 2/3 (NHE2/3) and Pendrin-like anion exchanger (Pd). Putative anion channels depicted in grey. Based on Wright and Wood (2015).   28             Figure 1.6 Salt excretion in the seawater teleost gill Presumed pathways for sodium and chloride excretion in the seawater teleost gill. Na+/K+-ATPase (NKA), Na+/K+/2Cl- cotransporter (NKCC), cystic fibrosis transmembrane conductance regulator (CFTR) and inward-rectifier K+ channel (Kir). Based on Edwards and Marshall (2012).   29             Figure 1.7 Ammonia excretion in the freshwater teleost gill Presumed pathways for ammonia excretion in the freshwater teleost gill. Carbonic anhydrase (CA), Na+/K+-ATPase (NKA), vacuolar-type H+-ATPase (VHA), Na+/H+ exchanger 2/3 (NHE2/3), anion exchanger 1 (AE1), and Rhesus glycoproteins B and C (Rhbg, Rhcg). Based on Hwang and Lin (2013).   30         Figure 1.8 Gill arch structure in hemichordates and cephalochordates Gill arch cross-sections of the hemichordate, Saccoglossus kowalevskii (A) and cephalochordate, Branchiostoma lanceolatum (B). Ciliated arches arranged as primary and secondary gill bars. Gill bars line the pharyngeal cavity. Sections stained with Mayer’s Haemotoxylin and Eosin Y. Scale bars = 250 µm (A), 30 µm (B). Images from Gillis et al. (2012).   31              Figure 1.9 Cladogram of major vertebrate taxa Colours indicate some key events in vertebrate gill evolution with relevance to gas exchange and ion regulation. Gills acquire a primary role in gas exchange (orange), gills acquire a primary role in ion regulation (blue), jaws develop from anterior pharyngeal gill arches (purple). Larval teleosts suggest that gills acquired a primary role in ion regulation (blue) before gas exchange (orange) in the vertebrate stem. References in text.   32 Chapter 2: The effects of body size and activity on gill recruitment for gas exchange and ion regulation in an early vertebrate representative  2.1 Introduction  As described in Chapter 1, gills are hypothesized to have become the primary site for gas exchange and ion regulation along the vertebrate stem (Gans and Northcutt, 1983; Brauner and Rombough, 2012). In this scenario, vertebrate ancestors were constrained to a small, worm-like existence by their reliance on the skin to breathe and to regulate acid-base ion balance. Shifting gas exchange and ion regulation to the gills is believed to have relaxed these constraints, facilitating the transition to larger, armoured and more active modes of life.  The link between this vertebrate transition and an enhanced capacity for gas exchange at gills is well supported by morphological data. As reviewed in Section 1.2.2, developmental studies indicate a stem-vertebrate origin for structures associated with enhanced gill diffusion and convection, including gill filaments, lamellae, pillar cells and a muscular pharyngeal pump (Mongera et al., 2013; Green and Bronner, 2014; Green et al., 2015; Gillis and Tidswell, 2017). Fossil data also indicate that gill filaments and the pharyngeal pump first appeared alongside larger, more fish-like body forms in the putative stem-vertebrates (Shu et al., 1999; Xian-guang et al., 2002; Morris and Caron, 2014) and before the bony dermal armour of ostracoderms (Janvier, 2008). However, the role for ion regulation at gills in this transition lacks developmental and fossil support, and the roles for both ion regulation and gas exchange lack functional support in representatives of early vertebrates and their ancestors.   With no functional support in ancestral representatives, most functional data linking gas exchange and ion regulation at gills to this vertebrate transition comes from larval teleost development. Like the presumed vertebrate ancestor, larval teleosts are believed to be initially constrained to a high body surface area to volume ratio (SA:V), thin dermis and low activity by a reliance on the skin for gas exchange and ion regulation. As gills develop, they replace the skin as the primary site for these processes while SA:V declines, the dermis thickens and activity increases. However, the gills of every larval teleost examined first replace the skin as the primary site for ion regulation (reviewed by Rombough, 2007; Brauner and Rombough, 2012). In rainbow trout, direct measurements in vivo show that gills become the primary site of sodium  33 uptake and ammonia excretion at 15 days post-hatch (dph) vs 28 for oxygen uptake (Fu et al., 2010; Zimmer et al., 2014; Zimmer and Wood, 2015). Furthermore, ionocytes appear before lamellae on the gills of all larval teleosts investigated (reviewed in Brauner and Rombough, 2012). This pattern led some to propose that early vertebrate gills acquired a primary role in ion regulation before gas exchange (Fu et al., 2010), and that early vertebrate size, dermal thickness and activity were perhaps more constrained by cutaneous ion regulation than by cutaneous gas exchange (Brauner and Rombough, 2012).  The teleost data are compelling, but limited in their relevance to early vertebrate evolution. Foremost, teleosts do not resemble the ancestral vertebrate morphology or lifestyle. The ancestral vertebrate was likely a worm-like burrower with filter-feeding gills (Cameron et al., 2000; Brown et al., 2008; Holland et al., 2015; Lowe et al., 2015), but the teleosts investigated are pelagic fishes that do not filter-feed. The demands and constraints associated with these different morphologies and lifestyles could result in very different gill and skin functions for gas exchange and ion regulation. Second, larval teleost development is an embryonic phase where rapid and dramatic changes to basic gill and body plan occur (reviewed by Finn and Kapoor, 2008). This makes it difficult to determine if gill recruitment is driven by changes to body SA:V, dermal thickness and activity, or other unrelated aspects of developmental programming. Lastly, all studies to date have only investigated resting animals in normoxia. Ecologically relevant challenges to oxygen supply and demand may increase gill recruitment for gas exchange (Holeton, 1971; Booth, 1978), potentially revealing an earlier shift from skin to gills that precedes ion regulation. To address the caveats associated with prior work in larval teleosts, this chapter characterized gill and skin function in the ammocoete phase of lamprey. Like the ancestral vertebrate, ammocoetes are worm-like burrowers that filter-feed with gills. They also spend several years in this post-embryonic phase, during which body mass slowly increases by 100-fold despite little change to basic gill and body plan (reviewed by Dawson et al., 2015). Ammocoetes are thus a more representative animal system in which to test the effects of body size, dermal thickness and activity on early vertebrate gill function, and they lack the confounding effects of embryonic development.  With this representative system, I hypothesized that (1) early vertebrate SA:V, dermal thickness and activity were constrained by cutaneous gas exchange and ion regulation, and that  34 (2) SA:V, dermal thickness and activity were more constrained by cutaneous ion regulation than by cutaneous gas exchange. To test this, I directly measured the flux of gases and ions at ammocoete gills and skin across a range of sizes in vivo using a divided chamber preparation. Measurements were taken at rest and following challenges to oxygen supply (hypoxia) and demand (high temperature) to ensure maximum gill recruitment for gas exchange. I predicted that (1) ammocoete gills would replace the skin as the primary site for gas exchange and ion regulation with increasing body size and challenges to oxygen supply and demand, and that (2) gills would replace the skin as the primary site for ion regulation at a smaller body size than for gas exchange in all conditions tested. These are the first functional data in an early vertebrate representative to test how gas exchange and ion regulation at gills might be linked to early vertebrate size, dermal thickness and activity. These results provide new insight into the evolutionary history of vertebrate gill function, and its proposed role in the transition to larger, armoured and more active modes of life.   35 2.2 Methods  Animal collection and husbandry Ammocoetes were collected by dip-net from the Salmon River near Langley, British Columbia (49o07'39.2"N, 122o34'55.5"W) and transported to the University of British Columbia’s Vancouver campus for experimentation between 2014-2018. Animals were held at 10oC on a 12:12 photoperiod in recirculating aquaria (96 L) filled with dechlorinated Vancouver city tapwater (in mM: Na+, 0.06; Cl-, 0.05; Ca2+, 0.03; Mg2+, 0.007; K+, 0.004; alkalinity in mg CaCO3·l-1, 3.3; pH 6.5; Metro Vancouver, 2018) and 5 cm of burrowing substrate. Ammocoetes were fed baker’s yeast twice weekly, and water changes were made every 5 days. All animals were held under these conditions for at least 2 weeks prior to experimentation and starved at least 48 h prior to experimentation.  Divided flux chambers Ammocoetes were placed in divided flux chambers similar to those used by Wells and Pinder (Wells and Pinder, 1996a, 1996b) to estimate the relative contributions of skin and gills to gas exchange and ion regulation (pictured in Figure 2.1). Briefly, lightly anesthetized animals (0.05 g · L-1 MS-222; tricaine methanesulfonate, Syndel, Canada) were passed through small holes in latex dental dam (Hygenic Corporation, USA). Latex dams were fitted immediately posterior to branchial pores to separate head and gills from body. Once fitted, anterior and posterior chamber halves were placed on opposing sides of the latex dams to encompass anterior and posterior portions of the animal. Each acrylic chamber half was fitted with a stir bar, overflow tube and two needle ports for oxygen probes and water sampling. Chamber dimensions varied to accommodate the large size range of animals tested, and were such that body mass approximated 5-20% of chamber volume in all but the smallest ammocoetes. Once in the flux chambers, animals were flushed with fresh water free of anesthetic and subjected to one of three experimental protocols as described below. At protocol completion, animals were euthanized with a lethal dose of MS-222, cut into anterior and posterior halves at the point of chamber division, weighed and analyzed for surface area as described below. All experiments were performed during daylight hours (08:00-19:00) under identical photoperiods.   36 Experimental protocols 1. Normoxia at 10oC (O2, CO2 NH3/4+, Na+ & Ca2+ fluxes) Ammocoetes in open flux chambers were acclimated for 2 h in an aerated water bath at 10oC. Following this acclimation period, chambers were sealed and water PO2 was recorded from anterior and posterior chamber halves simultaneously with needle-type optodes (Presens; Germany). Chamber halves were flushed when oxygen saturation fell below 80%, and PO2 measurements were repeated for three cycles. Following PO2 measurements, chambers were opened to the air and radioisotope (22Na or 45Ca) was added to either anterior or posterior halves. Water samples of 750 µl were taken from both chamber halves at 5 minutes, 2 h and 4 h after isotope addition. Five hundred µl of each sample was immediately frozen at -20oC for later measurement of total ammonia, and the remaining sample was analyzed for ion content and radioactivity. Surface area and wet mass for ammocoete halves were calculated following measurements for radioactivity. Representative CO2 and Ca2+ fluxes were also later measured in a single size class of lampreys under these conditions.  2. Normoxia at 26oC (O2 & CO2 flux) Animals in open flux chambers were subjected to an acute temperature ramp from 10oC to 26oC over 2 h in an aerated water bath. An upper temperature of 26oC was used because it yielded maximum ventilation rate (animals ceased ventilating above 27oC). Once at 26oC, chambers were sealed and PO2 recorded with needle-type optodes from both chamber halves simultaneously. From each chamber half, 20 µl water samples were taken from needle ports with a gas tight syringe (Hamilton; USA) five times before oxygen saturation fell below 80%. Water samples were immediately analyzed for toal CO2 (TCO2) as described below (see CO2 excretion). Chamber halves were flushed when oxygen saturation fell below 80%, and this cycle was repeated at least three times for each animal. Ventilation frequency was recorded for 1 minute during each cycle for all animals.  3. Hypoxia at 20oC (O2 flux) Animals in open flux chambers were subjected to acute hypoxia by bubbling nitrogen into the surrounding water bath (20oC). Water bath PO2 was reduced to ~4 kPa (~20% saturation) over 20 min, and animals were left to acclimate for 1 h before chamber halves were sealed.  37 Water PO2 was recorded by optodes in both chamber halves simultaneously once sealed, and ventilation frequency was recorded for 1 minute. Trials were terminated when chamber oxygen fell below 3% saturation.  Analyses O2 uptake Mean slopes were taken from the linear portions of 3 recorded O2 cycles for each chamber half in a trial. Background respiration from blank trials was then subtracted from mean slopes to yield a ∆PO2·h-1 (mm Hg O2·h-1). ∆PO2·h-1 was then used to calculate oxygen uptake for chamber halves as follows:  Equation 2.1             O2 uptake for chamber half = ∆PO2·h-1 • αO2 • (v - mp)  where O2 uptake is reported as µmols O2·h-1, “αO2” is O2 solubility (µmols O2·ml-1·mm Hg-1; Boutilier et al., 1984), “v” is chamber volume (ml) and “mp” is wet mass (g) of the ammocoete portion in the chamber half. Whole body oxygen uptake rate (ṀO2) was taken as the sum of anterior and posterior chamber half rates and expressed as a function of total ammocoete wet mass (g). ṀO2 is reported as µmols O2·g-1·h-1. The method for determining gill contributions to whole body O2 uptake is described further below (see Percent flux at gills).  CO2 excretion Water samples (20 µls) were analyzed for TCO2 as in Lee et al. (2018). Briefly, samples extracted from each chamber half with a gas tight syringe (Hamilton; USA) were immediately injected into an acidified N2 sparging column leading to an infrared CO2 analyzer (LI-7000; Licor, USA). Total CO2 was expressed as a concentration (µmols·ml-1) and all values for a given trial were plotted as a function of time. Slope of this line produced a ∆TCO2·h-1 (µmols CO2·ml-1·h-1), which was used to calculate CO2 excretion rate for chamber halves as follows:  Equation 2.2           CO2 excretion for chamber half = ∆TCO2·h-1 • (v - mp)   38 where CO2 excretion is reported as µmols CO2·h-1, “v” is volume (ml) of the chamber half and “mp” is wet mass (g) of the ammocoete portion in the chamber half. Whole body CO2 excretion rate (ṀCO2) was taken as the sum of rates for anterior and posterior chamber halves and expressed as a function of total ammocoete wet mass (g). ṀCO2 is reported as µmols CO2·g-1·h-1. The method for determining gill contributions to whole body CO2 excretion is described further below (see Percent flux at gills).  NH3/4+ excretion Water samples were analyzed for total ammonia (NH3/4+) colorometrically (Verdouw et al., 1978) using a SpectraMax 190 microplate reader (Molecular Devices, USA). The difference in ammonia concentrations between initial and final water samples was used to calculate ∆NH3/4+ (nmols NH3/4+·ml-1). NH3/4+ excretion rate for each chamber half was then calculated as follows:  Equation 2.3            NH3/4+ excretion for chamber half = ∆NH3/4+ • (v - mp) • t-1  where NH3/4+ excretion is reported as nmols NH3/4+·h-1, “v” is volume (ml) of the chamber half, “mp” is wet mass (g) of the ammocoete portion in the chamber half, and “t” is time between water samples (h). Whole body NH3/4+ excretion rate (ṀNH3/4+) was taken as the sum of rates for anterior and posterior chamber halves and expressed as a function of total ammocoete wet mass (g). ṀNH3/4+ is reported as nmols NH3/4+·g-1·h-1. The method for determining gill contributions to whole body NH3/4+ excretion is described further below (see percent flux at gills).  Na+ uptake Sodium uptake rates were measured as in Fu et al. (2010). Either anterior or posterior chamber halves were injected with 0.5 µCi of 22Na. Water samples were taken from both chamber halves as described above for Protocol 1. At trial completion, ammocoetes were rinsed three times with 5 mM cold NaCl to displace surface bound 22Na and once with deionized water. Whole ammocoetes and water samples were measured for radioactivity in counts per minute (cpm) with a gamma counter (PerkinElmer, USA). Trials were discarded if activity above background was found in water samples from the chamber half that did not receive isotope injection. Water Na+ concentration was determined by flame atomic absorption spectrometry  39 (Varian, Australia). Sodium uptake rates were reported as µmols Na+·g-1·h-1 and calculated as in Brauner & Wood (2002):  Equation 2.4                         Na+ uptake = Rfish • SAwater-1 • m-1 • t-1  where “Rfish” is the specific activity of the total ammocoete digest (cpm), “SAwater” is the average specific activity of the water (cpm·µmols Na+-1), “m” is ammocoete wet mass (g) and “t” is trial duration (h).  Ca2+ uptake Calcium uptake rates were measured as in Rudman et al. (2019) for a subset of lampreys approximating the smallest size in this study (0.08±0.003 g wet mass; n = 16). Analysis was identical to that for Na+ uptake save the following exceptions. At trial onset, 100 µl of 45Ca working solution was injected to either chamber half to yield ~0.4 µCi per ml of chamber water. At trial completion, lampreys were rinsed twice in a 10 mM cold CaCl2 solution, once in 50 mM EDTA and once in deionized water. Whole lampreys were digested overnight at 40oC in a 5:1 volume:mass ratio of 1 M nitric acid. Digest aliquots and water samples were then diluted five times in Ultima Gold AB liquid scintillation cocktail (Perkin Elmer, USA) and measured for radioactivity with a liquid scintillation counter (LSC-2000; Beckman Coulter Inc, USA). A quench curve was constructed with varying amounts of tissue digest so that counting efficiency could be corrected to be the same as that of water samples. Calcium uptake was reported as nmols Ca2+·g-1·h-1 and calculated as in Zimmer et al. (2019):  Equation 2.5                        Ca2+ uptake = Rfish • SAwater-1 • m-1 • t-1  where “Rfish“ is the specific activity of the total ammocoete digest (cpm), “SAwater“ is the average specific activity of the water (cpm·nmol Ca2+-1) , “m” is ammocoete wet mass (g), and “t” is trial duration (h).     40 Epidermal Surface Area At trial completion, euthanized ammocoete halves were fixed in 70% ethanol for at least 48 h following mass and length measurements. The epidermis was removed from each half, flattened under coverslips, and photographed with a dissecting scope. Surface area (mm2) for each half was then calculated using ImageJ (National Institute of Health, USA).  Epidermal Thickness  A separate set of 20 ammocoetes (0.043-1.787 g) was euthanized for measurements of epidermal thickness. Following euthanasia, ammocoetes were weighed, measured for length and prepared for fixation and sectioning as in Sackville et al. (2012). Briefly, ammocoetes were fixed in 4 % neutral buffered formalin for 24 h at 4oC. Samples were then dehydrated in an ethanol series, cleared in xylene and infiltrated/embedded in paraffin (Type 6; Richard-Allen Scientific, USA). For each individual, 5 µm transverse sections were made at midpoints of the pharynx, body and anus. Sections were collected on 3-aminopropyltriethoxysilane coated slides (Sigma, USA), dried and de-waxed in xylene. Epidermal thickness was measured at all three midpoints under a microscope with ImageJ (NIH, USA) and used to calculate a mean epidermal thickness (µm) for each individual. Mean epidermal thicknesses for all ammocoetes were then plotted as a function of body mass and fitted with an allometric curve (R2 = 0.92; Appendix Figure A.1). The equation describing the relationship between wet mass and epidermal thickness was calculated as:  Equation 2.6                                            t = 45.2(m)0.34  where “t” is epidermal thickness (µm) and “m” is wet mass (g).  Cutaneous Anatomical Diffusion Factor  To estimate the cutaneous anatomical diffusion factor (ADF), measurements of total body surface area (mm2) from ammocoetes sampled during experimentation were first expressed as a function of wet mass (g). These values were then divided by an approximate epidermal thickness calculated from Equation 2.6 for the corresponding wet mass and reported as mm2·µm-1·g-1.  41 Cutaneous ADF was estimated in this way because epidermal thickness and surface area could not be measured in the same animal with available methods.  Percent flux at gills Flux rates for anterior and posterior chamber halves were adjusted with epidermal surface area to represent flux at gills and skin, respectively (Fu et al., 2010; Zimmer et al., 2014). Following surface area correction, percent flux at gills was then calculated by dividing anterior chamber flux rates by total flux rates.  Statistics Data were analysed with Prism 8 for Mac OS X (Version 8.4; GraphPad Software Inc., USA). Percent gas fluxes at gills were compared using a two-way ANOVA and Tukey’s post-hoc test with treatment and body mass as factors. One-way ANOVA and Tukey’s post-hoc test were used to analyze all other data. All data are presented as means±s.e.m and P<0.05 was used throughout.   42 2.3 Results  Percent Na+ flux at gills was 100% in normoxia at 10oC for all sizes tested (Figure 2.2), and was thus not measured for further increases with temperature or hypoxia. Follow-up trials confirmed this pattern for ion flux was not unique to Na+, finding percent Ca2+ flux at gills to be 100% in normoxia at 20oC for a representative sample of the smallest ammocoetes (Figure 2.2; total uptake rate of 2.60±0.14 nmols Ca2+·g-1·h-1). Conversely, percent flux at gills for O2, NH3/4+ and CO2 never approached 100% at any size in any condition (Figure 2.2). However, gill recruitment for these fluxes did increase with body mass, temperature and hypoxia. In normoxia at 10oC, percent O2 uptake at gills increased from ~20% at 0.059 g to 35% at 1.62 g (Figure 2.2). Ammonia mirrored this pattern, as did the representative sample for CO2 (Figure 2.2). At 26oC, ṀO2 rose ~3-fold in the smallest ammocoetes (Figure 2.3A), increasing gill contributions to ~35% (Figure 2.2). This increased to ~50% in the largest ammocoetes, with CO2 again following a similar pattern (Figure 2.2). Finally, hypoxia further increased gill recruitment to ~45% in the smallest ammocoetes, reaching ~70% by 1.00 g (Figure 2.2). Two-way ANOVA did not find a significant interaction between body size and treatment for percent flux at gills for gases (P = 0.80) despite significant main effects for each factor (P = 0.0001 for body size and treatment). The increased gill recruitment with body mass for O2, NH3/4+ and CO2 fluxes was accompanied by a ~10-fold reduction in cutaneous ADF (Figure 2.4), and occurred despite reductions to whole animal mass-specific flux rates (Figure 2.3). Respiratory exchange ratios (RER), ammonia quotients (AQ), ventilation frequencies and Q10’s are reported in the Appendix (Figure A.2).    43 2.4 Discussion  Ammocoete gills replace the skin as the primary site for gas exchange with increasing body size and challenges to oxygen supply and demand. This supports the hypothesis that early vertebrate SA:V, dermal thickness and activity were constrained by cutaneous gas exchange. However, gills were responsible for 100% of ion uptake in all sizes and conditions tested. This lack of shift from skin to gills does not support the hypothesis that SA:V, dermal thickness and activity are constrained by cutaneous ion regulation, let alone more constrained than by cutaneous gas exchange. This complete and unexpected absence of cutaneous ion uptake in even the smallest ammocoetes instead suggests the gill’s role in ion regulation might be unrelated to post-embryonic changes in SA:V, dermal thickness and activity.  Gas exchange Gas exchange in post-embryonic ammocoetes followed a pattern similar to larval teleosts. All three respiratory gases moved primarily across the skin of our smallest animals, then shifted to gills with increasing body size as SA:V declined and the dermis thickened. Interestingly, the smallest ammocoetes in this study share a nearly identical oxygen uptake rate, SA:V and dermal thickness with newly hatched salmonids, and both animal systems acquire ~80% of oxygen cutaneously (~4 µmols O2·g-1·h-1 at ~18 µm and ~140 mm2 in ammocoetes vs ~4 µmols O2·g-1·h-1 at ~20 µm and ~150 mm2 in rainbow trout and Atlantic salmon; Wells and Pinder, 1996a, 1996b; Fu et al., 2010; Zimmer et al., 2014). The lone difference from larval teleosts appears to be that ammonia excretion is uncoupled from sodium uptake (see Ion regulation).  Rates of gas exchange are consistent with prior measurements in free-swimming ammocoetes, suggesting the experimental preparation did not affect gas exchange in any unexpected way. Wilkie et al. (2001) report resting ṀO2 and ṀNH3/4+ as ~1.0-2.0 µmols O2·g-1·h-1 and ~50-100 nmol NH3/4+·g-1·h-1, respectively, in unanesthetized 1.3-3.9 g ammocoetes at 15oC. Assuming a temperature effect (Q10) of ~2, these values agree with those observed here in ~1.5 g animals at 10oC (~1.3 µmols O2·g-1·h-1 and ~40 nmol NH3/4+·g-1·h-1). These are the first measurements of CO2 excretion in ammocoetes in water, but the respiratory exchange ratio (RER) of ~0.75 is within a reasonable range.  44 Gill recruitment for gas exchange increased as expected following exposure to an acute thermal maxiumum. Indeed, contributions for oxygen uptake and carbon dioxide doubled from ~20 to 40% in the smallest ammocoetes. Furthermore, rates of gas exchange during this challenge appear to approximate those of maximal exercise. Wilkie et al. (2001) found that ṀO2 in 1.3-3.9 g ammocoetes rose from ~1.0-2.0 to ~4.5 µmols O2·g-1·h-1 following chase to exhaustion at 15oC, which agrees with the increase from ~1.3 to ~6.5 µmols O2·g-1·h-1 observed here in similarly sized 10 oC animals acutely exposed to 26oC. As with resting values, no prior measurements for CO2 have been made in ammocoetes in water, but the RER’s of ~0.75-0.95 are again within a reasonable range.  Relative contributions to gas exchange by skin and gills were not measured at 20oC prior to hypoxic exposure. Consequently, a direct comparison to isolate the effects of hypoxia on gill recruitment is absent. However, the greater reliance on gills in hypoxia at 20oC than in normoxia at 26oC suggests that variation in oxygen supply alone is an important driver for gill recruitment. This is further supported by the observation that ventilation in ammocoetes at 20oC was only detectable below 8 kPa PO2.  Ion regulation Unlike gas exchange, ion regulation in post-embryonic ammocoetes followed a different pattern from that in larval teleosts. Rather than shifting from skin to gills with changes to SA:V, dermal thickness and activity, all ammocoete ion uptake occurred at the gills. This is especially striking given that the smallest ammocoetes had a similar SA:V, dermal thickness and resting sodium uptake rate as newly hatched salmonids that acquire 80% of sodium cutaneously (~14 nmols Na+·ammocoete-1·h-1 vs ~10 nmols Na+·larva-1·h-1 in rainbow trout; Fu et al., 2010; Wells and Pinder, 1996a). Calcium uptake also followed this same pattern, and although relative contributions by skin and gills in larval teleosts have not been measured, total uptake rates here are consistent with rainbow trout acclimated to similarly low levels of environmental calcium (~2.6 nmols Ca2+·g-1·h-1 in ammocoetes at 60 µM Ca2+ vs ~2 nmols Ca2+·g-1·h-1 in rainbow trout at 50 µM Ca2+; Perry and Wood, 1985). To my knowledge, these are the first direct measurements of ion uptake in lampreys at any life stage.  This increased reliance on gills for ion regulation in ammocoetes relative to larval teleosts might be related to differences in epidermal perfusion. Ammocoetes lack dermal  45 capillaries (Potter et al., 1995). Without extensive epidermal perfusion, ionocytes of the outer epithelium might be unable to maintain the simultaneous contact with blood and water typically required for trans-epithelial ion transport. This hypothesis is consistent with observations in larval teleosts, where cutaneous ionocytes occur almost exclusively at the vascularized epithelium of the yolk sac (Varsamos et al., 2002). Epidermal perfusion will also affect gas exchange, but not in the same way. Unlike ions, gases can diffuse across multiple cell layers, and are thus not as reliant upon direct contact between the circulation and outer epithelial cells. This may explain why ammocoetes are able to maintain high levels of cutaneous gas exchange without dermal capillaries. This notion is further supported by partitioned respirometry in larval Atlantic salmon at hatch, where the vascularized yolk sac and unperfused skin have similar area-specific rates of oxygen uptake (0.085 vs 0.089 µg O2·h-1·cm2-1 for yolk sac and skin, respectively; Wells and Pinder, 1996b). Thus, epidermal perfusion appears more critical to ion regulation than gas exchange, and might be a better indicator for cutaneous ion regulation than total body SA:V and dermal thickness.  This study was not designed to determine the mechanisms of sodium and calcium uptake in ammocoetes. However, the uncoupling of sodium uptake from ammonia excretion reveals a striking departure from current models for freshwater teleost ion regulation. In teleosts, ammonia excretion is believed to provide an important driving force for sodium uptake. In this pathway, intracellular ammonium deprotonates and exits ionocytes down its partial pressure gradient through an apical ammonia channel (Rhcg). Ammonia is then reprotonated on the water side, maintaining a favourable partial pressure gradient for its excretion. This “acid-trapping” mechanism on the apical water side also generates a favourable gradient for proton excretion, which is believed to help drive sodium uptake from the water by powering an apical sodium-proton exchanger (NHE2/3). Together, Rhcg and NHE2/3 are believed to form a metabolon that drives ammonium-dependent sodium uptake (reviewed by Wright and Wood, 2012).  In ammocoetes, ammonia excretion and sodium uptake appear uncoupled. The mechanism for ammonia excretion remains unknown, but diffusion is likely dependent on Rh glycoproteins distributed ubiquitously throughout skin and gills (Blair et al., 2017). It remains to be determined if this diffusion is further enhanced by other apical machinery that acidifies the boundary layer to promote a similar “acid-trapping” effect. The mechanism for sodium uptake also remains understudied, but may resemble an earlier and more controversial pathway  46 proposed for teleosts. This alternate pathway has sodium entering through an apical sodium channel (ENaC) down a negative electropotential generated by apical H+-ATPase (VHA). The controversy surrounding this pathway arose primarily because ENaC appears to be absent from teleost genomes despite pharmacological support for its presence (Esaki et al., 2007; Preest et al., 2005; Reid, 2003; Wilson et al., 2000). However, lampreys do possess an ENaC gene, and recent work suggests they may rely on this historically elusive teleost pathway (Ferreira-Martins et al., 2016). The mechanism for calcium uptake is also unknown in ammocoetes. Ammocoetes do, however, possess orthologues of the apical epithelial calcium channel (trpv6), basolateral sodium-calcium exchanger (ncx1a/b) and Ca2+-ATPase (pmca) implicated in teleost pathways (Smith et al., 2013). Future work might therefore reveal that ammocoetes share the same mechanism of calcium uptake as teleosts. Ammocoetes clearly represent an exciting and largely untapped comparative system for the study of freshwater ion regulation in vertebrates, which is especially intriguing given their ion and osmoregulating strategies likely evolved independently (Evans, 1984; Halstead and Lawson, 1985).  Evolutionary implications These are the first functional data in an early vertebrate representative to test how gas exchange and ion regulation at gills might be linked to early vertebrate size, dermal thickness and activity. These results support the long-held belief that gills facilitated the early vertebrate transition to larger, armoured and more active modes of life by replacing the skin as the primary site for gas exchange (Gans and Northcutt, 1983). However, the lack of shift from skin to gills observed here for ion uptake does not support a similar scenario for ion regulation. The complete absence of ion uptake at the skin of even the smallest ammocoetes suggests that gills instead acquired their primary role in ion regulation for reasons unrelated to body size, dermal thickness and activity –perhaps even before the evolution of vertebrates. Alternatively, the freshwater and osmoregulating life history of ammocoetes could be confounding these results. Early vertebrates and their ancestors were likely marine osmoconformers (Evans, 1984). The increased ionoregulatory demands associated with this derived aspect of ammocoete life history might therefore increase gill recruitment for ion regulation in a way that does not represent the ancestral condition.  47 Chapter 4 characterizes gill and skin function for ion regulation in ancestral representatives from cephalochordates and hemichordates. These marine osmoconformers provide critical insight into whether or not the pattern observed here for ion regulation in ammocoetes accurately reflects the ancestral condition.   48 2.5 Figures               Figure 2.1 Divided chamber Entosphenus tridentatus head and gills are separated from the rest of the body with a latex dam. Each acrylic chamber half is fitted with a stir bar, sampling ports and needle type O2 probes.   49         Figure 2.2 Percent of total gas and ion flux at gills of Entosphenus tridentatus Oxygen uptake (orange), carbon dioxide excretion (grey), total ammonia excretion (green), sodium uptake (blue) and calcium uptake (red) as a function of body mass in normoxia at 10oC (shaded circles), 26oC (open circles) and hypoxia at 20oC (open squares). Data presented as means±s.e.m., n=10. Letters indicate significant differences within fluxes, and asterisks indicate significant differences between fluxes within a size class (two-way ANOVA with Tukey’s post-hoc test, P<0.05). For clarity, only statistical comparisons for oxygen and sodium fluxes are presented.  0.0 0.5 1.0 1.5 2.0020406080100body	mass	(g)%	flux	at	gillsaAa*a,b b bb**A,BB,CC*******a,bbc*** *** *** *** *** 50          Figure 2.3 Whole body rates of gas and ion flux in Entosphenus tridentatus Oxygen uptake (A), carbon dioxide excretion (B), total ammonia excretion (C) and sodium uptake (D) as a function of wet mass in animals exposed to normoxia at 10oC (shaded circles), 26oC (open circles) and hypoxia at 20oC (open squares). Data presented as means±s.e.m.,	n=10. Letters indicate significant differences within treatments (one-way ANOVA with Tukey’s post-hoc test, P<0.05), asterisks indicate significant differences between treatments within a size class (oxygen only, two-way ANOVA with Tukey’s post-hoc test, P<0.05).  0.0 0.5 1.0 1.5 2.0051015body mass (g)µmols O2⋅ g ⋅ hr-1b,cc cabAA,BB,CCaa,bbb**********0.0 0.5 1.0 1.5 2.0050100150200body mass (g)nmols NH3/4+⋅ g ⋅ hr-1abb,cc c0.0 0.5 1.0 1.5 2.0051015body mass (g)µmols CO2⋅ g ⋅ hr-10.0 0.5 1.0 1.5 2.00100200300body mass (g)nmols Na+ ⋅ g ⋅ hr-1abb,cc,ddA BC D 51              Figure 2.4 Cutaneous anatomical diffusion factor (ADF) of Entosphenus tridentatus Data presented as means±s.e.m., letters indicate significant differences (n=10, one-way ANOVA with Tukey’s post-hoc test, P<0.05).  0.0 0.5 1.0 1.5 2.00.00.51.01.52.0body mass (g)ADF (cm2 ⋅ µm-1⋅ g-1)abcd 52 Chapter 3: Gas exchange in a hemichordate representative  3.1 Introduction  As described in Chapter 1, gills are hypothesized to have acquired a primary role in gas exchange along the vertebrate stem (Northcutt, 2005; Gillis and Tidswell, 2017). In this scenario, vertebrate ancestors were initially constrained to a small, worm-like existence by a reliance on the skin to breathe. Shifting gas exchange to gills is believed to have relaxed these constraints, thereby facilitating the evolution of increased size, dermal thickness and activity in early vertebrates. An increased capacity for gas exchange at gills in early vertebrates associated with larger, more active modes of life is well supported. As described in Section 1.2.2, developmental data point to a vertebrate origin for structures associated with enhanced gill diffusion and convection (reviewed by Green and Bronner, 2014; Green et al., 2015; Gillis and Tidswell, 2017), and fossil data suggest many of these structures first appeared in larger, fish-like body forms of the putative stem-vertebrates (Shu et al., 1999; Xian-guang et al., 2002; Morris and Caron, 2014). Despite strong support for an increased capacity for gas exchange at gills in the vertebrate stem, there is little support for the assumption that gills were not already the primary site for gas exchange before vertebrates evolved. As reviewed in Section 1.2.1, fossil and developmental studies suggest the worm-like morphology and burrowing, filter-feeding habits of the vertebrate ancestor most closely resembled those of extant cephalochordates and hemichordate enteropneust worms (Cameron et al., 2000; Brown et al., 2008; Holland et al., 2015; Lowe et al., 2015; Nanglu et al., 2015). The high body surface area to volume ratio (SA:V), simple gill bars and lack of pharyngeal pump in these fossil and extant representatives are qualitatively consistent with a negligible role for gills in gas exchange. However, the only quantitative data supporting this notion are morphometric measurements in the cephalochorate Branchiostoma lanceolatum, which estimate gills account for less than 5% of total body diffusive capacity (Schmitz et al., 2000). These data are robust, but more convincing conclusions would include similar estimates from enteropneust worms. Both representative groups are equally important to inferring the ancestral condition, and morphologies differ considerably between them (Figure 1.2, Chapter 1). Direct measurements of gas exchange at gills would also provide more  53 compelling results, as convection may alter epithelial contributions in ways that differ from calculated diffusive capacities. Unfortunately, cephalochordate gills cannot be isolated with a divided preparation for direct measurements of gas exchange as done for ammocoetes in Chapter 2. Unlike ammocoetes, cephalochordate gill bars span more than two thirds total body length, and the entire pharyngeal complex is enveloped by an atrial membrane with a single posterior pore for exhalant water flow (Figure 1.2B, Chapter 1). As a result, separating the ventral pharyngeal region from the dorsal cutaneous surfaces would be difficult. Furthermore, even if the atriopore exhalant could be isolated from cutaneous surfaces, estimates of gill contributions would be confounded by other tissue surfaces and organs within the atrium. Thus, direct measurements for gas exchange at cephalochordate gills were not made here. Fortunately, gill function in enteropneust worms can be isolated with a modified divided preparation. Enteropneust worms have a tripartite body plan composed of a proboscis, collar and trunk (Figure 1.2C, Chapter 1). Pharyngeal gills are confined to the anterior half of the trunk, which is surrounded by an outer muscular wall perforated with gill pores for exhalant water flow. This anterior “gilled” half of the animal can thus be easily separated from the posterior half containing the digestive tract. Acorn worms are too fragile to be fixed in a divided respirometer, but they possess a remarkable regenerative capacity that allows them to be partitioned into viable fragments with and without gills. Indeed, prior work examining regeneration in Saccoglossus kowalevskii partitioned whole animals in exactly this manner (Tweedell, 1961). The anterior fragments persisted indefinitely, exhibiting regeneration 3 days post-amputation. Posterior fragments did not regenerate, but still persisted as viable entities for approximately 1 week at 21oC. Viable halves of S. kowalevskii with and without gills can therefore be placed in respirometers separately.  Here, I hypothesized that gills of the hemichordate enteropneust worm, S. kowalevskii were not a primary site for gas exchange. To test this, I exploited the regenerative ability of S. kowalevskii to measure the rates of oxygen uptake and ammonia excretion with closed respirometers in whole worms, worm halves with gills, and worm halves without gills. I predicted that gill presence would not be associated with higher rates of gas exchange. To ensure maximum gill recruitment for gas exchange, measurements were taken during ecologically relevant hypoxic and thermal challenges. These are the first direct measurements estimating gill  54 contributions to gas exchange in a representative of the pre-vertebrate ancestor. Together with prior morphometric work in cephalochordates, these are the only quantitative data available to assess whether gills were the primary site of gas exchange before the evolution of vertebrates.   55 3.2 Methods  Animal collection and husbandry Saccoglossus kowalevskii were purchased from the Marine Biological Laboratory in Woods Hole, USA, and shipped to the University of British Columbia’s Point Grey campus in Vancouver, Canada. At the University of British Columbia, animals were held in static, 40 L seawater tanks (10 animals per tank; 34 ppt artificial seawater made from Instant Ocean salt mix, USA) at 10oC on a 12:12 light:dark photoperiod. Tanks were supplied with sandy substrate, and 30% water changes were performed twice weekly. All animals were held for at least one week prior to experimentation, and fasted for 24 h before entering one of three protocols described below. Mean wet mass of the animals used for experimentation was 0.105±0.009 g (n = 40). A second enteropneust species, Protoglossus graveolens was subjected to identical husbandry and experimental conditions. Results for this species mirrored those observed for S. kowalevskii and are listed in the Appendix (Figure B.1).  Experimental protocols 1. Normoxia at 10oC Ten whole animals were transferred to acrylic respirometers (4 ml) in an aerated water bath at 10oC. Each respirometer was fitted with a stir bar, overflow tube and needle type oxygen probe (Presens, Germany). Following a 1 h acclimation period in respirometers open to the water bath, respirometers were sealed and PO2 recorded until falling to ~80% saturation. Respirometers were then manually flushed, and the cycle was repeated twice more. Following the third cycle, animals were transferred to aerated, 10 ml chambers open to the air at the water bath surface. Empty respirometers were then re-sealed to measure background respiration rates. From the open, aerated chambers containing the animals, 1 ml water samples were taken every 30 min for 4 h and frozen at -20oC for later measurement of total ammonia. Following the final water sample, a razor blade was used to cut animals in half with a transverse section immediately posterior to the caudal gill pore. Animal halves were left to recover in separate, aerated chambers at 10oC for at least 12 hours post-separation. After this recovery period, animal halves were placed in 4 ml respirometers and subjected to the same measurements of PO2 and total ammonia as whole animals. Upon completion of these measurements, animal halves were euthanized with  56 a lethal dose of MS-222 (tricane methanesulfonate; Syndel, Vancouver) and measured for wet mass.  2. Normoxia at 20oC Animals were subjected to the same protocol as outlined for normoxia at 10oC, except all measurements were performed at 20oC. Ten whole animals were warmed to 20oC from 10oC in a water bath at 2.5oC·h-1 the night before experimentation. 20oC was chosen as an ecologically relevant thermal challenge that would significantly increase oxygen demand from 10oC, but not result in mortality over 24 h. Insufficient numbers of animals were available to experimentally determine the thermal maximum for S. kowalevskii in this experiment. However, selection of this temperature was based on prior work with summer acclimated worms that recommended rearing temperatures not exceed 24oC (Lowe et al., 2004).  3. Hypoxia at 20oC Ten whole animals at 10oC were brought up to 20oC as in the previous protocol. Animals were placed in submerged respirometers open to the water bath. Water PO2 in the bath was then reduced to ~20% atmospheric with a nitrogen sparge. Following a 1 h acclimation at this reduced PO2, respirometers were sealed and PO2 recorded as it dropped to ~1% saturation. Whole animals were then euthanized with a lethal dose of MS-222 (tricane methanesulfonate; Syndel, Vancouver) and measured for wet mass. A different, second set of ten whole animals at 10oC were partitioned into anterior and posterior halves as in the previous protocols. Animal halves were allowed to recover for at least 12 h, and then subjected to the same temperature ramp, experimental protocol and measurements as whole animals in hypoxia. This second, naïve set of animals was used for fragmentation to avoid any potential confounding effects that pre-exposure of whole animals to hypoxia might have on gas exchange.  Analyses To calculate oxygen uptake rates (ṀO2), slopes were taken from the linear portions of three recorded O2 cycles for each chamber in a trial. Mean slopes were then adjusted with background respiration values from blank trials to yield a ∆PO2·h-1 (mm Hg O2·h-1). ∆PO2 was used to calculate ṀO2 as follows:  57 Equation 3.1                               ṀO2 = ∆PO2 • αO2 • v • m-1  where ṀO2 is reported as µmols O2·g-1·h-1, “αO2” is O2 solubility (µmols O2·ml-1·mm Hg-1; Boutilier et al., 1984), “v” is chamber volume (ml) and “m” is ammocoete wet mass (g). For ammonia excretion rates, water samples were analyzed for total ammonia (NH3/4+) colourometrically (Verdouw et al., 1978) using a SpectraMax 190 microplate reader (Molecular Devices, USA). The difference in ammonia concentrations between initial and final water samples was used to calculate ∆NH3/4+ (µmols NH3/4+·ml-1). Ammonia excretion (ṀNH3/4+) was then calculated as follows:  Equation 3.2                            ṀNH3/4+ =  ∆NH3/4+ • v  • m-1 • t-1  where ṀNH3/4+ is reported as µmols NH3/4+·g-1·h-1, “v” is chamber volume (ml), “m” is acorn worm/fragment wet mass (g) and “t” is time (h) between water samples.  Statistics  Statistical analyses were performed with Prism 8 (Graphpad Software Inc, USA). All data are presented as means±sem and compared within treatments using one-way ANOVA (Tukey’s post-hoc test, P<0.05).    58 3.3 Results  In normoxia at 10oC, oxygen consumption rates did not differ significantly between whole worms (3.83±0.26 µmols O2·g-1·h-1), anterior (3.61±0.16 µmols O2·g-1·h-1) or posterior fragments (3.73±0.28 µmols O2·g-1·h-1; Figure 3.1). Rates of ammonia excretion also did not differ between whole worms, anterior or posterior fragments (Figure 3.2).  Similarly, in normoxa at 20oC, oxygen consumption rates did not differ between whole worms (6.01±0.30 µmols O2·g-1·h-1), anterior (5.87±0.30 µmols O2·g-1·h-1) or posterior fragments (6.19±0.48 µmols O2·g-1·h-1; Figure 3.1). Rates of ammonia excretion also did not differ between whole worms, anterior or posterior fragments (Figure 3.2).  In hypoxia at 20oC, the rate of oxygen consumption was higher in posterior fragments (2.48±0.25 µmols O2·g-1·h-1) than whole worms (1.59±0.29 µmols O2·g-1·h-1) or anterior fragments (1.60±0.18 µmols O2·g-1·h-1; Figure 3.1). Rates of oxygen consumption did not differ between whole worms and anterior fragments in hypoxia.    59 3.4 Discussion  These results support the hypothesis that gills of Saccoglossus kowalevskii are not a primary site for gas exchange. This is evidenced by the ability of worm segments without gills to maintain the same rates of oxygen uptake and ammonia excretion as segments and whole worms with gills, even when challenged with variation in oxygen demand (increased temperature) and supply (hypoxia).  To my knowledge, this is only the second measurement of oxygen uptake in a hemichordate and the first for ammonia excretion. Previously reported rates of oxygen uptake for similarly sized members of the enteropneust worm, G. crozieri at 25oC match those observed here for S. kowalevskii at 10oC (~4 µmols O2·g-1·h-1; Ditadi et al., 1997). This similarity despite a 15oC difference in experimental conditions is surprising, given that an average temperature effect (Q10) of ~2 would yield a ~2.5-fold change in metabolic rate over this range. However, differences in species biology, acclimation conditions and methodology could all contribute to this discrepancy. Methodological differences in particular could be playing a role, as Ditadi et al. (1997) measured oxygen uptake with an unstirred Warburg apparatus. Equilibration of oxygen within and between both water- and air-filled Warburg chamber sections would therefore be contingent on passive diffusion, and may underestimate ṀO2. Interestingly, rates of oxygen uptake for S. kowalevskii at 10oC closely resemble those reported for similarly sized resting ammocoetes at 10oC (Chapter 2). In this case, similar results were obtained when using the same methodology and experimental temperature for two different aquatic burrowers that rely primarily on cutaneous gas exchange. The observed ammonia quotient at 10oC of ~0.1 for S. kowalevskii is also within the expected range for marine invertebrates (Yu et al., 2012), further validating the experimental setup and animal condition.  Oxygen uptake rates predictably increased with temperature in the thermal challenge, but the observed Q10 of ~1.5 is lower than the expected ~2-3. Although ṀO2 would need to be measured at more than two temperatures for confirmation, this lower Q10 may indicate a slowing or plateau in the rise of ṀO2 at or near 20oC. Such a plateau would suggest that S. kowalevskii might be approaching a thermal limit, and would further support the use of 20oC as a thermal challenge for oxygen demand. Indeed, the generalized trend for ṀO2 in many ectotherms subjected to rising temperature is to increase to a thermal maximum with a Q10 of ~2-3, plateau,  60 and then sharply decrease (Pörtner, 2010; Schulte, 2015; Jutfelt et al., 2018). S. kowalevskii collected in the summer are recommended to be kept below ~25oC (Lowe et al., 2004), thus 20oC could very well be pushing these winter acclimated worms close to their limit for a 24 h exposure. This is further supported by ammonia excretion rates. Unlike ṀO2, ṀNH3/4+ in S. kowalevskii exhibits a higher Q10 of ~3.5 that results in a doubling of the ammonia quotient (AQ) from ~0.1 to ~0.2 at 20oC. Elevated rates of ammonia excretion relative to ṀO2 have been observed near the thermal limits for other aquatic ectotherms, possibly stemming from increased protein degradation associated with thermal stress or injury (Aldridge et al., 1995; Williams et al., 2016; Giacomin et al., 2017). Again, ṀO2 would need to be measured at additional temperatures for confirmation. However, the observed trends in ṀO2 and ṀNH3/4+, coupled with known upper temperatures for rearing summer worms, suggest that 20oC may approach a thermal limit for these winter acclimated worms.  Curiously, worm segments without gills maintained slightly higher rates of oxygen uptake in hypoxia than whole worms or segments with gills. The reasons for this unexpected result are unknown, but plausible explanations exist. First and perhaps most likely, the diffusion distance in posterior worm segments might simply be thinner, as the trunk body wall is generally thinner and less robust here than in anterior segments (Figure 1.2; Barrington, 1965). Second, whole worms and anterior segments might experience reduced oxygen demands in hypoxia relative to posterior segments. This could occur if different tissues respond differently to hypoxia. For example, if movement or activity is reduced as observed in some organisms exposed to hypoxia or low temperature (Speers-Roesch et al., 2018), anterior worm segments may experience greater reductions in oxygen uptake because they contain the more muscular and active proboscis and trunk wall. Alternatively, S. kowalevskii may undergo a coordinated reduction in whole body metabolic rate during hypoxia. If such a response were coordinated by a central processing centre and/or sensory neurons in the anterior segment, separated posterior segments may lack this response. Sensory perception and processing in hemichordates remain largely unstudied. However, concentrated cell bodies of neurons in the proboscis and collar of the anterior segment are speculated as possible processing centres that could play such a role (Nomaksteinsky et al., 2009; Miyamoto and Wada, 2018). Regardless of why ṀO2 is higher in posterior segments than whole worms and anterior segments in hypoxia, the results still suggest that gills are not a primary site for gas exchange.  61  The absence of gill recruitment for gas exchange in S. kowalevskii is somewhat unexpected given that the entire anterior trunk is lined with vascularized gill bars. This absence is even more surprising given that lamprey ammocoetes of similar size, burrowing lifestyle and oxygen demand recruit gills for gas exchange during similar thermal and hypoxic challenges (Chapter 2). A reduced gill diffusive capacity likely underlies this interspecific difference in recruitment, as acorn worms lack gill filaments and lamellae. However, circulatory system and mode of ventilation likely also play key roles. Indeed, S. kowalevskii has an open circulation in which peristalsis and weak contractile sacs move hemoglobin-less hemolymph through vessels lacking an endothelium (Barrington, 1965; Pardos and Benito, 1988). Such systems are generally characterized by slow circulation and low oxygen carrying capacity, and are thus likely far less effective in delivering oxygen to tissues than a closed ammocoete system with hemoglobin and a muscular heart. Cilia-driven ventilation in S. kowalevskii also generates much lower water flow through the pharynx than the muscular velum of ammocoetes, likely limiting oxygen uptake from the environment (Pardos, 1988; Gonzalez and Cameron, 2009). Thus, even if S. kowalevskii were to possess favourable gill morphology for gas exchange, it may lack the convective systems necessary to exploit it.  Evolutionary implications Hemichordate enteropneust worms are an important comparative group for testing the deuterostome ancestry of vertebrate gill function because of their phylogenetic position and resemblance to the ancestral morphology and lifestyle (Holland et al., 2015; Lowe et al., 2015; Simakov et al., 2015). Here, I provide the first and only measurements of gas exchange (or lack thereof) for hemichordate gills. These results are consistent with a vertebrate origin of gas exchange at gills. These findings and their implications for our understanding of how and when vertebrate gill function evolved are discussed further in Chapter 5.   62 3.5 Figures            Figure 3.1 Oxygen uptake rates of whole and fragmented Saccoglossus kowalevskii Black, grey and white circles represent whole animals, anterior fragments and posterior fragments, respectively. Measurements taken in normoxia at 10oC, 20oC and in hypoxia at 20oC. Data presented as means±sem, n=8. Asterisks indicate significant differences within treatments (P<0.05, one-way ANOVA with Tukey’s post-hoc test).  10oC 20oC hypoxia02468µmols O2⋅ g ⋅ hr-1* 63              Figure 3.2 Ammonia excretion rates of whole and fragmented Saccoglossus kowalevskii Black, grey and white circles represent whole animals, anterior fragments and posterior fragments, respectively. Measurements taken in normoxia at 10oC and 20oC. Data presented as means±sem, n=8. No significant differences within treatments (P>0.05, one-way ANOVA). Error bars at 10oC are within the span of the points.  10oC 20oC0.00.51.01.52.0μmols NH3/4+⋅ g ⋅ hr-1 64 Chapter 4: Ion regulation in hemichordate and cephalochordate representatives  4.1 Introduction  As outlined in Chapter 1, gills are believed to have replaced the skin as the primary site for ion regulation in the vertebrate stem. This is based mostly on the fact that gills are the primary site for ion regulation in adult fishes, and that gills replace the skin as the primary site for ion regulation in developing fishes (reviewed by Brauner and Rombough, 2012). However, there is no support in representatives of early vertebrates or their ancestors for the assumption that gills did not serve as the primary site for ion regulation before the evolution of vertebrates. To address this knowledge gap, this chapter characterized gill and skin function for ion regulation in representatives of ancestral hemichordates (Saccoglossus kowalevskii) and cephalochordates (Branchiostoma floridae). Ion regulation in hemichordates and cephalochordates is almost completely unexplored. Like all invertebrate deuterostomes, hemichordates are marine organisms believed to be osmoconformers (Barrington, 1965). However, it remains unknown if acid-base or hydromineral balance of the internal milieu is regulated to any degree, and neither gill nor skin function has been investigated in this regard. Enteropneust worms such as S. kowalevskii do have nephridia-like structures within the proboscis, but their function has not been determined (Balser and Ruppert, 1990). Furthermore, ionocytes have yet to be identified anywhere in the animal. Cephalochordate ion regulation has been explored further, but not much more is known. Like hemichordates, cephalochordates are also marine organisms believed to be osmoconformers. However, some species can tolerate ~20 ppt seawater for several weeks (Binyon, 1981; Webb and Hill, 1958), suggesting some osmoregulatory capacity. It remains unknown if this tolerance is associated with active regulation of the internal milieu by specialized epithelia, or if the body’s cells defend their osmotic balance independently. Furthermore, tolerance to hypercarbia or other acid-base challenges has yet to be tested. Studies investigating specific ion regulating epithelia in cephalochordates are equally inconclusive. Nephridia-like structures are intimately associated with the pharyngeal gill bars and their coelomic spaces, but function remains unknown (Moller and Ellis, 1974). Positive  65 immunoreactivity for key ion transport machinery (NKA, VHA, CA, NHE, CFTR and Pendrin) has been reported at multiple epithelia, including gills and skin (Pederzoli et al., 2014; Cuoghi et al., 2018). However, antibodies were not tested for non-specific binding, and relative contributions from different epithelia could not be estimated. Others found elevated gene expression for NKA and NKCC at gills relative to skin, but did not corroborate this signal with estimates of activity or relative protein abundance (Li et al., 2017). Thus, as in hemichordates, ionocytes and/or ion regulating epithelia have yet to be identified and located.  Ideally, roles that the skin and gills of S. kowalevskii and B. floridae play in ion regulation would be assessed with direct measurements of ion flux in vivo as done for ammocoetes in Chapter 2. Unfortunately, their marine residence hinders radioisotope use. Thus, three different indirect measurements were instead combined to assess ionocyte location and activity in vitro. These are (1) gene expression for putative ion transporters associated with vertebrate ionocytes, (2) ATPase activity and (3) gene expression for the vertebrate ionocyte marker Foxi. These three measurements can be unconvincing on their own, but together can provide robust indications of ionocyte location and activity. For example, gene expression of putative ion transporters can estimate which transport machinery is present at different tissues, and in what relative quantities. However, expression does not always accurately reflect protein abundance (Liu et al., 2016). ATPase activity can therefore corroborate observed expression patterns by estimating ion regulating capacity. Recalling Section 1.1.3, NKA and/or VHA power most ion transport pathways at vertebrate gill ionocytes (Hwang et al., 2011). Their activity is thus commonly used as a proxy for ion regulating capacity in vertebrate gills and other tissues (McCormick, 1993). However, ATPase’s and some ion transport machinery can also be elevated in electrically active cells of muscle and nervous tissue (Nikolic, 2016; Pirkmajer and Chibalin, 2016). In these instances, Foxi expression can serve as an additional marker for ionocyte presence. Foxi is a subclass of forkhead box transcription factors with three members in vertebrates (Foxi1, Foxi2 & Foxi3; Solomon et al., 2003). Each of these Foxi members is implicated in ionocyte specification for multiple ion regulating epithelia in multiple species, including the gills and skin of zebrafish (Danio rerio; Esaki et al., 2007; Hsiao et al., 2007; Cruz et al., 2013), the skin of xenopus (Xenopus laevis; Dubaissi and Papalopulu, 2011; Quigley et al., 2011) and the  66 kidney, lung, intestine, inner ear and epididymis of mouse and human (Mus musculus and Homo sapiens; Blomqvist et al., 2004, 2006; Vidarsson et al., 2009; Montoro et al., 2018). In all cases where DNA binding is characterized, Foxi members directly bind the promoter regions of ionocyte specific machinery, including ion transporters (VHA, AE4, AE1 and Pendrin; Kurth et al., 2006; Yang et al., 2007; Vidarsson et al., 2009; Singh et al., 2018) and other Foxi members (Cha et al., 2012). Genome scans for the consensus binding sequence of Foxi1 in mice also show putative binding sites in the promoter regions for other ionocyte related genes, such as mineralocorticoid receptors and cell adhesion molecules (Kurth et al., 2006; Overdier et al., 1997). Due to this ionocyte specificity and because expression is elevated in both progenitor and mature cells, Foxi has served as an important marker for vertebrate ionocytes (Montoro et al., 2018; Plasschaert et al. 2018). Foxi is also implicated in gene regulatory networks for the early development of pharyngeal arch derivatives such as jaws and the inner ear (Edlund et al., 2015; Birol et al., 2016). However, expression of these networks is mostly restricted to early development, and Foxi binding sites unrelated to ionocytes have not been found. Furthermore, many of these structures have ionocytes and/or develop in close proximity to them. Foxi has not been explored for a link to ion regulation in the invertebrate deuterostomes, but its involvement would bolster any test for homology with vertebrate ionocytes. Hemichordates and cephalochordates do possess a single Foxi gene (Yu et al., 2008; Shimeld et al., 2010), and expression has been observed in the developing gill pore of S. kowalevskii embryos (Fritzenwanker et al., 2014). However, function remains unknown, and expression has not been measured in adult hemichordates or in cephalochordates of any life stage. Here, I hypothesized that gills of S. kowalevskii and B. floridae were primary sites of ion regulation. To test this, gill and skin were measured for (1) gene expression of putative ion transporters associated with vertebrate ionocytes, (2) ATPase activity and (3) gene expression of the vertebrate ionocyte marker Foxi. Gills are predicted to have a greater signal than skin for all three measurements in both species. Maximum likelihood trees and multiple sequence alignments with characterized proteins from Homo sapiens and Danio rerio were used to assess homology and infer function in putative ion transporters from S. kowalevskii and B. floridae. To further explore the possible link between Foxi and ion regulation in S. kowalevskii and B. floridae, putative ion transport genes were scanned upstream for the consensus binding sequence of murine Foxi1.   67 4.2 Methods  Animal collection and husbandry Saccoglossus kowalevskii were purchased from the Marine Biological Laboratory in Woods Hole MA, USA. Animals were shipped to the University of British Columbia’s Point Grey Campus in Vancouver, Canada, where they held in static, 40 L seawater tanks at 10oC (10 animals per tank; 34 ppt artificial seawater made from Instant Ocean salt mix, USA). Tanks were aerated, supplied with sandy substrate and maintained on a 12:12 light:dark photoperiod. 30% water changes were performed twice weekly. Saccoglossus kowalevskii used in experiments had a mean wet mass of 0.105±0.009 g (n = 20).  Branchiostoma floridae were purchased from Gulf Specimen Marine Laboratories in Panacea FL, USA, and also shipped to the University of British Columbia’s Point Grey campus in Vancouver, Canada. Branchiostoma floridae were held in identical conditions to S. kowalevskii, but maintained at 20oC. Branchiostoma floridae used in experiments had a mean wet mass of 0.049±0.005 g (n = 20). All animals from both species were held for one week at the University of British Columbia under their respective conditions and fasted 24 h before sampling for tissue.  Tissue sampling  All animals were pinned to silicone-coated Petri dishes in seawater for dissection. Tissue samples were carefully excised with the aid of a dissecting microscope, transferred to RNase-free bullet tubes, flash frozen in liquid N2 and then stored at -80oC until further analysis. For B. floridae, tissue samples were taken for gills (all pharyngeal gill bars), liver (entire hepatic diverticulum), muscle (dorsal section immediately posterior to atriopore) and skin (outer dermis, but excluding that lining the atrium). For S. kowalevskii, tissue samples were taken for gills (all pharyngeal gill bars), intestine (entire gut tube posterior to hind-most gill pore), muscle (entire proboscis) and skin (outer dermis). Animals of a given species were all sampled within a 4 h period, and each animal dissection took no more than 5-10 minutes. A total of 20 animals per species were sampled (10 for ATPase measurements, 10 for gene expression). Only results for gill and skin are shown here, and the remaining tissue data are in the Appendix (Figures C.2, C.3 and C.4).  68 ATPase activity  Na+/K+-ATPase (NKA) and H+-ATPase (VHA) activities were measured using modified versions of the protocols outlined by McCormick (1993) and Tresguerres et al. (2005). Briefly, frozen tissue samples from 10 individuals were randomly selected for homogenization in ice-cold SEID buffer (150 mM sucrose, 50 mM imidazole, 10 mM EDTA, 0.1% sodium deoxycholate, pH 7.3) with a handheld glass homogenizer the day of assay. Homogenates were centrifuged at 5000 g for five minutes, and supernatants transferred to new bullet tubes on ice. Each sample supernatant was then run in triplicate on a SpectraMax 190 microplate reader (Molecular Devices, USA) at 25oC and 340 nm for 3 separate treatments: control (5 mM azide), ouabain (5 mM azide, 1 mM ouabain) and NEM (5 mM azide, 1 mM ouabain, 1 mM N-Ethylmaleimide). Activity of NKA was taken as the difference between control and ouabain treatments, and VHA activity as the difference between ouabain and NEM treatments. Preliminary measurements for each tissue type determined the concentrations of ouabain and NEM for maximum inhibition, and found no difference between inhibition by NEM and bafilomycin. Protein concentration of homogenates was subsequently measured in triplicate using Bradford reagent (Sigma-Aldrich, Canada) and bovine serum albumin (BSA) as a standard. NKA and VHA activities are expressed as µmols ADP·mg protein-1·h-1.   Gene expression  Frozen tissue samples from 10 individuals were randomly selected for homogenization at 4oC in TRIzol reagent (Life Technologies Inc, Canada) with a bullet blender (Next Advance Inc, USA) and 1.0 mm diameter zirconium oxide beads. Total RNA was extracted with TRIzol Reagent and further purified with RNeasy PowerClean Pro CleanUp Kit (Qiagen, Germany) according to the manufacturers’ protocols. Total RNA concentration and quality were then assessed with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA) and stored at -80oC until cDNA synthesis. Only RNA samples with A260/A280 and A260/A230 ratios of ~2.0 or higher were used to measure gene expression.  The cDNA was synthesized from 1 ug RNA for each tissue sample with a High Capacity cDNA Reverse Transcription Kit (Life Technologies, Canada) and T100 Thermal Cycler (Bio-Rad Laboratories, USA) according to the manufacturer’s protocol. Gene expression was then measured using quantitative real-time PCR (qRT-PCR) with a CFX96 Real-Time System (Bio- 69 Rad Laboratories, USA) and SYBR Green PCR Master Mix (Applied Biosystems, USA). Each 20 ul qRT-PCR reaction contained forward and reverse primers at a concentration of 20 nM (Integrated DNA Technologies, Canada), and was supplemented with 20 ug BSA (Life Technologies, Canada) to dampen any intrinsic inhibition. The PCR protocol consisted of 4 steps: (1) 95oC for 10 min, (2) 40 cycles of 95oC for 15 s and 55oC for 1 min, (3) 95oC for 10 s, and (4) a rise from 65 to 95oC by 0.5oC increments at 5 s each.  Target sequences were identified in B. floridae and S. kowalevskii with reciprocal best BLAST hits for characterized proteins in Danio rerio and/or Homo sapiens (Ward and Moreno-Hagelsieb, 2014). Sequence accession numbers are provided in the Appendix (Table C.1), and homology between species was further quantified as described below. Species specific primers for B. floridae and S. kowalevskii were then designed for corresponding transcripts from RefSeq mRNA databases (NCBI txid7739 and txid10224, respectively) with PrimerQuest (Integrated DNA Technologies, Canada) for use with qRT-PCR (primer sequences listed in Appendix Table C.2). Primers were validated for each gene with standard curves made with cDNA from whole-body RNA samples. Standard curves were included on each plate for quality control of qRT-PCR reactions. All primers had efficiency values of ~95-105%. Reported gene expression values are normalized to 18S and skin within respective species.  Identifying putative ion transport genes  As described above, putative ion transporters for B. floridae and S. kowalevskii were identified with reciprocal best BLAST hits for characterized proteins in D. rerio and/or H. sapiens. To further assess the homology of BLAST hits with proteins of known function, putative proteins were aligned with multiple sequences from D. rerio and H. sapiens for each target gene family. Where possible, representatives for each member of a target gene family were included. Foxi and Rh orthologues were previously identified in S. kowalevskii and B. floridae by others (Fritzenwanker et al., 2014; McDonald and Ward, 2016), but alignments were still performed here to assess sequence identity and conservation of key residues with characterized proteins in H. sapiens. Each BLAST hit analyzed here also matched significantly with the PFAM database for functional motifs associated with the assigned gene family. For each gene family, a multiple sequence alignment was completed with MUSCLE (Edgar, 2004) to construct a maximum likelihood tree in MEGA X (Stecher et al., 2020) using  70 the Neighbor-Joining method (500 bootstrap replicates). If putative proteins from B. floridae and S. kowalevskii clustered within the proteins of D. rerio and H. sapiens for a given family, a second alignment was performed. This second analysis aligned the putative proteins from B. floridae and S. kowalevskii with the three most closely related human proteins. From this second alignment, putative protein sequences were compared to those of H. sapiens for the presence of key active site residues and to calculate percent identity and similarity. Human sequences were used for comparison because they were often the best characterized at the amino acid level. For transmembrane proteins, only transmembrane regions were used to calculate percent identity and similarity.  Putative Foxi binding sites  As in Kurth et al. (2006), genes for putative ion transporters were scanned 1 kb upstream for matches with the M. musculus Foxi1 core consensus binding sequence (TRTTTRY; R = A or G, Y = T or C). Genes for putative NKA and VHA catalytic subunits were also scanned.  Statistics  Statistical analyses were performed with Prism 8 (Graphpad Software Inc, USA). All data are presented as means±s.e.m., and comparisons between skin and gill were made with unpaired t-tests (P<0.05).   71 4.3 Results  ATPase activity and gene expression  In B. floridae, NKA and VHA activities were ~10-fold higher in gill than skin (Figure 4.1A; P<0.001). Similarly, gill expression of AE, NHE, CA and Foxi was 2, 5, 7 and 10-fold higher than skin, respectively (Figure 4.2A; P<0.01). However, Rh expression exhibited the opposite pattern, and was ~30-fold higher in skin than gill (Figure 4.2A, P<0.001).  In S. kowalevskii, NKA and VHA activities did not differ between gill and skin (Figure 4.1B). Expression of Foxi was 10-fold higher in gill than skin (P<0.001), but no difference between tissues was observed for any other gene examined (Figure 4.2B).  Sequence alignment & phylogeny  Protein sequences from B. floridae and S. kowalevskii all clustered within their target gene families when aligned with multiple sequences from D. rerio and H. sapiens (Figures 4.3-4.7). Further comparisons between these putative proteins and their most closely related human sequences revealed a high degree of similarity. Percent identity was ~55-65% for transmembrane regions of AE, NHE and Rh (Table 4.1), with percent similarity at ~70-80%. Values were lower for CA and Foxi, but still at ~44-49% identity and ~63-74% similarity (Table 4.1). Furthermore, bfFOXi-like and skFOXi-like shared a 93.7 and 87.4% sequence identity, respectively, with the DNA binding domain of hsFOXi1 (Table 4.1).  Key residues from active sites in human proteins were also mostly conserved in sequences from B. floridae and S. kowalevskii (Figures 4.8-4.12). The only notable difference is in skCA-like, where Q92 is replaced with G (Figure 4.10). In human CA2, Q92 forms an important hydrogen bond with H94 that helps H94 properly position the zinc molecule in the active site (Boone et al., 2014). It is unknown if and how this substitution may affect putative carbonic anhydrase activity, but a glutamine-glycine substitution is not considered conservative.  Putative Foxi binding sites Multiple matches with the murine Foxi1 core consensus binding sequence were identified 1 kb upstream for all putative ion transport genes except for skAE-like, bfNKAα1-like and bfNKAβ1-like (Table 4.2). Only those matches closest to transcription start sites are reported.   72 4.4 Discussion  The results of this chapter support the hypothesis that gills of the cephalochordate Branchiostoma floridae are a primary site of ion regulation. This hypothesis is well supported by elevated gill ATPase activities, gene expression for putative ion transporters and gene expression for the ionocyte marker Foxi. The results are less clear for the hemichordate Saccoglossus kowalevskii, but data suggest their gills might also play an important role in ion regulation. However, this role might be shared with the skin and/or other tissues.  Branchiostoma floridae The gills of B. floridae displayed a strong signal for ion regulation across all measurements. At the enzyme level, gill Na+,K+-ATPase (NKA) and V-type H-ATPase (VHA) activities were 10-fold higher than those of skin, and consistent with values reported for many teleost and elasmobranch gills (1-2 and 0.2-1 µmols ADP·mg protein-1·h-1 for NKA and VHA, respectively; Richards et al., 2003; Tresguerres et al., 2005; Perry et al., 2006). These are the first measurements of NKA and VHA activities in B. floridae, and they suggest that B. floridae gills possess the capacity to drive ion regulation pathways similar to those found in vertebrate gills.  The gills of B. floridae also had elevated gene expression for other putative machinery implicated in vertebrate ion regulation pathways. Putative anion exchanger (AE-like), sodium-proton exchanger (NHE-like) and carbonic anhydrase (CA-like) expression was 2, 5 and 7-fold higher, respectively, at gills than skin. These proteins are all key players in multiple pathways described for trans-epithelial ion and pH regulation in vertebrate ionocytes (Hwang and Lin, 2013), and also often display higher baseline expression in ion regulating tissues. For example, CA2 and NHE2/3 expression in rainbow trout (Oncorhynchus mykiss) is anywhere from 4 to 1000-fold higher in gill than non-ion regulating tissues (Esbaugh, 2005; Ivanis et al., 2008). All of the putative proteins identified in B. floridae have yet to be characterized, but high sequence identity with related proteins in Homo sapiens and the conservation of key active site residues supports a similar general function. Together with NKA and VHA, this suite of upregulated ion transport machinery in B. floridae gills resembles the primary components for acid and base secreting ionocytes in marine elasmobranchs. Basolateral NKA, cytosolic CA2 and apical NHE3 are believed to co-localize in  73 “acid secreting” ionocytes, while basolateral VHA, cytosolic CA2 and a different apical anion exchanger (“Pendrin” or Slc26a6) are proposed to co-localize in distinct “base secreting“ ionocytes (see Figure 1.5, Chapter 1; Piermarini and Evans, 2001; Piermarini et al., 2002; Tresguerres et al., 2005; Choe et al., 2007; Gilmour and Perry, 2009; Reilly et al., 2011; Guffey et al., 2015; Roa and Tresguerres, 2017). The comparison with marine elasmobranchs is particularly relevant because they likely share an osmoconforming strategy with cephalochordates (Wright and Wood, 2015). The ion regulation demands of B. floridae might therefore be more similar to those of marine elasmobranchs, and they appear to be equipped with similar machinery.  The final signal for ion regulation in B. floridae was Foxi expression, which was ten times higher in gills than skin. Foxi is a transcription factor necessary for ionocyte specification in all vertebrates examined, and serves as an important marker of both progenitor and differentiated cells (Montoro et al., 2018; Plasschaert et al., 2018). A direct link between Foxi and ion regulation in B. floridae remains to be determined, but high sequence identity with the DNA binding domain of mammalian Foxi1 suggests it may target a similar core consensus sequence. Furthermore, with the exception of NKA, all of the putative ion transporters examined here for B. floridae possess at least one match for this mammalian consensus sequence in potential upstream promoter regions. Again, a direct link between Foxi and ion regulation in B. floridae has yet to be determined, but these similarities and the elevated co-expression with putative ion transporters are consistent with the role of Foxi in vertebrates. The only gene measured in B. floridae with significantly higher expression in skin than gills was a putative Rhesus glycoprotein, which was ~30 times higher. Rhesus glycoproteins serve as transmembrane channels for both gaseous and protonated forms of ammonia (reviewed by Wright and Wood, 2012) and possibly CO2 gas (Geyer et al., 2013). These data are thus consistent with the notion of cutaneous gas exchange in cephalochordates as suggested by prior morphometric measurements in Branchiostoma lanceolatum (Schmitz et al., 2000). Taken together, these results suggest that B. floridae gills possess a capacity (ATPase activity), machinery (ion transporter expression) and cell type (Foxi expression) similar to those associated with ion regulation at vertebrate gills. This consistency across multiple indices provides good support for B. floridae gills as a primary site for ion regulation homologous with vertebrate gills.  74 Saccoglossus kowalevskii S. kowalevskii gills are implicated in ion regulation as well, but results are less conclusive than for B. floridae. Equal and elevated ATPase activities for gills and skin suggest both tissues possess the capacity to drive vertebrate-like ion regulation pathways. VHA activity matches that for gills of teleosts and elasmobranchs, and although NKA activity is one third this value, it is still within range of those reported for many vertebrate gills (1-2 and 0.2-1 µmols ADP·mg protein-1·h-1 for NKA and VHA, respectively; Richards et al., 2003; Tresguerres et al., 2005). Equal expression between tissues for all ion transport machinery is also consistent with a shared capacity. However, because it is unknown if expression is equally high or low between tissues, specific transporters cannot be implicated in putative ion regulation pathways. To my knowledge, these are the first measurements of ATPase activity and ion transporter expression in a hemichordate. Shared capacities for ion regulation between skin and gills could indicate a number of different scenarios. First and perhaps most simply, epithelial ion regulation could be occurring at both tissues. However, Foxi expression is ten times higher at gills than skin. As with B. floridae, a link between Foxi and ion regulation in S. kowalevskii has yet to be determined despite high sequence identity with vertebrate orthologues. However, if Foxi is shown to be a reliable marker for ionocytes, it suggests the majority of these specialized cells are at the gills rather than distributed equally between tissues. Second, ATPase activites at the skin might be elevated by electrically active nervous and/or muscle tissue. Unlike B. floridae and cephalochordates, S. kowalevskii lacks an outer cuticle and has a less well-defined epidermal layer with a single cell thickness (Barrington, 1965). The epidermis houses one of the highest densities of nervous tissue in the animal (epidermal nerve plexus; Nomaksteinsky et al., 2009; Miyamoto and Wada, 2018) and sits directly on top of muscle in the outer trunk wall, which was likely also incorporated into sampled “skin” tissue. By comparison, the simple gill bars of S. kowalevskii are relatively devoid of such tissue, composed primarily of acellular collagen and rudimentary hemolymph vessels. ATPase activities at the gills are thus less likely than activities at the skin to be elevated by the presence of nervous or muscle tissue. Third and finally, S. kowalevskii might have ionocytes at gills for epithelial ion regulation, but retain a robust capacity for intracellular ion regulation at multiple tissues. This is  75 further supported by additional measurements of elevated ATPase activities in the intestine and muscular proboscis, which are similar to those of gills and skin (Appendix, Figure C.2A). Such an intracellular capacity would be independent of ionocyte distribution and Foxi expression, and predicted in S. kowalevskii given certain aspects of its biology. Indeed, independent and robust regulation of intracellular pH by tissues is common among CO2 tolerant fishes and some embryonic vertebrates (Shartau et al., 2016). This pattern is observed when extracellular pH regulation by gills or some designated organ becomes limited or insufficient during an acid-base disturbance (Sackville et al., 2018). Such a pattern might be necessary for S. kowalevskii’s regenerative ability, as fragments from different regions of the body survive and grow independently (Tweedell, 1961). Even in intact animals, independent tissue regulation might be required if S. kowalevskii’s rudimentary circulatory system lacks the speed and/or degree of perfusion to effectively regulate the extracellular compartment of distal regions with a central organ. This might be especially relevant in the intertidal burrows where S. kowalevskii resides if environmental PCO2 rises faster than the rate of extracellular pH compensation (Richards, 2011). Regardless of the reason for elevated ATPase activity at other tissues, these results suggest that gills of S. kowalevskii have a capacity (ATPase activity) and cell type (Foxi expression) for ion regulation similar to those of vertebrate gills.   Taken together, these results support the hypothesis that gills of B. floridae are a primary site for ion regulation, and are consistent with the hypothesis that gills of S. kowalevskii play a primary role as well. Additional work is still required to confirm the presence, function and machinery associated with putative, vertebrate-like ionocytes at these tissues, and future experiments should incorporate ion and acid-base challenges to stimulate a functional response in the metrics used. This is especially true for S. kowalevskii, which lacked a clear expression signal for ion transporters at any tissue. However, the data presented here are still compelling. Gill ATPase activities alone provide a strong signal for ion regulation at gills in both representative species. Perhaps most striking is that these activities resemble those measured in ammocoete gills (Appendix, Figure C.1). This is especially noteworthy given that the simple collagenous bars of B. floridae and S. kowalevskii are relatively devoid of the muscle and nervous tissue present in ammocoete gills, and are not also tasked with the additional ionoregulatory demands of life in freshwater. Elevated Foxi expression at B. floridae and S. kowalevskii gills also presents a strong case for the presence homologous, vertebrate-like  76 ionocytes. The link between Foxi and ionocytes has yet to be confirmed in these species, but homology with vertebrate ionocytes would require its expression. Furthermore, the high sequence identity with mammalian Foxi1 is encouraging, as are matches with its core consensus sequence in potential promoter regions for putative ion transporting genes.  Evolutionary implications As described in Chapter 1, B. floridae and S. kowalevskii represent important comparative groups for testing the deuterostome ancestry of vertebrate gill function because of their phylogenetic positions and resemblance to the ancestral morphology and lifestyle (Cameron et al., 2000; Brown et al., 2008; Holland et al., 2015; Lowe et al., 2015; Simakov et al., 2015). Here, I provide the first support for ion regulation at hemichordate gills and the most comprehensive and compelling support for ion regulation at cephalochordate gills. These results suggest that gills might have acquired their primary role in ion regulation before the evolution of vertebrates. These findings and their implications are discussed further in Chapter 5.   77 4.5 Figures   Figure 4.1 Skin and gill ATPase activities in B. floridae and S. kowalevskii Na+/K+-ATPase (NKA) and H+-ATPase (VHA) activity in skin (white) and gill (black) of B. floridae (A) and S. kowalevskii (B). Data presented as means ± sem, n=10. Asterisks indicate significant differences between tissues (P<0.01, unpaired t-test).  NKA VHA0.00.51.01.52.0µmols ADP ⋅ mg-1 ⋅ h-1NKA VHA0.00.51.01.52.0µmols ADP ⋅ mg-1 ⋅ h-1ABB. floridaeS. kowalevskii** 78    Figure 4.2 Skin and gill gene expression in B. floridae and S. kowalevskii Gene expression for putative ion transporters and ionocyte markers in skin (white) and gill (black) of B. floridae (A) and S. kowalevskii (B). Data presented as means ± sem, n=10. Asterisks indicate significant differences between tissues (P<0.01, unpaired t-test).  AE NHE CA Rh FOXi110100relative expression (18S-1) *****AE NHE CA Rh FOXi110100relative expression (18S-1)*ABB. floridaeS. kowalevskii 79      Figure 4.3 Anion exchanger phylogeny  Maximum likelihood tree depicting how putative anion exchangers from Branchiostoma floridae (bf) and Saccoglossus kowalevskii (sk) cluster relative to anion exchangers from the SLC4a family in Homo sapiens (hs) and Danio rerio (dr). Numbers indicate the percentage of 500 bootstrap replicates supporting a given node. Branch lengths drawn to scale and indicate divergence in number of substitutions per site. Sequence accession numbers in brackets. AE = Anion Exchanger; NBC = Sodium Bicarbonate Cotransporter.   80       Figure 4.4 Sodium-proton exchanger phylogeny Maximum likelihood tree depicting how putative sodium-proton exchangers (NHEs) from Branchiostoma floridae (bf) and Saccoglossus kowalevskii (sk) cluster relative to NHEs from the SLC9a family in Homo sapiens (hs) and Danio rerio (dr). Numbers indicate the percentage of 500 bootstrap replicates supporting a given node. Branch lengths drawn to scale and indicate divergence in number of substitutions per site. Sequence accession numbers in brackets.   81      Figure 4.5 Carbonic anhydrase phylogeny Maximum likelihood tree depicting how putative carbonic anhydrases (CA) from Branchiostoma floridae (bf) and Saccoglossus kowalevskii (sk) cluster relative to carbonic anhydrases in Homo sapiens (hs) and Danio rerio (dr). Numbers indicate the percentage of 500 bootstrap replicates supporting a given node. Branch lengths drawn to scale and indicate divergence in number of substitutions per site. Sequence accession numbers in brackets.   82      Figure 4.6 Ammonia transporter phylogeny Maximum likelihood tree depicting how putative Rhesus glycoproteins (Rh) from Branchiostoma floridae (bf) and Saccoglossus kowalevskii (sk) cluster relative to Rh proteins and ammonia transporters (AMT) in Homo sapiens (hs), Danio rerio (dr), Ciona intestinalis (ci) and Drosophila melanogaster (dm). Numbers indicate the percentage of 500 bootstrap replicates supporting a given node. Branch lengths drawn to scale and indicate divergence in number of substitutions per site. Sequence accession numbers in brackets.   83      Figure 4.7 Forkhead box phylogeny Maximum likelihood tree depicting how putative Foxi protein from Branchiostoma floridae (bf) and Saccoglossus kowalevskii (sk) cluster relative to Fox proteins in Homo sapiens (hs) and Danio rerio (dr). Numbers indicate the percentage of 500 bootstrap replicates supporting a given node. Branch lengths drawn to scale and indicate divergence in number of substitutions per site. Sequence accession numbers in brackets.  84   85   Figure 4.8 Multiple sequence alignment for anion exchangers Sequence alignment for putative anion exchangers in Branchiostoma floridae (bfAE-like) and Saccoglossus kowalevskii (skAE-like) with human AE1, 2 and 3. Transmembrane regions and critical active site residues for hsAE1 are indicated with horizontal bars and red asterisks, respectively (Reithmeier et al., 2016). Identical and chemically similar amino acids indicated by black and grey shading, respectively.   86    87            Figure 4.9 Multiple sequence alignment for sodium-proton exchangers Sequence alignment for putative sodium-proton exchangers in Branchiostoma floridae (bfNHE-like) and Saccoglossus kowalevskii (skNHE-like) with human NHE1, 2 and 3. Horizontal bars indicate transmembrane regions in hsNHE1 (Pedersen and Counillon, 2019). Identical and chemically similar amino acids indicated by black and grey shading, respectively.   88          Figure 4.10 Multiple sequence alignment for carbonic anhydrases Sequence alignment for putative carbonic anhydrases in Branchiostoma floridae (bfCA-like) and Saccoglossus kowalevskii (skCA-like) with human CA2, 5a and 8. Key active site residues for hsCA2 noted in red (“Z” = zinc binding, “P” = proton shuttle, and “*” = other residues from hydrophilic and proximal hydrophobic regions of active site; Boone et al., 2014). Identical and chemically similar amino acids indicated by black and grey shading, respectively.   89   Figure 4.11 Multiple sequence alignment for Rhesus glycoproteins Sequence alignment for putative Rhesus glycoproteins in Branchiostoma floridae (bfRh-like) and Saccoglossus kowalevskii (skRh-like) with human RhCG, RhAG and RhBG. Transmembrane regions (horizontal bars) and key residues (red letters) are indicated for RhCG (“g” = phenylalanine gates, “d” = histidine dyad, “*” = other residues implicated in proton and ammonium binding; Baday et al., 2015; Gruswitz et al., 2010). Identical and chemically similar amino acids indicated by black and grey shading, respectively.   90        Figure 4.12 Multiple sequence alignment for forkhead box I Sequence alignment for putative forkhead box I in Branchiostoma floridae (bFOXi-like) and Saccoglossus kowalevskii (skFOXi-like) with human FOXi1, 2 and 3. The horizontal bar indicates the DNA binding domain in hsFOXi1 (from PROSITE database; Sigrist et al., 2005, 2012). Identical and chemically similar amino acid residues indicated by black and grey shading, respectively. 91               Human protein Putative protein % Identity % Similarity hsAE1 (TM regions) bfAE-like skAE-like 58.9 65.9 78.7 80.5 hsNHE1 (TM regions) bfNHE-like skNHE-like 64.2 63.8 76.4 80.5 hsCA2 bfCA-like skCA-like 48.6 43.7 72.3 74.1 hsRhCG (TM regions) bfRh-like skRh-like 54.3 57.8 69.5 77.0 hsFOXI1a (DNA binding domain) bfFOXI-like skFOXI-like 93.7 87.4 95.8 90.5  Table 4.1 Percent identity between human and protovertebrate protein sequences   92         Gene  NCBI ID# Position Sequence bfAE-like 7253927 -554/-548 TATTTAT bfNHE-like 7212036 -623/-617 TATTTAC bfCA-like 7216702 -332/-326 TATTTAT bfNKAα1-like 7218356 none none bfNKAβ1-like 7217419 none none bfVHAa1-like 7220642 -421/-415 TATTTGC bfVHAb1-like 7219325 -212/-206 TATTTGC     skAE-like 100372733 none none skNHE-like 100370587 -766/-760 TATTTGC skCA-like 102809159 -319/-313 TGTTTAT skNKAα1-like 100370816 -170/-164 TATTTAT skNKAβ1-like 102808012 -300/-294 TGTTTGT skVHAa1-like 100370533 -355/-349 TATTTAT skVHAb1-like 100372746 -285/-279 TATTTGT     Mus musculus Foxi1 core consensus sequence: TRTTTRY (R = A or G; Y = T or C)  Table 4.2 Putative promoter binding sites for Foxi in B. floridae and S. kowalevskii Only first match within 2 kb upstream from transcription start site listed. Core consensus sequence from Kurth et al. (2006) and Overdier et al. (1997).   93 Chapter 5: General Discussion  5.1 Introduction  The primary goal of this thesis was to better understand how and when vertebrate gills acquired their primary roles in gas exchange and ion regulation. Currently accepted views propose that gills acquired these roles from the skin along the vertebrate stem. By replacing skin as the primary site for gas exchange and ion regulation, gills are thought to have facilitated the early vertebrate transition to larger, armoured and more active modes of life. The role for gas exchange in this scenario is well supported by morphological data from fossil and developmental studies. However, ion regulation lacks equivalent morphological support, and neither gas exchange nor ion regulation has functional support from representatives of early vertebrates or their ancestors. To address this knowledge gap, I characterized gill and skin function for gas exchange and ion regulation in representatives of ancestral vertebrates (ammocoetes, Entosphenus tridentatus), cephalochordates (amphioxus, Branchiostoma floridae) and hemichordates (acorn worms, Saccoglossus kowalevskii). Intraspecific comparisons between ammocoetes tested the effects of body size and activity on early vertebrate gill function, and interspecific comparisons between all three taxa tested the ancestral origins of gill function. These comparative approaches were bolstered by (1) gill arch homology among representative species and (2) resemblance of representative species to the ancestral morphology and lifestyle. The results are summarized in Figure 5.1. For gas exchange, only vertebrate gills played a primary role (Chapter 2). Furthermore, this role was acquired secondarily from the skin with increasing body size and challenges to oxygen supply and demand. Hemichordate gills were not associated with an enhanced capacity for gas exchange despite challenges to oxygen supply and demand (Chapter 3), and although cephalochordate gas exchange was not examined here, prior morphometric work suggests their gills also play a negligible role (~5% of total body diffusive capacity; Schmitz et al. 2000). For ion regulation, the gills of all three representatives were implicated. Direct measurements in ammocoetes found the gills responsible for all ion uptake at all body sizes and conditions tested, while the skin played no detectable role (Chapter 2). Gene expression and  94 enzyme activity for ionocyte markers displayed a similar pattern in amphioxus, supporting gills as the primary site for ion regulation in cephalochordates as well (Chapter 4). These same ionocyte markers suggest acorn worm gills might also play a primary role in ion regulation, but that this role might be shared with other tissues (Chapter 4). Taken together, these results support the long-held belief that gills acquired their primary role in gas exchange from the skin as early vertebrates increased body size, dermal thickness and activity. These are the first functional data in representatives of early vertebrates and their ancestors to support this established view, and they complement existing morphological data from fossil and developmental studies. However, ion regulation at gills appears unrelated to this vertebrate transition. This work instead suggests that gills acquired their primary role in ion regulation long before gas exchange in the last common ancestor of chordates or deuterostomes –perhaps near the inception of pharyngeal gill arches. The transition from skin to gills observed for ion regulation in larval teleosts might therefore be a derived phenomenon associated with embryonic development in vertebrates. The remainder of this chapter discusses some of the more interesting implications of these findings. I then conclude with suggestions for future experiments to address the limitations and interpretations of this work.  5.2 A deep origin for ion regulation at gills, but for what?  One of the most exciting discoveries of this thesis is that gills might have acquired their primary role in ion regulation before the evolution of vertebrates and perhaps in the last common ancestor of deuterostomes. This is close to the presumed origin of pharyngeal gill arches, suggesting that ion regulation might have been an original function of gills. However, the function of this ancestral ion regulation remains unknown. As described in Section 1.1.3, maintaining acid-base balance is widely regarded as the ancestral function of ion regulation at vertebrate gills. This is also a likely candidate for early deuterostomes, and further experimentation may reveal this to be the case. However, there is little evidence that extant invertebrate deuterostomes tightly regulate extracellular pH. In echinoderms, only very slow extracellular pH compensation occurs following days of exposure to low levels of hypercarbia (Stumpp et al., 2012; Collard et al., 2013, 2015; Stumpp and Hu,  95 2017). Furthermore, much of this compensation occurs by changes in the extracellular non-bicarbonate buffering capacity, rather than bicarbonate retention via acid-base ion regulation. Echinoderms might not be the best representation of the ancestral condition, but invertebrate deuterostomes in general lack circulatory adaptations that might be required to effectively regulate acid-base balance (Barrington, 1965; Boot et al., 2003; Green et al., 2015; Tang et al., 2019). The slow circulation and poor perfusion of distal tissues typical of invertebrate deuterostomes could easily hinder the effectiveness of extracellular pH regulation, as could increased permeability of the internal milieu associated with a poorly defined integument. This might have required early deuterostomes to rely on intracellular pH regulation instead, and I found some support for this capacity in the hemichordate S. kowalevskii in Chapter 4.  5.3 Was the ancestral form of ion regulation linked to filter-feeding?  Alternatively, the ancestral form of ion regulation at gills might have been linked to feeding. As described in Section 1.2.1, the ancestral deuterostome is presumed to have used gills to filter-feed. This is strongly supported by the fact that gills of all extant invertebrate deuterostomes are used for this purpose. Two aspects of filter-feeding may depend on epithelial ion regulation at gills. The first is active nutrient uptake. Crustaceans, bivalves and even the vertebrate hagfish have been shown to use gills for active uptake of amino acids and other nutrients from the surrounding water (Gomme, 2001; Glover et al., 2011; Blewett and Goss, 2017). In many of these organisms, uptake is linked to active sodium transport and associated with gill regions populated by ionocytes. This mechanism has yet to be explored in extant hemichordates and cephalochodates, but could easily be the ancestral form of ion regulation in early deuterostome gills. The second aspect of filter feeding that may depend on epithelial ion regulation at gills is mucin secretion. Filter-feeding gills are mucociliary complexes that rely on mucin to trap food particles from the water as it filters through the pharyngeal slits. Food-filled mucin is then transported down the gills to the digestive tract by cilia for digestion. Mucin is initially secreted as densely packed granules onto the gill surface where it can expand over 1000-fold (Ambort et al., 2012). This expansion is critical, as packed mucin is too dense and viscous for efficient particle entrapment and ciliary transport. For example, disruption of mucin expansion in the  96 mucociliary complex of human lungs can result in disease states such as cystic fibrosis, where thick masses of mucus accumulate and interfere with breathing (Quinton, 2008). Interestingly, mucin expansion may require acid-base ion regulation of the boundary layer. Indeed, mucin is held in its packed conformation by protons and calcium ions bound to its molecular surface (Ambort et al., 2012). In model systems such as mammalian lung and intestine, ionocytes actively secrete bicarbonate into the boundary layer to sequester these ions, thereby facilitating mucin expansion (Quinton, 2010; Gustafsson et al., 2012; Birchenough et al., 2015). Seawater pH might be sufficiently high to sequester mucin-bound protons at the gills of marine filter-feeders, but calcium ions likely still pose a problem. Studies demonstrate that mucin expansion in humans is severely impaired at 4 mM calcium (Espinosa, 2002), whereas seawater calcium is ~10 mM. This proposed function in mucin expansion is especially compelling because the ionocytes present in the amphibian and mammalian mucociliary complexes appear to be homologous with those of teleost fish gills. This is well supported by the fact that ionocyte differentiation and specification in all three epithelia is controlled by orthologues of Foxi (Hsiao et al., 2007; Quigley et al., 2011; Montoro et al., 2018). Furthermore, much of the same ion transport machinery is also present. Most notably, orthologues of the apical anion channel CFTR (cystic fibrosis transmembrane regulator), which secretes bicarbonate for mucin secretion in tetrapod lungs and chloride for hydromineral balance in marine teleost gills (Edwards and Marshall, 2012; Gustafsson et al., 2012). Neither presence nor homology of ionocytes is confirmed in the invertebrate deuterostomes, but the elevated expression of Foxi and other ionocyte markers observed in the gills of amphioxus and acorn worms in Chapter 4 is encouraging. It is tempting to speculate that ancestral ionocyte function served to facilitate mucin expansion for filter-feeding at the origin of gills in the stem-deuterostome. These cells and their machinery could have then served as an exaptation for more derived forms of ion regulation associated with homeostasis of the internal milieu, such as maintaining acid-base balance, hydromineral balance and ammonia excretion. The role of ionocytes in the lungs of terrestrial vertebrates might therefore more closely resemble an ancestral function in early deuterostomes rather than a derived condition associated with air breathing.  97 It is also possible that this ancestral ionocyte function preceded the origin of gills and deuterostomes. Mucin secretion is proposed as a key innovation associated with multicellularity at the base of the metazoan tree, where tissue formation is hypothesized to rely on a protective mucus layer to prevent bacterial invasion (Arendt et al., 2015; Bakshani et al., 2018). If ionocytes are indeed critical for early mucin function, they may prove to be an ancient cell type that played a key role in the origin of metazoans (discussed further in Section 5.4).  5.4 Did filter-feeding limit gas exchange at gills?  Filter-feeding might have also played an important role in the evolution of gas exchange at gills. However, in this case it may have been a primary constraint limiting gas exchange rather than selecting for it. This notion is supported by my work and that of others with ammocoetes. Ammocoetes resemble the vertebrate ancestor in being worm-like burrowers with filter-feeding gills. However, they also possess many derived respiratory adaptations that were acquired along the vertebrate stem to enhance gas exchange at gills (pillar cells, velar pump, etc, reviewed in Sections 1.2.2 and 1.3.1). Despite these adaptations, I show that ammocoetes rely on their skin for a large portion of gas exchange, even at larger body sizes. This might be a consequence of their worm-like body plan, but comparisons with adult lampreys suggest morphological features associated with filter-feeding may reduce gill diffusive capacity. As described in Section 1.3, ammocoetes undergo a true metamorphosis when transitioning to the adult phase. They become active, free-swimming organisms that no longer filter-feed, but retain their worm-like body plan. General gill structure and surface area in adults equals that of similarly sized ammocoetes, but lamellar diffusion distance is less than half (Lewis and Potter, 1976, 1982). Furthermore, this thinner diffusion distance is correlated with the loss of two ammocoete-specific gill structures with hypothesized roles in feeding: ammocoete mitochondrion-rich cells (AMRCs) and an enlarged intercellular space in the lamellar epithelium. The ammocoete mitochondrion-rich cell (AMRC) is a putative ionocyte specific to ammocoetes (Youson and Freeman, 1976). Bartels et al. (2009) demonstrate these cells are distinct from ionocytes that regulate hydromineral balance, and suggest they play a role in filter-feeding because of their absence in adults. No specific role in feeding was proposed or  98 confirmed, but they do possess carbonic anhydrase and mucin granules (Conley and Mallatt, 1988; Bartels et al., 1998; Bartels and Potter, 2004). Future work may therefore reveal a role in mucin secretion and/or nutrient uptake as proposed above. In freshwater teleosts, ionocytes typically account for less than 10% of the gill epithelium (Wilson and Laurent, 2002). However, ionocyte number and size can increase dramatically to maintain sufficient rates of ion uptake when challenged with ion-poor water. Because ionocytes are often larger than most cells in the gill epithelium, their proliferation can increase the diffusion distance and reduce the capacity for gas exchange (Perry, 1998). Known as the osmorespiratory compromise (Randall et al., 1972; Nilsson, 1986), this phenomenon is demonstrated in rainbow trout (Oncorhynchus mykiss) following acclimation to softwater. In this example, ionocyte proliferation and expansion doubles gill diffusion distance, requiring trout to increase ventilation rate by ~35% to maintain routine oxygen uptake (Greco et al., 1995, 1996). AMRC’s account for an impressive 60-80% of epithelial cells in ammocoete gills (Mallatt and Ridgway, 1984; Bartels and Potter, 2004). Thus, AMRC presence is most likely a major contributor to the increased diffusion distance in ammocoetes, and may constrain the gill’s capacity for gas exchange. The second gill structure that likely limits the capacity for gas exchange in ammocoetes is a pronounced intercellular space between the outer epithelium and lamellar blood channel. This space is devoid of red blood cells and accounts for ~15% of the blood-water diffusion distance in ammocoetes of G. australis (Lewis and Potter, 1982). Proposed roles for this structure include (1) secondary circulation for trans-epithelial exchange processes (Mittal and Munshi, 1971), perhaps for nutrient uptake as suggested above, and (2) hydrostatic support (Hughes and Munshi, 1979), which might be necessary to maintain gill structure during feeding. However, as with AMRC’s, this structure is mostly implicated in feeding because of its absence in adults, and function awaits confirmation. In addition to AMRC’s and the intercellular space, the act of filter-feeding itself may further limit gas exchange at gills. Indeed, coating the epithelium in a thick mucus layer with trapped detritus could increase diffusion distance and hinder interlamellar water flow. Furthermore, this may coincide with increased respiratory demands, as filter-feeding ammocoetes continuously transport food to the gut for simultaneous digestion (Mallatt, 1981). Specific dynamic action can be high in fishes, estimated to reach 70% of maximum oxygen consumption rates in rainbow trout (Alsop and Wood, 1997). Gill diffusive capacity might  99 therefore be most compromised at a time when gas exchange demands are high. This might increase selection for cutaneous gas exchange, further constraining ammocoetes to their worm-like body plan. Innovations in feeding mode have long been implicated in the evolution of early vertebrate size and activity (Northcutt, 2005). However, their hypothesized roles have been to provide greater caloric intake necessary for increased growth and activity. Ammocoetes suggest that a shift away from filter-feeding might have also facilitated increased body size and activity by relaxing the constraints on gill diffusive capacity. Additional work is required to confirm the proposed feeding functions for ancestral ionocytes, AMRC’s and the ammocoete intercellular space, but existing support for these ideas is compelling. Future work may reveal that filter-feeding was a primary selective pressure directly shaping the evolution of vertebrate gill function in both ion regulation and gas exchange.  5.5 Future Directions   This thesis provides compelling data regarding the ancestral origins of vertebrate gill function in gas exchange and ion regulation. As evidenced by this chapter, many different and exciting lines of research related to this work remain to be explored -some of which further test the conclusions made here, while others pursue new questions spurred by these findings. Some of the more important and exciting of these next steps are highlighted below.  1) Confirm pre-vertebrate origin of gill ionocytes  The conclusion that ion regulation at gills has a pre-vertebrate origin is based on proxies for ionocyte presence and homology at the gills of carefully chosen representative species. Tissue measurements of gene expression and enzyme activity support the presence of ionocytes in the gills of cephalochordate and hemichordate representatives, and Foxi expression suggests these putative ionocytes are homologous with those in vertebrates. These data are very encouraging, but do not provide unequivocal support for ionocyte presence or homology –especially in hemichordates. Unequivocal support requires colocalization of these markers in single cells and a common embryonic origin. Currently, ionocytes have yet to be identified in  100 any invertebrate deuterostome, and ionocyte homology among vertebrates is only confirmed within osteichthyes.  A more robust indication of ionocyte presence and homology would provide more definitive conclusions regarding the ancestral origins of vertebrate ionocytes. This would address most remaining uncertainty surrounding the proposed pre-vertebrate origin for ion regulation at gills. Furthermore, if ionocyte presence and homology are confirmed in these representatives, other exciting hypotheses regarding ionocyte origins and the evolution of ion regulation can then be tested. The most exciting of these are listed below.  2) A deep metazoan origin for ionocytes? If ionocytes are indeed confirmed to be present in the last common ancestor of deuterostomes, future work should test the possibility of an even earlier origin. Section 5.2 speculates that vertebrate-like ionocytes might be an ancient metazoan cell type that played a key role in mucin secretion and the advent of multicellularity. This hypothesis could be tested with a similar comparative approach to that employed here, where ionocyte presence and homology are investigated in carefully chosen extant representatives from protostome, cnidarian and poriferan clades (see Figure 5.2 for metazoan cladogram). This avenue of research is especially exciting because epithelial ionocytes in multiple protostome clades have already been identified and characterized (reviewed by Fehsenfeld and Weihrauch, 2017; Hu and Tseng, 2017; Leone et al., 2017), but homology has yet to be tested. Results might reveal a single ancient origin for ionocytes in metazoans, or multiple more recent origins. Regardless of the outcome, these results will provide unparalleled insight into the origin(s) and evolution of ionocytes, and provide a foundation upon which hypotheses regarding the evolution of ion regulation in metazoans can be based.  3) Determine ancestral ionocyte function  Once a roadmap for ionocyte origins and interrelationships in metazoans is constructed as described above, the next logical and most exciting step will be to explore the evolution of ionocyte function. A comparative approach similar to that employed by this thesis could characterize ionocyte function in carefully chosen extant species believed to represent different stages in animal evolution. This approach could attempt to test multiple hypotheses, including if  101 ionocytes first functioned in mucin secretion and were then coopted for their more derived function in ion regulation of the internal milieu. Protostomes again make this research avenue even more exciting by providing additional and potentially parallel systems in which to study the evolution of ionocytes and ion regulation. Crustaceans and mollusks have independently evolved filter-feeding gill structures functionally analogous to those of deuterostomes. These protostome “gills” also house ionocytes that in some clades are implicated in the same general forms of ion regulation as vertebrates. For example, crustaceans have independently invaded freshwater and rely on ionocytes in their gills to regulate acid-base balance, hydromineral balance and ammonia excretion in much the same way as teleost fishes (Fehsenfeld and Weihrauch, 2017; Leone et al., 2017). Did ionocyte function in crustaceans and other protostome clades follow a similar evolutionary trajectory as those in deuterostomes and vertebrates? Did ionocytes in protostomes and deuterostomes arise independently? If so, how? Pursuing these types of comparative studies will ideally lead to a better understanding of how ionocytes and their different adaptive functions arose, and what role these events might have played in the evolution of multiple animal clades.   102 5.6 Figures             Figure 5.1 Thesis summary Deuterostome cladogram summarizing the origins of gill function in ion regulation and gas exchange as supported by results from this thesis. A novel stem-deuterostome origin for gill function in ion regulation (blue) is proposed near the inception of pharyngeal gill arches (pink) and their role in filter-feeding (black). A later vertebrate origin for gill function in gas exchange (orange) with increasing body size, dermal thickness and activity is supported by this thesis and consistent with current views. With the exception of the urochordate larvaceans, clades in grey lack extant members that represent the ancestral condition and were thus not investigated here.    103            Figure 5.2 Metazoans and ionocyte origins Cladogram of major metazoan taxa depicting presence of known ionocytes (green), a stem-deuterostome origin for vertebrate ionocytes (blue; supported by this study) and multiple hypothetical origins for ionocytes (grey). Ctenophores, xenacoelomorphs and placozoans omitted for clarity. Phylogenetic relationships after Laumer et al. (2019).  104 References  Aldridge, D.W., Payne, B.S., and Miller, A.C. (1995). Oxygen consumption, nitrogenous excretion, and filtration rates of Dreissena polymorpha at acclimation temperatures between 20 and 32 °C. Can. J. Fish. Aquat. Sci. 52, 1761–1767. Alsop, D., and Wood, C. (1997). The interactive effects of feeding and exercise on oxygen consumption, swimming performance and protein usage in juvenile rainbow trout (Oncorhynchus mykiss). J. Exp. Biol. 200, 2337. Ambort, D., Johansson, M.E.V., Gustafsson, J.K., Nilsson, H.E., Ermund, A., Johansson, B.R., Koeck, P.J.B., Hebert, H., and Hansson, G.C. (2012). Calcium and pH-dependent packing and release of the gel-forming MUC2 mucin. Proc. Natl. Acad. Sci. 109, 5645–5650. Arendt, D., Benito-Gutierrez, E., Brunet, T., and Marlow, H. (2015). Gastric pouches and the mucociliary sole: setting the stage for nervous system evolution. Philos. Trans. R. Soc. B Biol. Sci. 370, 20150286. Baday, S., Orabi, E.A., Wang, S., Lamoureux, G., and Bernèche, S. (2015). Mechanism of NH4+ recruitment and NH3 transport in Rh proteins. Structure 23, 1550–1557. Bakshani, C.R., Morales-Garcia, A.L., Althaus, M., Wilcox, M.D., Pearson, J.P., Bythell, J.C., and Burgess, J.G. (2018). Evolutionary conservation of the antimicrobial function of mucus: a first defence against infection. Npj Biofilms Microbiomes 4, 14. Balser, E.J., and Ruppert, E.E. (1990). Structure, ultrastructure, and function of the preoral heart-kidney in Saccoglossus kowalevskii (Hemichordata, Enteropneusta) including new data on the stomochord. Acta Zool. 71, 235–249. Barrington, E.J. (1965). The Biology of Hemichordata and Protochordata (Edinburgh: Oliver and Boyd). Bartels, H., and Potter, I.C. (2004). Cellular composition and ultrastructure of the gill epithelium of larval and adult lampreys: Implications for osmoregulation in fresh and seawater. J. Exp. Biol. 207, 3447–3462. Bartels, H., Potter, I.C., Pirlich, K., and Mallatt, J. (1998). Categorization of the mitochondria-rich cells in the gill epithelium of the freshwater phases in the life cycle of lampreys. Cell Tissue Res. 291, 337–349. Bartels, H., Schmiedl, A., Rosenbruch, J., and Potter, I.C. (2009). Exposure of the gill epithelial cells of larval lampreys to an ion-deficient environment: a stereological study. J. Electron Microsc. (Tokyo) 58, 253–260. Bertrand, S., and Escriva, H. (2011). Evolutionary crossroads in developmental biology: amphioxus. Development 138, 4819–4830.  105 Bettex-Galland, M., and Hughes, G.M. (1973). Contractile filamentous material in the pillar cells of fish gills. J. Cell Sci. 13, 359. Binyon, J. (1981). The effects of lowered salinity upon Branchiostoma lanceolatum from the English Channel. J. Mar. Biol. Assoc. U. K. 61, 685–689. Birchenough, G.M.H., Johansson, M.E., Gustafsson, J.K., Bergström, J.H., and Hansson, G.C. (2015). New developments in goblet cell mucus secretion and function. Mucosal Immunol. 8, 712–719. Birol, O., Ohyama, T., Edlund, R.K., Drakou, K., Georgiades, P., and Groves, A.K. (2016). The mouse Foxi3 transcription factor is necessary for the development of posterior placodes. Dev. Biol. 409, 139–151. Blair, S.D., Wilkie, M.P., and Edwards, S.L. (2017). Rh glycoprotein immunoreactivity in the skin and its role in extrabranchial ammonia excretion by the sea lamprey (Petromyzon marinus) in fresh water. Can. J. Zool. 95, 95–105. Blewett, T.A., and Goss, G.G. (2017). A novel pathway of nutrient absorption in crustaceans: branchial amino acid uptake in the green shore crab (Carcinus maenas). Proc. R. Soc. B Biol. Sci. 284, 20171298. Blomqvist, S.R., Vidarsson, H., Fitzgerald, S., Johansson, B.R., Ollerstam, A., Brown, R., Persson, A.E.G., Bergström, G., and Enerbäck, S. (2004). Distal renal tubular acidosis in mice that lack the forkhead transcription factor Foxi1. J. Clin. Invest. 113, 1560–1570. Blomqvist, S.R., Vidarsson, H., Söder, O., and Enerbäck, S. (2006). Epididymal expression of the forkhead transcription factor Foxi1 is required for male fertility. EMBO J. 25, 4131–4141. Boone, C.D., Pinard, M., McKenna, R., and Silverman, D. (2014). Catalytic mechanism of α-class carbonic anhydrases: CO2 hydration and proton transfer. In Carbonic Anhydrase: Mechanism, Regulation, Links to Disease, and Industrial Applications, S.C. Frost, and R. McKenna, eds. (Dordrecht: Springer Netherlands), pp. 31–52. Boot, M.J., Gittenberger-De Groot, A.C., Van Iperen, L., Hierck, B.P., and Poelmann, R.E. (2003). Spatiotemporally separated cardiac neural crest subpopulations that target the outflow tract septum and pharyngeal arch arteries. Anat. Rec. 275A, 1009–1018. Booth, J.H. (1978). The distribution of blood flow in the gills of fish: Application of a new technique to rainbow trout (Salmo Gairdneri). J. Exp. Biol. 73, 119. Boutilier, R.G., Heming, T.A., and Iwama, G.K. (1984). Physicochemical parameters for use in fish respiratory physiology. In Fish Physiology, W.S. Hoar, and D.J. Randall, eds. (Academic Press), pp. 403–430. Brauner, C.J., and Rombough, P.J. (2012). Ontogeny and paleophysiology of the gill: New insights from larval and air-breathing fish. Respir. Physiol. Neurobiol. 184, 293–300.  106 Brauner, C.J., Shartau, R.B., Damsgaard, C., Esbaugh, A.J., Wilson, R.W., and Grosell, M. (2019). 3 - Acid-base physiology and CO2 homeostasis: Regulation and compensation in response to elevated environmental CO2. In Fish Physiology, M. Grosell, P.L. Munday, A.P. Farrell, and C.J. Brauner, eds. (Academic Press), pp. 69–132. Brauner, C.J., and Wood, C.M. (2002). Ionoregulatory development and the effect of chronic silver exposure on growth, survival, and sublethal indicators of toxicity in early life stages of rainbow trout (Oncorhynchus mykiss). J. Comp. Physiol. B. 172, 153–162.  Brown, F.D., Prendergast, A., and Swalla, B.J. (2008). Man is but a worm: Chordate origins. Genesis 46, 605–613. Cameron, C.B. (2005). A phylogeny of the hemichordates based on morphological characters. Can. J. Zool. 83, 196–215. Cameron, J.N. (1989). The respiratory physiology of animals (New York: Oxford University Press). Cameron, J.N., and Heisler, N. (1983). Studies of ammonia in the rainbow trout: Physico-chemical parameters, acid-base behaviour and respiratory clearance. J. Exp. Biol. 105, 107. Cameron, C.B., Garey, J.R., and Swalla, B.J. (2000). Evolution of the chordate body plan: New insights from phylogenetic analyses of deuterostome phyla. Proc. Natl. Acad. Sci. 97, 4469–4474. Caron, J.-B., Morris, S.C., and Cameron, C.B. (2013). Tubicolous enteropneusts from the Cambrian period. Nature 495, 503–506. Cha, S.-W., McAdams, M., Kormish, J., Wylie, C., and Kofron, M. (2012). Foxi2 Is an animally localized maternal mRNA in xenopus, and an activator of the zygotic ectoderm activator Foxi1e. PLoS ONE 7, e41782. Choe, K.P., Edwards, S.L., Claiborne, J.B., and Evans, D.H. (2007). The putative mechanism of Na+ absorption in euryhaline elasmobranchs exists in the gills of a stenohaline marine elasmobranch, Squalus acanthias. Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 146, 155–162. Collard, M., Laitat, K., Moulin, L., Catarino, A.I., Grosjean, P., and Dubois, P. (2013). Buffer capacity of the coelomic fluid in echinoderms. Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 166, 199–206. Collard, M., De Ridder, C., David, B., Dehairs, F., and Dubois, P. (2015). Could the acid-base status of Antarctic sea urchins indicate a better-than-expected resilience to near-future ocean acidification? Glob. Change Biol. 21, 605–617. Conley, D.M., and Mallatt, J. (1988). Histochemical localization of Na+,K+-ATPase and carbonic anhydrase activity in gills of 17 fish species. Can. J. Zool. 66, 2398–2405.  107 Cruz, S.A., Chao, P.-L., and Hwang, P.-P. (2013). Cortisol promotes differentiation of epidermal ionocytes through Foxi3 transcription factors in zebrafish (Danio rerio). Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 164, 249–257. Cuoghi, I., Lazzaretti, C., Mandrioli, M., Mola, L., and Pederzoli, A. (2018). Immunohistochemical analysis of the distribution of molecules involved in ionic and pH regulation in the lancelet Branchiostoma floridae (Hubbs, 1922). Acta Histochem. 120, 33–40. Damas, H. (1944). Recherches sur le développement de Lampetra fluviatilis L.-contribution à l’étude de la cephalogénèse des vertébrès. Arch Biol Paris 55, 1–289. Dawson, H.A., Quintella, B.R., Almeida, P.R., Treble, A.J., and Jolley, J.C. (2015). The ecology of larval and metamorphosing lampreys. In Lampreys: Biology, Conservation and Control, M.F. Docker, ed. (Dordrecht: Springer Netherlands), pp. 75–137. Ditadi, A.S.F., Mendes, E.G., and Bianconcini, M.S.C. (1997). Influence of body mass and environmental oxygen tension on the oxygen consumption rates of an enteropneust, Glossobalanus crozieri. Braz. J. Med. Biol. Res. 30, 1441–1444. Docker, M.F., and Potter, I.C. (2019). Life history evolution in lampreys: Alternative migratory and feeding types. In Lampreys: Biology, Conservation and Control, M.F. Docker, ed. (Dordrecht: Springer Netherlands), pp. 287–409. Dubaissi, E., and Papalopulu, N. (2011). Embryonic frog epidermis: a model for the study of cell-cell interactions in the development of mucociliary disease. Dis. Model. Mech. 4, 179–192. Dymowska, A.K., Hwang, P.-P., and Goss, G.G. (2012). Structure and function of ionocytes in the freshwater fish gill. Respir. Physiol. Neurobiol. 184, 282–292. Dymowska, A.K., Schultz, A.G., Blair, S.D., Chamot, D., and Goss, G.G. (2014). Acid-sensing ion channels are involved in epithelial Na+ uptake in the rainbow trout Oncorhynchus mykiss. Am. J. Physiol.-Cell Physiol. 307, C255–C265. Edgar, R.C. (2004). MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797. Edlund, R.K., Birol, O., and Groves, A.K. (2015). Chapter Fourteen - The role of Foxi family transcription factors in the development of the ear and jaw. In Current Topics in Developmental Biology, P.A. Trainor, ed. (Academic Press), pp. 461–495. Edwards, S.L., and Marshall, W.S. (2012). 1 - Principles and patterns of osmoregulation and euryhalinity in fishes. In Euryhaline Fishes, S.D. McCormick, A.P. Farrell, and C.J. Brauner, eds. (Academic Press), pp. 1–44. Esaki, M., Hoshijima, K., Kobayashi, S., Fukuda, H., Kawakami, K., and Hirose, S. (2007). Visualization in zebrafish larvae of Na+ uptake in mitochondria-rich cells whose  108 differentiation is dependent on foxi3a. Am. J. Physiol.-Regul. Integr. Comp. Physiol. 292, R470–R480. Esbaugh, A.J. (2005). Cytoplasmic carbonic anhydrase isozymes in rainbow trout Oncorhynchus mykiss: Comparative physiology and molecular evolution. J. Exp. Biol. 208, 1951–1961. Espinosa, M. (2002). Acidic pH and increasing [Ca2+] reduce the swelling of mucins in primary cultures of human cervical cells. Hum. Reprod. 17, 1964–1972. Evans, D. (1984). Gill Na+/H+ and Cl-/HCO3- exchange systems evolved before the vertebrates entered fresh water. J. Exp. Biol. 113, 465. 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. Fehsenfeld, S., and Weihrauch, D. (2017). Acid–base regulation in aquatic decapod crustaceans. In Acid-Base Balance and Nitrogen Excretion in Invertebrates, D. Weihrauch, and M. O’Donnell, eds. (Cham: Springer International Publishing), pp. 151–191. Ferreira-Martins, D., Coimbra, J., Antunes, C., and Wilson, J.M. (2016). Effects of salinity on upstream-migrating, spawning sea lamprey, Petromyzon marinus. Conserv. Physiol. 4, cov064. Finn, R.N., and Kapoor, B.G. (2008). Fish larval physiology (CRC Press), 736 pp. Fritzenwanker, J.H., Gerhart, J., Freeman, R.M., and Lowe, C.J. (2014). The Fox/Forkhead transcription factor family of the hemichordate Saccoglossus kowalevskii. EvoDevo 5, 17. Fu, C., Wilson, J.M., Rombough, P.J., and Brauner, C.J. (2010). Ions first: Na+ uptake shifts from the skin to the gills before O2 uptake in developing rainbow trout, Oncorhynchus mykiss. Proc. R. Soc. B Biol. Sci. 277, 1553–1560. Gans, C., and Northcutt, R.G. (1983). Neural crest and the origin of vertebrates: A new head. Science 220, 268. Gee, H. (2008). The amphioxus unleashed. Nature 453, 999–1000. Gerhart, J., Lowe, C., and Kirschner, M. (2005). Hemichordates and the origin of chordates. Curr. Opin. Genet. Dev. 15, 461–467. Geyer, R.R., Parker, M.D., Toye, A.M., Boron, W.F., and Musa-Aziz, R. (2013). Relative CO2/NH3 permeabilities of human RhAG, RhBG and RhCG. J. Membr. Biol. 246, 915–926.  109 Giacomin, M., Schulte, P.M., and Wood, C.M. (2017). Differential effects of temperature on oxygen consumption and branchial fluxes of urea, ammonia, and water in the dogfish shark (Squalus acanthias suckleyi). Physiol. Biochem. Zool. 90, 627–637. Gillis, J.A., and Tidswell, O.R.A. (2017). The origin of vertebrate gills. Curr. Biol. 27, 729–732. Gillis, J.A., Fritzenwanker, J.H., and Lowe, C.J. (2012). A stem-deuterostome origin of the vertebrate pharyngeal transcriptional network. Proc. R. Soc. B Biol. Sci. 279, 237–246. Gilmour, K.M., and Perry, S.F. (2009). Carbonic anhydrase and acid-base regulation in fish. J. Exp. Biol. 212, 1647–1661. Glover, C.N., Bucking, C., and Wood, C.M. (2011). Adaptations to in situ feeding: Novel nutrient acquisition pathways in an ancient vertebrate. Proc. R. Soc. B Biol. Sci. 278, 3096–3101. Gomme, J. (2001). Transport of exogenous organic substances by invertebrate integuments: The field revisited. J. Exp. Zool. 289, 254–265. Gonzalez, P., and Cameron, C.B. (2009). The gill slits and pre-oral ciliary organ of Protoglossus (Hemichordata: Enteropneusta) are filter-feeding structures. Biol. J. Linn. Soc. 98, 898–906. González, M.E., Blánquez, M.J., and Rojo, C. (1996). Early gill development in the rainbow trout, Oncorhynchus mykiss. J. Morphol. 229, 201–217. Greco, A.M., Gilmour, K.M., Fenwick, J.C., and Perry, S.F. (1995). The effects of softwater acclimation on respiratory gas transfer in the rainbow trout Oncorhynchus mykiss. J. Exp. Biol. 198, 2557. Greco, A.M., Fenwick, J.C., and Perry, S.F. (1996). The effects of soft-water acclimation on gill structure in the rainbow trout Oncorhynchus mykiss. Cell Tissue Res. 285, 75–82. Green, S.A., and Bronner, M.E. (2014). The lamprey: A jawless vertebrate model system for examining origin of the neural crest and other vertebrate traits. Differentiation 87, 44–51. Green, S.A., Simoes-Costa, M., and Bronner, M.E. (2015). Evolution of vertebrates as viewed from the crest. Nature 520, 474–482. Gruswitz, F., Chaudhary, S., Ho, J.D., Schlessinger, A., Pezeshki, B., Ho, C.-M., Sali, A., Westhoff, C.M., and Stroud, R.M. (2010). Function of human Rh based on structure of RhCG at 2.1 A. Proc. Natl. Acad. Sci. 107, 9638–9643. Guffey, S.C., Fliegel, L., and Goss, G.G. (2015). Cloning and characterization of Na+/H+ Exchanger isoforms NHE2 and NHE3 from the gill of Pacific dogfish Squalus suckleyi. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 188, 46–53.  110 Gustafsson, J.K., Ermund, A., Ambort, D., Johansson, M.E.V., Nilsson, H.E., Thorell, K., Hebert, H., Sjövall, H., and Hansson, G.C. (2012). Bicarbonate and functional CFTR channel are required for proper mucin secretion and link cystic fibrosis with its mucus phenotype. J. Exp. Med. 209, 1263–1272. Halstead, L.B., and Lawson, J.D. (1985). The vertebrate invasion of fresh water. Philos. Trans. R. Soc. Lond. B Biol. Sci. 309, 243–258. Hiroi, J., and McCormick, S.D. (2012). New insights into gill ionocyte and ion transporter function in euryhaline and diadromous fish. Respir. Physiol. Neurobiol. 184, 257–268. Hiroi, J., Kaneko, T., Seikai, T., and Tanaka, M. (1998). Developmental sequence of chloride cells in the body skin and gills of Japanese flounder (Paralichthys olivaceus) larvae. Zoolog. Sci. 15, 455–460. Hochachka, P.W., and Somero, G.N. (2002). Biochemical adaptation: mechanism and process in physiological evolution (New York: Oxford University Press). Holeton, G.F. (1971). Respiratory and circulatory responses of rainbow trout larvae to carbon monoxide and to hypoxia. J. Exp. Biol. 55, 683–694. Holland, L.Z. (2016). Tunicates. Curr. Biol. 26, R146–R152. Holland, N.D., and Holland, L.Z. (2017). The ups and downs of amphioxus biology: A history. Int. J. Dev. Biol. 61, 575–583. Holland, N.D., Holland, L.Z., and Holland, P.W.H. (2015). Scenarios for the making of vertebrates. Nature 520, 450–455. Hsiao, C.-D., You, M.-S., Guh, Y.-J., Ma, M., Jiang, Y.-J., and Hwang, P.-P. (2007). A positive regulatory loop between foxi3a and foxi3b is essential for specification and differentiation of zebrafish epidermal ionocytes. PLoS ONE 2, e302. Hu, M., and Tseng, Y.-C. (2017). Acid–base regulation and ammonia excretion in Cephalopods: An ontogenetic overview. In Acid-Base Balance and Nitrogen Excretion in Invertebrates, D. Weihrauch, and M. O’Donnell, eds. (Cham: Springer International Publishing), pp. 275–298. Hughes, G.M., and Morgan, M. (1973). The structure of fish gills in relation to their respiratory function. Biol. Rev. 48, 419–475. Hughes, G.M., and Munshi, J.S.D. (1979). Fine structure of the gills of some Indian air-breathing fishes. J. Morphol. 160, 169–193. Hwang, P.P. (1990). Salinity effects on development of chloride cells in the larvae of ayu (Plecoglossus altivelis). Mar. Biol. 107, 1–7.  111 Hwang, P.P., and Lin, L.Y. (2013). Gill ionic transport, acid-base regulation, and nitrogen excretion. In The Physiology of Fishes, (CRC Press, Balkema), pp. 205–234. 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. Ivanis, G., Esbaugh, A.J., and Perry, S.F. (2008). Branchial expression and localization of SLC9A2 and SLC9A3 sodium/hydrogen exchangers and their possible role in acid-base regulation in freshwater rainbow trout (Oncorhynchus mykiss). J. Exp. Biol. 211, 2467–2477. Janvier, P. (2008). Early jawless vertebrates and cyclostome origins. Zoolog. Sci. 25, 1045–1056. Jiang, X., Rowitch, D.H., Soriano, P., McMahon, A.P., and Sucov, H.M. (2000). Fate of the mammalian cardiac neural crest. Dev. Camb. Engl. 127, 1607–1616. Jutfelt, F., Norin, T., Ern, R., Overgaard, J., Wang, T., McKenzie, D.J., Lefevre, S., Nilsson, G.E., Metcalfe, N.B., Hickey, A.J.R., et al. (2018). Oxygen- and capacity-limited thermal tolerance: Blurring ecology and physiology. J. Exp. Biol. 221, jeb169615. Katoh, F., Shimizu, A., Uchida, K., and Kaneko, T. (2000). Shift of chloride cell distribution during early life stages in seawater-adapted killifish, Fundulus heteroclitus. Zoolog. Sci. 17, 11–18. Kirby, M., Gale, T., and Stewart, D. (1983). Neural crest cells contribute to normal aorticopulmonary septation. Science 220, 1059–1061. Kumai, Y., and Perry, S.F. (2011). Ammonia excretion via Rhcg1 facilitates Na+ uptake in larval zebrafish, Danio rerio, in acidic water. Am. J. Physiol.-Regul. Integr. Comp. Physiol. 301, R1517–R1528. Kuratani, S.C., and Kirby, M.L. (1991). Initial migration and distribution of the cardiac neural crest in the avian embryo: An introduction to the concept of the circumpharyngeal crest. Am. J. Anat. 191, 215–227. Kurth, I., Hentschke, M., Hentschke, S., Borgmeyer, U., Gal, A., and Hübner, C.A. (2006). The forkhead transcription factor Foxi1 directly activates the AE4 promoter. Biochem. J. 393, 277–283. Kwan, G.T., Wexler, J.B., Wegner, N.C., and Tresguerres, M. (2019). Ontogenetic changes in cutaneous and branchial ionocytes and morphology in yellowfin tuna (Thunnus albacares) larvae. J. Comp. Physiol. B 189, 81–95. Laumer, C.E., Fernández, R., Lemer, S., Combosch, D., Kocot, K.M., Riesgo, A., Andrade, S.C.S., Sterrer, W., Sørensen, M.V., and Giribet, G. (2019). Revisiting metazoan phylogeny with genomic sampling of all phyla. Proc. R. Soc. B Biol. Sci. 286, 20190831.  112 Lee, D.J., Gutbrod, M., Ferreras, F.M., and Matthews, P.G.D. (2018). Changes in hemolymph total CO2 content during the water-to-air respiratory transition of amphibiotic dragonflies. J. Exp. Biol. 221, jeb181438. Leone, F.A., Lucena, M.N., Garçon, D.P., Pinto, M.R., and McNamara, J.C. (2017). Gill ion transport ATPases and ammonia excretion in aquatic crustaceans. In Acid-Base Balance and Nitrogen Excretion in Invertebrates, D. Weihrauch, and M. O’Donnell, eds. (Cham: Springer International Publishing), pp. 61–107. Lewis, S.V., and Potter, I.C. (1976). Gill morphometrics of the lampreys Lampetra fluviatilis (L.) and Lampetra planeri (Bloch). Acta Zool. 57, 103–112. Lewis, S.V., and Potter, I.C. (1982). A light and electron microscope study of the gills of larval lampreys (Geotria australis) with particular reference to the water-blood pathway. J. Zool. 198, 157–176. Li, J., Eygensteyn, J., Lock, R., Verbost, P., Heijden, A., Bonga, S., and Flik, G. (1995). Branchial chloride cells in larvae and juveniles of freshwater tilapia Oreochromis mossambicus. J. Exp. Biol. 198, 2177–2184. Li, M., Jiang, C., Zhang, Y., and Zhang, S. (2017). Activities of amphioxus GH-like protein in osmoregulation: Insight into origin of vertebrate GH family. Int. J. Endocrinol. 2017, 1–13. Liu, Y., Beyer, A., and Aebersold, R. (2016). On the dependency of cellular protein levels on mRNA abundance. Cell 165, 535–550. Lowe, C.J., Tagawa, K., Humphreys, T., Kirschner, M., and Gerhart, J. (2004). Hemichordate embryos: Procurement, culture, and basic methods. In Methods in Cell Biology, (Elsevier), pp. 171–194. Lowe, C.J., Clarke, D.N., Medeiros, D.M., Rokhsar, D.S., and Gerhart, J. (2015). The deuterostome context of chordate origins. Nature 520, 456–465. Mallatt, J. (1981). The suspension feeding mechanism of the larval lamprey Petromyzon marinus. J. Zool. 194, 103–142. Mallatt, J., and Ridgway, R.L. (1984). Ultrastructure of a complex epithelial system: The pharyngeal lining of the larval lamprey Petromyzon marinus. J. Morphol. 180, 271–296. Manzon, R.G., Youson, J.H., and Holmes, J.A. (2015). Lamprey metamorphosis. In Lampreys: Biology, Conservation and Control, M.F. Docker, ed. (Dordrecht: Springer Netherlands), pp. 139–214. Martik, M.L., Gandhi, S., Uy, B.R., Gillis, J.A., Green, S.A., Simoes-Costa, M., and Bronner, M.E. (2019). Evolution of the new head by gradual acquisition of neural crest regulatory circuits. Nature 574, 675–678.  113 McCormick, S.D. (1993). Methods for Nonlethal Gill Biopsy and Measurement of Na+, K+-ATPase Activity. Can. J. Fish. Aquat. Sci. 50, 656–658. McDonald, T.R., and Ward, J.M. (2016). Evolution of electrogenic ammonium transporters (AMTs). Front. Plant Sci. 7. Metro Vancouver (2018). Water quality control annual report. Water Serv. Rep. Resour. 1. Mittal, A.K., and Munshi, J.S.D. (1971). A comparative study of the structure of the skin of certain air-breathing fresh-water teleosts. J. Zool. 163, 515–532. Miyamoto, N., and Wada, H. (2018). Hemichordate nervous system. In Oxford Research Encyclopedia of Neuroscience, (Oxford University Press), p. Moller, P.C., and Ellis, R.A. (1974). Fine structure of the excretory system of Amphioxus (Branchiostoma floridae) and its response to osmotic stress. Cell Tissue Res. 148. Mongera, A., Singh, A.P., Levesque, M.P., Chen, Y.-Y., Konstantinidis, P., and Nusslein-Volhard, C. (2013). Genetic lineage labeling in zebrafish uncovers novel neural crest contributions to the head, including gill pillar cells. Development 140, 916–925. Montoro, D.T., Haber, A.L., Biton, M., Vinarsky, V., Lin, B., Birket, S.E., Yuan, F., Chen, S., Leung, H.M., Villoria, J., et al. (2018). A revised airway epithelial hierarchy includes CFTR-expressing ionocytes. Nature 560, 319–324. Morris, S.C., and Caron, J.-B. (2012). Pikaia gracilens Walcott, a stem-group chordate from the Middle Cambrian of British Columbia. Biol. Rev. 87, 480–512. Morris, S.C., and Caron, J.-B. (2014). A primitive fish from the Cambrian of North America. Nature 512, 419–422. Nanglu, K., Caron, J.-B., and Cameron, C.B. (2015). Using experimental decay of modern forms to reconstruct the early evolution and morphology of fossil enteropneusts. Paleobiology 41, 460–478. Newstead, J.D. (1967). Fine structure of the respiratory lamellae of teleostean gills. Z. Für Zellforsch. Mikrosk. Anat. 79, 396–428. Nikolic, L.M. (2016). Regulation of Na+/K+-ATPase activity in the nervous system. In Regulation of Membrane Na+/K+-ATPase, S. Chakraborti, and N.S. Dhalla, eds. (Cham: Springer International Publishing), pp. 295–309. Nilsson, S. (1986). Control of gill blood flow. In Fish Physiology: Recent advances, S. Nilsson, and S. Holmgren, eds. (Dordrecht: Springer Netherlands), pp. 86–101. Nomaksteinsky, M., Röttinger, E., Dufour, H.D., Chettouh, Z., Lowe, C.J., Martindale, M.Q., and Brunet, J.-F. (2009). Centralization of the deuterostome nervous system predates chordates. Curr. Biol. 19, 1264–1269.  114 Northcutt, G.R. (2005). The new head hypothesis revisited. J. Exp. Zoolog. B Mol. Dev. Evol. 304B, 274–297. Overdier, D.G., Ye, H., Peterson, R.S., Clevidence, D.E., and Costa, R.H. (1997). The winged helix transcriptional activator HFH-3 is expressed in the distal tubules of embryonic and adult mouse kidney. J. Biol. Chem. 272, 13725–13730. Pardos, F. (1988). Fine structure and function of pharynx cilia in Glossobalanus minutus Kowalewsky (Enteropneusta). Acta Zool. 69, 1–12. Pardos, F., and Benito, J. (1988). Blood vessels and related structures in the gill bars of Glossobalanus minutus (Enteropneusta). Acta Zool. 69, 87–94. Pedersen, S.F., and Counillon, L. (2019). The SLC9A-C mammalian Na+/H+ exchanger family: Molecules, mechanisms and physiology. Physiol. Rev. 99, 2015–2113. Pederzoli, A., Mandrioli, M., and Mola, L. (2014). Expression of carbonic anhydrase, cystic fibrosis transmembrane regulator (CFTR) and V-H+-ATPase in the lancelet Branchiostoma lanceolatum (Pallas, 1774). Acta Histochem. 116, 487–492. Perry, S.F. (1998). Relationships between branchial chloride cells and gas transfer in freshwater fish. Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 119, 9–16. Perry, S.F., and Wood, C.M. (1985). Kinetics of branchial calcium uptake in the rainbow trout: Effects of acclimation to various external calcium levels. J. Exp. Biol. 116, 411. Perry, S.F., Rivero-Lopez, L., McNeill, B., and Wilson, J. (2006). Fooling a freshwater fish: How dietary salt transforms the rainbow trout gill into a seawater gill phenotype. J. Exp. Biol. 209, 4591–4596. Piermarini, P.M., and Evans, D.H. (2001). Immunochemical analysis of the vacuolar proton-ATPase B-subunit in the gills of a euryhaline stingray (Dasyatis sabina): effects of salinity and relation to Na+/K+-ATPase. J. Exp. Biol. 204, 3251. Piermarini, P.M., Verlander, J.W., Royaux, I.E., and Evans, D.H. (2002). Pendrin immunoreactivity in the gill epithelium  of a euryhaline elasmobranch. Am. J. Physiol.-Regul. Integr. Comp. Physiol. 283, R983–R992. Pirkmajer, S., and Chibalin, A.V. (2016). Na+/K+-ATPase regulation in skeletal muscle. Am. J. Physiol.-Endocrinol. Metab. 311, E1–E31. Pisam, M., Massa, F., Jammet, C., and Prunet, P. (2000). Chronology of the appearance of β, A, and α mitochondria-rich cells in the gill epithelium during ontogenesis of the brown trout (Salmo trutta). Anat. Rec. 259, 301–311. Plasschaert, L.W., Žilionis, R., Choo-Wing, R., Savova, V., Knehr, J., Roma, G., Klein, A.M., and Jaffe, A.B. (2018). A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte. Nature 560, 377–381.  115 Pörtner, H.-O. (2010). Oxygen- and capacity-limitation of thermal tolerance: a matrix for integrating climate-related stressor effects in marine ecosystems. J. Exp. Biol. 213, 881–893. Potter, I.C., Welsch, U., Wright, G.M., Honma, Y., and Chiba, A. (1995). Light and electron microscope studies of the dermal capillaries in three species of hagfishes and three species of lampreys. J. Zool. 235, 677–688. Potter, I.C., Gill, H.S., Renaud, C.B., and Haoucher, D. (2015). The taxonomy, phylogeny, and distribution of lampreys. In Lampreys: Biology, Conservation and Control, M.F. Docker, ed. (Dordrecht: Springer Netherlands), pp. 35–73. Preest, M.R., Gonzalez, R.J., and Wilson, R.W. (2005). A pharmacological examination of Na+ and Cl− transport in two species of freshwater fish. Physiol. Biochem. Zool. 78, 259–272. Quigley, I.K., Stubbs, J.L., and Kintner, C. (2011). Specification of ion transport cells in the Xenopus larval skin. Development 138, 705–714. Quinton, P.M. (2008). Cystic fibrosis: Impaired bicarbonate secretion and mucoviscidosis. The Lancet 372, 415–417. Quinton, P.M. (2010). Role of epithelial HCO3- transport in mucin secretion: Lessons from cystic fibrosis. 299, 12. Randall, D.J., and Daxboeck, C. (1982). Cardiovascular changes in the rainbow trout (Salmo gairdneri Richardson) during exercise. Can. J. Zool. 60, 1135–1140. Randall, D.J., Baumgarten, D., and Malyusz, M. (1972). The relationship between gas and ion transfer across the gills of fishes. Comp. Biochem. Physiol. A Physiol. 41, 629–637. Rangroo Thrane, V., Thrane, A.S., Wang, F., Cotrina, M.L., Smith, N.A., Chen, M., Xu, Q., Kang, N., Fujita, T., Nagelhus, E.A., et al. (2013). Ammonia triggers neuronal disinhibition and seizures by impairing astrocyte potassium buffering. Nat. Med. 19, 1643–1648. Reid, S.D. (2003). Localization and characterization of phenamil-sensitive Na+ influx in isolated rainbow trout gill epithelial cells. J. Exp. Biol. 206, 551–559. Reilly, B.D., Cramp, R.L., Wilson, J.M., Campbell, H.A., and Franklin, C.E. (2011). Branchial osmoregulation in the euryhaline bull shark, Carcharhinus leucas: a molecular analysis of ion transporters. J. Exp. Biol. 214, 2883. Reithmeier, R.A.F., Casey, J.R., Kalli, A.C., Sansom, M.S.P., Alguel, Y., and Iwata, S. (2016). Band 3, the human red cell chloride/bicarbonate anion exchanger (AE1, SLC4A1), in a structural context. Biochim. Biophys. Acta BBA - Biomembr. 1858, 1507–1532. Richards, J.G. (2011). Physiological, behavioral and biochemical adaptations of intertidal fishes to hypoxia. J. Exp. Biol. 214, 191–199.  116 Richards, J.G., Semple, J.W., Bystriansky, J.S., and Schulte, P.M. (2003). Na+/K+-ATPase isoform switching in gills of rainbow trout (Oncorhynchus mykiss) during salinity transfer. J. Exp. Biol. 206, 4475–4486. Roa, J.N., and Tresguerres, M. (2017). Bicarbonate-sensing soluble adenylyl cyclase is present in the cell cytoplasm and nucleus of multiple shark tissues. Physiol. Rep. 5, e13090. Rombough, P. (2002). Gills are needed for ionoregulation before they are needed for O2 uptake in developing zebrafish, Danio rerio. J. Exp. Biol. 205, 1787–1794. Rombough, P. (2007). The functional ontogeny of the teleost gill: Which comes first, gas or ion exchange? Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 148, 732–742. Rombough, P.J. (1999). The gill of fish larvae: Is it primarily a respiratory or an ionoregulatory structure? J. Fish Biol. 55, 186–204. Rottinger, E., and Lowe, C.J. (2012). Evolutionary crossroads in developmental biology: Hemichordates. Development 139, 2463–2475. Rudman, S.M., Goos, J.M., Burant, J.B., Brix, K.V., Gibbons, T.C., Brauner, C.J., and Jeyasingh, P.D. (2019). Ionome and elemental transport kinetics shaped by parallel evolution in threespine stickleback. Ecol. Lett. ele.13225. Sackville, M., Wilson, J.M., Farrell, A.P., and Brauner, C.J. (2012). Water balance trumps ion balance for early marine survival of juvenile pink salmon (Oncorhynchus gorbuscha). J. Comp. Physiol. B 182, 781–792. Sackville, M.A., Shartau, R.B., Damsgaard, C., Hvas, M., Phuong, L.M., Wang, T., Bayley, M., Thanh Huong, D.T., Phuong, N.T., and Brauner, C.J. (2018). Water pH limits extracellular but not intracellular pH compensation in the CO2-tolerant freshwater fish Pangasianodon hypophthalmus. J. Exp. Biol. 221, jeb190413. Saunders, R.L. (1962). The irrigation of the gills in fishes: II. Efficiency of oxygen uptake in relation to respiratory flow activity and concentrations of oxygen and carbon dioxide. Can. J. Zool. 40, 817–862. Schmitz, A., Gemmel, M., and Perry, S.F. (2000). Morphometric partitioning of respiratory surfaces in amphioxus (Branchiostoma lanceolatum Pallas). J. Exp. Biol. 203, 3381. Schulte, P.M. (2015). The effects of temperature on aerobic metabolism: Towards a mechanistic understanding of the responses of ectotherms to a changing environment. J. Exp. Biol. 218, 1856–1866. Shartau, R.B., Baker, D.W., Crossley, D.A., and Brauner, C.J. (2016). Preferential intracellular pH regulation: Hypotheses and perspectives. J. Exp. Biol. 219, 2235–2244.  117 Shih, T.-H., Horng, J.-L., Liu, S.-T., Hwang, P.-P., and Lin, L.-Y. (2011). Rhcg1 and NHE3b are involved in ammonium-dependent sodium uptake by zebrafish larvae acclimated to low-sodium water. Am. J. Physiol.-Regul. Integr. Comp. Physiol. 302, R84–R93. Shimeld, S.M., and Donoghue, P.C.J. (2012). Evolutionary crossroads in developmental biology: Cyclostomes (lamprey and hagfish). Development 139, 2091–2099. Shimeld, S.M., Degnan, B., and Luke, G.N. (2010). Evolutionary genomics of the Fox genes: Origin of gene families and the ancestry of gene clusters. Genomics 95, 256–260. Shu, D.-G., Luo, H.-L., Conway Morris, S., Zhang, X.-L., Hu, S.-X., Chen, L., Han, J., Zhu, M., Li, Y., and Chen, L.-Z. (1999). Lower Cambrian vertebrates from south China. Nature 402, 42–46. Sigrist, C.J.A., De Castro, E., Langendijk-Genevaux, P.S., Le Saux, V., Bairoch, A., and Hulo, N. (2005). ProRule: A new database containing functional and structural information on PROSITE profiles. Bioinformatics 21, 4060–4066. Sigrist, C.J.A., de Castro, E., Cerutti, L., Cuche, B.A., Hulo, N., Bridge, A., Bougueleret, L., and Xenarios, I. (2012). New and continuing developments at PROSITE. Nucleic Acids Res. 41, D344–D347. Simakov, O., Kawashima, T., Marlétaz, F., Jenkins, J., Koyanagi, R., Mitros, T., Hisata, K., Bredeson, J., Shoguchi, E., Gyoja, F., et al. (2015). Hemichordate genomes and deuterostome origins. Nature 527, 459–465. Singh, S., Jangid, R.K., Crowder, A., and Groves, A.K. (2018). Foxi3 transcription factor activity is mediated by a C-terminal transactivation domain and regulated by Protein Phosphatase 2A (PP2A) complex. Sci. Rep. 8, 17249. Smith, A.B. (2005). The pre-radial history of echinoderms. Geol. J. 40, 255–280. Smith, A.B. (2008). Deuterostomes in a twist: The origins of a radical new body plan. Evol. Dev. 10, 493–503. Smith, J.J., Kuraku, S., Holt, C., Sauka-Spengler, T., Jiang, N., Campbell, M.S., Yandell, M.D., Manousaki, T., Meyer, A., Bloom, O.E., et al. (2013). Sequencing of the sea lamprey (Petromyzon marinus) genome provides insights into vertebrate evolution. Nat. Genet. 45, 415–421. Solomon, K.S., Logsdon, J.M., and Fritz, A. (2003). Expression and phylogenetic analyses of three zebrafish FoxI class genes. Dev. Dyn. 228, 301–307. Speers-Roesch, B., Norin, T., and Driedzic, W.R. (2018). The benefit of being still: Energy savings during winter dormancy in fish come from inactivity and the cold, not from metabolic rate depression. Proc. R. Soc. B Biol. Sci. 285, 20181593.  118 Stecher, G., Tamura, K., and Kumar, S. (2020). Molecular Evolutionary Genetics Analysis (MEGA) for macOS. Mol. Biol. Evol. 37, 1237–1239. Stenslokken, K.O., Sundin, L., and Nilsson, G.E. (1999). Cardiovascular and gill microcirculatory effects of endothelin-1 in Atlantic cod: evidence for pillar cell contraction. J. Exp. Biol. 202, 1151. Stockard, C.R. (1906). The development of the mouth and gills in Bdellostoma stouti. Am. J. Anat. 5, 481–517. Stumpp, M., and Hu, M.Y. (2017). pH regulation and excretion in echinoderms. In Acid-Base Balance and Nitrogen Excretion in Invertebrates, D. Weihrauch, and M. O’Donnell, eds. (Cham: Springer International Publishing), pp. 261–273. Stumpp, M., Trübenbach, K., Brennecke, D., Hu, M.Y., and Melzner, F. (2012). Resource allocation and extracellular acid–base status in the sea urchin Strongylocentrotus droebachiensis in response to CO2 induced seawater acidification. Aquat. Toxicol. 110–111, 194–207. Sumrall, C.D., and Wray, G.A. (2007). Ontogeny in the fossil record: Diversification of body plans and the evolution of “aberrant” symmetry in Paleozoic echinoderms. Paleobiology 33, 149–163. Sundin, L., and Nilsson, G.E. (1998). Endothelin redistributes blood flow through the lamellae of rainbow trout gills. J. Comp. Physiol. B 168, 619–623. Swalla, B.J., and Smith, A.B. (2008). Deciphering deuterostome phylogeny: Molecular, morphological and palaeontological perspectives. Philos. Trans. R. Soc. B Biol. Sci. 363, 1557–1568. Tang, W., Martik, M.L., Li, Y., and Bronner, M.E. (2019). Cardiac neural crest contributes to cardiomyocytes in amniotes and heart regeneration in zebrafish. ELife 8, e47929. Tassia, M.G., Cannon, J.T., Konikoff, C.E., Shenkar, N., Halanych, K.M., and Swalla, B.J. (2016). The global diversity of hemichordata. PLOS ONE 11, e0162564. Tresguerres, M., Katoh, F., Fenton, H., Jasinska, E., and Goss, G.G. (2005). Regulation of branchial V-H+-ATPase, Na+/K+-ATPase and NHE2 in response to acid and base infusions in the Pacific spiny dogfish (Squalus acanthias). J. Exp. Biol. 208, 345–354. Tweedell, K.S. (1961). Regeneration of the enteropneust, Saccoglossus kowalevskii. Biol. Bull. 120, 118–127. Varsamos, S., Diaz, J.P., Charmantier, G., Flik, G., Blasco, C., and Connes, R. (2002). Branchial chloride cells in sea bass (Dicentrarchus labrax) adapted to fresh water, seawater, and doubly concentrated seawater. J. Exp. Zool. 293, 12–26.  119 Varsamos, S., Nebel, C., and Charmantier, G. (2005). Ontogeny of osmoregulation in postembryonic fish: A review. Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 141, 401–429. van der Meer, D.L.M., van den Thillart, E., Witte, F., de Bakker, M., Besser, J., Richardson, M.K., Spaink, H.P., Leito, J., and Bagowski, C.P. (2005). Gene expression profiling of the long-term adaptive response to hypoxia in the gills of adult zebrafish. Am. J. Regul. Integr. Comp. Physiol. 289, R1512–R1519.   Verdouw, H., Van Echteld, C.J.A., and Dekkers, E.M.J. (1978). Ammonia determination based on indophenol formation with sodium salicylate. Water Res. 12, 399–402. Vidarsson, H., Westergren, R., Heglind, M., Blomqvist, S.R., Breton, S., and Enerbäck, S. (2009). The forkhead transcription factor Foxi1 is a master regulator of vacuolar H-ATPase proton pump subunits in the inner ear, kidney and epididymis. PloS One 4, e4471–e4471. Vo, M., Mehrabian, S., Étienne, S., Pelletier, D., and Cameron, C.B. (2019). The hemichordate pharynx and gill pores impose functional constraints at small and large body sizes. Biol. J. Linn. Soc. 127, 75–87. Ward, N., and Moreno-Hagelsieb, G. (2014). Quickly finding orthologs as reciprocal best hits with blat, last, and ublast: How much do we miss? PLoS ONE 9, e101850. Warga, R., and Nüsslein-Volhard, C. (1999). Origin and development of the zebrafish endoderm. Dev. Camb. Engl. 126, 827–838. Webb, J.E., and Hill, M.B. (1958). The ecology of Lagos Lagoon. IV. On the reactions of Branchiostoma nigeriense Webb to its environment. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 241, 355–391. Wells, P., and Pinder, A. (1996a). The respiratory development of Atlantic salmon. I. Morphometry of gills, yolk sac and body surface. J. Exp. Biol. 199, 2725. Wells, P., and Pinder, A. (1996b). The respiratory development of Atlantic salmon. II. Partitioning of oxygen uptake among gills, yolk sac and body surfaces. J. Exp. Biol. 199, 2737. Wilkie, M.P., Bradshaw, P.G., Joanis, V., Claude, J.F., and Swindell, S.L. (2001). Rapid metabolic recovery following vigorous exercise in burrow‐dwelling larval sea lampreys (Petromyzon marinus). Physiol. Biochem. Zool. 74, 261–272. Williams, C.M., Buckley, L.B., Sheldon, K.S., Vickers, M., Pörtner, H.-O., Dowd, W.W., Gunderson, A.R., Marshall, K.E., and Stillman, J.H. (2016). Biological impacts of thermal extremes: Mechanisms and costs of functional responses matter. Integr. Comp. Biol. 56, 73–84.  120 Wilson, J.M., and Laurent, P. (2002). Fish gill morphology: Inside out. J. Exp. Zool. 293, 192–213. Wilson, J.M., Laurent, P., Tufts, B.L., Benos, D.J., Donowitz, M., Vogl, A.W., and Randall, D.J. (2000). NaCl uptake by the branchial epithelium in freshwater teleost fish: An immunological approach to ion-transport protein localization. J. Exp. Biol. 203, 2279–2296. Wright, P.A., and Wood, C.M. (2012). Seven things fish know about ammonia and we don’t. Respir. Physiol. Neurobiol. 184, 231–240. Wright, P.A., and Wood, C.M. (2015). 5 - Regulation of ions, acid–base, and nitrogenous wastes in elasmobranchs. In Physiology of Elasmobranch Fishes: Internal Processes, R.E. Shadwick, A.P. Farrell, and C.J. Brauner, eds. (Academic Press), pp. 279–345. Xian-guang, H., Aldridge, R.J., Siveter, D.J., Siveter, D.J., and Xiang-hong, F. (2002). New evidence on the anatomy and phylogeny of the earliest vertebrates. Proc. R. Soc. Lond. B Biol. Sci. 269, 1865–1869. Yang, T., Vidarsson, H., Rodrigo-Blomqvist, S., Rosengren, S.S., Enerbäck, S., and Smith, R.J.H. (2007). Transcriptional control of SLC26A4 is involved in Pendred syndrome and nonsyndromic enlargement of vestibular aqueduct (DFNB4). Am. J. Hum. Genet. 80, 1055–1063. Youson, J.H., and Freeman, P.A. (1976). Morphology of the gills of larval and parasitic adult sea lamprey, Petromyzon marinus L. J. Morphol. 149, 73–103. Yu, J.-K., Mazet, F., Chen, Y.-T., Huang, S.-W., Jung, K.-C., and Shimeld, S.M. (2008). The Fox genes of Branchiostoma floridae. Dev. Genes Evol. 218, 629–638. Yu, Z., Qi, Z., Hu, C., Liu, W., and Huang, H. (2012). Effects of salinity on ingestion, oxygen consumption and ammonium excretion rates of the sea cucumber Holothuria leucospilota. Aquac. Res. 44, 1760–1767.  Zimmer, A.M., and Wood, C.M. (2015). Ammonia first? The transition from cutaneous to branchial ammonia excretion in developing rainbow trout is not altered by exposure to chronically high NaCl. J. Exp. Biol. 218, 1467–1470. Zimmer, A.M., Wright, P.A., and Wood, C.M. (2014). What is the primary function of the early teleost gill? Evidence for Na+/NH+4 exchange in developing rainbow trout (Oncorhynchus mykiss). Proc. R. Soc. B Biol. Sci. 281, 20141422. Zimmer, A.M., Brix, K.V., and Wood, C.M. (2019). Mechanisms of Ca2+ uptake in freshwater and seawater-acclimated killifish, Fundulus heteroclitus, and their response to acute salinity transfer. J. Comp. Physiol. B 189, 47–60.    121 Appendices  Appendix A  -   Chapter 2 supplemental material           Figure A.1 Epidermal thickness of Entosphenus tridentatus Wet mass (g) vs epidermal thickness (µm) of Entosphenus tridentatus. Data fitted with an allometric curve (y = 45.2x0.034, R2 = 0.92).   0.0 0.5 1.0 1.5 2.0020406080mass (g)epidermal thickness (µm)y = 45.2x0.34R2 = 0.92 122           Figure A.2 AQ, Q10, RER and ventilation rate in Entosphenus tridentatus Ammonia quotients (A), temperature effects (B), respiratory exchange ratios (C) and ventilation rates in Entosphenus tridentatus. Data collected from divided chamber trials in normoxia at 10oC (shaded circles), 26oC (open circles) and hypoxia at 20oC (open squares). Data presented as means ± sem, n=10, except for temperature effects (B) which are estimated from mean ṀO2 values between different groups of ammocoetes at 10oC and 26oC.  0.0 0.5 1.0 1.5 2.00.000.020.040.060.080.10body mass (g)ammonia quotient (mol NH3/4+⋅ mol O2-1 )0.0 0.5 1.0 1.5 2.00.00.20.40.60.81.0body mass (g)respiratory exchange ratio (RER)0.0 0.5 1.0 1.5 2.00255075100125150body mass (g)ventilation (beats ⋅ min-1)0.0 0.5 1.0 1.5 2.0012345body mass (g)temperature effect (Q10)A BC D 123 Appendix B  -   Chapter 3 supplemental material   Figure B.1 Gas exchange in whole and fragmented Protoglossus graveolens Rates of oxygen uptake (A) and ammonia excretion (B). Black, grey and white circles represent whole animals, anterior fragments and posterior fragments, respectively. Measurements taken in normoxia at 10 and 20oC. Data presented as means±sem, n=8. Letters indicate significant differences within treatments (P<0.05, one-way ANOVA with Tukey’s post-hoc test).  10oC 20oC012345μmols O2⋅ g ⋅ hr-1aa,bb10oC 20oC0.000.250.500.751.00μmols NH 3/4+ ⋅ g ⋅ hr-1AB 124 Appendix C  -   Chapter 4 supplemental material            Figure C.1 Skin and gill NKA activity in S. kowalevskii, B. floridae and E. tridentatus Na+,K+-ATPase (NKA) activity of skin (white) and gill (blue) in S. kowalevskii, B. floridae and E. tridentatus. Data presented as means ± sem, n=10. Asterisks indicate significant differences between tissues (P<0.01, unpaired t-test).  S. kowalevskii B. floridae E. tridentatus0.00.51.01.52.0µmols ADP ⋅ mg-1 ⋅ h-1skingill** 125     Figure C.2 All tissue ATPase activity in S. kowalevskii, B. floridae and E. tridentatus Na+/K+-ATPase (NKA; blue; A, C, E) and H+-ATPase (VHA; red; B, D) activity in gill, skin, muscle and intestine of S. kowalevskii (A, B), B. floridae (C, D) and E. tridentatus (E). Data presented as means ± sem, n=10. Letters indicate significant differences between tissues (P<0.05, one-way ANOVA with Tukey’s post-hoc test).  gill skin muscle intestine0.00.51.01.52.0acbbµmols ADP ⋅ mg-1 ⋅ h-1gill skin muscle intestine0.00.51.01.52.0acbaµmols ADP ⋅ mg-1 ⋅ h-1gill skin muscle intestine0.00.51.01.52.0acbbµmols ADP ⋅ mg-1 ⋅ h-1gill skin muscle intestine0.00.51.01.52.0aa,bb bµmols ADP ⋅ mg-1 ⋅ h-1gill skin muscle intestine0.00.51.01.52.0µmols ADP ⋅ mg-1 ⋅ h-1A BCEDNKANKANKAVHAVHA 126     Figure C.3 All tissue gene expression in S. kowalevskii Gene expression in S. kowalevskii gill, skin, muscle and intestine for anion exchanger (AE; A), sodium-proton exchanger (NHE; B), carbonic anhydrase (CA; C), rhesus glycoprotein (Rh; D) and forkhead box protein I (Foxi; E). Data presented as means ± sem, n=8-10. Letters indicate significant differences between tissues (P<0.05, one-way ANOVA with Tukey’s post-hoc test).  gill skin muscle intestine110100relative expression (18S-1)Rha a,bba,bgill skin muscle intestine110100relative expression (18S-1)AEaaaabgill skin muscle intestine110100relative expression (18S-1)Foxiabc cgill skin muscle intestine110100relative expression (18S-1)CAgill skin muscle intestine110100relative expression (18S-1)NHEa abaA BCEDS. kowalevskii 127     Figure C.4 All tissue gene expression in B. floridae Gene expression in B. floridae gill, skin, muscle and intestine for anion exchanger (AE; A), sodium-proton exchanger (NHE; B), carbonic anhydrase (CA; C), rhesus glycoprotein (Rh; D) and forkhead box protein I (Foxi; E). Data presented as means ± sem, n=10. Letters indicate significant differences between tissues (P<0.05, one-way ANOVA with Tukey’s post-hoc test).  gill skin intestine muscle110100relative expression (18S-1)CAabbbgill skin intestine muscle110100relative expression (18S-1)AEabccgill skin intestine muscle110100relative expression (18S-1)abc cFoxigill skin intestine muscle110100relative expression (18S-1)NHEabb,ccgill skin intestine muscle110100relative expression (18S-1)Rhacba,bA BCEDB. floridae 128                      protein, gene D. rerio B. floridae S. kowalevskii Carbonic anhydrase II, Ca2 NP_954685 XP_002594302.1 XP_006814117.1 Anion exchanger 1b, Slc4a1b NP_001161738.1 XP_002605315.1 XP_002736672.2 Rhesus glycoprotein C, Rhcg1 AAM90586.1 XP_002597118.1 XP_006824549.1 Na+/H+ exchanger, Slc9a3.2 XP_009292331.1 XP_002597646.1 XP_002741674.2 Forkhead box protein i3a, Foxi3a NP_944599.2 XP_002608285.1 XP_002734694.1  Table C.1 Target genes and NCBI accession numbers    129     gene primer sequences bfAE-like F: 5’- CTA CTA CGA AGA AAG CAT AG -3’ R: 5’- GCA TTC TTC TCG AGT TAT T -3’ skAE-like F: 5’- TCT CTG GTG TTC ATT CTG TG -3’ R: 5’- TCT TTG CCA CAT CAT CAT CA -3’ bfRh-like F: 5’- GTA CTG GCC ATC CTT TAA CA -3’ R: 5’- CTA GAG ACA GGT ACG TGT TG -3’ skRh-like F: 5’- GGG AAA CTC ACA TTG GTA CT -3’ R: 5’- GTT CCA AGT TGT TCT GTT CG -3’ bfNHE-like F: 5’- CGA GAA GAA GGC CAT GAT TA -3’ R: 5’- TAC CTG TCT CTC CAG TAG TG -3’ skNHE-like F: 5’- TAC CAA CTA CGG ACT CTG AT -3’ R: 5’- GCA ACA GGG TAT CTT TCG TA -3’ bfFoxi-like F: 5’- TAG CAT GGC GAT GTA TT -3’ R: 5’- TAT TCA GGT ATG GAT TTG TG -3’ skFoxi-like F: 5’- CTC CAA GCT ATC TAA GTA ATC-3’ R: 5’- GGA ACG TAC CCA GTA TTA -3’ bf18S F: 5’- TTC TCC TTG GTG CTC TTA AC -3’ R: 5’- ACC GAG GTC CTA TTC CAT TA -3’ sk18S F: 5’- AGT CCA GCA TGG AAT AA -3’ R: 5’- ACG ATC CAA GAA TTT CAC -3’  Table C.2 Primer sequences for RT-qPCR  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.24.1-0394140/manifest

Comment

Related Items