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Mechanisms of ion regulation in the euryhaline killifish Fundulus heteroclitus : molecular and evolutionary… Scott, Graham R. 2004

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M E C H A N I S M S OF ION R E G U L A T I O N IN T H E E U R Y H A L I N E K I L L I F I S H FUND UL US HETEROCLITUS: M O L E C U L A R AND E V O L U T I O N A R Y PERSPECTIVES  By Graham R. Scott B.Sc, McMaster University, 2002 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept this, thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA August 2004  © Graham R. Scott, 2004  JUBCL THE  UNIVERSITY OF BRITISH C O L U M B I A  FACULTY OF G R A D U A T E STUDIES  Library Authorization  In p r e s e n t i n g this t h e s i s in partial fulfillment o f t h e r e q u i r e m e n t s for a n a d v a n c e d d e g r e e at t h e U n i v e r s i t y of British C o l u m b i a , I a g r e e that t h e L i b r a r y s h a l l m a k e it freely a v a i l a b l e for r e f e r e n c e a n d study. I further a g r e e that p e r m i s s i o n for e x t e n s i v e c o p y i n g of this t h e s i s for s c h o l a r l y p u r p o s e s m a y b e g r a n t e d b y t h e h e a d of m y d e p a r t m e n t o r b y his o r h e r r e p r e s e n t a t i v e s . It is u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n of this t h e s i s for f i n a n c i a l g a i n s h a l l not b e a l l o w e d without m y written p e r m i s s i o n .  G CO, i\ N a m e of A u t h o r (please print)  Title o f T h e s i s :  D a t e (dd/mm/yyyy)  t <, ^  YI&C-UA^  6$  -rkt  Id^y  35-  Degree:  Year:  200  ^  34-  D e p a r t m e n t of  T h e U n i v e r s i t y of British C o l u m b i a Vancouver, B C  S  Canada  na  grad.ubc.ca/forms/?formlD=THS  —'  page 1 of 1  last updated: 20-Jul-04  ABSTRACT  A comprehensive approach was employed to study various aspects of ionoregulatory physiology in the euryhaline killifish Fundulus heteroclitus. I first characterized the changes in gene expression in the gills of individuals from a northern population of killifish after abrupt transfer from near-isosmotic brackish water to either freshwater or seawater. Many changes in response to seawater transfer were transient: increased mRNA expression occurred 1 day after transfer for Na ,K -ATPase ctia (3+  +  fold), Na ,K ,2Cr-cotransporter 1 (NKCC1) (3-fold), and glucocorticoid receptor (1.3+  +  fold). In contrast, expression of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl" channel generally remained elevated (2-fold) in seawater. The responses to freshwater transfer were quite different than to seawater transfer. In particular, freshwater transfer caused a greater increase in Na ,K -ATPase a\ mRNA expression, but had no +  +  a  effect on V-type Ff-ATPase expression. I also examined the ionoregulatory physiology of both northern and southern killifish populations after freshwater transfer to understand possible mechanisms of freshwater adaptation. Pronounced differences in freshwater tolerance existed between northern (2% mortality) and southern (19% mortality) killifish populations. Differences in Na regulation between each population were small and likely cannot account for this +  large difference in mortality, as plasma Na , Na ,K -ATPase mRNA expression and +  +  +  activity in the gills, and Na flux rates were all similar between populations after +  freshwater transfer. Large differences in Cl" regulation existed between populations and likely contributed to the marked differences in mortality after freshwater transfer. Plasma  Ill  Cl" decreased rapidly and remained low following freshwater transfer in southerns, but not in northerns, corresponding to a higher rate of Cl" loss from southerns after transfer. Elevated Cl" loss from southern fish in freshwater was possibly due to a persistence of seawater gill morphology, as paracellular permeability and apical crypt density in the gills were both higher than in northern fish. Taken together, my data demonstrate that killifish use an array of regulatory strategies, including gene expression, to modulate ion transport in response to changing environmental salinity. Salinity tolerance is not uniform within the species, however, as intraspecific variation exists suggesting that northerns are better adapted to freshwater.  iv THESIS ORGANIZATION A N D F O R M A T  The present thesis is written in a manuscript-based format approved by the University of British Columbia and consists of a total of 5 chapters. Chapter 1 provides a general introduction and outlines objectives of the thesis. Chapters 2 and 3 are manuscripts that have been published or accepted for publication in peer-reviewed journals. Chapter 4 provides a summary of findings, conclusions, and perspectives. Chapter 5 contains all the references cited throughout the thesis.  Chapter 1:  Introduction  Chapter 2:  Changes in gene expression in gills of the euryhaline killifish Fundulus heteroclitus after abrupt salinity transfer.  Authors:  Graham R. Scott, Jeff G. Richards, Biff Forbush, Paul Isenring, and Patricia M. Schulte (referred to as Scott et al., 2004a)  Date Accepted:  March 20, 2004  Journal:  American Journal of Physiology- Cell Physiology  Comments:  This study was conducted by G.R.S. under the supervision of P.M.S. J.G.R provided advice and technical support. B.F. and P.I. provided gene sequence information.  Chapter 3:  Intraspecific divergence of ionoregulatory physiology in the euryhaline teleost Fundulus heteroclitus: possible mechanisms of freshwater adaptation.  Authors:  Graham R. Scott, Joseph T. Rogers, Jeff G. Richards, Chris M. Wood, and Patricia M. Schulte (referred to as Scott et al., 2004b)  Date Accepted:  May 27, 2004  Journal:  Journal of Experimental Biology  Comments:  This study was conducted by G.R.S. under the supervision of C.M.W. and P.M.S. J.T.R. and J.G.R provided advice and technical support.  Chapter 4:  Summary, Conclusions, and Perspectives  Chapter 5:  Bibliography  VI  TABLE O F CONTENTS  Abstract  11  Thesis Organization and Format  1 V  Table of Contents  v i  List of Figures  x  List of Tables  x i i  List of Abbreviations  x  Acknowledgements  x v  m  Chapter 1 Introduction  1  Ion Transport by Seawater Fish Gills  1  Ion Transport by Freshwater Fish Gills  4  Modulation of Ion Transport During Fluctuating Salinity  7  Killifish Ionoregulatory Physiology  8  Objectives  10  Chapter 2 Changes in Gene Expression in Gills of the Euryhaline Killifish Fundulus heteroclitus After Abrupt Salinity Transfer Introduction Materials and Methods  12 12 15  vii Experimental Animals  15  Total RNA Extraction, Reverse Transcription, and Cloning  15  Salinity Transfer Protocol  19  Real-Time PCR Analysis of Gene Expression  20  Na ,K -ATPase Activity Assay and Western Blotting  22  +  +  Plasma Variables  24  Statistical Analyses  24  Results  26  Plasma Sodium  '.  26  Expression of Genes in Killifish Gills  26  Na ,K -ATPase  26  Na,K,2Cl"-Cotransporter  28  CFTR Cl" Channel  33  +  +  +  +  V-Type H -ATPase +  '.  33  Plasma Cortisol and Glucocorticoid Receptor  36  Correlation of Ion Transporter mRNA Expression  36  Discussion  40  Ion Transporter Expression After Seawater Transfer  41  Ion Transporter Expression After Freshwater Transfer  43  Cortisol and Salinity Transfer  Chapter 3  46  Intraspecific Divergence of Ionoregulatory Physiology in the Euryhaline Teleost Fundulus heteroclitus: Possible Mechanisms of Freshwater Adaptation  49  Introduction  49  Materials and Methods  53  Experimental Animals  53  Salinity Transfer Experiment  53  Total RNA Extraction and Reverse Transcription  54  Real-Time PCR Analysis of Gene Expression  55  Na ,K -ATPase Activity  56  Freshwater Flux Experiments  57  Ion and Radioactivity Measurements  61  Scanning Electron Microscopy  62  Statistical Analyses  62  +  +  Results  64  Survival  64  Plasma Ions  64  Gene Expression and Protein Activity  67  Ion Fluxes and PEG-4000 Clearance Rates  70  Gill Morphology  73  Discussion  78  Regulation of Na Balance  78  Regulation of Cl" Balance  81  Possible Mechanisms of Freshwater Adaptation  85  +  IX  Chapter 4 Summary, Perspectives, and Conclusions  87  Gene Regulation After Salinity Transfer  87  Gene Regulation and Ion Transport  89  Mechanisms of Ion Transport in Freshwater Killifish Gills  90  Physiological Mechanisms of Freshwater Adaptation  94  Overall Conclusions  96  Chapter 5 Bibliography  97  X  LIST O F FIGURES  Fig. 1 -1. Proposed mechanism of ion secretion by fish gills in seawater  3  Fig. 1-2. Proposed mechanisms of ion absorption by fish gills in freshwater  6  Fig. 2-1. Plasma sodium in killifish before and after transfer from brackish water to either brackish water, seawater, or freshwater  27  Fig. 2-2. Na,K-ATPase 0Ci mRNA expression and Na,K-ATPase activity in killifish gills a  before and after transfer from brackish water to either brackish water, seawater, or freshwater  29  Fig. 2-3. NKCC1 mRNA expression and NKCC protein abundance in killifish gills before and at several times after transfer from brackish water to either brackish water, seawater, or freshwater  31  Fig. 2-4. CFTR mRNA expression and CFTR protein abundance in killifish gills before and after transfer from brackish water to either brackish water, seawater, or freshwater  34  Fig. 2-5. Plasma Cortisol and glucocorticoid receptor mRNA expression in gills in killifish before and after transferfrombrackish water to either brackish water, seawater, or freshwater  37  Fig. 2-6. NKCC1 and CFTR mRNA expression correlated to Na,K-ATPase oci mRNA a  expression in killifish gills at several times after transfer to seawater  39  Fig. 3-1. Survival of northern and southern killifish after transfer to either brackish water or freshwater...  ; 65  xi Fig. 3-2. Plasma sodium and chloride levels in northern and southern killifish before and after transfer from brackish water to either brackish water or freshwater  66  Fig. 3-3. Fold-change in Na ,K -ATPase a mRNA expression as well as Na ,K +  +  +  +  ]a  ATPase activity in northern and southern killifish gills before and after transfer from brackish water to either brackish water or freshwater  68  Fig. 3-4. Total flux and unidirectional fluxes of Na and total Cl" flux in northern and +  southern killifish after transfer from brackish water to freshwater  72  Fig. 3-5. Representative scanning electron micrographs of the gills of northern and southern killifish before and 8 days after transfer from brackish water to freshwater  75  Fig. 3-6. Freshwater-type mitochondria-rich cell, 'intermediate' cell, and apical crypt density on the apical surface of northern and southern killifish gills before and 8 days after transfer from brackish water to freshwater  76  Fig. 4-1. Na ,K -ATPase oci mRNA expression and unidirectional Na influx after +  +  +  a  transfer of killifish from near-isosmotic brackish water to freshwater  91  Fig. 4-2. Proposed mechanism of ion absorption by killifish gills in freshwater  93  LIST O F T A B L E S  Table 2-1. Primers used for cloning of ion transport genes  17  Table 2-2. Primers used for qRT-PCR of ion transport genes  21  Table 2-3. Gill mRNA expression after salinity transfer measured by qRT-PCR  30  Table 3-1. Fold-changes in gill mRNA expression after freshwater transfer of seawater ion transporter genes measured by qRT-PCR  71  Table 3-2. Unidirectional Cl" influx, extrarenal and renal [H]PEG-4000 clearance rates, 3  and urination frequency 8 days after freshwater transfer  74  Xlll  LIST O F ABBREVIATIONS  ADP  adenosine diphosphate  GFR  glomerular filtration rate  AE  anion exchanger  GR  glucocorticoid receptor  AE1  AE isoform 1  h  hour  ATP  adenosine triphosphate  Jin  unidirectional influx  BW  brackish water  J in,Na  unidirectional Na influx  CA  carbonic anhydrase  Jin,Cl  unidirectional Cl" influx  CA2  CA isoform 2  Jnet  net flux  cDNA  complementary DNA  J net,ion  net flux of an ion (Na or  CFTR  cystic fibrosis  +  +  Cl")  transmembrane  Jout  unidirectional efflux  conductance regulator Cl"  Jout,ion  unidirectional efflux of an  channel  ion (Na or Cl") +  Ct  threshold cycle  MR  mitochondria-rich  cpm  counts per minute  mRNA  messenger RNA  DNA  deoxyribonucleic acid  NADH  nicotinamide adenine  dNTP  deoxynucleotide  dinucleotide, reduced  triphosphate  form  ECR  extrarenal clearance rate  EFloc  elongation factor la  ENaC FW  NBC  Na ,HC03 cotransporter  NBC1  NBC isoform 1  epithelial Na channel  NHE  Na ,H exchanger  freshwater  NHE2  NHE isoform 2  +  +  +  _  +  xiv NKCC  Na ,K ,2C1" cotransporter  SLC4  solute carrier family 4  NKCC1  NKCC isoform 1  SLC24  solute carrier family 24  PEG  polyethylene glycol  SW  seawater  PCR  polymerase chain reaction  t  time  PKA  protein kinase A  V  mean flux chamber  PKC  protein kinase C  PNA  peanut lectin agglutinin  V-ATPase  vacuolar-type H -ATPase  PNA+  PNA positive  W  body weight  PNA-  PNA negative  [ion]  ppt  parts per thousand  concentration in the water  qRT-PCR  quantitative real-time  at the end of a flux period  +  +  PCR  volume  f  [ion]i  +  ion (Na or Cl") +  ion (Na or Cl") +  RCR  renal clearance rate  concentration in the water  RNA  ribonucleic acid  at the start of a flux  s  second  period  SA  mean external specific  Efish  total radioactivity in the  activity  •  slope of the regression  SEM  SLC  scanning electron  line of radioactivity  microscopy  versus time  solute carrier  XV  ACKNOWLEDGEMENTS  I have been fortunate in the last 2 years to have worked with such an interesting and stimulating group of people. I would like to give my sincerest thanks to my supervisor Dr. Trish Schulte for her support and guidance. Trish always made herself available to help in any way she could, and was a never-ending source of motivation. Furthermore, I would like to thank Trish for her excellent ability to foster confidence and mould the intellectual maturity of her students. I would also like to thank my committee members Dr. Bill Milsom and Dr. Colin Brauner for their support. Dr. Chris Wood also deserves special mention for providing exceptional advice and giving me the opportunity to return to his lab in collaboration. I have also received technical assistance from several people throughout my degree who deserve special attention. In particular, I would like to thank Joe Rogers and Linda Diao from McMaster University for some late hours spent analyzing samples. Rosalind Leggatt also provided expert technical instruction, for which I am grateful. Of the many new and old friends I have worked with here at UBC, Dr. Jeff Richards is worthy of my sincerest thanks. Sometimes reminiscent of a supervisor, I would like to thank Jeff for his friendship and technical assistance (in its many forms); his advice has likely been more influential than any others' so far in shaping my academic future. I would also like to thank my friends and labmates Nann Fangue, Heather Bears, and Anne Todgham for many good times, and for teaching me many important things (despite my occasional resistance!). The undergraduate students Lisa Aird, Tina Ting Fan, Rich Gardiner, Julie Hathaway, Ben Herring, Lara Hooker, Karolyn  xvi Keir, Milica Mandic, Misty Newman, and Julia Wierzchowski are thanked for their friendship and support, as well as masters students Matt Bibeau, Amelia Grant, and Myriam Hofmeister. I would also like to thank everyone else in the comparative physiology group in the department of zoology for several shared laughs and shared (and possibly spilt) beers. Allison Barnes also deserves more credit than she ever gets for taking care of all the little things that can drive graduate students (me^ anyway) insane. Thanks as well to the rest of the administrative staff in zoology, and to several anonymous janitorial staff members for cleaning up my fish room mishaps. I was supported by a scholarship from the Natural Sciences and Engineering Research Council of Canada (to which my pocketbook is grateful). <T would like to thank my parents Melonie and Gary, and my brother Geoff, for their love and support. Finally, I would like to thank Angela Magel for everything that really matters.  CHAPTER 1  INTRODUCTION  Maintaining ionic homeostasis is an important physiological challenge for the majority of animals. Both protein synthesis and function are extremely susceptible to fluctuations of inorganic osmolytes (Weber et al., 1977; Bowlus and Somero, 1979), so regulation of intracellular ions is essential for normal cellular function. Many organisms protect intracellular ion levels by homeostatically regulating the ionic composition of their extracellularfluids.For example, aquatic animals can effectively maintain the ionic homeostasis of their extracellularfluids,even during transition between drastically different environmental salinities. This is particularly true for teleost fish, which maintain ion balance in both seawater and freshwater largely due to the transport capacity of their gills (see reviews by Wood and Marshall, 1994; Perry, 1997; Evans et al., 1999; Marshall, 2002). Through the coordinated actions of the gills and other organs, fish have been able to colonize environments ranging from dilute to salinities greater than seawater.  Ion Transport by Seawater Fish Gills  In order to maintain ionic homeostasis in seawater environments, fish must counteract their tendency to gain salts and lose water by passive diffusion in seawater environments. This is accomplished by drinking seawater to counteract water efflux and  actively excreting excess ions across the gills (see reviews by Wood and Marshall, 1994; Evans et al., 1999; Marshall, 2002). The gill epithelium is heterocellular, but in the context of ion secretion it contains three important cell types: pavement cells mitochondria-rich (MR) cells, and accessory cells (Wilson and Laurent, 2002). The first cell type covers the majority of the gill surface, while the latter two are less abundant. Despite their low density, MR cells are thought to be the primary contributor to ion , transport. Seawater MR cells (or 'chloride cells') are located along the afferent-vascular (trailing) edge of the gillfilamentsand in interlamellar spaces. Accessory cells are similarly distributed, and tend to be associated with MR cells. The morphology of MR cells varies appreciably with salinity, but includes several definable features in seawater (reviewed by Pisam and Rambourg, 1991; Perry, 1997; Wilson and Laurent, 2002). MR cells are commonly ovoid, and in seawater their apical surfaces are relatively smooth, concave, and form apical crypts. The basolateral surface of seawater MR cells has a pronounced tubular system. Seawater MR cells exist in a multicellular conformation and share shallow cell-cell junctions with accessory cells (Karnaky, 1992). Seawater gill ion regulation involves the coordinated action of an active transporter, a passive cotransporter, and several ion channels (reviewed by Wood and Marshall, 1994; Evans et al., 1999; Marshall, 2002) (Fig. 1-1). High-activity Na ,K +  +  ATPase transports sodium across the basolateral cell membrane and creates the primary driving force for ion excretion, producing an electrochemical gradient that favours passive Na efflux through shallow ('leaky') paracellular junctions. The predominant +  form of this multi-subunit enzyme likely includes the oti- and pVisoforms of the catalytic and regulatory subunits, respectively (e.g., Seidelin et al., 2001b; Feng et al., 2002;  Fig. 1-1. Proposed mechanism of ion secretion by fish gills in seawater. Basolateral Na ,K -ATPase creates the electrochemical gradient favouring Na secretion via the paracellular pathway and Cl' secretion via basolateral Na ,K ,2C1" -cotransporter 1 (NKCC1) and apical cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel. +  +  +  +  +  Semple et al., 2002; Hirose et al., 2003; Perry et al., 2003b; Richards et al., 2003). Intracellular K ions then migrate towards the serosa down their diffusive gradient +  through K channels in the basolateral membrane. +  Not only do electrochemical gradients favour passive Na efflux through +  paracellular junctions, this gradient also facilitates transport of Cl" into the ionoregulatory cell from the serosa via Na ,K ,2C1" cotransporter (NKCC isoform 1, Tipsmark et al., +  +  2002). Intracellular chloride is thus elevated, which produces a diffusive gradient for Cl" migration through a cystic fibrosis transmembrane conductance regulator (CFTR) Cl" channel in apical membrane crypts (Singer et al., 1998).  Ion Transport by Freshwater Fish Gills  In contrast to ion regulation in seawater, aquatic organisms tend to lose salts and gain water by passive diffusion when in freshwater. Teleosts have therefore developed mechanisms of hyperionic regulation whereby they actively absorb ions from the dilute environment across the gills and excrete extremely dilute urine. In contrast to the seawater gill, freshwater gills do not contain accessory cells, and MR cells may exist on gill filaments and lamellae (reviewed by Pisam and Rambourg, 1991; Perry, 1997; Wilson and Laurent, 2002). M R cells are commonly unicellular and form deep tight junctions with pavement cells, which reduces paracellular permeability and makes the freshwater gill epithelium relatively impermeable to passive ion efflux (Karnaky, 1992). The apical surface of freshwater M R cells is highly interdigitated and slightly convex.  Tubular and tubulovesicular systems invaginate the basolateral and apical surfaces, respectively, and likely facilitate membrane proximity. The most accepted model of Na and Cl" absorption by MR cells in freshwater +  fish gills involves the coordinated action of 2 active transporters, a passive antiporter, and several ion channels (reviewed by Marshall, 2002; Perry et al., 2003b) (Fig. 1-2A). An electrochemical gradient is created across the gill epithelium by apical H -ATPase +  (vacuolar-type, Perry et al., 2000), which actively pumps protons across the apical surface out of the MR cell. This gradient favours Na uptake across the apical membrane +  into the MR cell through an epithelial Na channel. Na ,K -ATPase (ai- and Pi-isoforms) +  +  +  on the basolateral surface actively transports intracellular Na across the basolateral +  membrane in exchange for K ions. K is then cycled back across the basolateral +  +  membrane by an unidentified K channel. +  Through the actions of the enzyme carbonic anhydrase (isoform 2, Hirata et al., 2003), equimolar amounts of H C O 3 " ions are produced from CO2 when protons are supplied to H -ATPases in the apical membrane. Bicarbonate efflux is therefore favoured +  to maintain acid-base balance when H -ATPase activity is high. Chloride uptake across +  the apical surface occurs in exchange for H C O 3 " via a C1-HCO3" anion exchanger. Anion exchangers from solute carrier families 4 (SLC4al-4: anion exchangers 1-4) (Sullivan et al., 1996; Perry et al, 2003b) and 26 (SLC26a4: Pendrin) (Piermarini et al., 2002; Perry et al., 2003b) may play this role. Chloride subsequently moves across the basolateral membrane through a Cl" channel, which is likely CFTR (Marshall and Singer, 2002). Evidence in support of another model for ion uptake by fish gills has recently been gaining attention (Fig. 1-2B). In this model, Na uptake (and acid excretion) and Cl" +  Fig. 1-2. Proposed mechanisms of ion absorption by fish gills in freshwater. (A) One mitochondria-rich (MR) cell model. (B) Two MR cell model. Apical H -ATPase creates the electrochemical gradient favouring apical Na uptake through epithelial Na channels (ENaC), and apical Cl" uptake occurs in exchange for bicarbonate via 'Pendrin' (SLC24a4) anion exchanger. Carbonic anhydrase 2 (CA2) provides H and HCO3" to facilitate ion uptake. CFTR, cystic fibrosis transmembrane conductance regulator Cl" channel; NBC1, Na ,HC0 "-cotransporter 1; PNA, peanut lectin agglutinin; ????, unknown molecular identity. +  +  +  +  +  3  uptake (and base excretion) are accomplished by two different subpopulations of MR cells (Goss et al., 2001; Galvez et al., 2002; Perry et al., 2003b; Reid et al., 2003; Hawkings et al., 2004). The Na /acid transporting cell is functionally analogous to the oc+  intercalated cell of the mammalian kidney, while the Cl'/base transporting cell is analogous to the P-intercalated cell and expresses peanut lectin agglutinin (PNA). This model is slightly more complicated than the former, as the products of carbonic anhydrase are sequestered in opposite directions in either subpopulation. 'PNA-negative' (PNA-) cells express apical H -ATPase, apical ENaC and basolateral Na ,K -ATPase to +  +  +  facilitate Na absorption, and move the bicarbonate produced by CA2 across the +  basolateral surface. The bicarbonate transporter involved is unknown, though a Na ,HC0 " cotransporter (NBC isoform 1, Hirata et al., 2003), a C1"-HC0 " anion +  3  3  exchanger (Perry et al., 2003b), and a HC0"-permeable Cl" channel (CFTR, Marshall et 3  al., 2002) have all been suggested (NBC1 is shown in Fig. 1-2A). PNA+ cells likely express H -ATPase to pump protons across the basolateral surface, such that apical CT+  HCO3" exchange maintains acid-base balance and facilitates Cl" absorption.  Modulation of Ion Transport During Fluctuating Salinity  Some fish species have the capacity to modulate ion flux in response to fluctuations in environmental salinity. Not surprisingly, these euryhaline species alter the cellular and molecular characteristics of their gills to maintain ion balance (reviewed by Sakamoto et al., 2001). These changes typically involve partial or complete transformation between seawater and freshwater ionoregulatory physiologies, including  altered ion transporter expression and activity, cytoskeletal reorganization, and alterations to gill surface morphology. Direct regulation may occur in response to changes in cell volume, but may also respond to neural and hormonal control systems. Because euryhaline fish modulate ion transport so effectively in response to salinity change, they often experience only small transient changes in plasma ion levels in the face of large changes in external salinity. A good example of one such species is the killifish Fundulus heteroclitus, which tolerates abrupt transfer between freshwater and seawater with minimal disruption to plasma osmolality (e.g., Jacob and Taylor, 1983). Because of their superb euryhalinity, the mechanisms that killifish modulate ion transport in response to osmotic change are frequently studied (Marshall, 2003). This species is an excellent model for the physiological adjustments to salinity change in fish gills, and is therefore the subject of this thesis.  Killifish Ionoregulatory Physiology  The common killifish Fundulus  heteroclitus inhabits  salt marshes and estuaries  along the east coast of North America. Due to the nature of these habitats, killifish are subject to routine fluctuations in environmental conditions. Environmental variations in temperature, dissolved oxygen, and salinity are common (e.g., Power et al., 2000; Albaret et al., 2004), and in certain locations may occur in the presence of anthropogenic contaminants (Weis, 2002). As such, killifish are extremely tolerant of environmental fluctuations, and are thus a frequent model for studies of physiological acclimatory responses. In particular, the euryhalinity of killifish has made these animals important  subjects for studying ionoregulatory physiology in fish. Their salinity tolerance is exceptional, rangingfromfreshwaterto nearly 4 times the salinity of seawater, and although they are generally brackish water and seawater residents, some isolated populations inhabit freshwater (Samaritan and Schmidt, 1982). In many respects, the seawater ionoregulatory physiology of killifish is typical of most other seawater-dwelling species. Indeed, studies on intact killifish and in vitro preparations of the killifish opercular epithelium led to much of our current understanding of the mechanisms of hypoosmotic ionoregulation in fish. Some of these early findings include the relationship of gill morphology and chloride cells with ion transport (Copeland, 1948; Philpott, 1965), drinking in seawater (Potts and Evans, 1967), and cortisol-mediated regulation of Na ,K -ATPase activity and ion secretion (Pickford +  +  et al., 1970). More recently, killifish have proven a useful model for studying the molecular biology of seawater fish gills, such as the cloning, characterization, expression patterns, and electrophysiology of apical CFTR (Marshall et al., 1995; Singer et al., 1998). In contrast to seawater, the freshwater ionoregulatory physiology of killifish is anything but typical. The current dogma assumes that Na ,K -ATPase activity is greatest +  +  in the gills of seawater fish, primarily because it is the only electrogenic pump responsible for ion transport, unlike the case in freshwater where both Na ,K -ATPase +  +  and H -ATPase are involved. In killifish, however, activity of this enzyme is frequently +  the same in bothfreshwaterand seawater (Marshall et al., 1999; Katoh et al., 2001). This is likely due to higher freshwater Na ,K -ATPase activity levels and not lower seawater +  +  levels, as the importance of Na ,K -ATPase for ion secretion in seawater killifish is +  +  already established (Wood and Marshall, 1994). Apical sodium transport by killifish may also occur by divergent mechanisms. Amiloride treatment causes concurrent inhibition of Na and Fl* fluxes in freshwater +  killifish; such tight coupling is characteristic of apical NHE rather than apical H*ATPase/ENaC exchange of sodium for protons (Patrick and Wood, 1999). In fact, NHE2 expression increases after freshwater transfer in killifish gills (Claiborne et al., 2002). Furthermore, V-type H -ATPase localizes to the basolateral membrane of MR cells in +  freshwater killfish gills (Katoh et al., 2003), unlike many otherfishspecies that demonstrate an apical localization (e.g., Wilson et al., 2000). Although the molecular mechanisms of Na absorption may differ from some +  other species, killifish still transport Na at an appreciable rate in freshwater (Patrick et +  al., 1997; Patrick and Wood, 1999; Wood and Laurent, 2003). In contrast, killifish in freshwater absorb Cl' at much lower rates, and have negligible base excretion after NaHC03 injection, suggesting unimportant or absent CI/HCO3" exchange (Patrick et al., 1997; Patrick and Wood, 1999). As a result, Wood and Laurent (2003) have suggested that killifish must maintain Cl" balance through the diet when in freshwater environments.  Objectives  The overall objectives of the present thesis were to employ a comprehensive approach to study various aspects of ionoregulatory physiology in a euryhalinefish.In  this regard, I used molecular, biochemical, morphological, and physiological methods to explore the following specific objectives:  1. Characterize the changes in gene expression in the gills of killifish after abrupt salinity transfer (from near-isosmotic brackish water to either freshwater or seawater). 2. Examine the relationship between gene regulation and ion flux after freshwater transfer. 3. Determine whether there is intraspecific variation in freshwater ionoregulatory physiology within F. heteroclitus, and in doing so, define possible mechanisms of freshwater adaptation in fish.  CHAPTER 2  C H A N G E S I N G E N E E X P R E S S I O N IN G I L L S O F T H E E U R Y H A L I N E K I L L I F I S H  H  E  T  E  R  O  C  L  I  T  U  S  A F T E R A B R U P T SALINITY T R A N S F E R  F U N D U L U S  1  Introduction  Ion transport is an essential function performed by epithelia in many animal tissues. Inherent to the function of ion transporting epithelia is the ability to modulate ion flux in response to changing internal and environmental conditions. For example, the euryhaline killifish Fundulus heteroclitus experiences routine fluctuations in environmental salinity in its natural habitat, during which the gill epithelium rapidly adjusts ion flux rates to help maintain ion balance (Wood and Laurent, 2003). This allows killifish to tolerate salinities ranging from freshwater (FW) to nearly four times seawater (SW) (Griffith, 1974). Gill ion transport is facilitated by mitochondria-rich (MR) cells in the gill epithelium, which can individually transform between ion absorption and ion secretion states in response to salinity change (Katoh and Kaneko, 2003). Such plasticity makes the gills of killifish an excellent model for investigating how epithelia alternate between absorptive and secretory functions. The principal transporters responsible for ion movement across gill epithelia have been the subject of numerous reviews (Wood and Marshall, 1994; Perry, 1997; Evans et al., 1999; Sakamoto et al., 2001; Marshall, 2002). Similar to the mechanisms described  1  Chapter 2 has been previously published (Scott et al., 2004a)  for several other secretory epithelia (Boucher and Larsen, 1988; Nauntofte, 1992; Riordan et al., 1994), ion secretion by fish gills requires a basolateral Na ,K -ATPase, +  +  which creates an electrochemical gradient favouring ion movement, a basolateral Na ,K ,2Cr-cotransporter (NKCC) and an apical Cl" channel (cystic fibrosis +  +  transmembrane conductance regulator, CFTR). Ion absorption by fish gills is not as well understood, but likely involves a basolateral Na ,K -ATPase, either an apical V-type Ff+  +  ATPase coupled to a Na channel or an apical Na,Ff-exchanger (NHE), and in some +  +  +  species an apical C 1 " , H C 0 3 " anion exchanger (AE). The ability of killifish to move between SW and F W environments requires modulation of ion flux across the gills, and this is partly controlled by changes in the activity of ion transporters. Short-term regulation of ion transporter activity (and thus ion flux) appears to involve changes in cell volume. For example, an increase in Cl" secretion in SW is mediated by cell shrinkage (PKC regulation of NKCC) and cAMP (PKA regulation of CFTR) (Hoffmann et al., 2002), and involves trafficking of ion transporters to their respective membranes (Marshall et al, 2002). On the other hand, a rapid reduction of Cl" secretion in FW is mediated by cell swelling (tyrosine kinase inhibition of CFTR) (Marshall et al., 2000) and sympathetic innervation via 0C2-adrenergic receptors (acting through Ca /inositol triphosphate pathway) (Marshall et al., 1993; Marshall et 2+  al., 1998). In contrast to short-term regulation, relatively few studies have assessed transcriptional and translational regulation of ion transporters after salinity transfer in killifish (Singer et al., 1998; Claiborne et al., 1999). Such regulatory mechanisms appear nevertheless important. For example, CFTR mRNA expression is known to be  14  upregulated in the gills of this species after transfer from FW to SW (Singer et al., 1998). Because the killifish is a common model for studying short-term regulation of ion transport by gill epithelia, a good understanding of the integrated responses to salinity transfer can be obtained by studying these additional regulatory aspects. Furthermore, knowledge of the mechanisms underlying modulation of ion fluxes during changing osmotic requirements may lead to the identification of conserved patterns applicable to other ion transporting epithelia. The objective of this study was to examine mRNA expression patterns and protein activity or abundance of several ion transporters in killifish gills after salinity transfer. To accomplish this, fish were transferred from near-isosmotic brackish water (BW, 10 ppt) to either FW or SW. Isosmotic BW, at which the gradients favouring passive ion flux are minimized, is the preferred salinity for F. heteroclitus (Fritz and Garside, 1974), and transfer from BW to either extreme of salinity may be more environmentally representative of the conditions killifish naturally encounter in estuaries.  Materials and Methods  Experimental Animals  Adult killifish (Fundulus heteroclitus macrolepidotus) were captured from estuaries in Hampton, New Hampshire (for cloning and salinity transfer) or in the region of Mount Desert Island, Maine (for cloning). For the salinity transfer studies, fish were held in static filtered glass aquaria filled with 10 ppt synthetic BW (Deep Ocean, Energy Savers) made up in dechlorinated Vancouver city tap water ([Na ], 0.17 mM; hardness, +  30 mg/1 as CaC03; pH 5.8-6.4). Before sampling,fishwere maintained for at least 30 days in these aquaria at an ambient temperature of 24 °C and a 14L.10D photoperiod. Fish were fed commercial trout chow (PMI Nutrition International) daily. All animal care and experimentation was conducted according to University of British Columbia animal care protocol #A01-0180.  Total RNA Extraction, Reverse Transcription, and Cloning  Genes of interest were cloned from multiple killifish tissues, including gill, liver, heart, kidney, and spleen, which were sampled after rapid decapitation of the fish. For most cloning experiments, total RNA was extracted from tissues (approximately 20 mg) using Tripure isolation reagent (Boeringer Mannheim), following the manufacturer's instructions. RNA concentrations were determined spectrophotometrically and RNA integrity was verified by agarose gel electrophoresis (-1% [wt/vol] agarose:TAE).  Extracted RNA samples were stored at -80°C following isolation. First strand cDNA was synthesized by reverse transcribing 3 p.g total RNA using 10 pmoles oligo(dTi8) primer and 20U RevertAid H Minus M-MuLV reverse transcriptase (MBI Fermentas) following the manufacturer's instructions. For some of the cloning experiments, polyA RNA was isolated directly using an alternative method (Isenring et al., 1998). Briefly, tissue was homogenized and digested for lh in 200 mg/1 proteinase K (0.5% SDS, 100 mM NaCl, 20 mM TrisCl, 1 mM EDTA, pH 8.0) at 37°C. After adjusting [NaCl] to 400 mM, tissue homogenates were incubated with oligo-dT cellulose for 4h at room temperature. PolyA RNA was eluted in 1 mmol.1"  1  EDTA, 0.05% SDS, pH 8.0, and concentrated by ethanol precipitation. In these experiments, the template was primed with a gene-specific anti-sense oligonucleotide and extended with the enzyme AMV and 0.4 uM dNTPs in an appropriate reaction buffer. Several genes of potential importance for ion regulation in fish were cloned using a PCRbased approach. Multiple alignments of previously published cDNA sequences were constructed using ClustalW (Thompson et al., 1994) to identify conserved gene regions, from which primers were then designed (Table 2-1). Specific sequences within Na ,K +  +  ATPase 0Cib, glucocorticoid receptor (GR), band 3 anion exchanger 1 (AE1), elongation factor la (EFla), Na,K,2Cl"-cotransporter 1 (NKCC1) and NKCC2 were amplified +  +  from killifish gill (Na ,K -ATPase ai , GR, and NKCC1), liver (EFla), spleen (AE1), +  +  b  heart (NKCC1), or kidney (NKCC1 and NKCC2) using Taq polymerase (MBI Fermentas) or a combination of Taq and PWO (Invitrogen). Each PCR consisted of 30 to 40 cycles of 30 to 40s at 94 °C, 30 to 50s at the lower annealing temperature for each respective primer set (see Table 2-1), and 60s for every lOOObp of expected product at 72  c o o  oo  m t~m vo  Q  <  B Q  o S  O  3 EX,  '3  T f T f  OO T f  u  o  00  ,—1 T f  N  o  OQ  o o m o  ON  o vo CM  ON T f  T f  IH  in  's o  3 5r  < U U U  a  <  < o a 4) ICO  u u u u tin"  u < u u <  2  c3 © ir>  < <  u o o <  U  <:  o o H H U  a  P o u  <  u  < o  O  < o u  O  H  H H U H  H  H  a  u  O O O  P o  u  U U H  P  H  U  < o  u u H  P O  a  H U U  Pi  o  u  $  o < o  u <: < a o P 0  &  o u o p o  U  H  P  O  U  o  H H  U  o o  H  fl O  o  <  P  co CS  u  c  g  CO  H U H H  a P u ? P u u o  IT '--1  S  6  <  00 00  o m N  5 a  ON  o p  00  vo o 00 00  <§  00  o m  .0  3 -a: o o  00  <  I I  tN  <  < a o  c -s:  s  I o  5  Os */->  O 00 CN  Cs  m o  <  O  o  00 in  o PQ  o  <  p  o oo CN  CN in  Cs  00  m ro  3  3  o o  Os  CN  VO  o  VO  m  3  3  3  3 ro vo  a u  05  3  CN vo  a s to  5  o c  b  a  1  v.  Q  ro  in  o o  u  o  <  < u  < o  o o o  H H  u u <  < o  u  o o u o  <  o  <  p  < o o H  o  o < o p  o o o  o u o  P H  P4  H  u  o o  < u H  o u H  u u  < o < H  o o  u H  o o u  T3  a si  s  -s; o o  a •5 a  •S o  is.  CO  u  H  g 3 Q  Q  o H o u <  o  Os  3"  R S  a a  CN co oo  .3  s  •SJ  o  CN  u u  P H  3  -3 a  1  60  3 -«  S  t  o  °C. PCR products were verified by electrophoresis on 1% agarose gels containing ethidium bromide and cloned into a pGEM-T Easy (Promega) or pCR2.1 (Invitrogen) vector plasmid. Multiple clones of each fragment were sequenced bidirectionally and consensus sequences were submitted to Genbank database (Na ,K -ATPase a n , , Acc. No. +  +  AY430089; GR, Acc. No. AY430088; AE1, Acc. No. AY430090; EFla, Acc. No. AY430091; NKCC1, Acc. No. AY533706; NKCC2, Acc. No. AY533707). The other killifish gene sequences used in this work were from Na ,K -ATPase a i (Acc. No. +  +  a  AY057072), Na ,K -ATPase a (Acc. No. AY057073), cystic fibrosis transmembrane +  +  2  conductance regulator Cl" channel (CFTR; Acc. No. AF000271), and V-type FT-ATPase subunit A (V-ATPase; Acc. No. AB066243).  Salinity Transfer Protocol  After at least 1 month acclimation to a salinity of 10 ppt, 8 controlfishwere sampled, and those remaining were quickly transferred by net to aquaria containing FW (0 ppt), BW (10 ppt), or SW (35 ppt). Individualfishwere subsequently sampled by netting at 3h, 8h, 24h, 96h, 14 days, and 30 days after transfer from BW. Blood samples were collected in heparinized capillary tubes from the severed caudal peduncle and fish were killed by rapid decapitation. Blood was centrifuged at 13000g for 10 minutes and plasma was frozen in liquid nitrogen. Second and third gill arches were isolated and immediately frozen in liquid nitrogen. Gills were not perfused beforefreezing,because blood has been previously shown to contribute little to whole-gill gene expression (Perry et al., 2000). All tissues were stored at -80°C until analysed.  Real-Time PCR Analysis of Gene Expression  Messenger RNA was extracted and reverse transcribed from killifish gills using the first method of reverse transcription described above. Gene expression was assessed using quantitative real-time PCR (qRT-PCR) on an ABI Prism 7000 sequence analysis system (Applied Biosystems). Primers for all genes were designed using Primer Express software (version 2.0.0, Applied Biosystems; see Table 2-2). PCR reactions contained 1 p.1 of cDNA, 4 pmoles of each primer and Universal SYBR green master mix (Applied Biosystems) in a total volume of 21 pi. All qRT-PCR reactions were performed as follows: 1 cycle of 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min (set annealing temperature of all primers). PCR products were subjected to melt curve analysis and representative samples were electrophoresed to verify that only a single product was present. Control reactions were conducted with no cDNA template or with non-reverse transcribed RNA to determine the level of background and genomic DNA contamination, respectively. Genomic contamination was below 1:49 starting cDNA copies for all templates except AE1, NKCC2, and Na ,K +  +  ATPase 0C2 genes. The latter were not detected above background in gills (data not shown) and were subjected to no further analysis. Negligible expression of AE1 in gill samples likely indicates low relative contamination by erythrocytes, in which AE1 is highly expressed infish(Hubner et al., 1992). A randomly selected control sample was used to develop a standard curve for each primer set, and all results were expressed relative to these standard curves. Results  21  Table 2-2. Primers Usedfor qRT-PCR of Ion Transport Genes  Gene  Sequence (5'-3')  Na ,K -ATPase a +  +  ] a  Na ,K -ATPase a i +  +  Na ,K -ATPase oc +  +  2  NKCC1  NKCC2  CFTR1  GR  V-ATPase  AE1  EFloc  b  F:  AAG ATC ATG GAG TCC TTT AAG AAT CTG  R:  CAC CTC CTC TGC ATT GAT GCT  F:  CAG TCA TGG GTC GGA TTG CT  R:  TGG AGT GCG TCC AAC CTC TAG  F:  GCG TTT TAC TTC CGG GCT TT  R:  TGC AAT ATT ACA TCT GAC AGA CAC ACA  F:  CCC GCA GCC ACT GGT ATT  R:  GCC ATC TGT GGG TCA GCA A  F:  GCC GTG GCC CTT TGC  R:  CCA GGT CGG TTG TGT TTC CT  F:  AAT CGA GCA GTT CCC AGA CAA G  R:  AGC TGT TTG TGC CCA TTG C  F:  GTA CCA AAA GAA GGC CTG AAG TG  R:  CCT TGA TGT AAG TCA TCC TGA TCT CA  F:  TGA AGT TCA AGG ACC CGG TTA  R:  CTG CGC GTA CTC GCC TTT  F:  TGA TTG TGA GCA AAC CTG AGA GA  R:  GGA GGA GCA GGT CAA AAT GAA A  F:  GGG AAA GGG CTC CTT CAA GT  R:  ACG CTC GGC CTT CAG CTT  were then normalized to EFla, a gene for which mRNA expression in the gills does not change following salinity transfer (data not shown). For clarity in graphing, post-transfer samples were expressed relative to the pre-transfer BW control samples. All samples were run in duplicate (coefficients of variation were <10%). To avoid unnecessary analysis, pre-transfer controls, 24h, 96h, and 14 day time points were analysed first. The expression at remaining time points was only analysed for those genes that were affected consistently by changes in salinity. The absolute level of mRNA expression of each gene examined was estimated semi-quantitatively 24h after transfer (BW, FW, and SW) using the following formula: Efficiency"  0  (1)  Here, efficiency is determined from the slope of the standard curve and Ct corresponds to the threshold cycle number. These results were subsequently normalized to the estimated absolute expression of EFla. Due to the nature of analysis using qRT-PCR, these data can only be used for approximate comparison of expression levels between genes.  Na ,K -A TPase Activity Assay and Western Blotting +  +  Na ,K -ATPase activity was determined by coupling ouabain-sensitive ATP +  +  hydrolysis to pyruvate kinase- and lactate dehydrogenase-mediated NADH oxidation as outlined by McCormick (McCormick, 1993). For this assay, second and third gill arches were homogenized in 500 pi of SEI (150 mM sucrose, 10 mM EDTA, 50 mM imidazole, pH 7.3) containing 0.1% Na-deoxycholate and centrifuged at 5000g for 30s at 4 °C. Supernatents were immediately frozen in liquid nitrogen and stored at -80°C until  23  analysed. ATPase activity was determined in the presence or absence of 0.5 mM ouabain using 10 ul supernatent thawed on ice and was normalized to total protein content (measured using the bicinchoninic acid method, Sigma-Aldrich). All samples were run in triplicate (coefficients of variation were <10%). Ouabain-sensitive ATPase activity is expressed as umol ADP/mg protein/h. NKCC and CFTR protein abundance was measured by Western immunoblotting according to Marshall et al. (2002). Gill homogenates were prepared as outlined above and denatured for 3 min in boiling SDS-sample buffer (Laemmli, 1970). Eight percent SDS-polyacrylamide gels were loaded with total gill homogenates (20 ug protein/lane) and protein was transferred to nitrocellulose membranes (Bio-Rad) using a Trans-Blot semi-dry transfer cell (Bio-Rad). Blots were first incubated for lh with 1.0 ug.mr of 1  primary antibody diluted in TTBS buffer (17.4 mM Tris-HCl, 2.6 mM Tris base, 500 mM NaCl, 2.0 mM sodium azide, 0.05% Tween-20, pH 7.5) containing 2% skim milk; the primary antibody used against killifish NKCC protein was a polyclonal mouse antihuman NKCC (T4; Iowa Hybridoma Bank, University of Iowa) (Lytle et al., 1992) and the primary antibody used against killifish CFTR protein was a monoclonal mouse antihuman CFTR (R&D Systems). Blots were subsequently incubated for lh with goat antimouse IgG secondary antibody (alkaline phosphatase conjugated; Stressgen) diluted 1:3000 in TTBS. Membranes were developed in alkaline phosphatase buffer containing 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro-blue tetrazolium (NBT) (SigmaAldrich). Band intensity was quantified using a FluorChem 8800 imager (Alpha Innotech) assisted by AlphaEaseFC software (v.3.1.2; Alpha Innotech). Samples are expressed relative to a randomly chosen protein standard (included on each gel to control  24  for transfer efficiency) and normalized to pre-transfer BW control samples. In these studies, the sensitivity of Western immunoblotting for quantification of protein content was verified through dose-response analyses of both antibodies using homogenates from 2 fish.  Plasma Variables  Plasma sodium was determined using flame atomic absorption spectrophotometry (SpectrAA-220FS, Varian) with Fisher certified standards. Plasma Cortisol was determined by enzyme-linked immunosorbant assay following the manufacturer's instructions (Neogen).  Statistical Analyses  Data are expressed as means ± SEM. For gene expression, protein abundance, transporter activity, and plasma variables, Friedman's 2-factor non-parametric analysis of variance (ANOVA) was used to determine whether the effect of salinity over time differed between BW, SW, and FW. Kruskal-Wallis H non-parametric ANOVA was used to ascertain overall differences as a function of either salinity (within each time point) or time (for each salinity). Because measured variables could have changed due to handling of fish alone, the effect of SW or FW transfer was assessed by comparison with the matched BW control using Mann-Whitney U non-parametric comparisons at time points  where salinity effects were detected by ANOVA. All statistical analyses were conducted using SPSS version 10.0 and a significance level of p<0.05 was used throughout.  Results  Plasma Sodium  Changes in plasma Na as a function of salinity over time were significantly different among salinity groups (Fig. 2-1). Plasma Na did not change relative to matched BW controls at any time point sampled after transfer to SW. In contrast, transfer to FW decreased plasma Na compared to BW controls 1 day after transfer.  Expression of Genes in Killifish Gills  Estimated absolute mRNA expression level (determined using equation 1, measured 1 day after transfer to all salinities) followed the order from highest to lowest (shown relative to EFla in parentheses): Na ,K -ATPase a (10"), NKCC1 (IO ), +  +  1  2  )a  Na ,K -ATPase a, (IO ), GR (410 ), V-ATPase (2-10"), and CFTR (3-10 ). For most +  +  -2  3  3  -4  b  of these genes, changes in mRNA expression, protein abundance, or transporter activity as a function of salinity over time was significantly different among the three salinity groups (see Figs. 2-2 to 2-5). Additionally, most of these variables also changed significantly as a function of time within each salinity group including the BW controls. For this reason, data reported here will focus on the effect of SW or FW transfer compared to BW controls within each time point.  Na ,K -ATPase +  +  27  180 H  E 160  ro £ £  140H  120  pre  2  4  10  20  30  Time (days)  Fig. 2-1. Plasma sodium (n > 5) in killifish before (pre) and after transferfrombrackish water (BW, 10 ppt) to either BW (square), seawater (SW, downward triangle), or freshwater (FW, upward triangle). Data are expressed as means ± SE. * Significant difference from time-matched BW control (p < 0.05).  Patterns of Na ,K -ATPase 0Ci mRNA expression in the gills differed between +  +  a  SW and FW transfers (Fig. 2-2A). Whenfishwere transferred from BW to SW, Na ,K +  +  ATPase oci expression increased transiently 1 day after transfer, to nearly 3-fold above a  BW controls. There was also a small, but statistically significant increase at 14 days. In contrast to SW, the increase in Na ,K -ATPase oci mRNA expression infishtransferred +  +  a  from BW to FW was more prolonged, peaking 6-fold above BW controls at 4 days (Fig. 2-2A). In fact, the increase in expression 4 days after FW transfer was higher than that at any point in SW. Interestingly, although mRNA of the Na ,K -ATPase aib isoform was +  +  expressed at high levels (see above), expression did not change in SW and was only slightly decreased (0.8-fold) 14 days into FW (Table 2-3). Na ,K -ATPase activity changed in a similar manner to Na ,K -ATPase +  +  +  +  oci  a  expression after both SW and FW transfer (Fig. 2-2B). A transient 2-fold increase in Na ,K -ATPase activity was observed 1 day into SW compared to matched BW controls. +  +  Na ,K -ATPase activity underwent a more prolonged increase after FW transfer, up 2+  +  fold above BW controls from 1 to 4 days.  Na,K*,2Cr -Cotransporter  NKCC1 mRNA expression patterns in the gills were very similar to those for Na ,K -ATPase 0Ci after SW transfer, showing comparable transient changes (Fig. 2+  +  a  3A). Expression of this gene increased 3-fold above BW controls 1 day after transfer,  pre  0  1  2  4  10  20  30  10  20  30  Time (days)  B  0  pre  1  2  4  Time (days)  Fig. 2-2. Na,K-ATPase a mRNA expression (n > 7) (A) and Na,K-ATPase activity (n > 6) (B) in killifish gills before (pre) and after transfer from brackish water (BW, 10 ppt) to either BW (square), seawater (SW, downward triangle), or freshwater (FW, upward triangle). Expression is relative to elongation factor la and is normalized to pre-transfer controls. FW activities are offset for clarity at 3h, 8h, and 24h. Data are expressed as means ± SE. * Significant difference from time-matched BW control (p < 0.05). ]a  30  Table 2-3. Gill mRNA Expression After Salinity Transfer Measured by qRT-PCR  Salinity Gene Na ,K -ATPase 0 C i +  +  b  V-type H -ATPase +  Time  BW  FW  SW  24h  0.40 ± 0.02  0.38 ±0.01  0.44 ± 0.03  96h  0.42 ±0.07  0.30 ±0.03  0.23 ±0.04  14d  0.31 ±0.02  0.25 ±0.02*  0.27 ±0.01  24h  0.71 ±0.03  0.63 ±0.03  0.87 ±0.05*  96h  0.42 ±0.02  0.53 ±0.07  0.55 ±0.08  14d  0.48 ±0.04  0.48 ±0.04  0.61 ±0.06  '  Values are means ± SE. n > 7. 'Significant difference from brackish water control at the respective time point (p<0.05). qRT-PCR expression data are normalized to pre-transfer controls.  31  Fig. 2-3. NKCC1 mRNA expression (n > 7) (A) and NKCC protein abundance (n > 6) (C) in killifish gills before (pre) and at several times after transfer from brackish water (BW, 10 ppt) to either BW (square), seawater (SW, downward triangle), or freshwater (FW, upward triangle). Expression is relative to elongation factor la and is normalized to pre-transfer controls. (B) Representative western blots at 14 days. Protein abundance was quantified at approximately 170 kDa using mouse anti-human NKCC antibody (see Materials and Methods for details). This molecular mass corresponds to fiillyglycosylated NKCC protein. Data are expressed as means + SE. * Significant difference from time-matched BW control (p < 0.05).  0  1  1  pre  — n  0  1  ,  r  1  2  4  Time (days)  10  20  30  33  with a small subsequent rise at 14 days. In contrast, NKCC1 mRNA expression decreased 2.5-fold 1 day into FW. Western blots using the T4 antibody (mouse anti-human NKCC) revealed a single 170 kDa immunoreactive band, likely corresponding to fully-glycosylated NKCC protein (Moore-Hoon and Turner, 1998) (Fig. 2-3B). NKCC abundance estimated at this band increased 2-fold 14 and 30 days after SW transfer, but there was no change after FW transfer (Fig. 2-3C).  CFTR Cl Channel  Changes in CFTR mRNA expression were more prolonged than those for Na ,K +  +  ATPase a i and NKCC1 in SW. CFTR expression increased rapidly 3h after transfer to a  SW (2-fold above BW controls), and remained elevated until returning to control levels 30 days later (Fig. 2-4A). On the other hand, CFTR mRNA expression was generally reduced in FW, decreasing as much as 10-fold below BW controls at 1 day post-transfer. CFTR abundance on immunoblots was estimated at an approximate size of 160 kDa, corresponding to the mature protein (Fig. 2-4B). A second faint band at 120 kDa was not quantified, and probably represents immature unglycosylated protein (Bertrand and Frizzell, 2003). CFTR protein abundance at 160 kDa did not change over time in SW, and no differences from matched BW controls were detected (Fig. 2-4C). FW transfer increased CFTR protein levels at 1 day post-transfer (Fig. 2-4C).  V-Type LT-ATPase  Fig. 2-4. CFTR mRNA expression (n > 7) (A) and CFTR protein abundance (n > 6) (C) in killifish gills before (pre) and after transfer from brackish water (BW, 10 ppt) to either BW (square), seawater (SW, downward triangle), or freshwater (FW, upward triangle). Expression is relative to elongation factor la and is normalized to pre-transfer controls. (B) Representative western blots at 1 day. Protein abundance was quantified at approximately 160 kDa using mouse anti-human CFTR antibody (see Materials and Methods for details), corresponding to the molecular mass of mature CFTR protein. A faint immunoreactive band at approximately 120 kDa was not quantified, and likely represents immature unglycosylated protein. Data are expressed as means ± SE. * Significant difference from time-matched BW control (p < 0.05).  Time (days)  There were only minor 2-3). SW transfer increased while  FW transfer had  effects of salinity on V-ATPase mRNA expression (Table  expression above  no effect on  BW controls (1.2-fold) 1 day post-transfer,  V-ATPase expression compared to BW  controls  at  any time point.  Plasma Cortisol and Glucocorticoid Receptor  Plasma Cortisol responded 5A). Transfer from BW to BW  in a similar manner to each salinity over time  transiently increased plasma Cortisol, likely as a result  handling. Plasma Cortisol also increased transiently after both changes were more pronounced than for matched  BW  to SW, plasma Cortisol was nearly 3-fold above BW in a similar  SW  controls.  controls  and  of  FW transfer, but  After transfer from BW  at 3h. FW transfer  resulted  3-fold rise in plasma Cortisol 3h after transfer.  GR mRNA expression tended to decrease immediately salinities, followed by a transient rise in expression at changes with time, however, both transfer compared increased  (Fig. 2-  to BW  controls  after transfer to all three  1 day (Fig. 2-5B). Despite these  FW and SW transfer reduced GR expression 8h after  (1.7-fold below BW  controls), and SW transfer  GR expression 1 day post-transfer (1.3-fold above BW  Correlation of Ion Transporter mRNA Expression  controls).  37  10  20  30  Time (days)  B  c o  2H  •</> w CD  i_  a x m  E O  pre  2  4  —i 10  1  1  20  30  Time (days)  Fig. 2-5. Plasma Cortisol (n > 6) (A) and glucocorticoid receptor (GR) mRNA expression in gills (n > 7) (C) in killifish before (pre) and after transfer from brackish water (BW, 10 ppt) to either BW (square), seawater (SW, downward triangle), or freshwater (FW, upward triangle). Freshwater data are offset for clarity at 3h and 8h. Expression is relative to elongation factor la and is normalized to pre-transfer controls. Data are expressed as means ± SE. * Significant difference from time-matched BW control (p < 0.05).  Following SW transfer Na ,K -ATPase +  +  oti  a  and NKCC1 mRNA expression were  very well correlated (r = 0.872, p < 0.001) (Fig. 2-6A). Expression was related such that 2  a 3-fold increase in NKCC1 coincided with a 2-fold increase in Na ,K -ATPase +  (slope of 1.4 ± 0.1). Na ,K -ATPase +  +  0Ci  a  +  0Ci  a  and CFTR were also positively correlated in  SW, but this relationship was not as strong (p < 0.001, r = 0.309, slope of 0.5 ± 0.1) (Fig. 2  2-6B). There was a similar association between NKCC1 and CFTR after SW transfer (r  2  = 0.202, p < 0.001; data not shown). Expression of ion transporters after FW transfer was not correlated to the same degree (data not shown): Na ,K -ATPase a.\ and CFTR +  +  a  showed a slight negative correlation post-transfer (r = 0.186, p = 0.002), but expression 2  of neither gene correlated with NKCC1 (p > 0.448).  0  i 1  1 2  Na,K-ATPase a  0  -I  1  0  1  :  •  1 a  1 2  1  1  1  3  4  5  mRNA Expression  1  1  1  3  4  5  N a , K - A T P a s e cx m R N A E x p r e s s i o n 1a  Fig. 2-6. NKCC1 (A) and CFTR (B) mRNA expression correlated to Na,K-ATPase ai mRNA expression in killifish gills at several times after transfer to seawater. mRNA expression is relative to elongation factor la and is normalized to pre-transfer controls. (A) r = 0.872, p < 0.001, y = (1.4 ± 0.1) - (0.7 ± 0.2); (B) r = 0.309, p < 0.001, y = (0.5 ± 0.1) - (1.1 ± 0.2). Hatched curves represent 95% confidence limits of regression. a  2  2  40  Discussion  Killifish typically encounter fluctuations in environmental salinity on a daily basis in their natural habitat. Rapid modulation of ion flux across the gills is thus important for maintaining ion balance, and likely occurs through post-translational processes. These include changes in the subcellular localization and activity of various ion transporters (Towle et al., 1977; Marshall et al., 1993, 1998, 2000, 2002; Hoffmann et al., 2002), as well as rapid changes in gill epithelium morphology (Daborn et al., 2001), leading to immediate changes in transepithelial ion currents. The rapid adjustments that occur shortly after salinity change likely allow these animals to maintain ionic homeostasis in the face of fluctuations in estuarine salinity that are transient and somewhat unpredictable. Salinity change may persist for a longer duration, however, such as when killifish move up and down tributaries between seasons (Halpin, 1997) and when populations invade freshwater (Powers et al., 1993). In these situations, additional mechanisms may be important for long-term adjustment to salinity change. For example, prolonged transfer to SW or FW appears to involve changes in the abundance of mitochondria-rich cells in the gill epithelium (Katoh and Kaneko, 2003). In the current study, we have also observed unique patterns of ion transporter expression in the gills of killifish after transfer to either SW or FW. Gene regulation and cell proliferation likely contribute to the further increases in ion flux that are observed one or more days after salinity change (Wood and Laurent, 2003). Our work and that of others therefore demonstrates very clearly that the gills of killifish are able to employ an array of regulatory strategies, including those at transcriptional and translational levels, to regulate  ion balance in various environments. As a result, even after large changes in environmental salinity, killifish experience only small, transient changes in plasma ion levels (this study and Jacob and Taylor, 1983; Marshall et al., 1999; Katoh and Kaneko, 2003).  Ion Transporter Expression after Seawater Transfer  Consistent with the physiological model for ion secretion by gill epithelia, SW transfer increased the expression of Na ,K -ATPase cti , NKCC1, and CFTR at the +  +  a  mRNA level. The gill epithelium is heterogeneous, so the 2- to 3-fold changes in ion transporter expression we report here in whole gills may have been much higher in individual ion transporting cells. Interestingly, the increase in expression was transient for Na ,K -ATPase a and NKCC1, but prolonged for CFTR. Furthermore, Na ,K +  +  +  +  u  ATPase 0Ci and NKCC1 expression patterns were tightly correlated after SW transfer (r  2  a  = 0.872), but expression of both genes was poorly correlated (r < 0.309) with CFTR. 2  Differentially sensitive salt- or hormone-responsive regulatory elements for Na ,K +  +  ATPase 0Ci , NKCC1, and CFTR genes could explain these results. Alternatively, a  basolateral (Na ,K -ATPase (Xi and NKCC1) and apical (CFTR) ion transporter genes +  +  a  may be regulated by different transcription factors. Future experiments are required to determine the factors regulating transcription of ion transporter genes in killifish gills. Changes in protein abundance are often assumed to parallel changes in mRNA expression. Consistent with this assumption, Na ,K -ATPase activity, although not +  +  necessarily an indicator of protein abundance, increased in parallel with Na ,K -ATPase +  +  42  ocia  mRNA expression. This was not the case for NKCC after SW transfer, however,  because increased NKCC1 gene expression preceeded a prolonged increase in NKCC protein abundance. The prolonged increase in NKCC protein, coupled with the transient increase in NKCC1 mRNA levels, suggests that NKCC protein turnover in the gill may be low. Parallel changes in CFTR mRNA and protein levels were also absent. In fact, CFTR protein abundance did not change following SW transfer, even though mRNA expression was elevated for a prolonged period. This lack of correlation suggests posttranscriptional regulation of protein abundance. Elevated chloride conductance in SW might therefore be modulated primarily by post-translational mechanisms (e.g., intracellular trafficking, see Marshall et al., 2002; Bertrand and Frizzell, 2003), and might not involve changes in CFTR protein abundance. Taken together, our data suggest that the dynamic between gene transcription and protein abundance is not always straightforward and may depend on numerous factors (see reviews by Derrigo et al., 2000; Wilson and Cerione, 2000). Some similarity exists between the patterns of gene expression reported here for killifish and those reported in previous studies for other species. For example, a prolonged increase in CFTR expression has also been observed in the gills of Atlantic salmon (Salmo salar) transferred from FW to SW (Singer et al., 2002). Similarly, NKCC protein has been shown to increase in the gills of Atlantic salmon (Pelis et al., 2001) and tilapia (Oreochromis mossambicus) (Wu et al., 2003) after SW acclimation. Despite some similarities in gene expression among species, our data also demonstrate that the patterns of gene expression among various fish species are not always the same. For example, Na ,K -ATPase oti and NKCC1 mRNA expression +  +  a  increase only transiently in the gills of killifish after SW transfer, whereas expression of these genes increase for more prolonged periods in the gills of Atlantic salmon (D'Cotta et al., 2000; Seidelin et al., 2000; Tipsmark et al., 2002), brown trout (Salmo trutta) (Madsen et al., 1995; Tipsmark et al., 2002), or European sea bass (Dicentrarchus labrax) (Jensen et al., 1998). Transient changes in killifish gill Na ,K -ATPase activity +  +  after SW transfer were also observed in this study and others (Towle et al., 1977; Mancera and McCormick, 2000), while more prolonged increases in Na ,K -ATPase +  +  activity are typical of manyfishspecies (Madsen et al., 1995; Jensen et al., 1998; Seidelin et al., 2000; Seidelin et al., 2001a; Lee et al., 2003; Richards et al., 2003). The transient nature of many changes observed in gill protein expression and activity appears to be an important response of killifish to SW transfer, and may reflect greater tolerance to salinity change. Plasma Na did not change significantly after transferfromBW to +  SW, so prolonged changes in expression may be unnecessary for maintaining ion balance. Different physiological responses of fish to changes in salinity may also have arisen from the effects of alternative life histories (e.g., anadromy versus euryhalinity) or evolutionary histories (i.e., freshwater versus seawater ancestry).  Ion Transporter Expression after Freshwater Transfer  Recent work has suggested that the mechanism of ion uptake by killifish gills in FW differsfromthe prevalent model, which was developed predominantly in salmonids. In these species, it is proposed that Na uptake through epithelial Na channels (ENaC) is +  +  driven by the electrical gradient produced by apical V-ATPase (Lin and Randall, 1993;  44  Perry et al., 2000; Reid et al., 2003). In killifish, however, Patrick and Wood (1999) demonstrated that Na uptake and tf efflux in FW is inhibited by both amiloride and low +  water pH to the same degree, suggesting that apical Na flux is primarily mediated by +  Na /H -exchangers (NHE). Similarly, in a discussion of some unpublished observations, +  +  Claiborne et al. (2002) noted that NHE2 mRNA expression increased in the gills of killifish after FW transfer. Consistent with thesefindings,our data show that transcription of V-ATPase was unaffected by salinity transfer, even though it was expressed at high levels in the gills. Taken together, these data suggest that NHEs may be important for Na absorption across the apical membrane of the gills of FW killifish. Interestingly, +  recent work by Katoh et al. (2003) immunolocalized V-ATPase to the basolateral membrane of mitochondria-rich cells in the gills of freshwater killifish, and immunoreactivity increased in dilutefreshwater.Thus, basolateral V-ATPase may play a role in ion absorption, regulated primarily through post-transcriptional mechanisms. Freshwater elasmobranch gills express V-ATPase on the basolateral surface of mitochondria-rich cells as well (Piermarini and Evans, 2001), so in this way the mechanisms of ion transport in the gills of killifish and some elasmobranchs may be similar. Na,K -ATPase a mRNA expression and Na ,K -ATPase activity increased +  +  u  after FW transfer, and maximal changes above BW controls coincided with the reestablishment of plasma Na. Increased Na ,K -ATPase transcription may occur in +  +  conjunction with chloride cell hypertrophy, as previously described for killifish in FW (Marshall et al., 1999; Katoh and Kaneko, 2003). Interestingly, both expression and activity were upregulated to a greater extent after FW transfer than SW transfer in  45  killifish, similar to findings reported for some other species (e.g., milkfish, Chanos chanos) (Lin et al., 2003). In contrast, for many species (e.g., salmonids, tilapia, and others; discussed above) Na ,K -ATPase mRNA expression and activity increase to their +  +  greatest extent after SW transfer. These species have also been reported to upregulate apical V-ATPase in FW (Lin and Randall, 1993; Hiroi et al., 1998; Seidelin et al., 2001b). The discrepancy in Na ,K -ATPase regulation between killifish and these species +  +  may therefore relate to the above suggestion that apical NHEs mediate Na absorption in +  killifish, rather than ENaC coupled to V-ATPase. Without the electrochemical gradient created by active apical extrusion of protons by V-ATPase, higher Na ,K -ATPase +  +  activity may be required in the gills of killifish to maintain a favourable Na gradient +  across the apical membrane. As was the case after SW transfer, mRNA expression patterns after FW transfer were not necessarily followed by predictable changes in protein abundance. For example, both NKCC1 and CFTR mRNA expression decreased in killifish gills after FW transfer, whereas protein abundance did not. In fact, CFTR abundance (measured at 160 kDa) actually increased one day after FW transfer. A second isoform such as exists in Atlantic salmon (Singer et al., 2002) could account for this increase in CFTR abundance. Our real-time PCR primers may have failed to amplify a second isoform, while our antibody may have cross-reacted with both isoforms. Interestingly, CFTR immunofluorescence in the cytoplasm and/or on the basolateral surface of pavement cells in killifish opercular epithelium increases in FW (detected using the same antibody used in this study, Marshall et al., 2002), possibly indicating that a FW-specific isoform exists.  46  Cortisol and Salinity Transfer  Elevation of plasma Cortisol is recognized as an important part of the endocrine signaling response that occurs shortly after SW transfer and has been observed in several fish species, including killifish (Jacob and Taylor, 1983; Marshall et al., 1999). In the present study, we have observed a similar rise in plasma Cortisol during the 24h following SW transfer. This response may in fact account for the upregulation of Na ,K -ATPase in +  +  fish gills that also occurs in SW as suggested by McCormick (McCormick, 2001). In this study, plasma Cortisol also increased transiently after FW transfer, as previously reported in killifish (Jacob and Taylor, 1983). In contrast to SW transfer, however, the role of this hormone infishduring the early stages of FW acclimation is poorly understood. Interestingly,  Cortisol  treatment promotes ion uptake both in vivo and across  cultured gill epithelia, and has also been shown to increase Na ,K -ATPase activity as +  +  well as transepithelial resistance, and to reduce passive ion fluxes (McCormick, 2001; Kelly and Wood, 2002; Zhou et al., 2003). Cortisol may have therefore contributed to the Na ,K -ATPase upregulation observed after FW transfer. To this effect, it is interesting to +  +  note that Na ,K -ATPase mRNA expression is increased by glucocorticoids in some +  +  mammalian absorptive tissues, including lung (Ingbar et al., 1997) and renal tubule (Celsi etal., 1991) epithelia. GR expression was observed to decrease early after BW, FW, and SW transfers, during which time plasma Cortisol increased. Furthermore, the plasma Cortisol elevation observed 3h after both SW and FW transfers was followed by decreased GR expression at 8h compared to BW controls. These changes in GR mRNA expression observed over  47  time suggest negative feedback regulation by Cortisol, as has been previously suggested for salmonids (Uchida et al., 1998; Shrimpton and McCormick, 1999). The changes in GR expression may not have been solely due to negative feedback regulation, however. Increased expression of this receptor occurred after 1 day in SW compared to BW and FW, and this was not preceded by decreased plasma Cortisol. Hence, other specific effects of SW transfer could have brought about changes in GR expression. These factors may be better appreciated if the potentially interactive effects of Cortisol and osmotic change are eliminated. The transient rise in GR expression after SW transfer is another interesting result in this study. Indeed, this behaviour contrasts with that observed in chum salmon (Oncorhynchus keta), for which GR expression in gills increases progressively with SW acclimation (Uchida et al., 1998). Differences between the responses of anadromous and euryhaline fish to salinity transfer may therefore apply beyond the expression and function of ion transporters to various regulatory systems. The data presented here indicate that responses to salinity transfer differ between killifish and salmonids. Differences in life histories between these taxa may have contributed to these varied physiological strategies used to cope with variation in environmental salinity. Salmonids are anadromous and generally prepare for migration between salinities at specific stages of their life cycle, so it is perhaps foreseeable that they rely strongly on long-term changes in transcription and translation of ion transporters to maintain ion balance. In contrast, killifish encounter daily variations in estuarine salinity that are somewhat unpredictable, so they regulate ion transporters in various ways, many of which are rapid and/or transient.  In summary, we have quantified the expression patterns of several ion transport genes in the gills of killifish after transfer from intermediate salinity to FW and SW; such transfers are probably representative of what many estuarinefishnormally encounter in their natural habitats. Interestingly, we observed that the expression patterns varied appreciably as a function of salinity, and that changes in mRNA expression were not always matched by changes in protein abundance. This work advances our understanding of the factors regulating ion transport in euryhalinefish,and demonstrates important mechanisms of functional plasticity in transport epithelia.  CHAPTER 3  INTRASPECIFIC D I V E R G E N C E O F I O N O R E G U L A T O R Y P H Y S I O L O G Y IN T H E E U R Y H A L I N E TELEOST  FUNDULUS HETEROCLITUS:  ADAPTATION  POSSIBLE M E C H A N I S M S O F F R E S H W A T E R  2  Introduction  The common killifish Fundulus heteroclitus inhabits brackish water estuaries and salt marshes along the eastern coast of North America. The species distribution is latitudinal, from Newfoundland to Florida, and thus spans a cline of environmental temperatures. Correspondingly, many previous studies have investigated thermal adaptations in populations across the range. Differences between populations include latitudinal differences in glycolytic enzyme expression and activity (Powers et al., 1986; Pierce and Crawford, 1996), endocrinology (DeKoning et al., 2004; Picard and Schulte, 2004), metabolism (Podrabsky et al., 2000), morphology, and behaviour (Powers et al., 1993), and as a result, these fish are sometimes divided into 2 subspecies, F.h. macrolepidotus (northern) and F.h. heteroclitus (southern). In contrast, few studies have assessed whether intraspecific physiological differences exist between populations of F. heteroclitus in response to other environmental factors (e.g., tidal cycle, DiMichele and Westerman, 1997).  2  Chapter 3 has been accepted for publication (Scott et al., 2004b)  Species within the genus Fundulus are suggested to have arisen from brackish water ancestors, and there is substantial variation in both the salinity of their native habitats (rangingfromfreshwaterto seawater) and their salinity tolerance (Griffith, 1974). Intraspecific differences in salinity tolerance and distribution also appear to exist within some Fundulus species. For example, northern populations of F. heteroclitus have higher fertilization success and larval survival in hyposmotic salinities than southern populations (Able and Palmer, 1988). Furthermore, the proportion of northern genotypes increases infreshwaterhabitats, even at latitudes and temperatures that are typical for the southern subspecies (Powers et al., 1993). It is therefore likely that molecular or physiological differences exist within F. heteroclitus that form the basis for variation in freshwater tolerance. Past habitat availability may have selected for differences infreshwatertolerance between northern and southern individuals of F. heteroclitus. After previous glaciation events, new freshwater and estuarine habitats would have opened due to glacial retreat from previously ice-covered areas (Powers et al., 1986). Fundulus heteroclitus populations able to colonize northern habitats therefore faced opportunities for freshwater invasion without competitionfromindigenous fish species, in contrast to the situation further south. The role of natural selection infreshwaterinvasion events has been explicitly demonstrated (Lee and Petersen, 2002) and numerous accounts attest to the selective advantage of euryhalinity (Lee and Bell, 1999). However, the physiological adaptations necessary for brackish water and marine fish to invade freshwater are unclear. Intraspecific comparison of the mechanisms maintaining ion balance in F.  heteroclitus populations in freshwater may identify factors of selective importance for freshwater adaptation. The hyposmotic nature of freshwater environments (typically <10 mOsmol l") 1  favours ion efflux from fish, because they maintain substantially higher body fluid osmolality (300-350 mOsm l"). The largest component of ion efflux in freshwater fish 1  likely occurs across the gills due to their high surface area. Fish in freshwater therefore decrease the paracellular permeability across the gill epithelium, primarily by increasing the thickness of tight junctions between mitochondria-rich (MR) cells and neighbouring cells (Sardet et al., 1979; Ernst et al., 1980). Fish also decrease transcellular permeability by inactivating ion secretion pathways (Marshall et al., 1993, 1998, 2000; Scott et al., 2004a and Chapter 2 of this thesis). To counteract ion efflux and maintain ionic homeostasis, fish in freshwater absorb ions across the gills (see reviews by Wood and Marshall, 1994; Perry, 1997; Evans et al., 1999; Marshall, 2002). Sodium absorption by killifish gills likely involves a basolateral Na ,K -ATPase and an apical Na ,H -exchanger (Patrick and Wood, 1999; Claiborne et +  +  +  +  al., 2002; Scott et al., 2004a and Chapter 2), but may also involve basolateral H*-ATPase (Katoh et al., 2003). Unlike the majority offishin freshwater, F. heteroclitus does not actively absorb chloride, and thus maintains chloride balance through unique and yet undefined mechanisms (Patrick and Wood, 1999). The objective of this study was to compare the ionoregulatory ability of individuals from northern and southern populations of F. heteroclitus after direct salinity transfer. The ionoregulatory ability of each population was assessed by measuring survival, plasma ions, mRNA expression, protein activity, ion flux, paracellular  52  permeability, gill morphology, and aspects of renal function after transfer from nearisosmotic brackish water (10 g l") to freshwater. 1  Materials and Methods  Experimental Animals  Adult killifish (Fundulus heteroclitus L.) of the northern subspecies (F.h. macrolepidotus) were captured from Hampton, New Hampshire. Adults of the southern subspecies (F.h. heteroclitus) were captured from either Whitney Island, Florida (salinity transfer and flux experiments) or New Brunswick, Georgia (microscopy experiment). For the salinity transfer and microscopy studies, fish were maintained in indoor holding facilities in synthetic brackish water (10 g 1"; Deep Ocean, Energy Savers, Carson, CA, 1  USA) made up in dechlorinated Vancouver city tap water ([Na ], 0.17 mmol l" ; [Cl"], +  1  0.21 mmol l" ; hardness, 30 mg l" as CaCOs; pH 5.8-6.4) in static filtered glass aquaria. 1  1  Before sampling, fish were maintained for at least 30 days in this brackish water at an ambient temperature of 21-24 °C and a 14L:10D photoperiod. Fish were fed commercial trout chow (PMI Nutrition International, Brentwood, MO, USA: 2.2% calcium, 0.8% chloride, 0.5% sodium, 0.5% potassium, 0.2% magnesium) at an approximate daily ration of 1-2% (food mass/body mass). Treatment of animals was conducted according to University of British Columbia and McMaster University animal care protocols #A01-. 0180 and #02-10-61, respectively.  Salinity Transfer Experiment  54  Some of the data collected in this salinity transfer experiment have been previously reported for the northern subspecies alone (Scott et al., 2004a and Chapter 2). Eight northern and southern control fish were sampled after acclimation to 10 g l" , after 1  whichfishfrom each population were quickly transferred by net to aquaria containing freshwater (0 g I"; composition as above) or brackish water (10 g l"). Individual fish 1  1  were subsequently sampled by netting at 3h, 8h, 24h, 96h, 14 days, and 30 days after transfer from brackish water. Thefishwere stunned by cephalic blow, blood samples were collected in heparinized capillary tubes from the severed caudal peduncle, and the fish were then killed by rapid decapitation. Blood was centrifuged at 13000 x g for 10 minutes and plasma was frozen in liquid nitrogen. Second and third gill arches were immediately frozen in liquid nitrogen. All tissues were stored at -80°C until analysed.  Total RNA Extraction and Reverse Transcription  Total RNA was extracted from tissues (approximately 20 mg) using Tripure isolation reagent (Roche Diagnostics, Montreal, QC, Canada), following the manufacturer's instructions. RNA concentrations were determined spectrophotometrically and RNA integrity was verified by agarose gel electrophoresis (~1% [wt vol"] agarose:Tris-acetate EDTA). Extracted RNA samples were stored at 1  80°C following isolation. First strand cDNA was synthesized by reverse transcribing 3 ng total RNA using 10 pmoles oligo(dTig) primer and 20U RevertAid H Minus M-MuLV reverse transcriptase (MBI Fermentas Inc., Burlington, ON, Canada) following the manufacturer's instructions.  Real-Time PCR Analysis of Gene Expression  Primers for killifish Na ,K -ATPase +  +  <Xi  a  (Acc. No. AY057072), cystic fibrosis  transmembrane conductance regulator (CFTR) Cl" channel (Acc. No. AF000271), Na,K,2Cl"-cotransporter 1 (NKCC1; Acc. No. AY533706), and elongation factor la +  +  (EFla, expression control; Acc. No. AY430091) were designed using Primer Express software (version 2.0.0, Applied Biosystems Inc., Foster City, CA, USA) and are reported in Scott et al. (2004a) and Chapter 2 of this thesis. Gene expression was quantified using quantitative real-time PCR (qRT-PCR) on an ABI Prism 7000 sequence analysis system (Applied Biosystems). PCR reactions contained 1 pi of cDNA, 4 pmoles of each primer and Universal SYBR green master mix (Applied Biosystems) in a total volume of 21 pi. All qRT-PCR reactions were performed as follows: 1 cycle of 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min (set annealing temperature of all primers). PCR products were subjected to melt curve analysis to confirm the presence of a single amplicon and representative samples were electrophoresed to verify that only a single band was present. Control reactions were conducted with no cDNA template or with non-reverse transcribed RNA to determine the level of background or genomic DNA contamination, respectively. Genomic contamination was below 1:49 starting cDNA copies for all templates. A randomly selected control sample was used to develop a standard curve for each primer set, and all results were expressed relative to these standard curves. Results were then standardized to EFla, a gene for which mRNA expression in the gills does not  change following salinity transfer (data not shown), and were expressed relative to the matched brackish water controls within each time point. A l l samples were run in duplicate (coefficients of variation were <10%). Because significant changes in gene expression due to freshwater transfer were observed in the northern population only at 24h, 96h, and 14 days post-transfer by Scott et al. (2004a, see Chapter 2), gene expression was only quantified at these time points (and pre-transfer) in the southern population.  Na ,K*-ATPase Activity  Na ,K -ATPase activity was determined by coupling ouabain-sensitive ATP +  +  hydrolysis to pyruvate kinase- and lactate dehydrogenase-mediated N A D H oxidation as outlined by McCormick (1993). For this assay, second and third gill arches were homogenized in 500 pi of SEI buffer (150 mmol l" sucrose, 10 mmol l" EDTA, 50 mmol 1  1  l" imidazole, pH 7.3) containing 0.1% Na-deoxycholate and centrifuged at 5000 x g for 1  30s at 4 °C. Supernatants were immediately frozen in liquid nitrogen and stored at -80°C until analyzed. ATPase activity was determined in the presence or absence of 0.5 mmol f 1  ouabain using 10 ul supernatant thawed on ice and was normalized to total protein  content (measured using the bicinchoninic acid method, Sigma-Aldrich, Oakville, ON, Canada). A l l samples were run in triplicate (coefficients of variation were always <10%). Na ,K -ATPase activity, measured as ouabain-sensitive ATPase activity is expressed as +  +  umol ADP mg protein" h" . 1  1  Freshwater Flux Experiments  Killifish were held at McMaster University for at least 30 days before experimentation in 10 g l" brackish water (made up in dechlorinated Hamilton city tap 1  water) at room temperature (-21 °C), and were maintained at a 14L:10D photoperiod. Freshwater was prepared to approximate Vancouver city tap water by mixing appropriate amounts of dechlorinated Hamilton water and reverse-osmosis water (measured final composition, [Na ], 0.17 mmol l" ; [Cl"], 0.20 mmol l" ). Static polyethylene flux +  1  1  chambers were, fitted with a lid and aeration line, and were wrapped in black plastic to minimize disturbance of fish. Each chamber contained 270 ml of freshwater and 37 kBq of either N a (NEN Life Science Products Inc., Boston, M A , USA) or C1 (ICN 22  36  Biomaterials, Irvine, CA, USA) isotope at the start of each flux period. Unidirectional and net N a flux rates and net Cl" flux rates were measured over +  the first 8h after transfer to freshwater and at 1 and 4 days post-transfer in one experiment, and also at 8 and 14 days post-transfer in a second experiment. Unidirectional Na influx was measured by monitoring the disappearance of N a isotope 22  from the water. In the first experiment, ten fish from each population were transferred to individual freshwater flux chambers containing isotope and duplicate water samples (2 X 5 ml for N a radioactivity measurements) were taken every 30 minutes for the first 8h 22  after transfer. Additional flux measurements were made 1 and 4 days after transfer on the same fish. For these flux periods, duplicate water samples were taken every hour for 4h after the addition of isotope. In the second experiment, ten killifish from each population were first transferred to static aerated freshwater tanks for 7 days and then moved to flux  58  chambers. Flux periods at 8 and 14 days after transfer were then conducted as before, with water samples being taken every hour for 4h after the addition of isotope on days 8 and 14. In both experiments, water in the flux chambers was replaced between flux periods with clean freshwater (containing no radioactivity, at least once daily) to remove waste and excess isotope. Unidirectional Cl" influxes from F. heteroclitus in freshwater are normally extremely low and cannot be determined by measuring the disappearance of isotope from the water. They must instead be measured by quantifying isotope appearance in the fish, which is a more sensitive technique (Patrick et al., 1997; Wood and Laurent, 2003). Unidirectional Cl" influx was therefore determined 8 days after freshwater transfer by measuring whole-animal uptake of C1. Eight killifish from each population were first 36  held in static aerated freshwater tanks for 8 days, then moved to flux chambers containing isotope. Water samples were taken immediately after the addition of C1 (just before fish 36  were added), andfishand water were sampled 4h later. At the end of both Na and C1 22  36  experiments,fishwere rinsed by allowing live animals to ventilate their gills (for at least 5s) in freshwater containing no radioactivity, and were then sacrificed. Radioactivity (counts per minute, cpm) was determined in eachfishand in each water sample, as was total water Na and Cl' concentrations. +  Unidirectional Na influx rates +  (Ji , a) n  N  in umol kg" h" were calculated as  S J in,Na  SAxW  1  1  (1)  where E is the slope of the regression line of radioactivity versus time (in cpm h" ), SA is the mean external specific activity (in cpm pmol"), and W is the body weight in (kg). 1  Unidirectional Cl" influx rates (Ji ,ci) were calculated as n  Efish Jin.ci  (2)  —  SA x W x t where Zf h is the total radioactivity in the fish (in cpm) and t is the flux period duration ls  (inh). Average net Na and Cl" flux rates +  (J t,ion) ne  in pmol kg" h" were calculated as 1  1  [ion]; - [ion]  (3)  f  Jnet.ion  V  Wxt where [ion]i and [ion]f are the concentrations of Na or Cl" in the water at the start and +  end of the flux period (in pmol l' ), respectively, V is the mean flux chamber volume (in 1  1). By conservation of mass, unidirectional efflux rates  (J t,ion) ou  in urnol kg" h" were 1  1  calculated as Jout,ion  Jnet,ion " Jin,ion  (4)  Because the above calculations assume there is no "backflux" of radioisotopefromthe fish into the surrounding media, which can be a significant source of error, internal specific activity within the fish must remain low compared to external specific activity of the medium. At the end of each flux experiment the ratio of internal to external specific activity was therefore verified to be <10% (Kirschner, 1970). A method adapted from Curtis and Wood (1991) was employed to study the diffusive permeability of thefishto a paracellular permeability marker 8 days after freshwater transfer. The technique monitors the appearance of radiolabeled polyethylene  glycol ([H]PEG-4000) in the external water relative to the radioactivity of a terminal 3  plasma sample, from which PEG-4000 clearance rates can be calculated. Radioactivity appearing in discrete pulses represents bouts of urination from the urinary bladder (renal PEG-4000 clearance), whereas radioactivity not appearing in pulses represents diffusion across the gills and body surface (extra-renal PEG-4000 clearance). [H]PEG-4000 is the 3  marker of choice for glomerular filtration rate infish(Beyenbach and Kirschner, 1976), and its renal clearance rate is considered equivalent to the glomerularfiltrationrate (GFR). On day 7, approximately 16h prior to measurements on day 8, killifish were injected intraperitoneally with 1 |ll g" of [H]PEG-4000 (NEN Life Science Products) in 1  3  140 mmol l" NaCl (111 kBq uT ) and left in their individual containers for the label to 1  1  equilibrate overnight throughout the extracellular compartment. On day 8, the water was changed, the volume set to 250 ml, the aeration set to produce good mixing, and the fish left to settle for a further 60 minutes. Thereafter, a 1 ml water sample was drawn from eachfishcontainer at exactly 5 minute intervals for the next 6-7 h, after which a blood sample was taken and plasma separated as described earlier. Plots of total water [ H] radioactivity against time revealed clear step-wise 3  increases attributable to bouts of urination, and the sum of all radioactivity (cpm) appearing in these pulses (by renal excretion) was subtracted from the total appearance (cpm) over the 6-7 h period to yield extrarenal excretion (cpm). Dividing each of these values by plasma radioactivity (cpm uT ), time (h), and body mass (kg) yielded renal 1  clearance rate of PEG-4000 (equivalent to glomerularfiltrationrate, in ml kg" h") and 1  1  extrarenal clearance rate of PEG-4000 (in ml kg" h"), the latter providing an index of the 1  1  diffusive permeability of thefishto a paracellular permeability marker. As terminal urine  61  samples for radioactivity counting could not be obtainedfromthese smallfish,urine flow rate could not be determined (c.f., Curtis and Wood, 1991), but urination frequency (bursts h") could be calculated. 1  Ion and Radioactivity Measurements  Sodium concentrations of plasma and water samples were determined using flame atomic absorption spectrophotometry (SpectrAA-220FS, Varian, Mulgrave, VC, Australia) with Fisher Scientific (Nepean, ON, Canada) certified standards. Chloride concentrations of plasma and water samples were measured colorimetrically (Zall et al., 1956) with Radiometer (Copenhagen, Denmark) certified standards. Na radioactivities 22  infishand water samples were determined using a Minaxi Autogamma 5000 counter (Packard Instruments, Downers Grove, IL, USA). For C1 radioactivities in whole fish, 36  animals were digested in three volumes of 1 mol l"  1  HNO3  at 60 °C for 48 h. These  samples were centrifuged, supernatants (1 ml) were added to 5 ml of an acid-compatible scintillation cocktail (Ultima Gold; Packard Bioscience, Meriden, CT, USA), and radioactivity was measured by scintillation counting (Rackbeta 1217; LKB Wallac, Turku, Finland). C1 radioactivities in water samples (5 ml) were also measured by 36  scintillation counting in 10 ml of scintillation fluid (ACS; Amersham, Piscataway, NJ, USA), and data were corrected for the slight difference in counting efficiencies between the two scintillation fluors. H radioactivities in water (1 ml) and plasma (10-20 pi) 3  samples were measured by scintillation counting in a standard volume ratio of 1 ml of water (or diluted plasma) to 5 ml ACS (quench was shown to be uniform across samples).  62  Scanning Electron Microscopy  Second and third gill arches of killifishfromeach population were sampled before (n = 3) and 8 days after transfer to Vancouverfreshwater(n = 5), as described above. Gills were fixed for 24h in 0.1 mol l" phosphate-buffered saline (pH 7.4) containing 2% 1  paraformaldehyde and 2% glutaraldehyde. After fixation, samples were post-fixed in 0.1 mol f cacodylate buffer (pH 7.4) containing 1% OsCU. Tissues were dehydrated 1  progressively in ethanol (70, 85, 95, and 100%) for 10 min in each solution. Gill arches were dried in hexamethyldisilazane and sputter-coated with gold. Images collected by scanning electron microscopy were analyzed using a method similar to Daborn et al. (2001). Random locations on the afferent-vascular edge of gill filaments were observed at 3000X magnification. Apical crypts, freshwater-type MR cells ('chloride cells'), and 'intermediate' cells were counted for at least 10 different locations throughout the gills. Averages were calculated for eachfishand expressed as density mm" .  Statistical Analyses  Data are expressed as means ± SE. Kruskal-Wallis H non-parametric ANOVA was used to determine overall differences as a function of time (for each salinity) for each variable except survival. Mann-Whitney U non-parametric comparisons were then used to compare between salinities (within populations at each time) or between populations  (at each salinity and time). Survival between sampling times was compared using the one-tailed Fisher's exact test. Statistical analyses were conducted using SPSS version 10.0 and a significance level of p<0.05 was used throughout.  64  Results  Survival  There were pronounced differences between the survival of northern and southern killifish (Fig. 3-1). Individuals from the northern population had very low mortality after both freshwater (98% survival after 30 days) and brackish water control (100% survival) transfer. In contrast, southern individuals suffered substantial mortality after freshwater transfer compared to brackish water controls (94% survival), starting 5 days into freshwater and stabilizing after 11 days (81% survival). The mortalities experienced by brackish water-transferred northerns, brackish water-transferred southerns, and freshwater-transferred northerns were statistically indistinguishable; significantly elevated mortality was detected between 4 and 14 day sampling times in freshwatertransferred southerns (p < 0.05).  Plasma Ions  The effect of freshwater transfer on plasma ions differed between populations in a manner consistent with the differences in mortality (Fig. 3-2). Freshwater transfer decreased plasma Na compared to brackish water controls in both populations, but this only occurred 1 day after transfer of northern killifish while southern killifish had decreased plasma levels 3h, 8h, and 1 day into freshwater (Fig. 3-2A). Plasma Na balance was re-established 4 days after transfer in both populations.  65  Fig. 3-1. Survival of northern (black) and southern (grey) killifish after transfer to either brackish water (BW; 10 g l") or freshwater (FW). 1  66  A  pre  0  1  2  4  10  20  30  10  20  30  Time (days)  B  pre  0  1  2  4  Time (days)  Fig. 3-2. Plasma sodium (n > 5) (A) and chloride (n > 5) (B) levels in northern (black) and southern (grey) killifish before (pre) and after transfer from brackish water (BW; 10 g l" ) to either brackish water (square symbol) or freshwater (FW; triangle). Data are expressed as means ± SE. * Significant difference from time-matched brackish water control (p < 0.05). 1  67  Differences in plasma Cl between populations after freshwater transfer were more pronounced than differences in plasma Na (Fig. 3-2B). Northern killifish maintained plasma Cl balance at all times after freshwater transfer. In contrast, southern killifish in freshwater had lower plasma Cl at 1, 4, and 14 days after transfer compared to brackish water controls.  Gene Expression and Protein Activity  Na ,K -ATPase regulation in the gills afterfreshwatertransfer differed between +  +  northern and southern populations of F. heteroclitus. There was a prolonged increase in the relative Na ,K -ATPase 0Ci mRNA expression in individualsfromthe northern +  +  a  population, which peaked at 4 days in freshwater at nearly 6-fold above time-matched brackish water controls (Fig. 3-3A). Individualsfromthe southern population increased Na ,K -ATPase +  +  oci  a  expression to a lesser extent, to only 4-fold controls at 4 days after  transfer. Unlike northern killifish, southern killifish did not increase expression at 1 or 14 days into freshwater. Changes in Na ,K -ATPase activity in the gills as a result offreshwatertransfer +  +  also differed between populations. Activity increased 2-fold at 1 and 4 days following freshwater transfer in northern killifish (Fig. 3-3B), while no significant increases occurred after transfer in southern killifish (Fig. 3-3C). Activity also reached higher absolute levels in northern killifish (3.5 + 0.5 and 2.9 ± 0.3 urnol mg protein" h" at 8h in 1  northerns and southerns, respectively).  1  Fig. 3-3. Fold-change in Na ,K -ATPase oti mRNA expression (n > 7) (A) as well as Na ,K -ATPase activity (n > 6) (B,C) in northern (black) and southern (grey) killifish gills before (pre) and after transfer from brackish water (BW; 10 g l" ; square symbol) to either brackish water or freshwater (FW; triangle). Expression data are standardized to elongation factor la, and are relative to time-matched brackish water controls (see Materials and Methods). Data are expressed as means ± SE. * Significant difference from time-matched brackish water control. + Significant difference from northern population (p < 0.05). +  +  a  +  +  1  Time (days)  70  Relative expression of the seawater ion transporters NKCC1 and CFTR decreased at 1 and 4 days after freshwater transfer in both populations, dropping to approximately 2.5- and 10-fold below brackish water controls 1 day after transfer, respectively (Table 31). Decreased expression of these genes persisted longer in southern individuals, however, remaining below controls 14 days into freshwater.  Ion Fluxes and PEG-4000 Clearance Rates  There were only small differences in Na fluxes between northern and southern +  populations following freshwater transfer. Both northern and southern fish decreased Na  +  efflux rapidly after freshwater transfer (Fig 3-4A). Unidirectional efflux and negative net flux were significantly decreased 4-8h after transfer compared to the 0-4h flux period, and this decrease persisted until at least 14 days after transfer. Both populations also increased unidirectional Na influx progressively after freshwater transfer. By 14 days +  after freshwater transfer, however, northern killifish had significantly higher unidirectional influx and net flux. In fact, northernfishincreased Na influx by 5-fold 14 +  days post-transfer compared to the 0-4h flux period, while southerns increased influx by only 3.3-fold. Net flux was also slightly higher in northernfishafter 96h in freshwater. We observed large differences in net Cl" flux between northern and southern killifish (Fig. 3-4B). Althoughfishfrom both populations decreased Cl" loss initially, northern killifish eliminated loss by 1 day after transfer and this was maintained at least 14 days in freshwater. In contrast, southern killifish did not appear to decrease Cl" loss below 100 umol kg" h", and significantly differed from northern killifish at 1 and 14 1  1  71  Table 3-1. Fold-Changes in Gill mRNA Expression After Freshwater Transfer of Seawater Ion Transporter Genes Measured by qRT-PCR  Time Gene NKCC1  CFTR  Population  24h  96h  14d  Northern  0.44 ± 0.06*  0.72 ± 0.09*  1.34 ±0.21  Southern  0.47 ± 0.09*  0.55 ±0.15*  0.59±0.04*'  Northern  0.11 ±0.01*  0.59 ± 0.07*  0.96 ± 0.20  Southern  0.14 ±0.03*  0.64 ±0.15*  0.38 ± 0.08*  +  ,+  Values are means ± SE. n > 7. qRT-PCR expression data are relative to time-matched brackish water control at the respective time point. * Significant differencefrombrackish water control at the respective time point (p<0.05). Significant differencefromnorthern population (p<0.05). +  72  A  -1000  J-T  1  1  1  0  1  1  R  8  4  14  Time (days)  B 200  1 I  a, -200  +  « -—»  •  or X  =  LL  £  -1  -400 -\  -600  Northern Net Flux Southern Net Flux  IT 4  8  14  Time (days)  Fig. 3-4. Total (net) flux and unidirectional fluxes of Na (A) and total Cl" flux (B) in northern (black and white) and southern (grey and hatched white) killifish after transfer from brackish water (10 g l") to freshwater (n > 9). Positive values represent influx (J ). + Significant difference from northern population (p < 0.05). +  1  in  days after transfer. Unidirectional Cl" influx was small (less than 3% of unidirectional Na influx, Fig. 3-4A) and identical between populations at 8 days post-transfer (Table 3+  2). Patterns of PEG-4000 clearance differed substantially between killifish populations at 8 days post-transfer (Table 3-2). Extrarenal clearance rates, which represent the general paracellular permeability of the gills and body surface, were 3-fold higher in southern killifish. Renal clearance was higher in northern killifish, which had 1.6-fold greater glomerularfiltrationrates (i.e., renal PEG-4000 clearance rates) and 1.5fold more frequent bursts of urination, though actual urine flow could not be calculated.  Gill Morphology  The gills of both northern and southern killifish had a similar morphology in brackish water (Fig. 3-5A,B). Apical crypts were abundant in both populations (-2000 mm" ), while freshwater-type MR cell density remained low (Fig. 3-6). 'Intermediate' cells, characterized by features that are midway between seawater (apical crypt) and freshwater (flat surface equipped with microvilli) morphologies were equally abundant between populations and salinities. Northern and southern killifish had different gill morphologies after transfer to freshwater (Fig. 3-5C,D). Apical crypt density in northern killifish gills was 15-fold lower in freshwater than in brackish water, while southern killifish in freshwater had an apical crypt density only 3-fold lower than those in brackish water (Fig. 3-6). The  74  Table 3-2. Unidirectional Ct Influx, Extrarenal and Renal ^H]PEG-4000 Clearance Rates, and Urination Frequency 8 days After Freshwater Transfer  Northern Population  Southern Population  Cl" Influx (umol kg" h")  9.2 ±2.3  6.6 ± 1.7  ECR (ml kg" h")  1.3 ±0.2  4.3 ± 0.3  RCR (ml kg" h")  6.3 ±0.8  3.9±0.5  1  1  1  1  1  1  Urination Frequency (bursts h") 1  1.1 ±0.1  +  +  0.7±0.1  +  Values are means ± SE. n = 8. Unidirectional Cl" influx was measured by monitoring the appearance of C1 in the whole body of the fish. Significant difference from northern 36  +  population (p<0.05). ECR, extrarenal clearance rate (an index of paracellular permeability); RCR, renal clearance rate (glomerular filtration rate).  Fig. 3-5. Representative scanning electron micrographs of the gills of northern (A,C) and southern (B,D) killifish before ( A , B ) and 8 days after transfer from brackish water (10 g F ) to freshwater ( C , D ) . Apical crypts (arrow), freshwater-type mitochondria-rich cells (arrowhead), and cells having intermediate morphology (asterisk) are apparent (scale bar, 10 um). (E) Enlarged view of freshwater-type mitochondria-rich cell (scale bar, 1 um).  76  BW  FW  FW Chloride Cells  BW  FW  Intermediate  BW  FW  SW Apical Crypts  Cell Type  Fig. 3-6. Freshwater-type mitochondria-rich (chloride) cell, 'intermediate' cell, and apical crypt density on the apical surface of northern (black) and southern (grey) killifish gills before (n = 3) and 8 days after transfer from brackish water (10 g l") tofreshwater(n = 5). Data are expressed as means ± SE. * Significant difference from brackish water control. + Significant difference from northern population (p < 0.05). 1  abundance of freshwater-type MR cells increased significantly in both populations after freshwater transfer (-3000-4000 mm"). 2  Discussion  Killifish typically encounter fluctuations in environmental salinity on a daily and seasonal basis in their natural habitat, and must therefore modulate ion flux across the gills to maintain ion balance (Marshall, 2003). In addition, we have observed substantial intraspecific variation between populations of F. heteroclitus, leading to large differences in freshwater tolerance. Individuals of the southern population suffered significantly greater mortality after prolonged freshwater transfer than did northerns. The eventual stabilization of mortality suggests that some southern individuals may be more freshwater tolerant than others, but even those southern individuals that survived experienced greater fluctuations in plasma ion levels (particularly plasma Cl") than did individuals of the northern population. The mechanistic differences between these populations that could have accounted for greater freshwater tolerance in northern killifish were numerous, and suggest that the coordinated physiological response after freshwater transfer is somewhat reduced in the southerns. We speculate that northern killifish are better adapted to freshwater environments, and that these identifiable differences between populations provide insight into the mechanisms of freshwater adaptation in fish.  Regulation of Na Balance +  There were small differences in the ability of northern and southern killifish populations to regulate Na balance after freshwater transfer. Plasma Na levels in +  +  individuals from the southern population decreased for a longer period after transfer  79  when compared to those from the northern population, and this decrease appeared to be of greater magnitude. The cause of this difference is unclear, as Na fluxes did not differ +  between populations until 96h after transfer. Differences in water flux across the gills (Robertson and Hazel, 1999), drinking rates (Potts and Evans, 1967), or renal function (see below) might have accounted for differences in plasma Na between populations. +  Rapid re-establishment of plasma Na balance in northern killifish after +  freshwater transfer has been observed numerous times, demonstrating the excellent euryhalinity of these animals. Transfer from seawater to freshwater initially decreases plasma Na as early as 4h after transfer, but pre-transfer seawater levels are quickly +  restored 1-2 days into freshwater (Jacob and Taylor, 1983; Katoh and Kaneko, 2003). Northern killifish transferredfromfreshwaterto seawater re-establish plasma Na with +  equal rapidity (Jacob and Taylor, 1983; Marshall et al., 1999). We have observed similar results after transferfromnear-isosmotic brackish water to eitherfreshwateror seawater (Scott et al., 2004a and Chapter 2), suggesting that individuals of the northern population canfreelymove betweenfreshwaterand seawater environments. Northern killifish increased both the relative mRNA expression (ai -isoform) and a  protein activity of gill Na ,K -ATPase by a greater magnitude and for a longer duration +  +  after freshwater transfer than did southern killifish. Mitochondria-rich cells in killifish gills are known to proliferate after freshwater transfer (Katoh et al., 2003). An increase in the abundance of cells expressing high levels of Na ,K -ATPase may have therefore +  +  contributed to the Na ,K -ATPase upregulation infreshwater.Furthermore, differences in +  +  relative Na ,K -ATPase expression between populations may have arisen from lower cell +  +  proliferation in southernfishafterfreshwatertransfer. In support of this hypothesis,  80  northern killifish gills had a high density of freshwater-type MR cells (as previously reported by Hossler et al., 1985), while density appeared to be lower in the gills of southern killifish in freshwater. The regulation of ion flux across fish gills by changes in ion transporter expression is generally assumed but infrequently tested (e.g., Sullivan et al., 1995; 1996). In this regard, we speculate that the differences in Na ,K -ATPase gene regulation +  +  between northern and southern killifish were at least partly responsible for the observed differences in Na flux. Northern killifish had greater changes in Na ,K -ATPase +  +  +  expression, which occurred concurrent with more positive net flux at 1 and 14 days and greater unidirectional Na influx at 14 days after freshwater transfer. Therefore, these +  data provide evidence for how active ion flux can be modulated by ion transporter gene regulation. Even though differences in Na ,K -ATPase expression and activity appear to exist +  +  between F. heteroclitus populations after freshwater transfer, the resultant differences in Na flux were small. Both populations initially suffered high Na efflux, at rates +  +  comparable to other Fundulus species (Pang et al., 1974); interestingly, our initial rates in F. heteroclitus are intermediate between F. diaphanus, afreshwaterspecies, and F. majalis, a seawater species. Efflux decreased rapidly after freshwater transfer, however, such that net loss was nearly eliminated by 24h after transfer in both F. heteroclitus populations. Similarly rapid reductions in Na efflux have been previously observed for +  F. heteroclitus (Motais et al., 1966; Potts and Evans, 1966; Pic, 1978; Wood and Laurent, 2003) as well as F. kansae (Potts and Fleming, 1971) after transferfromsaline water to freshwater.  81  Along with reductions in passive Na efflux, Na influx increased progressively in +  +  both populations after freshwater transfer, and by 14 days reached levels 3- to 5-fold above the initial influx. These influx rates are somewhat lower than previous reports in northern killifish, both shortly after transfer (Wood and Laurent, 2003) and after freshwater acclimation (Potts and Evans, 1966; 1967; Patrick and Wood, 1999). This difference is undoubtedly explained by the lower Na levels in our freshwater (0.17 mmol +  l") compared to these other studies (2-5-fold higher), because Na influx is critically 1  +  dependent on environmental Na in this concentration range (Patrick et al., 1997). Taken +  together, the results discussed above suggest that differences in Na regulation between +  populations of F. heteroclitus are small, and are unlikely to account for the pronounced differences in mortality in freshwater.  Regulation of Ct Balance  Northern and southern killifish populations differ significantly in their ability to regulate Cl" in freshwater, which likely contributes to the large differences in mortality they experienced after transfer. Northernfishactually appear to regulate Cl" levels more strictly than Na levels, as plasma Cl" was maintained for at least 30 days after freshwater +  transfer. This is in agreement with previous reports for northern killifish. For example, in a study by Jacob and Taylor (1983), transfer from seawater to freshwater decreased serum osmolality transiently, which was almost entirely accounted for by changes in serum Na . In contrast, we observed that southern killifish rapidly lost Cl" balance after +  freshwater transfer, as plasma levels fell quickly after transfer and were not re-  established. It is possible that these decreases in Cl" levels were sufficient to cause mortality: similar decreases in plasma Cl" levels have been observed in long-horned sculpin (Myoxocephalus octodecimspinosus) after transfer to hyposmotic environments, and these decreases were associated with greater mortality (Claiborne et al., 1994). In addition, Cl" imbalance would have created a 'strong ion' difference in the plasma of southern killifish (i.e., [Na ] > [Cl"]). To maintain charge neutrality, compensatory +  increases in plasma [ H C O 3 ] and pH may have occurred in southern fish, so the resulting blood alkalosis might have also contributed to the differences in mortality between populations. The observed differences in plasma Cl" between northern and southern killifish were likely due to differences in Cl" flux. Because unidirectional Cl" influx is extremely small in F. heteroclitus in freshwater (this study and Patrick et al., 1997; Patrick and Wood, 1999; Wood and Laurent, 2003), changes in total Cl" flux after freshwater transfer are primarily dictated by changes in unidirectional Cl" efflux. Shortly after transfer, individuals from both populations reduced passive Cl" loss, which is consistent with previous results (Pic, 1978). Northern populations continued to decrease Cl" efflux, such that Cl" loss was eliminated by 24h following freshwater transfer and remained negligible thereafter. This rapid elimination of Cl" loss likely accounts for the ability of northern killifish to maintain plasma Cl" balance in spite of negligible branchial uptake. Southern killifish did not eliminate Cl" efflux, which remained consistently greater than 100 umol kg" h", so they were unable to preserve Cl balance. 1  1  In order to eliminate Cl" efflux, killifish entering freshwater must eliminate both active and passive routes of Cl" excretion. Active secretion of ions across fish gills as  occurs in seawater involves a basolateral Na ,K -ATPase, a basolateral Na ,K ,2Cf+  +  +  +  cotransporter (NKCC1), and an apical cystic fibrosis transmembrane conductance regulator (CFTR) Cl" channel (see reviews by Wood and Marshall, 1994; Perry, 1997; Evans et al., 1999; Marshall, 2002). One and four days after freshwater transfer, individualsfromboth populations decreased gill mRNA expression of the seawater transporters NKCC1 and CFTR. In fact, this suppression actually persisted for a longer duration in southern killifish, so differences in active Cl" secretion are unlikely to account for the observed differences in Cl" efflux. Populations of F. heteroclitus appear to differ in their ability to eliminate passive Cl" loss. The higher extrarenal clearance rate of PEG-4000 in southern killifish indicates that the paracellular permeability of their gills is higher; increased Cl" loss may have therefore occurred through the paracellular pathway. The observed differences in gill morphology between populations are consistent with this hypothesis. A typical morphological feature of the gills of fish in seawater is the presence of apical crypts, which are formed by multicellular complexes of MR cells that share shallow junctions with high solute permeability. Both populations have equal abundance of apical crypts in brackish water, at densities approximately 7-8-fold lower than in seawater-acclimated animals (Hossler et al., 1985). However, southern killifish have significantly more apical crypts in freshwater, and thus maintain morphological features of the seawater gill. After transfer of northern killifish to hyposmotic environments, apical crypts are either covered over by pavement cells (Daborn et al., 2001), or are widened to uncover freshwater-type MR cells equipped with microvilli (Katoh and Kaneko, 2003). Freshwater MR cells typically form tight junctions with neighbouring cells that have low paracellular  84  permeability to solutes (Sardet et al., 1979; Ernst et al., 1980). These morphological transformations occur in conjunction with rapid reductions in Cl" secretion (Daborn et al., 2001). More apical crypts on the gills of southern killifish therefore suggests that fewer crypts were covered and/or converted into freshwater-type MR cells; incidentally, there was a trend towards greater freshwater MR cell density in northern killifish. As well as the structural reorganization of cell-cell junctions that occurs during transformation between seawater and freshwater gill morphologies, associated changes occur in the actin cytoskeleton within MR cells. These morphological alterations to cytoskeletal elements are important for the changes in transepithelial conductance that occur after salinity change (Daborn et al., 2001). Interestingly, interactions between actin and Na ,K - ATPase appear essential for tight junction formation in epithelia +  +  (Rajasekaran and Rajasekaran, 2003), so differences in Na ,K -ATPase gene regulation +  +  between killifish populations may be related to differences in Cl' efflux through the paracellular pathway. In addition to greater Cl" efflux and paracellular permeability, southern killifish exhibited lower glomerular filtration rates and lower urination burst frequencies than did northerns in freshwater. High glomerular filtration rates and urination frequencies are characteristic of freshwater fish, while the opposite are characteristic of seawater fish (e.g., Hickman and Trump, 1969; Curtis and Wood, 1991; Sloman et al., 2004). These observations suggest that slower or incomplete acclimation of renal function may have been another factor contributing to the poorer ionoregulatory performance and survival of southerns in freshwater.  85  Possible Mechanisms of Freshwater Adaptation  Our data suggest that southern killifish are less tolerant of freshwater transfer than are northerns because the coordinated response of their gills and kidney is less effective at maintaining ion balance. This may include small differences in Na regulation, +  suggested by small differences between populations in plasma Na , Na ,K -ATPase +  +  +  expression and activity in the gills, ion flux, and possibly gill MR cell abundance. A more convincing cause for differences in mortality, however, is the differences in Cl" regulation. Southern killifish experience large decreases in plasma Cl", have significant Cl" efflux, and maintain a moderate density of apical crypts after freshwater transfer. In contrast, northern killifish maintain plasma Cl", eliminate Cl" efflux, have lower paracellular permeability, have very few apical crypts in freshwater, and more typical freshwater renal function. Taken together, southern killifish seem to preserve some elements of seawater ionoregulatory physiology, while northerns are better able to make the necessary adjustments forfreshwateracclimation. A great deal of evidence suggests that northern populations of F. heteroclitus have evolved greater freshwater ionoregulatory ability than have southern populations. This is indicated by differences in distribution patterns (Powers et al., 1993), reproductive success (Able and Palmer, 1988), and adult survival (this study) in hyposmotic environments. These differences may have arisenfromselection acting on pre-existing variability within F. heteroclitus, or possiblyfromintrogression of allelesfromsympatric freshwater species (e.g., F. diaphanus) (Dawley, 1992). Although the evolutionary pressures accounting for these differences in salinity tolerance are still uncertain, our data  suggest that minimizing Cl" imbalance was an essential evolutionary step allowing northern killifish to survive in freshwater. Freshwater acclimation was possible in northern killifish despite no apparent mechanism for branchial Cl" uptake. These animals may instead be able to survive in freshwater habitats and maintain Cl" balance by minimizing Cl" efflux and meeting Cl" demands through the diet (Wood and Laurent, 2003).  CHAPTER 4  SUMMARY, PERSPECTIVES, AND CONCLUSIONS  By undertaking a comprehensive analysis of the molecular and physiological responses of killifish to salinity transfer, and by comparing some of these characteristics between populations with different salinity tolerances, several important results were obtained. The patterns of gene expression in killifish gills following seawater and freshwater transfer give insight into mechanisms of ion transporter gene regulation, as well as how genomic responses complement other physiological changes that modulate ion transport. Gene regulation may in fact play a pronounced role in modulating ion transport rates during chronic salinity change. The patterns of gene regulation after salinity transfer also seem to support current suggestions for the mechanisms of ion transport by killifish gills. However, many of these and other physiological characteristics differ between killifish populations, and suggest possible mechanisms of freshwater adaptation in fish from northern populations.  Gene Regulation After Salinity Transfer  Rapid modulation of ion flux across the gills is an important response of killifish to acute salinity change, such as what might occur on a daily basis in estuaries (Marshall, 2003). These rapid changes in flux likely occur through post-translational processes, including intracellular trafficking and phosphorylation of various ion transporters  88  (Marshall et al., 2000, 2002; Hoffmann et al., 2002), and rapid changes in gill epithelium morphology (Daborn et al., 2001). When salinity change persists for a longer duration, however, additional mechanisms regulating ion transport may become important. For example, prolonged transfer to seawater or freshwater appears to involve changes in the abundance of mitochondria-rich cells in the gill epithelium (Katoh and Kaneko, 2003). In addition, by performing a comprehensive analysis of ion transporter expression in the gills of northern killifish after both seawater and freshwater transfer (Chapter 2), I have demonstrated that gene regulation may be especially important for regulating ion balance after prolonged salinity transfer. The patterns of gene expression I observed after seawater transfer suggest that the mechanisms of gene regulation might differ for different ion transporters. The expression of two transporters, Na ,K -ATPase and NKCC1, were well correlated, but showed +  +  patterns of expression different from the CFTR Cl" channel. This disparity suggests that there may be different mechanisms of regulation between the basolateral and apical transporters. Interestingly, in seawater-acclimated Hawaiian goby (Stenogobius hawaiiensis), all MR cells expressing Na ,K -ATPase also expressed NKCC, but only a +  +  subset of Na ,K -ATPase-positive MR cells expressed CFTR (McCormick et al., 2003). +  +  Furthermore, increasing salinity had little effect on Na ,K -ATPase and NKCC +  +  immunoreactive cell number, but substantially increased the number of cells expressing CFTR. Taken together, these results agree with mine to suggest that there is differential regulation of apical (CFTR) and basolateral (Na ,K -ATPase and NKCC) ion transporters +  in seawater.  +  89  Freshwater transfer decreased the expression of NKCC 1 and CFTR, suggesting that the predominant role for these ion transporters is in salt secretion. This likely occurs along with intracellular trafficking that leads to transporter internalization (Marshall et al, 2002) and decreases in NKCC and CFTR activity (Zadunaisky et al., 1995; Marshall et al., 1998; Marshall et al., 2000). The gills of killifish therefore reduce ion transport by these proteins in freshwater by several mechanisms, which decreases transcellular conductance and thus ion efflux (Marshall, 2003). In addition to downregulation of pathways responsible for ion secretion, freshwater transfer also increases the expression of ion transporters involved in ion absorption. Indeed, Na ,K -ATPase expression increases for a prolonged period after +  +  freshwater transfer. Other proteins that are likely involved in ion absorption by killifish gills may also increase in expression after freshwater transfer, such as carbonic anhydrase 2 and Na ,H -exchanger 2 (Scott et al., unpublished observations; Claiborne et al., 2002). +  +  Gene Regulation and Ion Transport  Na ,K -ATPase likely creates the electrochemical gradient for Na absorption by +  +  +  killifish gills (discussed in Chapter 2 as well as in the next section), so changes in the expression of this enzyme may relate to changes in Na influx after freshwater transfer. +  Unidirectional Na influx in northern killifish increased progressively until at least 14 +  days after transfer. Interestingly, this was preceded by a prolonged increase in gill Na ,K -ATPase mRNA expression, which peaked 4 days after freshwater transfer. The +  +  relationship between Na influx and gill Na ,K -ATPase mRNA expression is shown in +  +  +  Fig. 4-1, which combines data from Figs. 2-2 and 3-4A. The temporal patterns illustrated suggest that mRNA expression of Na ,K -ATPase increases early after transfer, and that +  +  a (presumed) subsequent increase in the abundance of this protein is important for the observed changes in unidirectional Na influx. The differences between northern and +  southern killifish were also consistent with a direct relationship between Na ,K -ATPase +  +  expression and Na influx: northerns had greater changes in Na ,K -ATPase expression 1 +  +  +  to 14 days into freshwater, and had higher unidirectional Na influx at 14 days. It +  therefore seems likely that Na ,K -ATPase gene regulation acts as an important +  +  modulator of active ion flux across killifish gills.  Mechanisms of Ion Transport in Freshwater Killifish Gills  Ion absorption infreshwaterby many teleost fish species (e.g., salmonids, tilapia, etc.) involves the coordinated action of several ion transporters in MR cells of the gill epithelium (Perry et al., 2003b, see Fig. 1-2). Na uptake through apical ENaC occurs +  down the electrochemical gradient produced by H -ATPase, while Cl" uptake across the +  apical surface involves a CT/HCO3" exchanger. Basolateral Na ,K -ATPase and +  +  intracellular CA also appear important for uptake of both ions. These key elements underlie both proposed mechanisms of Na and Cl" absorption, involving either one +  common (Fig. 1-2A) or two distinct (Fig. 1-2B) MR cell subtypes for transporting each ion. Despite some notable exceptions (e.g., euryhaline killifish, Patrick and Wood, 1999; European eel, Grosell et al., 2000) this model of ion absorption byfishgills is well supported.  —I  pre  1—I—I  0  1 2  1  1  4  8  1—>  14  I  I  30  Time (days)  Fig. 4-1. Na ,K -ATPase a mRNA expression and unidirectional Na influx after transfer of killifishfromnear-isosmotic brackish water (BW) tofreshwater(FW). * Significant differencefromtime-matched BW control (expression) orfrominitial influx (p<0.05). u  Unlike most otherfishspecies studied to date, however, many of my observations and those of others suggest that killifish have divergent mechanisms of ion transport in freshwater. Na ,K -ATPase expression and activity increased by a greater magnitude and +  +  longer duration after freshwater transfer than after seawater transfer in northern killifish gills. In addition, V-type FF-ATPase expression was unchanged by freshwater transfer, and localizes to the basolateral instead of apical membrane of MR cells (Katoh et al., 2003). Without the electrochemical gradient produced by apical Ff^-ATPase, high Na ,K -ATPase abundance and activity may be necessary for Na absorption. NHEs may +  +  +  facilitate apical Na transport, as NHE2 abundance increases after freshwater transfer in +  killifish gills (Claiborne et al., 2002). Although the rate of Na influx in killifish is high, active Cl" absorption seems to +  be absent in freshwater. This suggests that Cl" absorbing MR cells, similar to the PNAMR cell subtype in salmonids (Fig. 1-2B), may be absent or non-functional in killifish gills in freshwater. I speculate that Na absorption by killifish gills is accomplished by a +  singular freshwater-type MR cell, and that uptake is driven by basolateral Na ,K +  +  ATPase (Fig. 4-2). Apical NHE2 likely transports Na into the MR cell in exchange for +  H , which are produced by CA2. Bicarbonate may be transported with Na across the +  +  basolateral surface via NBC1, and is subsequently excreted in the urine. Although the involvement of CA2 and NBC1 in ion absorption remains poorly defined, studies in other fish species have suggested their potential roles (Hirata et al., 2003; Perry et al., 2003a). When environmental sodium concentrations are extremely low, basolateral Vtype H -ATPase may also become important for ion transport (e.g., Katoh et al., 2003, +  which used a substantially lower water [Na] than in this thesis, see Discussion in Chapter  93  Fig. 4-2. Proposed mechanism of ion absorption by killifish gills in freshwater. Apical Na ,H -cotransporter facilitates Na uptake, driven by low intracellular [Na ] created by basolateral Na ,K -ATPase. Carbonic anhydrase 2 (CA2) provides H for exchange with Na , and the resulting bicarbonate is cleared across the basolateral surface by Na,HCCV-cotransporter 1 (NBC 1).The shaded portion represents an additional possible uptake route of Na when external [Na ] becomes extremely low. ENaC, epithelial Na channel. +  2 (NHE2)  +  +  +  +  +  +  +  +  +  +  +  2 for details). In some fish species (e.g., Atlantic stingray), basolateral H -ATPase facilitates Cl" uptake by extruding protons and thus driving apical C17HCCV exchange to maintain intracellular acid-base balance (Piermarini and Evans, 2001; Piermarini et al., 2002); killifish do not absorb Cl" in freshwater, however, so the role of basolateral H +  ATPase in their gills is unclear. Because tubular extensions of the apical and basolateral surfaces of MR cells are likely proximate (Wilson and Laurent, 2002), basolateral proton extrusion could create a negative intracellular potential near the apical surface. If this is indeed the case, Na uptake may be favoured through apical ENaC in killifish gills +  (shaded portion of Fig. 4-2). Interestingly, recent evidence in zebrafish suggests that lowering freshwater ion levels shifts the gill ion uptake strategy from NHE-mediated (passive, neutral) to H -ATPase/ENaC-mediated (active) (Boisen et al., 2003). It is +  therefore plausible that only NHE functions in 'ion-rich' freshwater, and that H -ATPase +  and ENaC abundances increase in dilute freshwater to maintain sufficient Na uptake +  rates. Furthermore, regulation of basolateral H -ATPase abundance may be post+  transcriptional, as suggested in Chapter 2.  Physiological Mechanisms of Freshwater Adaptation  Although northern and southern killifish populations differ in various aspects of Na regulation, differences in Cl" regulation between populations are likely responsible +  for their large difference in freshwater tolerance (Chapter 3 of this thesis). Changes in plasma Na are modest and similar between populations after abrupt freshwater transfer. +  Plasma Cl" dropped rapidly in southern killifish and was not re-established, however,  whereas northerns maintained plasma Cl" for at least 30 days in freshwater. This difference probably occurred due to differences in passive Cl" efflux across the gills of southern killifish. In contrast to northerns, southerns appearred to maintain elements of seawater ionoregulatory physiology that would be detrimental to Cl" balance in freshwater, including higher paracellular permeability and apical crypt density. At the molecular level, differences in the gene structure, abundance, or organization of appropriate cytoskeletal and intercellular junction proteins in the gills may account for some of the physiological differences between northern and southern killifish (e.g., actin, Daborn et al., 2001; claudins, Colegio et al., 2002; occludins, Schneeberger and Lynch, 2004), but this speculation remains to be tested. It has previously been suggested that northern killifish populations have adapted to freshwater environments, while southern killifish have not (Able and Palmer, 1988; Powers et al., 1993). Although the evolutionary pressures accounting for differences in salinity tolerance are uncertain (see Discussion in Chapter 3), the physiological differences between northern and southern populations in freshwater may give insight into possible mechanisms offreshwateradaptation. In this regard, the physiological modifications that northerns experience to eliminate Cl" loss afterfreshwaterentry appear particularly important. Southerns are unable to make similar modifications, as they maintain several features of seawater ionoregulatory physiology. Whether selection has caused the evolution of a few key regulatory systems in northern killifish, or whether several individual characteristics have been concurrently selected for, has yet to be determined. Interestingly, several other adaptive differences exist between populations of F. heteroclitus that may influence ionoregulatory ability,  96  particularly hormonal differences. Southern killifish appear to be more responsive to stress, including both plasma Cortisol and enzyme expression/activity responses to stress (DeKoning et al., 2004; Picard and Schulte, 2004). Differences between killifish populations in corticosteroid signalling systems may therefore influence ionoregulation after freshwater transfer, along with differences in other hormonal systems believed to influence ion transport (e.g., growth hormone, prolactin, etc., McCormick, 2001).  Overall Conclusions  Killifish use an array of regulatory strategies to modulate ion transport in response to changing environmental salinity. This includes changes in gene expression, which occurs after both freshwater and seawater transfer, and likely influences the transport rates of ions. Indeed, the capacity to regulate the expression of ion transport genes in response to salinity change may be fundamental to the ability of killifish to tolerate substantial salinity fluctuations. While F. heteroclitus has evolved salinity tolerance that is beyond the capability of many other fish species, northern populations of killifish likely continued to evolve even greater tolerance of freshwater environments than their southern counterparts. The evolutionary changes required to alter ionoregulatory ability are certainly complex, and extend well beyond the expression of ion transporters. Even just considering the gills, systems involved in regulating cell-cell junctions, cell structure, and cell proliferation seem important, and could be subject to selective forces. By further understanding how these systems have changed through evolution, we will undoubtedly heighten our knowledge of how selection acts on complex physiological systems.  97  CHAPTER 5  BIBLIOGRAPHY  Able, K . W. and Palmer, R. E . (1988). Salinity effects on fertilization success and larval mortality of Fundulus heteroclitus. Copeia, 345-350. Albaret, J . J., Simier, M . , Darboe, F. S., Ecoutin, J . M . , Raffray, J . and de Morais,  L . T. (2004). Fish diversity and distribution in the Gambia Estuary, West Africa, in relation to environmental variables. Aquat. Living Resour. YI, 35-46. Bertrand, C . A . and Frizzell, R. A . (2003). The role of regulated CFTR trafficking in epithelial secretion. Am. J. Physiol. Cell Physiol. 285, C1-C18. Beyenbach, B. J . and Kirschner, L . B. (1976). The unreliability of mammalian glomerular filtration markers in teleostean renal studies./. Exp. Biol. 64, 369-378. Boisen, A . M . Z., Amstrup, J., Novak, I. and Grosell, M . (2003). Sodium and chloride  transport in soft water and hard water acclimated zebrafish (Danio rerio). Biochim. Biophys. Acta 1618, 207-218.  Boucher, R. C. and Larsen, E . H . (1988). Comparison of ion transport by cultured secretory and absorptive canine airway epithelia. Am. J. Physiol. Cell Physiol. 254, C535-C547. Bowlus, R. D. and Somero, G. N . (1979). Solute compatibility with enzyme function and structure: rationales for the selection of osmotic agents and end-products of anaerobic metabolism in marine invertebrates. J. Exp. Zool. 208, 137-152.  98  Celsi, G., Nishi, A., Akusjarvi, G . and Aperia, A . (1991). Abundance of Na -K +  +  ATPase mRNA is regulated by glucocorticoid hormones in infant rat kidneys. Am. J. Physiol. Renal Fluid Electrolyte Physiol. 260, F192-F197. Claiborne, J . B., Blackston, C. R., Choe, K . P., Dawson, D. C , Harris, S. P.,  Mackenzie, L . A. and Morrison-Shetlar, A . I. (1999). A mechanism for branchial acid excretion in marinefish:identification of multiple Na /H +  +  antiporter (NHE) isoforms in gills of two seawater teleosts. J. Exp. Biol. 202, 315324. Claiborne, J . B., Edwards, S. L . and Morrison-Shetlar, A . I. (2002). Acid-base  regulation infishes:cellular and molecular mechanisms. J. Exp. Zool. 293, 302319. Claiborne, J . B., Walton, J . S. and Compton-McCullough, D. (1994). Acid-base  regulation, branchial transfers and renal output in a marine teleostfish(the longhorned sculpin Myoxocephalus octodecimspinosus) during exposure to low salinities. J. Exp. Biol. 193, 79-95. Colegio, O. R., Van Itallie, C. M . , McCrea, H . J., Rahner, C. and Anderson, J . M .  (2002). Claudins create charge-selective channels in the paracellular pathway between epithelial cells. Am. J. Physiol. Cell Physiol. 283, C142-C147. Copeland, D. E. (1948). The cytological basis of chloride transfer in the gills of Fundulus heteroclitus. J. Morphol. 82, 201-227.  Curtis, B. J . and Wood, C. M . (1991). The function of the urinary bladder in vivo in the freshwater rainbow trout. J. Exp. Biol. 155, 567-583.  99  D'Cotta, H . , Valotaire, C , Le Gac, F. and Prunet, P. (2000). Synthesis of gill Na -K +  +  ATPase in Atlantic salmon smolts: differences in a-mRNA and oc-protein levels. Am. J. Physiol. Regul. Integr. Comp. Physiol. 278, R101-R110.  Daborn, K., Cozzi, R. R. F. and Marshall, W. S. (2001). Dynamics of pavement cellchloride cell interactions during abrupt salinity change in Fundulus heteroclitus. J.Exp. Biol.lM, 1889-1899. Dawley, R. M . (1992). Clonal hybrids of the common laboratory fish Fundulus heteroclitus. Proc. Nat. Acad. Sci. USA 89, 2485-2488. DeKoning, A. B. L . , Picard, D. J., Bond, S. R. and Schulte, P. M . (2004). Stress and  interpopulation variation in glycolytic enzyme activity and expression in a teleost fish Fundulus heteroclitus. Physiol. Biochem. Zool. 11, 18-26. Derrigo, M . , Cestelli, A., Savettieri, G . and D i Liegro, I. (2000). RNA-protein  interactions in the control of stability and localization of messenger RNA (review). Int. J. Mol. Med. 5, 111-123. DiMichele, L . and Westerman, M . E . (1997). Geographic variation in development rate between populations of the teleost Fundulus heteroclitus. Mar. Biol. 128, 1-7. Ernst, S. A., Dodson, W. C. and Karnaky, K . J . (1980). Structural diversity of occluding junctions in the low-resistance chloride-secreting opercular epithelium of seawater-adapted killifish (Fundulus heteroclitus). J. Cell Biol. 87, 488-497.  Evans, D. H . , Piermarini, P. M . and Potts, W. T. W. (1999). Ionic transport in the fish gill epithelium. J. Exp. Zool. 283, 641-652. Feng, S. H . , Leu, J . H . , Yang, C. H . , Fang, M . J., Huang, C. J . and Hwang, P. P.  (2002). Gene expression of Na -K -ATPase al and a3 subunits in gills of the +  +  100  teleost Oreochromis mossambicus, adapted to different environmental salinities. Mar. Biotechnol. 4, 379-391. Fritz, E. S. and Garside, E. T. (1974). Salinity preferences of Fundulus heteroclitus and  F. diaphanus (Pisces: Cyprinodontidae): their role in geographic distribution. Can. J. Zool. 52, 997-1003. Galvez, F., Reid, S. D., Hawkings, G. and Goss, G . G . (2002). Isolation and  characterization of mitochondria-rich cell types from the gill of freshwater rainbow trout. Am. J. Physiol. Regul. Integr. Comp. Physiol. 282, R658-R668.  Goss, G. G., Adamia, S. and Galvez, F. (2001). Peanut lectin binds to a subpopulation of mitochondria-rich cells in the rainbow trout gill epithelium. Am. J. Physiol. Regul. Integr. Comp. Physiol. 281, R1718-R1725.  Griffith, R. W. (1974). Environment and salinity tolerance in the genus Fundulus. Copeia 1974, 319-331. Grosell, M . , Hogstrand, C., Wood, C. M . and Hansen, H . J . M . (2000). A nose-to-  nose comparison of the physiological effects of exposure to ionic silver versus silver chloride in the European eel (Anguilla anguilld) and the rainbow trout (Oncorhynchus mykiss). Aquat. Toxicol. 48, 327-342.  Halpin, P. M . (1997). Habitat use patterns of the mummichog, Fundulus heteroclitus, in New England .1. Intramarsh variation. Estuaries 20, 618-625. Hawkings, G. S., Galvez, F. and Goss, G. G. (2004). Seawater acclimation causes independent alterations in Na /K - and H -ATPase activity in isolated +  +  +  mitochondria-rich cell subtypes of the rainbow trout gill. J. Exp. Biol. 207, 905912.  Hickman, C. P. and Trump, B. F. (1969). The kidney. In Fish Physiology, vol. 1 (eds.  W. S. Hoar and D. J. Randall), pp. 91-239. New York, USA: Academic Press. Hirata, T., Kaneko, T., Ono, T., Nakazato, T., Furukawa, N . , Hasegawa, S., Wakabayashi, S., Shigekawa, M . , Chang, M . H . , Romero, M . F. and Hirose,  S. (2003). Mechanism of acid adaptation of a fish living in a pH 3.5 lake. Am. J. Physiol. Regul. Integr. Comp. Physiol. 284, R1199-R1212. Hiroi, J., Kaneko, T., Uchida, K . , Hasegawa, S. and Tanaka, M . (1998).  Immunolocalization of vacuolar-type H -ATPase in the yolk-sac membrane of +  tilapia (Oreochromis mossambicus) larvae. Zoolog. Sci. 15, 447-453. Hirose, S., Kaneko, T., Naito, N . and Takei, Y. (2003). Molecular biology of major components of chloride cells. Comp. Biochem. Physiol. B 136, 593-620. Hoffmann, E . K . , Hoffmann, E., Lang, F. and Zadunaisky, J . A . (2002). Control of  Cl" transport in the operculum epithelium  Fundulus heteroclitus: long- and  short-term salinity adaptation. Biochim. Biophys. Acta 1566, 129-139. Hossler, F. E., Musil, G., Karnaky, K . J . and Epstein, F. H . (1985). Surface  ultrastructure of the gill arch of the killifish, Fundulus heteroclitus, from seawater and freshwater, with special reference to the morphology of apical crypts of chloride cells. J. Morphol. 185, 377-386. Hubner, S., Michel, F., Rudloff, V . and Appelhans, H . (1992). Amino acid sequence of  band-3 protein from rainbow trout erythrocytes derived from cDNA. Biochem. J. 285, 17-23. Ingbar, D. H . , Duvick, S., Savick, S. K . , Schellhase, D. E., Detterding, R., Jamieson,  J. D. and Shannon, J . M . (1997). Developmental changes of fetal rat lung Na-K-  102  ATPase after maternal treatment with dexamethasone. Am. J. Physiol. Lung Cell. Mol. Physiol. 271, L665-L672. Isenring, P., Jacoby, S. C , Payne, J . A. and Forbush, B. (1998). Comparison of Na-K-  Cl cotransporters NKCC1, NKCC2, and the HEK cell Na-K-Cl cotransporter. J. Biol. Chem. 273, 11295-11301.  Jacob, W. F. and Taylor, M . H . (1983). The time course of seawater acclimation in Fundulus heteroclitus L. J. Exp. Zool. 228, 33-39.  Jensen, M . K., Madsen, S. S. and Kristiansen, K . (1998). Osmoregulation and salinity effects on the expression and activity of Na ,K -ATPase in the gills of European +  +  sea bass, Dicentrarchus labrax (L.). J. Exp. Zool. 282, 290-300.  Karnaky, K . J . (1992). Teleost osmoregulation: changes in the tight junction in response to the salinity of the environment. In Tight Junctions, (ed. M . Cereijido), pp. 175185. Boca Raton, USA: CRC Press. Katoh, F., Hasegawa, S., Kita, J., Takagi, Y . and Kaneko, T. (2001). Distinct seawater  andfreshwatertypes of chloride cells in killifish, Fundulus heteroclitus. Can. J. Zool. 79, 822-829. Katoh, F., Hyodo, S. and Kaneko, T. (2003). Vacuolar-type proton pump in the basolateral plasma membrane energizes ion uptake in branchial mitochondria-rich cells of killifish Fundulus heteroclitus, adapted to a low ion environment. J. Exp. Biol. 206, 793-803. Katoh, F. and Kaneko, T. (2003). Short-term transformation and long-term replacement of branchial chloride cells in killifish transferredfromseawater to freshwater,  103  revealed by morphofunctional observations and a newly established 'timedifferential double fluorescent staining' technique. J. Exp. Biol. 206, 4113-4123. Kelly, S. P. and Wood, C. M . (2002). Cultured gill epithelia from freshwater tilapia (Oreochromis niloticus):  effect of Cortisol and homologous serum supplements  from stressed and unstressed fish. J. Membr. Biol. 190, 29-42. Kirschner, L . B. (1970). The study of NaCl transport in aquatic animals. Am. Zool. 10,  365-376. Laemmli, U . K . (1970). Cleavage of structural proteins during assembly of the head bacteriophage T4. Nature 227, 680-685. Lee, C. E . and Bell, M . A. (1999). Causes and consequences of recent freshwater invasions by saltwater animals. Trends Ecol. Evol. 14, 284-288. Lee, C. E . and Petersen, C. H . (2002). Genotype-by-environment interaction for salinity tolerance in the freshwater-invading copepod Eurytemora affinis. Physiol. Biochem. Zool. 75,  335-344.  Lee, T. H . , Feng, S. H . , L i n , C. H . , Hwang, Y . H . , Huang, C. L . and Hwang, P. P.  (2003). Ambient salinity modulates the expression of sodium pumps in branchial mitochondria-rich cells of Mozambique tilapia, Oreochromis mossambicus. Zoolog. Sci. 20,  29-36.  Lin, H . and Randall, D. J . (1993). H -ATPase activity in crude homogenates of fish gill +  tissue: inhibitor sensitivity and environmental and hormonal regulation. J. Exp. Biol. 180,  163-174.  104  Lin, Y . M . , Chen, C. N . and Lee, T. H . (2003). The expression of gill Na,K-ATPase in milkfish, Chanos chanos, acclimated to seawater, brackish water and fresh water. Comp. Biochem. Physiol. A 135, 489-497. Lytle, C , X u , J . C , Biemesderfer, D., Haas, M . and Forbush, B. (1992). The Na-K-Cl  cotransport protein of shark rectal gland . 1. Development of monoclonal antibodies, immunoaffinity purification, and partial biochemical characterization. J. Biol. Chem. 267, 25428-25437. Madsen, S. S., Jensen, M . K . , Nehr, J . and Kristiansen, K . (1995). Expression of Na +  K -ATPase in the brown trout, Salmo trutta: in vivo modulation by hormones and +  seawater. Am. J. Physiol. Regul. Integr. Comp. Physiol. 38, R1339-R1345.  Mancera, J . M . and McCormick, S. D. (2000). Rapid activation of gill Na ,K -ATPase +  +  in the euryhaline teleost Fundulus heteroclitus. J. Exp. Zool. 287, 263-274. Marshall, W . S. (2002). Na , Cl", Ca and Zn transport byfishgills: retrospective +  2+  2+  review and prospective synthesis. J. Exp. Zool. 293, 264-283. Marshall, W. S. (2003). Rapid regulation of NaCl secretion by estuarine teleost fish: coping strategies for short-duration freshwater exposures. Biochim. Biophys. Acta 1618, 95-105. Marshall, W. S., Bryson, S. E. and Garg, D. (1993). oc-Adrenergic inhibition of Cl" 2  transport by opercular epithelium is mediated by intracellular Ca . Proc. Nat. Acad. Sci. USA 90, 5504-5508. Marshall, W . S., Bryson, S. E . and Luby, T. (2000). Control of epithelial Cl" secretion by basolateral osmolality in the euryhaline teleost Fundulus heteroclitus. J. Exp. Biol. 203, 1897-1905.  Marshall, W. S., Bryson, S. E., Midelfart, A. and Hamilton, W. F. (1995). Low-  conductance anion channel activated by cAMP in teleost Cl'-secreting cells. Am. J. Physiol. Regul. Integr. Comp. Physiol. 37, R963-R969. Marshall, W . S., Duquesnay, R. M . , Gillis, J . M . , Bryson, S. E . and Liedtke, C. M .  (1998) . Neural modulation of salt secretion in teleost opercular epithelium by a 2  adrenergic receptors and inositol 1,4,5-trisphosphate. J. Exp. Biol. 201, 19591965. Marshall, W . S., Emberley, T. R., Singer, T. D., Bryson, S. E . and McCormick, S. D.  (1999) . Time course of salinity adaptation in a strongly euryhaline estuarine teleost, Fundulus heteroclitus: A multivariable approach. J. Exp. Biol. 202, 15351544. Marshall, W . S., Lynch, E . A. and Cozzi, R. R. F. (2002). Redistribution of  immunofluorescence of CFTR anion channel and NKCC cotransporter in chloride cells during adaptation of the killifish Fundulus heteroclitus to sea water. J. Exp. Biol. 205, 1265-1273. Marshall, W . S. and Singer, T. D. (2002). Cystic fibrosis transmembrane conductance regulator in teleost fish. Biochim. Biophys. Acta 1566, 16-27. McCormick, S. D. (1993). Methods for nonlethal gill biopsy and measurement of Na ,K -ATPase activity. Can. J. Fish. Aquat. Sci. 50, 656-658. +  +  McCormick, S. D. (2001). Endocrine control of osmoregulation in teleost fish. Am. Zool. 41,781-794. McCormick, S. D., Sundell, K., Bjornsson, B . T., Brown, C. L . and Hiroi, J . (2003).  Influence of salinity on the localization of Na /K -ATPase, Na /K /2C1" +  +  +  +  106  cotransporter (NKCC) and CFTR anion channel in chloride cells of the Hawaiian goby (Stenogobius hawaiiensis). J. Exp. Biol. 206, 4575-4583.  Moore-Hoon, M . L . and Turner, R. J . (1998). Molecular and topological characterization of the rat parotid Na^,K,2Cl"-cotransporter. Biochim. Biophys. +  Acta 1373, 261-269. Motais, R., Garcia Romeau, F. and Maetz, J . (1966). Exchange diffusion effect and euryhalinity in teleosts. J. Gen. Physiol. 50, 391-422. Nauntofte, B. (1992). Regulation of electrolyte and fluid secretion in salivary acinar cells. Am. J. Physiol. Gastrointest. Liver Physiol. 263, G823-G837. Pang, P. K . T., Griffith, R. W., Schreibman, M . P. and Sawyer, W . H . (1974).  Environmental salinity and pituitary control of sodium balance in killifishes. Am. J.Physiol. 227, 1139-1142. Patrick, M . L . , Part, P., Marshall, W. S. and Wood, C. M . (1997). Characterization of  ion and acid-base transport in thefreshwater adapted mummichog (Fundulus heteroclitus). J. Exp. Zool. 279, 208-219.  Patrick, M . L . and Wood, C. M . (1999). Ion and acid-base regulation in the freshwater mummichog (Fundulus heteroclitus): a departurefromthe standard model for freshwater teleosts. Comp. Biochem. Physiol. A 122, 445-456. Pelis, R. M . , Zydlewski, J . and McCormick, S. D . (2001). Gill Na -K -2C1" +  +  cotransporter abundance and location in Atlantic salmon: effects of seawater and smolting. Am. J. Physiol. Regul. Integr. Comp. Physiol. 280, R1844-R1852.  Perry, S. F. (1997). The chloride cell: Structure and function in the gills of freshwater fishes. Annu. Rev. Physiol. 59, 325-347.  107  Perry, S. F., Beyers, M . L . and Johnson, D. A. (2000). Cloning and molecular characterisation of the trout (Oncorhynchus mykiss) vacuolar H -ATPase B +  subunit. J. Exp. Biol. 203, 459-470. Perry, S. F., Furimsky, M . , Bayaa, M . , Georgalis, T., Shahsavarani, A., Nickerson, J .  G. and Moon, T. W. (2003a). Integrated responses of Na /HC0 " cotransporters +  3  and V-type H -ATPases in the fish gill and kidney during respiratory acidosis. +  Biochim. Biophys. Acta 30, 175-184. Perry, S. F., Shahsavarani, A., Georgalis, T., Bayaa, M . , Furimsky, M . and Thomas,  S. L . Y . (2003b). Channels, pumps, and exchangers in the gill and kidney of freshwater fishes: their role in ionic and acid-base regulation. J. Exp. Zool. 300A, 53-62. Philpott, C. W. (1965). Halide localization in the teleost chloride cell and its identification by selected area electron diffraction: direct evidence supporting an osmoregulatory function for the seawater adapted chloride cell of Fundulus. Protoplasma 60, 7-23.  Pic, P. (1978). A comparative study of the mechanisms of Na and Cl" excretion by the +  gill of Mugil capito and Fundulus heteroclitus: effects of stress. J. Comp. Physiol.  123, 155-162. Picard, D. J . and Schulte, P. M . (2004). Variation in gene expression in response to stress in two populations of Fundulus heteroclitus. Comp. Biochem. Physiol. A  137, 205-216. Pickford, G . E., Pang, P. K . T., Weinstein, E., Torretti, J., Hendler, E . and Epstein,  F. H . (1970). Response of the hypophysectomized cyprinodont Fundulus  108  heteroclitus to replacement therapy with Cortisol: effects on blood serum and  sodium-potassium activated triphosphatase in the gills, kidney, and intestinal mucosa. Gen. Comp. Endocrinol. 14, 524-534. Pierce, V. A. and Crawford, D. L . (1996). Variation in the glycolytic pathway: the role of evolutionary and physiological processes. Physiol. Zool. 69, 489-508. 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 sabind): effects of salinity and relation to Na /K -ATPase. J. Exp. Biol. 204, 3251-3259. +  +  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.  Pisam, M. and Rambourg, A. (1991). Mitochondria-rich cells in the gill epithelium of teleost fishes: an ultrastructural approach. Int. Rev. Cytol. 130, 191-232. Podrabsky, J . E., Javillonar, C , Hand, S. C . and Crawford, D. L . (2000).  Intraspecific variation in aerobic metabolism and glycolytic enzyme expression in heart ventricles. Am. J. Physiol. Regul. Integr. Comp. Physiol. 279, R2344-R2348.  Potts, W. T. W. and Evans, D. H . (1966). The effects of hypophysectomy and bovine prolactin on salt fluxes infresh-water-adaptedFundulus heteroclitus. Biol. Bull. 131, 362-368. Potts, W. T. W. and Evans, D. H . (1967). Sodium and chloride balance in the killifish Fundulus heteroclitus. Biol. Bull. 133, 411-425.  Potts, W. T. W. and Fleming, W. R. (1971). The effect of environmental calcium and ovine prolactin on sodium balance in Fundulus kansae. J. Exp. Biol.  109  Power, M . , Attrill, M . J . and Thomas, R. M . (2000). Environmental factors and interactions affecting the temporal abundance of juvenile flatfish in the Thames estuary. J. Sea Res. 43, 135-149. Powers, D. A., Ropson, L , Brown, D. C , Vanbeneden, R., Cashon, R., Gonzalez-  Villasenor, L . I. and DiMichele, J . A . (1986). Genetic variation in Fundulus heteroclitus: geographic distribution. Am. Zool. 26, 131-144. Powers, D. A., Smith, M . , Gonzalez-Villasenor, I., DiMichele, J., Crawford, D. L . ,  Bernardi, G. and Lauerman, T. (1993). A multidisciplinary approach to the selectionist/neutralist controversy using the model teleost, Fundulus heteroclitus. In Oxford Survey in Evolutionary Biology, vol. 9 (eds. D. Futuyama and J.  Antonovics), pp. 43-107. Oxford, UK: Oxford University Press. Rajasekaran, A . K . and Rajasekaran, S. A . (2003). Role of Na-K-ATPase in the assembly of tight junctions. Am. J. Physiol. Renal Physiol. 285, F388-F396. Reid, S. D., Hawkings, G . S., Galvez, F. and Goss, G. G . (2003). Localization and  characterization of phenamil-sensitive Na influx in isolated rainbow trout gill +  epithelial cells. J. Exp. Biol. 206, 551-559. Richards, J . G., Semple, J . W., Bystriansky, J . S. and Schulte, P. M . (2003). Na /K +  ATPase a-isoform switching in gills of rainbow trout (Oncorhynchus mykiss) during salinity transfer. J. Exp. Biol. 206, 4475-4486. Riordan, J . R., Forbush, B. and Hanrahan, J . W. (1994). The molecular basis of chloride transport in shark rectal gland. J. Exp. Biol. 196, 405-418.  +  110  Robertson, J . C. and Hazel, J . R. (1999). Influence of temperature and membrane lipid composition on the osmotic water permeability of teleost gills. Physiol. Biochem. Zool. 72, 623-632. Sakamoto, T., Uchida, K . and Yokota, S. (2001). Regulation of the ion-transporting mitochondrion-rich cell during adaptation of teleost fishes to different salinities. Zoolog.Sci. 18, 1163-1174. Samaritan, J . M . and Schmidt, R. E . (1982). Aspects of the life history of a freshwater population of the mummichog, Fundulus heteroclitus (Pisces: Cyprinodontidae), in the Bronx river, New York, U.S.A. Hydrobiologia 94, 149-154. Sardet, C , Pisam, M . and Maetz, J. (1979). The surface epithelium of teleostean fish gills: cellular and junctional adaptations of the chloride cell in relation to salt adaptation. J. Cell Biol. 80, 96-117. Schneeberger, E . E . and Lynch, R. D. (2004). The tight junction: a multifunctional complex. Am. J. Physiol. Cell Physiol. 286, C1213-C1228. Scott, G . R., Richards, J . G., Forbush, B., Isenring, P. and Schulte, P. M . (2004a).  Changes in gene expression in gills of the euryhaline killifish Fundulus heteroclitus after abrupt salinity transfer. Am. J. Physiol. Cell Physiol. In press. Scott, G . R., Rogers, J . T., Richards, J . G., Wood, C. M . and Schulte, P. M . (2004b).  Intraspecific divergence of ionoregulatory physiology in the euryhaline teleost Fundulus heteroclitus: possible mechanisms offreshwateradaptation. J. Exp. Biol. Accepted. Seidelin, M . , Brauner, C . J., Jensen, F. B. and Madsen, S. S. (2001a). Vacuolar-type  H -ATPase and Na,K -ATPase expression in gills of Atlantic salmon (Salmo  Ill  salar) during isolated and combined exposure to hyperoxia and hypercapnia in fresh water. Zoolog. Sci. 18, 1199-1205. Seidelin, M . , Madsen, S. S., Blenstrup, H . and Tipsmark, C . K . (2000). Time-course  changes in the expression of Na ,K -ATPase in gills and pyloric caeca of brown +  +  trout (Salmo truttd) during acclimation to seawater. Physiol. Biochem. Zool. 73,  446-453. Seidelin, M . , Madsen, S. S., Cutler, C. P. and Cramb, G . (2001b). Expression of gill  vacuolar-type H -ATPase B subunit, and Na ,K -ATPase 0Ci and (3i subunit +  +  +  messenger RNAs in smolting Salmo salar. Zoolog. Sci. 18, 315-324. Semple, J . W., Green, H . J . and Schulte, P. M . (2002). Molecular cloning and characterization of two Na/K-ATPase isoforms in Fundulus heteroclitus. Mar. Biotechnol. 4, 512-519. Shrimpton, J . M . and McCormick, S. D. (1999). Responsiveness of gill Na /K +  +  ATPase to Cortisol is related to gill corticosteroid receptor concentration in juvenile rainbow trout. J. Exp. Biol. 202, 987-995. Singer, T. D., Clements, K . M . , Semple, J . W., Schulte, P. M . , Bystriansky, J . S.,  Finstad, B., Fleming, I. A. and McKinley, R. S. (2002). Seawater tolerance and gene expression in two strains of Atlantic salmon smolts. Can. J. Fish. Aquat. Sci. 59,125-135. Singer, T. D., Tucker, S. J . , Marshall, W . S. and Higgins, C . F. (1998). A divergent  CFTR homologue: highly regulated salt transport in the euryhaline teleost F. heteroclitus. Am. J. Physiol. Cell Physiol. 43, C715-C723.  112  Sloman, K . A., Scott, G. R., McDonald, D. G. and Wood, C. M . (2004). Diminished  social status affects ionoregulation at the gills and kidney in rainbow trout (Oncorhynchus mykiss). Can. J. Fish. Aquat. Sci. In press.  Sullivan, G . V., Fryer, J . N . and Perry, S. F. (1995). Immunolocalization of proton pumps (H -ATPase) in pavement cells of rainbow trout gill. J. Exp. Biol. 198, +  2619-2629. Sullivan, G. V., Fryer, J . N . and Perry, S. F. (1996). Localization of mRNA for the proton pump (H -ATPase) and C17HCCV exchanger in the rainbow trout gill. +  Can. J. Zool. 74, 2095-2103. Thompson, J . D., Higgins, D. G . and Gibson, T. J . (1994). ClustalW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673-4680. Tipsmark, C. K . , Madsen, S. S., Seidelin, M . , Christensen, A . S., Cutler, C. P. and  Cramb, G . (2002). Dynamics of Na ,K ,2C1" cotransporter and Na ,K -ATPase +  +  +  +  expression in the branchial epithelium of brown trout (Salmo trutta) and Atlantic salmon (Salmo salar). J. Exp. Zool. 293, 106-118.  Towle, D. W., Gilman, M . E. and Hempel, J . D. (1977). Rapid modulation of gill Na+K-dependent ATPase activity during acclimation of killifish Fundulus +  +  heteroclitus to salinity change. J. Exp. Zool. 202, 179-185. Uchida, K., Kaneko, T., Tagawa, M . and Hirano, T. (1998). Localization of Cortisol  receptor in branchial chloride cells in chum salmon fry. Gen. Comp. Endocrinol. 109,175-185.  113  Weber, L . A., Hickey, E . D., Maroney, P. A . and Baglioni, C. (1977). Inhibition of  protein synthesis by Cl". J. Biol. Chem. 252, 4007-4010. Weis, J. S. (2002). Tolerance to environmental contaminants in the mummichog,  Fundulus heteroclitus. Hum. Ecol. Risk Assess. 8, 933-953. 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. Wilson,  K.  F. and Cerione, R. A. (2000). Signal transduction and post-transcriptional  gene expression. Biol. Chem. 381, 357-365. Wood, C. A . and Laurent, P. (2003). Na versus Cl" transport in the intact killifish after +  rapid salinity transfer. Biochim. Biophys. Acta 1618, 106-119. Wood, C. M . and Marshall, W. S. (1994). Ion balance, acid-base regulation, and  chloride cell function in the common killifish, Fundulus heteroclitus - a euryhaline estuarine teleost. Estuaries YI, 34-52. Wu, Y. C , Lin, L . Y. and Lee, T. H. (2003). Na ,K ,2Cl"-cotransporter: A novel marker +  +  for identifying freshwater- and seawater-type mitochondria-rich cells in gills of the euryhaline tilapia, Oreochromis mossambicus. Zool. Stud. 42, 186-192. Zadunaisky, J . A., Cardona, S., Au, L . , Roberts, D. M . , Fisher, E . , Lowenstein, B., Cragoe, E . J. and Spring,  K.  R. (1995). Chloride transport activation by plasma  osmolality during rapid adaptation to high salinity  of Fundulus heteroclitus. J.  Membr. Biol. 143, 207-217. Zhou, B. S., Kelly, S. P., Ianowski, J. P. and Wood, C. M. (2003). Effects of Cortisol and prolactin on Na and Cl" transport in cultured branchial epithelia from FW +  rainbow trout.  Am. J. Physiol. Regul. Integr. Comp. Physiol. 285, R1305-R1316  

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