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Thermal acclimation and adaptation in the common killfish, Fundulus heteroclitus : thermal reaction norms… Fangue, Nann A. 2007

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T H E R M A L A C C L I M A T I O N A N D A D A P T A T I O N IN T H E C O M M O N KILLIFISH, FUNDULUS HETEROCLITUS: T H E R M A L R E A C T I O N N O R M S A N D "~" U N D E R L Y I N G M E C H A N I S M S by Nann A. Fangue B.S., The University of West Florida, 1999 M.S., The University of West Florida, 2001 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES (Zoology) THE UNIVERSITY OF BRITISH COLUMBIA September 24, 2007 © Nann A. Fangue, 2007 A B S T R A C T I have quantified the effects of temperature on the performance of killifish (Fundulus heteroclitus) from the northern and southern ends of the species' distribution along the East coast of North America, and examined potential mechanisms that could be involved in shaping these traits. I demonstrated that upper and lower thermal tolerance limits were higher in southern fish by ~1.5°C, consistent with adaptation to warmer habitat temperatures (Chapter Two). Thermal tolerance limits were, however, plastic in both populations, changing by >10°C with temperature acclimation, In thermal preference experiments, the thermally tolerant southern killifish chose lower temperatures than the less tolerant northern fish (29.0°C versus 30.6°C; Chapter Three), consistent with countergradient variation. Swimming performance ( U c r j t ) trials on thermally acclimated and acutely challenged killifish revealed a broad zone (~7-34°C) over which performance varied little with temperature in both populations (Chapter Four). Taken together, these data showed that although there were differences in thermal tolerance limits between populations, there was little evidence of local thermal adaptation in other measured traits. As a candidate mechanism underlying differences in thermal tolerance, I examined the role of heat shock proteins (Hsps) and found significant differences between populations in hsp mRNA expression and across multiple isoforms (Chapter Two), but these differences were not obviously consistent with the thermal tolerance differences between killifish populations. I also explored the adjustment of aerobic capacity at the level of the mitochondria (Chapter Five) as a potential mechanism defining an organism's thermal niche, as proposed in the hypothesis of oxygen limited thermal tolerance. In response to cold acclimation, both killifish populations increased mitochondrial oxidative capacity and content, with northern fish increasing both to a greater degree. With warm acclimation, however, killifish mitochondrial function and content were /very similar between populations. These data suggest the possibility of evolutionary modulation at the level of the mitochondria to enhance aerobic performance in the cold. Overall, I have shown that there are differences between northern and southern killifish at the physiological, biochemical, and molecular levels. These data highlight the importance of considering multiple mechanisms that could underlie differences in whole-organism performance when investigating local thermal adaptation. m T A B L E O F C O N T E N T S Abstract ii Table of Contents iv List of Tables viii List of Figures ix List of Abbreviations xii Co-Authorship Statement xv Chapter One: Introduction 1 Overview 1 Temperature and Biogeography 1 Thermal Reaction Norms 2 Time Scales of Thermal Responses 4 Biochemical and Molecular Determinants of Thermal Reaction Norms 5 Differences in the Thermal Stability of Proteins 6 Heat Shock Proteins and the Heat Shock Response 8 Changes in Membrane Properties 13 Oxygen Supply and Demand 15 Need for an Integrative Approach 18 Killifish as an Experimental Model 19 Killifish Habitat 19 Killifish Reproduction 22 Killifish Genetic Variation 22 Thesis Objectives 25 Objective One: Quantification of Thermal Tolerance in Fish 26 Variation in thermal tolerance limits in fishes 27 Intraspecific variation in thermal tolerance 29 Objective Two: Estimating the Shape of Thermal Performance Niches 30 Swimming performance 31 Oxygen consumption and the role of the mitochondria 34 Objective Three: Location of the Thermal Optima Using Thermal Preference Data 36 Thesis Organization 38 Chapter Two 38 Chapter Three 39 Chapter Four 39 Chapter Five ....40 References 42 Chapter Two: Intraspecific Variation in Thermal Tolerance and Heat Shock Protein Gene Expression in Common Killifish, Fundulus heteroclitus 60 Introduction 60 Materials and Methods 63 Experimental Animals 63 Upper and Lower Lethal Limits 64 Thermal Tolerance Methodology 65 Effects of Acclimation 65 Intraspecific Variation 66 Identification and Sequencing of hsp Genes 66 Isolation of genomic DNA, Total RNA extraction, and reverse-transcriptase PCR amplification 66 Sequence variation in hsc70 and hsp70 isoforms 69 Phylogenetic analysis 70 Relationship between Thermal Tolerance and Heat Shock Proteins..... 71 Heat shock experiment 71 Quantitative Real-time PCR analysis of hsc70, hsp70 and hsp90 gene expression 71 Statistical Analyses 72 Results 73 Thermal Tolerance in Killifish Populations 73 Effects of Body Size on Thermal Tolerance 74 Intra- and Interpopulation Variation in Thermal Tolerance 74 Sequence Variation in Fundulus Hsps 76 Variation in hsp Expression 77 Discussion 78 Killifish Thermal Tolerance 78 Hsps and Thermal Tolerance 80 References 86 Chapter Three: Countergradient Variation in Temperature Preference in Populations of Killifish, Fundulus heteroclitus 102 Introduction 102 Materials and Methods 104 Experimental Animals 104 Thermal Gradient Apparatus , 105 Analysis of Selected Temperatures 106 Statistics 108 Results 109 Size, Condition, and Sex 109 Fish Behavior in the Thermal Gradient 109 Acute Thermal Preferenda (T°C a c u t e) 110 Time (Timesei) and Selected Temperatures (T°C s ei) H I Selected Temperature as a Function of Time I l l Final Thermal Preferenda (T°Cf i n ai) 112 Discussion 113 The Final Thermal Preferenda (T°Cf i n ai) Differ Between Killifish Populations 113 Countergradient Variation in Final Thermal Preferenda 114 Low Thermal Responsiveness in Northern Killifish 114 Relationship Between T°Cf i n ai and Thermal Habitat 115 Relationship Between Critical Thermal Limits and Final Thermal Preferenda 116 Conclusions 117 References 119 Chapter Four: Intraspecific Variation in Swimming Performance and Energetics as a Function of Temperature in Killifish, Fundulus heteroclitus 129 v Introduction 129 Material and Methods 132 Experimental Animals 132 Critical Velocity Measures 133 Standardized Exercise Challenge 135 Analytical Techniques 136 Statistical Analysis 137 Results..... 137 Body Size 137 Experiment 1: Effects of Acclimation 138 Experiment 2: Performance in Acute Challenge 138 Experiment 3: Standardized Exercise Challenge 139 Other muscle metabolites 139 Liver glucose and glycogen 140 Plasma glucose and lactate 140 Experiment 4: Multiple Populations 141 Experiment 5: Standardized Exercise Challenge 141 Experiment 6: Low Temperature Acclimation 142 Discussion 143 Temperature Independent Swimming Performance 143 Intrinsic Temperature Insensitivity During Acute Thermal Challenge 145 Plasticity in Swimming Performance 147 Biochemical Correlates of Swimming Performance 149 Conclusions 151 References 153 Chapter Five: Inter-population Variation in the Effects of Thermal Acclimation on Mitochondrial Properties in Killifish (Fundulus heteroclitus) 167 Introduction 167 Materials and Methods 170 Experimental Animals 170 Tissue Sampling 171 Total R N A Extraction 172 Quantitative Real-Time PCR Analysis of Gene Expression 172 Protein Isolation and Enzyme Assays 173 Isolation of Mitochondria 174 Mitochondrial Respiration Measurements 174 Statistical Analyses 176 Results 176 General Mitochondrial Characteristics 176 Thermal Sensitivity of Mitochondrial Respiration 177 Maximum oxidative capacity (State III) 177 Proton leak (State IV0,) 179 Mitochondrial efficiency (State III-IVol) 179 Variation in Enzyme Activity 180 Variation in Gene Expression 181 Discussion 182 Acclimation Affects Mitochondrial Content 182 Relationship Between mRNA and Protein Levels 184 vi Acclimation Affects Mitochondrial Function 185 Proton Leak (IVol) 189 Patterns of Acclimation and Adaptation in Eurytherms 189 Implications for Whole-Organism Performance 191 references 193 Chapter Six: General Discussion and Conclusions 207 Overview 207 Summary of Salient Findings 208 Location of the Thermal Optimum 209 Quantification of Thermal Niche Limits 210 Mechanistic Basis of Niche Limits 215 Estimating the Shape of the Niche 219 Acute Thermal Performance of Mitochondria in Response to Thermal Acclimation 221 Conclusions 222 Future Directions 223 References 225 Appendix A 228 Appendix B 229 L I S T O F T A B L E S T A B L E 2.1 PRIMERS USED FOR Q R T - P C R OF HEAT SHOCK PROTEIN GENES 93 T A B L E 2.2 SIMPLE LINEAR REGRESSION EQUATIONS AND MODEL COMPARISONS OF R 2 94 T A B L E 2.3 CRITICAL THERMAL MAXIMA AND MINIMA ( ° C ) , TOTAL LENGTH (CM), AND MASS (G) FOR NORTHERN ( N H ) AND SOUTHERN ( G A ) KILLIFISH 95 T A B L E 3.1 M E A N MODAL SELECTED TEMPERATURES FOR NORTHERN AND SOUTHERN KILLIFISH ACCLIMATED TO 5 , 1 5 , AND 2 5 ° C 124 T A B L E 4.1 M U S C L E , LIVER, AND PLASMA METABOLITES FOR KILLIFISH AT REST OR AFTER A STANDARDIZED EXERCISE CHALLENGE 158 T A B L E 4.2 RESTING METABOLITES FOR MULTIPLE KILLIFISH POPULATIONS ACCLIMATED TO 2 3 ° C 159 T A B L E 4.3 T H E EFFECTS OF A STANDARDIZED EXERCISE CHALLENGE ON METABOLITES IN KILLIFISH 160 T A B L E 4.4 T H E EFFECTS OF LOW TEMPERATURE ACCLIMATION ( 7 ° C ) ON U C RI T AND METABOLITES IN KILLIFISH 161 T A B L E 5.1 PRIMERS USED FOR Q R T - P C R OF MITOCHONDRIAL GENES 205 TABLE 5.2 CYTOCHROME C OXIDASE, SUBUNIT TWO ( C O X I I ) , CITRATE SYNTHASE (CS), AND A T P SYNTHASE B-CHAIN (ATPSYN) M R N A LEVELS 206 T A B L E 6.1 T H E CALCULATED C T M A X TEMPERATURES FOR 5 , 1 5 , AND 2 5 ° C ACCLIMATED KILLIFISH COMPARED TO THE UPPER THERMAL LIMITS FOR ISOLATED MITOCHONDRIAL STATE III RATES 215 v i n L I S T O F F I G U R E S FIGURE 1.1 GENERALIZED THERMAL REACTION NORM 3 FIGURE 1.2 VARIATION IN THERMAL REACTION NORM POSITION AND SHAPE 4 FIGURE 1.3 C O A S T A L , INSHORE WATER TEMPERATURES FOR REPRESENTATIVE NORTHERN ( W E L L S INLET, MAINE) AND SOUTHERN (MATANZA, FLORIDA) KILLIFISH HABITATS 21 FIGURE 1.4 T H E R M A L REACTION NORM RELATING TEMPERATURE TO PERFORMANCE OPTIMA, LIMITS, AND BREADTH 26 FIGURE 1.5 COMPARATIVE ECOLOGICAL THERMAL TOLERANCE POLYGONS IN FISHES 28 FIGURE 1.6 PREDICTED PATTERNS OF SWIM PERFORMANCE IN KILLIFISH POPULATIONS ACCLIMATED TO A WIDE TEMPERATURE RANGE 40 FIGURE 2.1 CHRONIC THERMAL MAXIMA AND MINIMA FOR NORTHERN AND SOUTHERN KILLIFISH 96 FIGURE 2.2 CRITICAL THERMAL MAXIMA AND MINIMA FOR KILLIFISH ACCLIMATED TO TEMPERATURES BETWEEN 2.3 AND 3 4 . 0 ° C 97 FIGURE 2.3 CRITICAL THERMAL MAXIM AND MINIMA FOR THREE NORTHERN AND THREE SOUTHERN POPULATIONS OF KILLIFISH ACCLIMATED TO 2 2 ° C 98 FIGURE 2.4 PHYLOGENETIC RELATIONSHIPS AMONG VERTEBRATE HSC/HSP70 AMINO ACID SEQUENCES 99 FIGURE 2.5 BRANCHIAL HSC70, HSP70-1, AND HSP70-2 M R N A LEVELS IN NORTHERN AND SOUTHERN KILLIFISH IN RESPONSE TO HEAT SHOCK 100 FIGURE 2.6 BRANCHIAL mp90a AND HSP90PMRNX LEVELS FOR NORTHERN AND SOUTHERN KILLIFISH EXPOSED TO HEAT SHOCK 101 FIGURE 3.1 SELECTED TEMPERATURE AS A FUNCTION OF TIME FOR ONE REPRESENTATIVE 5 ° C ACCLIMATED NORTHERN FISH SHOWN AS AN EXAMPLE 125 ix FIGURE 3.2 MO D A L ACUTE THERMAL PREFERENCE, MODAL FINAL SELECTED TEMPERATURE, AND MODAL TIME TO FINAL SELECTED TEMPERATURE FOR NORTHERN AND SOUTHERN KILLIFISH ACCLIMATED TO 5, 15, AND 25°C 126 FIGURE 3.3 ME A N MODAL SELECTED TEMPERATURES AS A FUNCTION OF TIME IN NORTHERN AND SOUTHERN KILLIFISH ACCLIMATED TO 5, 15, AND 2 5 °C 127 FIGURE 3.4 MODAL AND MEAN FINAL THERMAL PREFERENDA FOR NORTHERN AND SOUTHERN KILLIFISH 128 FIGURE 4.1 CRITICAL SWIMMING SPEED (U C RI T ; BL-SEC" 1 ) FOR NORTHERN AND SOUTHERN KILLIFISH ACCLIMATED TO TEMPERATURES BETWEEN 5.2 AND 32.4°C 162 FIGURE 4.2 CRITICAL SWIMMING SPEED (UCRIT; BL-SEC" 1 ) FOR NORTHERN AND SOUTHERN KILLIFISH ACCLIMATED TO 18°C AND SWUM AT THE ACCLIMATION TEMPERATURE OR AT 5, 25, O R34°C 163 FIGURE 4.3 NORTHERN AND SOUTHERN KILLIFISH MUSCLE GLYCOGEN AND MUSCLE LACTATE CONCENTRATIONS FOR RESTING AND EXERCISING FISH ACCLIMATED TO 5, 15 AND 29°C 164 FIGURE 4.4 CRITICAL SWIMMING SPEED (U C RI T ; BL-SEC" 1 ) AND RESTING MUSCLE GLYCOGEN LEVELS FOR MULTIPLE NORTHERN AND SOUTHERN KILLIFISH POPULATIONS ACCLIMATED TO 23°C 166 FIGURE 5.1 TEMPERATURE DEPENDENCE OF STATE III RESPIRATION RATES, DETERMINED IN MITOCHONDRIA FROM NORTHERN AND SOUTHERN KILLIFISH ACCLIMATED TO 5, 15, AND 25°C • 198 FIGURE 5.2 STATE III RESPIRATION RATES DETERMINED IN MITOCHONDRIA FROM NORTHERN AND SOUTHERN KILLIFISH ACCLIMATED TO AND ASSAYED AT 5, 15, AND 25°C 200 FIGURE 5.3 TEMPERATURE DEPENDENCE OF STATE I V O L , DETERMINED IN MITOCHONDRIA FROM 5, 15, AND 2 5 °C ACCLIMATED NORTHERN AND SOUTHERN KILLIFISH 202 FIGURE 5.4 LIVER AND MUSCLE CYTOCHROME C OXIDASE (COX) AND CITRATE SYNTHASE (CS) ACTIVITY IN NORTHERN AND SOUTHERN KILLIFISH ACCLIMATED TO 5, 15, OR 25°C... 204 x FIGURE 6.1 T H E INTERPLAY BETWEEN TIMESCALE, PHYSIOLOGICAL AND BIOCHEMICAL MECHANISMS, AND BIOLOGICAL LEVELS OF ORGANIZATION WITH KILLIFISH PERFORMANCE 207 FIGURE 6.2 CONCEPTUAL SCHEME FOR CRITICAL TEMPERATURES AT WHICH AN ORGANISM'S PERFORMANCE IS ALTERED 208 XI L I S T O F A B B R E V I A T I O N S ACh acetylcholine AChE acetylcholine esterase ADP adenosine diphosphate A N C O V A analysis of covariance A N O V A analysis of variance ATP adenosine triphosphate ATP Syn ATP synthase B C A bicinchoninic acid BLs" 1 body lengths per second bp base pair °C degrees Celsius cDNA complementary DNA cms"1 centimeters per second COX cytochrome c oxidase CrP creatine phosphate CS citrate synthase C T M critical thermal methodology CTMax critical thermal maximum CTMin critical thermal minimum D N A deoxyribonucleic acid DTNB 5,5'-dithiobis-2-nitrobenzoic acid DTT dithiothreitol E D T A ethylenediaminetetraacetic acid E G T A ethyleneglycolbistetraacetic acid ETC electron transport chain FCCP carbonyl-cyanide p-trifluoro-methoxyphenyl hydrazone h hour Hsc heat shock cognate HSE heat shock element HSF1 heat shock factor-1 Hsp heat shock protein ILT incipient lethal temperature IMTG intramuscular triacylglycerol kDa kiloDalton K m Michaelis-Menten constant L liter LB Luria-Bertani L:D light:dark L D H lactate dehydrogenase L O E loss of equilibrium min minute M L E T median lethal exposure time mM millimolar mRNA messenger ribonucleic acid MS-222 tricaine methanesulfonate mtDNA mitochondrial deoxyribonucleic acid xii N A D H nicotinamide adenine dinucleotide, reduced N O A A National Oceanic and Atomospheric Administration OLTT oxygen limited thermal tolerance PCR polymerase chain reaction ppt parts per thousand Qio temperature coefficient qRT-PCR quantitative reverse transcription polymerase chain reaction R A C E rapid amplificatioin of cDNA ends RCR respiratory control ratio RFLP restriction fragment length polymorphism RNA ribonucleic acid RNAi RNA interference s second SD standard deviation SEM standard error of the mean SNK MRT Student-Newman-Keuls multiple range test 1 acute acutely selected temperature T°C s e l final selected temperature T°Cf,nal final thermal preferendum T c critical temperature T f failure temperature Tp pejus temperature Ton threshold induction temperature for gene expression T C A tricarboxylic acid cycle Time sei time to final selected temperature Ucrit critical swimming speed UTR untranslated region Vmax maximum enzyme velocity w watt xiii C O - A U T H O R S H I P S T A T E M E N T CHAPTER T W O : Authors: Date Accepted: Journal: Copyright policy: Comments: Intraspecific Variation in Thermal Tolerance and Heat Shock Protein Gene Expression in Common Killifish, Fundulus heteroclitus Nann A. Fangue, Myriam Hofmeister, and Patricia M . Schulte April 11 t h, 2006 Journal of Experimental Biology. 209:2859-2872. Articles in JEB are published under an exclusive, worldwide license granted to the publisher by the authors, who retain copyright. This study was conducted by N.A.F. under the supervision of P.M.S. M.H. was a visiting student who provided valuable technical assistance under N.A.F's supervision. CHAPTER T H R E E : Authors: Date Accepted: Journal: Copyright policy: Comments: Countergradient variation in temperature preference in populations of killifish, Fundulus heteroclitus Nann A. Fangue, Jason E. Podrabsky, Larry I. Crawshaw, and Patricia M . Schulte. To be submitted September 2007. A version of this chapter will be submitted to Ecology Letters Author retains copyright. This study was conducted by N.A.F. under the supervision of P.M.S. J.E.P and L.I.C. provided expert advice and experimental facilities. xiv CHAPTER FOUR: Intraspecific Variation in Swimming Performance and Energetics as a Function of Temperature in Killifish, Fundulus heteroclitus Authors: Nann A. Fangue, Milica Mandic, Jeffrey G. Richards, and Patricia M . Schulte Date Accepted: Journal: Copyright policy: Comments: Accepted pending revisions. Physiological and Biochemical Zoology. Authors have the non-exclusive right of republication of their papers, in whole or in part, in any book for which they are an author or an editor, provided that they give credit to the journal. This study was conducted by N.A.F. under the supervision of P.M.S. M . M . was a summer student who provided valuable technical assistance under N.A.F's supervision. J.G.R. provided expert advice. CHAPTER F I V E : Authors: Date Accepted: Journal: Copyright policy: Comments: Inter-population variation in the effects of thermal acclimation on mitochondrial properties in killifish (Fundulus heteroclitus) Nann A. Fangue, Jeffrey G. Richards, and Patricia M . Schulte To be submitted October 2007. A version of this chapter will be submitted to the Proceedings of the National Academy of Sciences. Author retains copyright. This study was conducted by N.A.F. under the supervision of P.M.S. J.G.R. provided expert advice. xv C H A P T E R O N E : I N T R O D U C T I O N OVERVIEW The goal of the research reported in this thesis is to understand the responses of organisms to temperature change on a variety of time scales. To address this goal, I have used populations of killifish (Fundulus heteroclitus), which are found in habitats that exhibit temperature variation on a variety of temporal and spatial scales. I have defined the responses to temperature of several critical organismal performance measures including thermal tolerance, thermal preference, and swimming performance and have begun to address the mechanistic basis of these responses. The purpose of this introductory chapter is to provide the background needed to place the chapters of this thesis into the context of the field of thermal biology and to provide justification for the experimental approaches that I have utilized in this research. TEMPERATURE AND BIOGEOGRAPHY Temperatures as low as - 89°C and as high as 350°C have been recorded on the earth's biosphere. Animals in metabolically active states, however, are restricted to a much narrower range of thermal conditions. Metazoans can be found at temperatures ranging from approximately - 6 0 ° C to 50°C, but comparative studies on organisms from diverse thermal environments have revealed that species differ dramatically in the ranges and absolute temperatures they can tolerate. Over evolutionary time scales, species have specialized to function within a specific range of temperatures, and thus, no single species can live across all temperatures where life is found (reviewed in Hochachka and Somero, 2002). This specialization is, in part, a reflection of the pervasive effects of temperature on biochemical reactions. Temperature is therefore considered to be one of, if not the, most important abiotic factors specifying organismal distributions (see for example Fry, 1947; Hochachka and Somero, 2002). Indeed, two important tenets have shaped the field of thermal biology: 1) temperature is 1 one of the most pervasive variables affecting biological processes, and 2) the laws of thermodynamics define the direction and rate of biochemical processes and thus underlie the effects of temperature on whole organismal performance. Because of the pervasive nature of the effects of temperature on biological processes, understanding temperature's role as a determinant of species distribution patterns has historically been a central goal in both ecological and physiological research (Valentine, 1966; Gaston, 2003; Somero, 2005; Osovitz and Hoffnann, 2007). This issue is particularly pressing because of growing concern regarding the effects of warming global temperatures on ecosystems, including potential changes in community structure and biogeographical patterning (Gilman et al., 2006; Harley et al., 2006). Predictions have been made suggesting that, as temperatures rise, the distribution and abundance of organisms will shift according to their thermal tolerance limits and their ability to adjust to new environmental temperatures (Fields et. al., 1993; Lubchenco et al., 1993). In fact, recent evidence from polar, temperate, and tropical ecosystems has shown that climate change has already affected the distribution and local abundance of many marine organisms (Hughes, 2000; Walther et al., 2002; Root et al., 2003; reviewed in Parmesan, 2006; Pdrtner and Knust, 2007). Because of the clear importance of temperature in specifying species' distributions and abundance, one of the primary goals of thermal biologists is to define the physiological and biochemical mechanisms that underlie an organism's thermal niche. T H E R M A L REACTION NORMS One approach to understanding the role of temperature in shaping an organism's thermal niche is to quantify thermal reaction norms. A thermal reaction norm is a curve that describes the effect of temperature on a measure of organismal performance (Huey and Kingsolver, 1979). Thermal reaction norms typically have a shape in which performance increases with increasing temperature, reaches a maximum or optimum at some intermediate temperature, and declines 2 again with further temperature increases (Figure 1.1). As performance declines away from the optimum, the organism eventually reaches temperatures where performance is poor but which the organism is capable of tolerating. Finally, the performance curve is bounded at low and high temperature extremes where an organism is incapable of maintaining function. Optimal Performance Temperature Figure 1.1 Generalized thermal reaction norm. While the thermal reaction norm is often assumed to have a normal distribution, the specific shape and position of these performance curves can vary among species and in response to thermal variation in an organism's environment. For example, vertical curve shifts alter the overall level of performance (Figure 1.2A), horizontal curve shifts change the temperature optimum of performance (Figure 1.2B), and adjustments to the width of the thermal niche alter the range of temperatures over which the organism is capable of maintaining performance (Figure 1.2C). As well, these vertical, horizontal, and width shifts are not mutually exclusive. Studies of ectothermic species found in contrasting thermal environments demonstrate variation in thermal niche specialization. For example, eurythermal species have a wide range of temperatures over which they are able to maintain performance (Figure 1.2C, dotted curve) 3 Temperature (°C) Figure 1.2 Variation in thermal reaction norm position and shape. A) Increase in maximum performance, B) Shift in thermal optimum, and C) Change in width (breadth) of the thermal reaction norm. Note: Because the shape of the thermal reaction norm is not known, I have represented the thermal optimum as a plateau for ease of visualization. relative to stenothermal species that specialize function across a much narrower range of temperatures (Figure 1 .2C, solid curve). As well, the position of the curve can be shifted to reflect hyper- (warm), temperate (moderate), or hypo- (cold) thermal niche specializations (Figure 1 .2B, solid, dashed, and dotted lines, respectively). Studies of niche variation provide insight into the patterns of thermal performance that are shaped by local thermal regimes. T I M E SCALES OF T H E R M A L RESPONSES Thermal reaction norms are shaped by processes occurring over several time scales. Over evolutionary time scales, persistent differences in local thermal conditions may result in adaptive changes in local populations allowing for thermal compensation of physiological function (Hochachka and Somero, 2002) . These adaptive differences can be seen when comparing fishes from very different thermal environments that exhibit a conservation of rates of physiological activity when measured at each fish's common habitat temperature. If, however, at a given locality, temperature varies on a daily or seasonal basis, fish may need to respond to temperature change on much shorter time scales. The process of temperature compensation over 4 weeks to months is termed acclimatization (or acclimation when applied under controlled laboratory conditions in response to a single, well-defined environmental parameter). Acclimatory responses are designed to maintain a constancy of energy production and functional performance as temperatures change over weeks to months (reviewed in Hazel and Prosser, 1974). Animals may also need to respond to acute thermal challenges occurring over seconds to hours. Behavioral responses are often critical on this time scale, but physiological responses such as changes in heart rate are also important, and even some biochemical responses (e.g. heat shock protein induction) can be relevant on these very short time scales. The combination of biochemical and physiological mechanisms involved in responding to each of these time scales of thermal challenge is integrated to influence an organism's overall thermal niche. BIOCHEMICAL AND M O L E C U L A R DETERMINANTS OF T H E R M A L REACTION NORMS The study of biochemical adaptation to temperature continues to be a central focus of research in comparative physiology, and recent advancements in genomics and proteomics are offering exciting new hypotheses about the role of temperature in shaping organismal performance (Hoffnann et al., 2005; Hofmann and Place, 2007; Sdrensen and Loeschcke, 2007). There are a number of physiological and biochemical mechanisms known to respond to thermal acclimation and adaptation, which have been proposed to play important roles in dictating the thermal performance niches of organisms (Hazel, 1995; Feder and Hofmann, 1999; Hochachka and Somero, 2002; Angilletta et al., 2006). This section of the introduction will provide a general review of four well-studied candidate mechanisms that contribute to thermal acclimation and adaptation of organisms: 1) the thermal stability of proteins, 2) heat shock proteins and the heat shock response, 3) membrane properties, and 4) oxygen supply and demand. 5 DIFFERENCES IN THE THERMAL STABILITY OF PROTEINS There is a large body of literature providing evidence that thermal denaturation of proteins occurs at physiologically relevant environmental temperatures (Hofmann and Somero, 1995; Feder and Hofmann, 1999; Place et al., 2004). Proteins have low net free energies of stabilization, which are necessary for rapid conformational changes required for function (for example binding of substrates and cofactors) but the resulting high conformational flexibility may compromise the protein's ability to remain thermally stable across all temperatures. Protein homologues from species adapted to different thermal environments exhibit variation in thermal stability. A positive relationship between the heat denaturation temperature of proteins and maximum body temperature has been shown across a diversity of taxa (reviewed in Somero and Hofmann, 1997). As well, the kinetic properties of enzymes, such as ligand binding events, are also strongly affected by temperature. Adaptive variation in these binding events could be due to either alterations in the amino acid residues that interact with the ligands or modifications in the conformational changes that occur with ligand binding. Comparative sequence analyses for homologous proteins have shown that the active site of these proteins is generally highly conserved (Powers et al., 1993; Hochachka and Somero, 2002). Therefore, it has been suggested that the differences in the kinetic properties of homologous proteins are more likely the result of differences in conformational flexibility. Michaelis-Menten constants (Km), used as an approximate index of the affinity between enzyme and substrate or cofactor, are highly conserved across species at their normal body temperatures. As well, fine-scale patterns of K m conservation have been shown in congeneric or populations of conspecifics adapted to different thermal environments. Studies of LDH-A homologues in barracuda species (Holland et al., 1997) as well as among Antarctic Notothenoids (Fields and Somero, 1997; 1998) have shown that very few amino acid substitutions involved in stabilizing the enzyme's conformation can account for differential stability and kinetics of this protein. 6 The discussion above suggests that enzymes cannot be both highly active at low temperatures as well as thermostable at warm temperatures, or that there is a tradeoff between thermal stability and functional capacity of proteins. A second interpretation for this implied trade-off between stability and activity in enzymes evolved for different temperatures, however, is not that beneficial changes to both protein catalytic efficiency and stability are incompatible, but simply that natural selection has exerted pressure on one but not both of these characteristics (Giver et al., 1998). Directed evolution studies have been used to try to distinguish between these two possibilities. Giver and coworkers (1998) have shown that in the mesophilic bacterium, Bacillus subtilis, an increase in the thermal stability of an esterase enzyme can be achieved with no compromise in catalytic efficiency. Similarly, Miyaki et al. (2000), using psychrophilic bacteria mutant libraries and thermal evolution screening techniques, showed that mutants could be found which increased the thermostability of protease subtilisin S41 with no cost to catalytic efficiency. One particularly interesting recent study used a gene replacement technique in which the adenylate kinase homologue from a mesophile was inserted into a thermophilic bacterial species (Counago et al., 2006). Laboratory evolution to temperature of this laboratory-engineered strain produced very few mutant alleles, but the ones that were produced increased both the thermostability and activity at higher temperatures and had a direct effect on organismal thermal fitness. The results from these studies suggest that the thermal range for stability and catalytic efficiency of proteins can be improved simultaneously. Givers et al. (1998), however, suggests that the mutations achieving both thermal stability and catalytic efficiency simultaneously are rare, making them unlikely to evolve in natural populations. Similarly, Miyazaki et al. (2000) suggests that the rarity of this phenomenon in nature is likely due to evolutionary effects rather than intrinsic limitations to the structure and function of proteins. 7 HEA T SHOCK PROTEINS AND THE HEA T SHOCK RESPONSE From the discussion above, it is clear that the function of proteins can be compromised at temperatures within the physiologically relevant range for an organism. The most direct evidence for this phenomenon comes from literature showing a well-documented correlation between an increase in heat shock protein (Hsp) levels and increasing thermal stress (Feder and Hofmann, 1999). Heat shock proteins were originally discovered in isolated Drosophila salivary glands. Upon exposure to thermal stress, chromosomes within these glands puff up at the DNA sites where hsp mRNAs are made (Ritossa, 1962). Heat shock proteins are highly conserved and have been found in all species studied to date (Feder and Hofmann, 1999). Heat shock proteins are now known to be encoded by multiple genes that are assigned to families based on sequence similarity and molecular mass: Hsp90 (85-90kDa), Hsp70 (68-73kDa) and low molecular weight Hsps (16-47kDa) (Gething, 1997; Basu et al., 2002). Heat shock proteins differ in thermal inducibility, intracellular location, and function both within and between gene families (Gething, 1997). In unstressed cells, heat shock proteins are expressed constitutively, assisting in the folding, assembly, and translocation of newly synthesized proteins, and the degradation of misfolded proteins (Gething and Sambrook, 1992; Haiti, 1996; Haiti and Hayer-Hartl, 2002). The importance of Hsps in coping with stress hinges upon two major functions, both designed to maintain the stability of the cellular protein pool: minimizing non-native protein aggregations and targeting these aggregations for either stabilization and repair or degradation and removal from the cell (Lindquist, 1986; Hightower, 1991; Morimoto, 1998; Feder and Hofmann, 1999). The induction of Hsps has been shown to occur in response to many stressors in addition to temperature, including pathogens (Ackerman and Iwama, 2001), heavy metals (Heikkila et al., 1982; Duffy et al. 1999), hypoxia (Airaksinen et al., 1998; Sorensen et al., 2003), osmotic shock (Smith et al., 1999), parasitism (Merino et al., 1998; Rinehart et al., 2002) and the presence of predators (Pauwels et al., 2005). Important discoveries related to the functional significance of 8 Hsps in stress tolerance were made in the early 1990's (Parsell and Lindquist, 1993). Sanchez and Linquist (1990) showed that yeast with a deleted hspl04 gene had a significant reduction in their thermal tolerance. Overexpression studies of hsp70 in Drosophila have demonstrated a marked improvement in organismal thermal tolerance with increased Hsp70 levels (Welte, 1993; Feder and Krebs, 1998). Together, these studies provide direct evidence that Hsp expression is an essential component of inducible thermal tolerance. Over the last decade, much progress in understanding the role of Hsps in stress tolerance has been made in insects, particularly using Drosophila as an experimental model for the study of thermal adaptation (Hoffmann et al., 2003; Sorensen and Loeschcke, 2007). Laboratory selection experiments have shown that the nature of the heat shock treatment is a critical determinant of the levels of Hsps observed. For example, lines of Drosophila selected for survival after repeated exposures to Hsp-inducing temperature stress (i.e. heat-hardening) resulted in flies with elevated basal Hsp70 levels and increased resistance to thermal stress (Sorensen et al. 1999). Experimental fly lines derived following long-term exposure to continual high temperature stress, however, resulted in decreased basal Hsp70 expression levels (Bettencourt et al., 2002; Sorensen et al., 1999). At the time, the decrease in Hsp70 level in high-temperature selected lines was considered to be counterintuitive, but further experiments have demonstrated that the maintenance of high Hsp70 levels is costly. Several costs of Hsp expression have been identified, including poor fecundity (Krebs and Loeschcke, 1994), and negative effects on developmental rates and survivorship (Krebs and Feder, 1998). Similar patterns of costs and benefits of heat hardening have been confirmed in wild populations of Drosophila exposed to latitudinal and altitudinal gradients in environmental temperatures (reviewed in Hoffmann et al, 2003; Sorensen et al , 2003; 2005) suggesting an important balance between the positive and negative consequences of heat hardening events (Loeschcke and Hoffmann, 2007). These data have been used to argue that punctuated extreme thermal stress 9 events (i.e. maximum and minimum habitat temperatures) rather than those of mean annual temperatures drive the evolution of Hsp expression for thermal adaptation (Sorensen and Loeschcke, 2007). Although Hsps have been identified as ecological and evolutionary targets of thermal adaptation (Parsell and Lindquist, 1993; Feder and Hofmann, 1999; Hoffmann et al., 2003), fundamental questions remain about the role of Hsps in conferring thermal tolerance in natural populations, particularly in non-model organisms. The interpretation of the role of Hsps in conferring thermal tolerance to natural populations will have to consider the complexity of different climatic parameters to be fully understood. In aquatic systems, it is widely accepted that Hsps play a role in the cellular thermal stress response, and their expression likely varies with the organism's thermal history (Hightower, 1991; Dietz and Somero, 1992; Buckley et al., 2001; Nakano and Iwama, 2002). Correlation between the expression of Hsps and an increase in thermal tolerance has been shown in a number offish cell lines, primary cell cultures, and in intact organisms held under both laboratory and field conditions (for reviews see Iwama et al., 1998; Basu et al., 2002). It has been clearly shown that a species' threshold for Hsp expression is correlated with the levels of thermal stress they naturally experience (Dilorio et al, 1996; Feder and Hofmann, 1999; Tomanek and Somero, 1999). For example, field-acclimatized gobies, Gillichthys mirabilis, have been shown to increase both Hsp levels and induction temperatures such that these values are positively correlated with seasonal temperature increases (Dietz and Somero, 1992). These data also suggest that natural fluctuations in water temperature are sufficient to elicit the heat shock response, and make Hsps good candidates as ecologically relevant mechanisms used by animals to ameliorate thermal stress (Dietz and Somero, 1992; Fader et al, 1994; Hofmann and Somero, 1995; Somero and Hofmann, 1997; Buckley et al., 2001; Todgham et al., 2006). In the laboratory, it has been confirmed that Hsp expression patterns are correlated with an organism's 10 thermal history and thermal tolerance limits (White et al., 1994; Nakano and Iwama, 2002; Todgham et al., 2006). The Hsp response is highly regulated allowing for the rapid and preferential synthesis of Hsps under protein denaturing conditions (DiDomenico et al., 1982, Yost et al., 1990, Wu et al., 1990). Inducible hsp gene expression is transcriptionally regulated by the binding of the heat shock transcription factor (specifically HSF1) to promoter regions (heat shock elements or HSEs) in the hsp genes. Briefly, the model for the role of HSF1 in the regulation of heat shock genes involves the trimerization of the monomeric HSF1 giving HSF1 DNA-binding ability, hyperphosphorylation and transactivation of HSF1, and the translocation of the HSF1 trimer into the nucleus (Sarge et al., 1993). It has been demonstrated that hsp induction results primarily from the binding of an activated HSF to HSEs upstream of hsp genes (Morimoto et al., 1992), and Hsp levels inhibit their own synthesis through a negative feedback loop, mediating changes in the activity of HSF 1 (Morimoto, 1998). Several mechanisms promote the preferential synthesis of Hsps in the presence of protein denaturing conditions. Some of these include the absence of introns in the case of hsp70 (Ingolia et al., 1980), special sequences in the 3' UTR of the gene that promote hsp mRNA stability (Petersen and Lindquist, 1988), as well as sequences in the 5' UTR of the gene that promote translational efficiency (McGarry and Lindquist 1986). The cellular heat shock response has been studied in a number of comparative systems (Tomanek and Somero, 1999; 2002; Nakano and Iwama, 2002; Tomanek and Sanford, 2003). The utility of this approach can be seen in experiments involving congeneric species with thermally distinct distributions, because they offer the possibility to further refine our understanding of the links between biogeographical patterning and protein adaptation to temperature (Hofmann and Somero, 1996; Stillman and Somero, 1996; Tomanek and Somero, 1999; 2002; Tomanek, 2005). This approach has shown that slight changes in environmental temperature are sufficient to favor proteins with different thermal sensitivities and expression 11 patterns, and that these changes may be especially well-developed in organisms already living at or near their lethal temperature limits (Hochachka and Somero, 2002). While it is well established that multiple Hsp families containing multiple members are induced in response to thermal stress (Feder and Hofmann, 1999), we still have a poor understanding of the true biochemical diversity of the Hsps involved in an organism's response to thermal stress. Much of the early work on Hsp induction and expression was performed using one-dimensional gel electrophoresis, which often fails to discriminate among related Hsps within a family. As a result, most of the studies on the relationship between the heat shock response, organism thermal tolerance and population distribution and abundance patterns have been conducted on a few well-studied model species (Feder and Hofmann, 1999; Sorensen et al., 1999; 2001; 2005; Michalak et al, 2001; Sorensen and Loeschcke, 2007; although see also White et al., 1994; Norris et al., 1995; Tomanek, 2005). Considerations of the complexity of the heat shock response including analysis of multiple Hsp isoforms may be critical in understanding the true role of these proteins in whole-organism traits such as thermal tolerance. It has been suggested that organisms express different isoforms as a mechanism to maintain physiological flexibility in the face of fluctuating conditions (Hightower, 1991; Richards et al., 2003; reviewed in Schulte, 2004). The multiple Hsp isoform system could be an important mechanism used by ectotherms to maintain flexibility in their response to fluctuating thermal conditions. While experiments investigating multiple Hsp isoforms are limited, cDNA microarray results are revealing very complex patterns of mRNA expression with thermal acclimation in a variety of fish species (Podrabsky and Somero, 2004; Gracey et al., 2004; Buckley et al., 2006). As well, experiments at the protein level are detecting the upregulation of numerous Hsp proteins with heat shock (Tomanek, 2005). While it is not known whether these protein variants are the products of many different genes or whether they are due to post-12 transcriptional modification, it is clear that a high degree of protein variation could contribute to plasticity of thermal phenotypes. CHANGES IN MEMBRANE PROPERTIES Membranes play a variety of essential roles acting as physical barriers, controlling transport of molecules, establishing proton gradients across the inner mitochondrial membrane, and being involved in membrane-based cell signaling processes and neural activity. Structural lipids in biological membranes display complex phase behaviors and physical properties that are very sensitive to temperature (Hazel, 1995). Membrane lipids also have dramatic effects on protein conformations and position within the membrane in turn affecting protein function (Wodtke, 1981; Guderley, 2004). Therefore, the adaptive modulation of the lipid environment to maintain the function of membrane proteins is a critical aspect of thermal adaptation. Thermal effects on membrane composition, particularly the increases in membrane unsaturation with short term exposure to cold as well as long term thermal adaptation, are well documented (Hazel and Williams, 1990). Modification of the phase transition temperatures of biological membranes are achieved by alterations in lipid head group composition, changes in the types and positions of acyl chains, and changes in the amount of cholesterol in the membranes (Hazel and Williams, 1990; Hazel, 1995). Alterations to these three membrane characteristics are among the most consistent adjustments seen in organisms in response to temperature fluctuations. Because one of the primary consequences for ectothermic organisms exposed to changing thermal conditions is the perturbation of membrane organization, membrane physical structure has been proposed as a weak link in setting thermal tolerance limits. Few studies, however, have tested the causal link between increased membrane unsaturation and the acquisition of cold tolerance. A recent study tested this contention in Caenorhabditis elegans where membrane phospholipid saturation was manipulated and cold tolerance tested (Murray et al., 2007). RNAi 13 suppression techniques were used to knock down the mRNA expression of A9-acyl desaturase genes. Desaturases introduce double bonds into saturated fatty acids, and desaturase activity is upregulated in the cold in a variety of organisms (Fujii and Fulco, 1977; Suzuki et al., 2000). Murray and co-workers (2007) showed the expected positive relationship between cold tolerance and membrane unsaturation in wild-type worms as well as a reduced cold tolerance in A9 desaturase knock-down worms with more saturated membranes, suggesting the increases in unsaturation with cold are in fact involved in cold tolerance. But, more interestingly, lipid unsaturation differences between wild-type worms acclimated to 25 and 10°C were much smaller than those in the knock-down worms, yet these knockdown worms could not make a full transition between cold tolerant and susceptible phenotypes. These authors suggest that cold-induced changes in lipid saturation only contribute -16% to the cold tolerance phenotypes in their experiment and the naturally acquired cold tolerance in wild-type worms must involve other unknown mechanisms beyond membrane unsaturation (Murray et al., 2007). A second particularly sensitive function of membranes seems to be their role in synaptic transmission. Synaptic failure at both high and low thermal extremes has been suggested as a possible weak link in thermal tolerance. Support for this idea comes from comparative studies in fishes showing that under conditions of thermal stress, fish lose equilibrium (LOE) and engage in short bouts of uncoordinated swimming at temperatures very near those resulting in thermal death. In fact, L O E is the endpoint of choice used in many thermal tolerance studies in fishes (Cox, 1974; Beitinger et al., 2000). Concurrent with the temperatures where uncoordinated swimming occurs, the rate of the neurotransmitter, acetylcholine (ACh), released from synaptic vesicles rises sharply (MacDonald et al., 1988). Synaptosomal membranes show changes to their membrane physical states with thermal acclimation, and a possible consequence of possessing highly fluid membranes for function in the cold may be changes to membrane fluidity with warming and associated ACh release (Cossins et al, 1977; Logue et al., 2000). As well, 14 acetylcholine esterase (AChE, the enzyme that is responsible for the degradation of ACh) has a K m that is very temperature sensitive such that at the thermal extremes of an organism's physiological tolerance, the affinity of AChE decreases very rapidly (Baldwin, 1971). The result is an increase in the amount of ACh released and a loss in the binding ability of the degradation enzyme (AChE), and these changes could be key to understanding the failure of neural function at thermal extremes. OXYGEN SUPPLY AND DEMAND Up to this point, my discussion of the mechanisms involved in determining an organism's thermal niche has focused on mechanisms operating at the cellular level. However, mechanisms at the level of the organism may be of equal or greater importance in specifying the shape of an organism's thermal niche. A common observation in biology is that as organizational complexity increases from prokaryotes to metazoans, the maximum heat tolerance of each group decreases and their metabolic rate increases (reviewed in Pdrtner, 2002). While it is unknown if the rise in complexity causes the decrease in thermal tolerance, it is interesting to entertain the possibility that the limits of thermal tolerance are set at the highest level of organizational complexity (i.e. at the level of co-ordination of organismal components resulting in a functional organism). The concept of symmorphosis (developed originally to describe mammalian respiratory systems) suggests that animals maintain just enough 'structure' to support oxygen flux rates at their maximum oxygen uptake rates and to avoid excess capacity in any single component (Taylor and Weibel, 1981). H.O. Pdrtner (2002) has adopted this concept in the context of thermal limitation and suggests that oxygen limitations at both high and low temperatures may be a unifying principle defining the boundaries of organismal thermal intolerance. Pdrtner (2002) has suggested that an organism's thermal tolerance limits are linked to adjustments in the aerobic scope and aerobic capacity of the organism. The supporting data 15 come from experiments on water breathing ectotherms showing that at both high and low critical thermal tolerance thresholds, organisms make a time-limited transition from an aerobic to an anaerobic mode of metabolism before death (Pdrtner et al., 1998; 2000; Pdrtner, 2001). Pdrtner (2002) also cites supporting evidence from older descriptive studies in fish showing that decreased oxygen availability coincided with a decrease in thermal tolerance (Alabaster and Welcomme, 1962), and hyperoxia exposure resulted in increased thermal tolerance limits (Weatherley, 1970). These findings have led to the development of the "oxygen-limited thermal tolerance (OLTT)" hypothesis as a unifying theory of the effects of temperature on organismal performance limits (Pdrtner, 2002). The OLTT hypothesis proposes that at both low and high temperatures, organismal performance is limited by the inability to supply oxygen to the respiring mitochondria, i.e. that there is a mismatch between oxygen supply and demand. Increasing temperatures result in increased oxygen demand by the mitochondria, and at some critical temperature threshold (termed pejus=getting worse, Shelford, 1931) the mitochondrial oxygen demand outstrips the ability of the circulatory and ventilatory systems to supply oxygen. As a result, aerobic scope declines, causing a reduction in performance. As temperature (and thus metabolic rate) continues to increase, a critical temperature is eventually reached at which even standard metabolic rate cannot be maintained and system failure results. Decreasing temperatures, in contrast, cause declines in the ability of the mitochondria to produce ATP, thus compromising the ability to perform normal physiological functions including the function of the ventilatory muscles and the circulatory pumps that are needed in order to supply oxygen to the working tissues. Thus, acclimation or adaptation to the cold must involve increases in either mitochondrial density or changes in their functional properties to improve function in the cold. Under the OLTT framework, however, this increase in overall mitochondrial capacity in the cold sets a higher baseline oxygen demand resulting in a cost in 16 response to acute warming. At warm temperatures, not only is metabolic rate increased, but there is also an associated increase in oxygen radical production. Having a higher oxygen demand at warm temperatures would be expected to decrease the upper thermal limits of the organism. Thus, Pdrtner (2001) has suggested that "adjustments of mitochondrial densities and their functional properties appear as a critical process in defining and shifting thermal tolerance windows". In principle, adjustments in an organism's overall mitochondrial capacity in response to temperature could be due to changes in mitochondrial content (e.g. density of mitochondria in a tissue) as well as in the functional properties of the mitochondria (e.g. oxidative capacity modulated by lipid bilayer composition, types of enzymes/isoforms present, and membrane protein composition). Unifying principles describing the patterns of mitochondrial content and function adjustments with thermal acclimation and adaptation, however, have yet to be completely elucidated. Increases in oxidative function, mitochondrial densities, and/or mitochondrial enzyme activities with cold acclimation have been shown for a number of fish species (Guderley et al., 1997; Johnston et al., 1994; 1998; St. Pierre et al., 1998; Guderley and St. Pierre, 2002; Lannig et al., 2003; Kraffe et al., 2007). Higher mitochondrial densities and increases in the activities of mitochondrial enzymes within the mitochondria have also been associated with adaptation to cold temperatures (Johnston et al., 1998; Lannig et al, 2003; Lucassen et al., 2003; Lucassen et al., 2006). The data implicating functional adjustments to the mitochondria as adaptations to temperature over evolutionary time, however, are mixed. Evidence in stenothermic species suggests that there is no evolutionary modification of function with cold adaptation (Johnston et al., 1998), but more recent evidence in eurythermal species suggests that function may, in fact, be upregulated in cold-adapted groups (Tschischka et al., 2000; Sommer and Pdrtner, 2004). 17 Overall, however, as suggested by Pdrtner et al. (2007), the oxygen-limited thermal tolerance framework might be a very good tool to try to integrate thermal performance measures in fishes and address some of the outstanding questions in thermal biology. While the OLTT is complicated, the benefit of this framework is that it considers the relationships between whole organism aerobic performance as well as the underlying molecular and cellular mechanisms that affect the aerobic function and capacity adjustments. As well, this concept considers the tradeoffs of thermal adaptation and acclimation that occur in response to temperature and result in shifts in thermal performance niches of species. N E E D FOR AN INTEGRATIVE APPROACH From the discussion above, it is clear that multiple mechanisms operating at different levels of biological organization are involved in the acclimatory and adaptive responses of organisms to heterogeneous thermal environments. It is also clear that perspectives taken from both evolutionary biology and physiology are necessary to resolve how organisms respond and adapt to their natural thermal environments. Reductionist approaches (dissecting cells into individual components) combined with experiments at increasing levels of biological organization that test the function of cellular components within their normal physiological background are complementary means to achieving a detailed and synthetic understanding of thermal biology. In virtually every recent synthetic review in the thermal biology literature, there is a call for integrative approaches (Feder and Hofmann, 1999; Hochachka and Somero, 2002; Hoffmann et al., 2003; Sorensen and Loeschcke, 2007; Angilletta et al., 2007). Integrative approaches, however, are inherently complex, as the mechanisms involved at each level of biological organization do not simply sum up to adequately describe an organism's thermal response. As well, results from a single level of investigation cannot be used to predict the outcome at other levels of biological organization. It is these emergent properties and the 18 interactions between the mechanisms occurring at all levels of biological organization, as well as the temporal scale over which they occur, that make studying responses to temperature challenging. The goal of my thesis research has been to examine various mechanisms involved in thermal adaptation and acclimation/acclimatization to try to gain new insights into the physiological processes that set the limits of an organism's thermal performance niche. KILLIFISH AS AN EXPERIMENTAL M O D E L To address my thesis objectives, I have used populations of the common killifish (Fundulus heteroclitus), which are found in marshes and estuaries along the Atlantic coast of North America. Killifish demonstrate remarkable tolerance to challenging environmental conditions, most notably temperature and salinity extremes. Within the species, substantial variation exists in morphological, molecular, genetic, and physiological traits between populations (Morin and Able, 1983; Mitton and Koehn, 1976; Powers et al., 1986; Powers et al., 1993; Powers and Schulte, 1998; Schulte, 2001; Scott et al., 2004). This variation shows significant directional change with temperature/coastal latitude such that two distinct regional subspecies have been suggested - the northern form, Fundulus heteroclitus macrolepidotus, occurring from the Gulf of St. Lawrence, Canada to New Jersey, USA, and the southern form, Fundulus heteroclitus heteroclitus, distributed from Virginia, USA to the Northeastern coast of Florida, USA (Morin and Able, 1983). KILLIFISH HABIT A T Killifish are the most abundant fish species in tidal marshes along the east coast of North America (Taylor, 1999). Killifish daily movements are influenced by the tides with killifish moving into areas of the salt marsh flooded on incoming tides to feed on detritus, algae, amphipods, tanaids, copepods, and insects until the tide recedes (Allen et al., 1994; McMahon et 19 al., 2005). In addition to tidal influences, there is also some seasonality to killifish movements. Killifish have been shown to occupy different portions of the salt marsh in the fall and winter compared to the spring and summer. In the summer, killifish are found widely distributed throughout the salt marsh (Taylor, 1999). In the winter, however, Fritz and coworkers (1975) showed that Delaware killifish make a winter migration to deeper waters. Smith and Able (1994) studying southern New Jersey killifish (mixed northern and southern forms), however, found evidence that killifish remained in shallow, salt-marsh pools for the winter rather than being distributed in the intertidal and subtidal creeks that were typical of their summer range. These authors suggest a number of advantages for overwintering in the shallow pools including the potential for these fish to quickly raise their metabolic rates with increasing pool temperatures, minimal locomotory costs in these pools as the flow is very low, as well as the possibility that the pools may act as a refuge from predatory birds and blue crabs that leave these areas in the winter (Roundtree and Able, 1992; Burger, 1983). Even though killifish move to different habitats seasonally, their daily home ranges are estimated to be between 18 and 36 m in all seasons (Fritz et al., 1975; Lotrich, 1975; Smith and Able, 1994), and as a result any individual fish is confined to a very small geographic range along the Atlantic coast. In contrast, environmental water temperatures vary greatly across the complete species' range. Figure 1.3 compares seasonal mean and extreme water temperatures from Matanza, Florida (representative southern habitat) and Wells Inlet, Maine (representative northern habitat) from a single year (summarized from N O A A NERRS Data). Overall, environmental temperatures range from -1.4 to 31.6°C with temperatures between 7.6 and 21.2°C experienced by both northern and southern populations. Interestingly, these common temperatures that are experienced by both northern and southern populations occur in different seasons. For example, 20°C is an average temperature for a southern fish in the fall whereas northern fish experience temperatures of 20°C only in mid-summer. Mean monthly temperatures 20 are, on average, 13.3°C higher in Florida compared to Maine at any given time of year further emphasizing the differences in the thermal habitats of killifish populations. Moving away from these extreme latitudes, annual mean water temperature shifts by ~1°C with every degree change in latitude (NOAA NERRS Data). 35 30 25 U ° ~ 2 < H )-s « 15; a. E to u S-5 0 -5 O / ' / c • 8 C o m m o n Temperature Range 7 - 2 1 ° C Jan Feb Mar Apl May Jun Jul Aug Sep Oct Nov Dec Month (2001) ° Florida monthly high and low temperatures • Maine monthly high and low temperature Florida monthly average temperatures Maine monthly average temperatures Figure 1.3 Coastal, inshore water temperatures for representative northern (Wells Inlet, Maine) and southern (Matanza, Florida) killifish habitats (calculated from N O A A NERRS Data from 2001). In addition to seasonal temperature variation with latitude, daily temperature fluctuations of more than 5°C on either side of the daily mean temperature are common in most months and across the entire distribution range of killifish (calculated from N O A A NERRS Data). There is also significant spatial variation in local temperature regimes such that coastal, estuary, and tidally isolated pools could all differ in their thermal characteristics within a small geographic area. While the latitudinal, seasonal, daily, and spatial thermal characteristics of killifish environment are very complex, studying killifish populations from across this geographic range offers the opportunity to ask the question: how can local adaptation occur in these killifish populations in an environment that is itself inherently thermally variable? 21 KILLIFISH REPRODUCTION Killifish are semilunar spawners and lay their eggs high in the intertidal zone on the highest spring tides of new and full moons. The spawning season varies with latitude beginning in March to May and ending in July to September, with the longest season occurring in southern populations (Kneib and Stiven, 1978). The embyos develop aerially in 7-15 days and hatch upon immersion in water by the spring tide following the one on which they were laid. An individual female can lay several hundred eggs in a single spawning event, and females spawn multiple times during the reproductive season (Taylor, 1999). Killifish from southern habitats typically lay their eggs in protected areas like inside empty mussel shells or nestled among vegetation (Taylor, 1999), whereas northern fish tend to use areas with less protection like the surfaces of algal mats or buried in the sand (Taylor and DiMichele, 1983). Embryo developmental rates have been shown to differ between killifish populations from different latitudes in a countergradient manner such that at a common temperature, eggs from northern fish develop faster than those from southern fish (DiMichele and Westerman, 1997). This developmental pattern is thought to be an adaptive mechanism to account for differences in aerial development time due to differences in latitudinal aerial temperatures. This strategy allows killifish at all latitudes to synchronize development and hatching times to match the high tide cycle irrespective of aerial temperature differences with latitude (Taylor and DiMichele, 1983; DiMichele and Westerman, 1997). KILLIFISH GENETIC VARIATION There is substantial genetic variation between northern and southern killifish, as assessed using both mitochondrial and nuclear DNA loci, suggesting a complicated history of spatially variable selection and secondary integration (Bernardi et al., 1993; Gonzalez-Villasenor and Powers, 1990; Adams et al., 2006). There are significant directional changes in allele isozyme 22 frequencies in relation to latitude (i.e., a cline), and these clines are highly variable in both shape and width between loci, supporting the argument that the genetic variability is maintained by selection (Brown and Chapman, 1991; Powers et al., 1993). Mitochondrial and nuclear DNA data collectively suggest that there is a sharp break in the genetic variation between northern and southern killifish forms in northern New Jersey near the mouth of the Hudson river (Bernardi et al., 1993; Gonzalez-Villasenor and Powers, 1990; Adams et al., 2006). Mitochondrial DNA restriction fragment length polymorphism (RFLP) data suggests that the divergence time between the two subspecies was approximately 1 million years ago (Gonzalez-Villasenor and Powers, 1990), well before the last glaciation event -12,000 years ago. A recent analysis using 8 microsatellite markers in 15 killifish populations sampled from across the entire distribution range suggests that killifish have been broadly distributed throughout their current distribution range even during the last glacial event (Adams et al., 2006). The abrupt transition in allele frequency separating northern and southern populations may reflect regional disequilibrium conditions associated with the Post-Pleistocene colonization history of the east coast of North America (Adams et al., 2006). These studies place genetic variation in a historical context, but fail to determine the degree to which the variation observed is due to stochastic processes, such as genetic drift, or selective forces driving adaptation to local environmental conditions. Multidisciplinary approaches designed to detect adaptations, including phylogenetic independent contrasts, population genetic analysis, and selection experiments, have all shown that the physiological specializations and genetic variation between killifish subspecies are likely to be adaptive responses to temperature or some other factor correlated with latitude (reviewed in Powers and Schulte, 1998; Schulte, 2001). Mitton and Koehn (1975) examined the extent to which variation in biochemical phenotypes reflects adaptation to environmental temperature using three populations of killifish- northern, southern, and a northern population from the heated effluent of a power plant which were exposed to temperatures much more similar to those 23 of the southern population. They found that the artificially heated northern fish resembled the southern population much more closely in their isoenzyme patterns (allele frequencies), suggesting that these protein polymorphisms are an adaptive genetic response to temperature. This argument is further supported by shifts in gene frequencies in several loci at other localities where temperature anomalies are found (Powers et al., 1986). Extensive work on the lactate dehydrogenase-B (Idh-B) locus of killifish has provided the most conclusive demonstration of temperature's role as a selective factor. Lactate dehydrogenase-B is involved in the interconversion of pyruvate and lactate and is important in anaerobic glycolysis and gluconeogenesis. LDH-B isozymes are known to differ in their kinetic properties such that L D H - B b (northern genotype) has greater catalytic efficiency than L D H - B 3 (southern geneotype) at low temperatures and vice versa (Place and Powers, 1979; 1984). Ldh-B genotype is tightly correlated with latitude/temperature, with the ldh-Bb genotype found in northern populations, and the ldh-Ba genotype being dominant in southern fish (Powers et al., 1991). Pierce and Crawford (1997) used phylogenetic analysis to test functional significance of glycolytic enzyme variation across the Fundulus genus. When the influence of phylogeny was removed, high cardiac LDH-B levels were always correlated with more northern habitats suggesting an adaptive, compensatory strategy in Fundulus in response to temperature. Schulte et al. (1997) used population genetic analysis to examine sequence variation in the promoter region of Idh-B genes, and found that the distribution of sequence variation was not consistent with the expectation for a neutral model of molecular evolution, again suggesting that Idh-B has undergone selection. Common garden experiments on northern and southern killifish reared in the lab have shown differential mortality in the response to temperature during development, consistent with the prediction that southern fish should survive higher temperature regimes than northern fish (DiMichele and Powers, 1991). Embryonic development rate (DiMichele and Westerman, 1997), time to hatching (DiMichele and Powers, 1982a), and swimming 24 performance (DiMichele and Powers, 1982b) experiments also collectively support, and strongly suggest, that the functional differences between Idh-B genotypes are adaptations to environmental temperature or another tightly linked correlate. THESIS OBJECTIVES Despite the large body of literature demonstrating intraspecific variation in a number of physiological performance measures in killifish and the clear potential for the adaptive significance of this variation, most previous studies on these fish have been conducted at a single experimental temperature. Surprisingly, what was lacking when I began the work presented in this thesis was a careful investigation of variation in killifish physiological performance across a wide range of thermal acclimation temperatures in both killifish subspecies. The purpose of my thesis was to estimate the effects of thermal acclimation and adaptation on killifish thermal performance. The three main objectives of my thesis research were to: 1) quantify thermal tolerance limits in killifish populations, use these measures to define the limits of the thermal performance niche, and to assess the role of Hsps in establishing these limits, 2) estimate the shape of the thermal reaction norm, and assess the role of metabolic factors, including fuel use and mitochondrial metabolism, in establishing this shape, and 3) determine the location of the thermal optimum for each killifish population (Figure 1.4). 25 Performance Optimum Temperature Figure 1.4 Thermal reaction norm relating temperature to performance. The hypothetical locations of the thermal optimum, high and low thermal limits and overall thermal performance breadth are identified. OBJECTIVE ONE: QUANTIFICATION OF THERMAL TOLERANCE IN FISH Laboratory estimates of thermal tolerance limits in fish typically employ either static or dynamic methods. Static methods acutely expose fish to a series of near-lethal, constant temperatures for a predetermined time period (typically between 12 hours and 7 days). Upper or lower incipient lethal temperatures, (ILT or temperatures lethal to 50% of the population), and median lethal resistance times (MLET or time to 50% fish mortality at a given plunge temperature) are then estimated (Bennett & Beitinger, 1997). Static methods require large numbers of fish, relatively long exposure times, employ unnatural acute thermal challenges, and are now considered unethical as death is the experimental endpoint (Fry, 1947; Bennett & Beitinger, 1997; Beitinger et al., 2000). Critical thermal methodology (CTM) is a dynamic method for the determination of thermal tolerance limits and is the method I chose to use in my thesis. The critical thermal maximum (CTMax) for upper limits, or minimum (CTMin) for lower limits are defined as the temperatures at which fish lose the ability to escape conditions that will ultimately lead to death (Cox, 1974; Lutterschmidt and Hutchison, 1997; Becker and Genoway, 1979; Beitinger et al., 26 2000). During a C T M trial, water temperatures are constantly increasing or decreasing at rates slow enough to quickly equilibrate with the fish's body temperature, but fast enough to prevent thermal acclimation during the experimental trial. Trials continue until a predefined C T M endpoint is reached. The endpoint must be repeatable, ecologically and ethically defensible, and represent a lethal endpoint in nature, but be non-lethal in the laboratory as the fish must recover when returned to acclimation temperatures (Cox, 1974; Beitinger et al., 2000). Loss of equilibrium (LOE) is the most common endpoint in fish thermal tolerance studies (Paladino et al., 1980; Beitinger et al., 2000). Dynamic tolerance determinations are rapid, require relatively few fish, and the temperature changes used more closely approximate those seen in nature (Cox, 1974; Bennett & Beitinger, 1997). Chronic maximum and minimum determinations can also be made to define a fish's upper and lower thermal acclimation limits. Chronic trials use slow rates of water temperature change to allow the fish's thermal acclimation to keep pace with the temperature change. Bennett et al. (1997) suggested a rate of 0.5°C per day for chronic experiments as it fulfills the above criteria and is an ecologically realistic rate of temperature change. The respective chronic thermal maximum or minimum value is taken as the high or low temperature at which 50% mortality is observed (Fields et al., 1987; Bennett and Beitinger, 1997). Variation in thermal tolerance limits in fishes A synthesis of upper and lower thermal tolerance limits for a species collected over the organism's entire thermal acclimation range can be modeled into ecological thermal tolerance polygons where the size (°C 2 ) , position, and shape of these polygons reflects a fish's specific thermal tolerance niche. 27 I ' I 1 I ' 1 1—I r — 0 TO 20 30 40 Acclimation Temperature ( ° C j Figure 1.5 Comparative ecological thermal tolerance polygons adapted from Fangue and Bennett (2003). For example, the Antarctic notothenioid (Trematomus sp.) inhabits very cold, stable environmental temperatures and consequently has one of the smallest polygons measured to date (Somero and DeVries, 1967) (Figure 1.5). In contrast, eurythermal fish have large, centrally positioned polygons. For example, the eurythermal sheepshead minnow has a polygon area of 1470°C 2 (Bennett and Beitinger, 1997) and the Atlantic stingray has a polygon area of 1068°C 2 (Fangue and Bennett, 2003). Hyperthermal specialists like the redbellied pacu, Piaractus brachypomus, have polygons positioned at warm temperatures compared to hypothermal specialist like the Trematomus sp. and the brown trout, Salmo trutta, with polygons shifted towards colder temperatures (Cooper and Bennett, pers. comm.; Elliott, 1981). Polygon shape is also species-specific, and thermal acclimation can dramatically shift upper and lower thermal tolerance limits. For example, Atlantic stingray acclimated to constant temperatures ranging from 5 to 37°C can adjust their upper and lower thermal tolerance limits by more than 8°C across this thermal acclimation range (Fangue and Bennett, 2003). Other species such as Antarctic 28 notothenioids make minimal adjustments in their thermal limits with thermal acclimation resulting in polygons that are very flat and nearly independent of thermal acclimation (Figure 1.5). Intraspecific variation in thermal tolerance Studies aimed at trying to understand the finescale patterns of the relationship between environmental temperature and variation in thermal tolerance using intraspecific comparisons have produced mixed results. Studies across a wide variety of taxa have shown that in some cases geographic comparisons of thermal tolerance traits indicate that differences among populations exist consistent with what is predicted by local environmental thermal regimes (Krebs and Loeschcke, 1995; Guerra et al., 1997; Sdrensen et al., 2001; Lee et al., 2003b). Other comparisons where latitudinal variation in thermal tolerance is expected have failed to reveal such differences (Davidson, 1990; Kimura et al., 1994). In fishes, many studies have shown that populations of a species sampled from differing thermal environments and acclimated to common temperatures have thermal tolerance limits such that fish from cooler latitudes exhibit lower tolerance limits than their warm-water counterparts (Hart, 1952; McCauley, 1958; Otto, 1973; Fields et al., 1987; Lohr et al., 1996; Strange et al., 2002). In contrast, other studies have failed to show thermal tolerance differences in populations from thermally contrasting environments (Brown and Feldmeth, 1971; Elliott et al, 1994; Smale and Rabeni, 1995). Thus, in contrast to the ample evidence for interspecific variation in thermal tolerance related to thermal habitat, the evidence for intraspecific variation in thermal tolerance linked to habitat variation is mixed. 29 OBJECTIVE Two: ESTIMATING THE SHAPE OF THERMAL PERFORMANCE NICHES Once the thermal limits of the performance niche are defined, the next step is to try to understand how organisms perform within these thermal bounds. Defining the shape of the thermal performance niche of a species involves measuring physiological performance across a wide range of temperatures. This task may sound straightforward, but early workers in the field of thermal biology such as F.E.J. Fry and G.E. Hutchison quickly realized that defining a fish's thermal niche was no simple task (Fry, 1947; Hutchinson, 1957). Fry classified the influence of temperature on an organism's thermal niche such that temperature could act as a controlling, limiting, directing, or lethal factor (Fry, 1947). For example, temperature can establish physiological rates (controlling factor), determine organismal distribution patterns (limiting factor), dictate preference or avoidance responses (directing factor) or destroy metabolic integration (lethal factor) (Fry, 1947). Therefore, these factors in combination delimit the thermal niche of a species. Hutchinson (1957) viewed an organism's niche as a multidimensional world of biotic and abiotic gradients that describe the combinations of conditions where an organism could eke out its living. According to Hutchison, to fully quantify an organism's niche, all factors and their interactions must be considered. Very quickly, however, describing a niche beyond 3 dimensions becomes overwhelming experimentally. In the hopes of defining an organism's realized thermal niche (the niche occupied by an organism in nature), experiments defining niches based on a single measure of performance across temperatures (fundamental niche) are typically used. The results from numerous measures can then be used to try to integrate up to the overall realized niche for a species. With this goal in mind, there are several approaches commonly used to define the breadth of the thermal reaction norm in fishes including: measures of growth rates and fecundity, the quantification of swimming performance, and metabolic rate measures. The latter two of these categories were investigated in this thesis. 3 0 Swimming performance Swimming performance is considered a main character determining survival in many species of fish, and maximal swimming performance may strongly influence fitness (Jones et al., 1974; Rome et al., 1992; Young and Cech, 1993; Swanson, 1998). The profound effects of temperature on fish swimming performance and bioenergetics have been studied at many levels of biological organization and have been the subject of extensive literature reviews (Bennett, 1990; Randall and Brauner, 1991; Kieffer, 2000; Lee et al., 2003a; 2003b; Farrell, 2007). Many fundamental physiological and biochemical processes directly involved in a fish's swimming ability are strongly affected by acclimation temperature and suggest that temperature could be a key limiter to a fish's swimming performance. Fish swimming performance can be classified into three categories: burst (20 seconds or less), prolonged (endurance of up to 200 min ending in fatigue) and sustained (cruising speeds sustained for greater than 200 min) (Brett, 1967; Beamish, 1966). Critical swimming speed (Ucrjt) was the measure used to investigate prolonged swim performance in my thesis. Critical swimming speed quantifies the theoretical maximum prolonged swimming speed in fish (Brett, 1964) and provides an ecologically relevant measure of a fish's ability to survive ecological challenges (reviewed in Plaut, 2001). Critical swimming speeds are determined using aquatic swim tunnels capable of delivering stepwise increases in water velocity until the fish fatigues and can no longer swim against the current. In fish, Ucrit generally increases with increasing acclimation/swim temperature up to a temperature where performance is maximal. This performance maximum can be maintained across several degrees, and the breadth and location of the performance plateau can vary depending on the species. Further increases in acclimation temperature away from the optima result in a decline in performance, and at both cold and warm thermal extremes, a failure in swimming occurs (see Figure 1.4). This general bell-shaped performance curve has been demonstrated for many fish species, but the particular shape of the curve and the thermal optima have been shown to be 31 species specific and often reflective of the fish's thermal habitat regimes (Heap and Goldspink, 1986; Randall and Brauner, 1991; O'Steen and Bennett, 2003; Lee et al. 2003b; MacNutt et al. 2004). Previous swimming performance studies in killifish have found U c r j t values between 3.6 and 5.8 body lengths per second, and these swimming speeds are fairly comparable to those of size-matched salmonids (DiMichele and Powers, 1982b; Katzman and Cech, 2001). Killifish sampled from Delaware (in the center of the hybrid zone) and genotyped for Ldh-B differ in their swimming ability such that northern fish (Ldh-Bb genotype) outperform southern fish (Ldh-Ba genotype) at low temperatures (10°C), but the swimming performance of the two populations converges at warmer temperatures (25°C) (DiMichele and Powers, 1982b). These authors suggested that differences in Ldh-B genotype cause differences in glycolytic flux in erythrocytes, resulting in differences in ATP levels and blood haemoglobin oxygen affinities between populations such that northern fish have a greater ability to deliver oxygen to tissues at low temperatures thereby improving swimming performance. While swimming performance increased with temperature in both northern and southern killifish, the magnitude of this increase was modest (DiMichele and Powers, 1982b). At acclimation temperatures between 10 and 25°C, the Qjo for Ucrit in northern and southern killifish was 1.2 and 1.3, respectively (calculated from DiMichele and Powers, 1982b). Targett (1978) also showed that killifish metabolism is relatively insensitive to temperature changes. The Qjo of respiration rate was just over 1 at temperatures from 13-30°C, but below these temperatures, oxygen consumption declined steeply and showed a strong effect of temperature with a Qjo = 4.4 between 13 and 5°C (Targett, 1978). While the effects of very low temperatures on swimming performance are unknown, the large Q i 0 effect on metabolism below 13°C suggests that aerobic swimming might be compromised at low temperatures. 32 Biochemical studies of glycolytic enzymes have shown that metabolic organization may also differ between killifish populations. Northern killifish have higher lactate dehydrogenase (LDH) activity in heart muscle (Pierce and Crawford, 1997; Podrabsky et al., 2000), as well as twice the hepatic L D H activity at any acclimation temperature (Segal and Crawford, 1994) when compared to southern fish. Lactate dehydrogenase is thought to have undergone selection during thermal adaptation, as it has been shown to have functional consequences in the intact organism, and is correlated to survival and reproduction (Powers and Schulte, 1998; Pierce, 2000). Taken together, these data suggest that differences in L D H between killifish populations could differentially influence metabolic flux and carbohydrate metabolism in killifish populations, and thus potentially affect swimming performance. A number of studies have looked at the metabolism of resting and exercising fish acclimated to various temperatures. These studies have shown that fish maintain activity at different temperatures by altering their metabolic fuel use. For example, Alsop et al. (1999) found that Nile tilapia, Oreochromis niloticus, increased carbohydrate oxidation more than 2-fold and both lipid and protein oxidation decreased by half in fish acclimated to 5°C relative to those acclimated to 15°C. Similarly, Kieffer et al. (1998) have shown that aerobically exercised rainbow trout (45% U c rit) changed the relative contributions of lipids, carbohydrates, and proteins with acclimation temperature. At 5°C, swimming trout increased both carbohydrate and lipid oxidation, but decreased protein use relative to resting fish. Trout exercised at 15°C, however, increased lipid oxidation, slightly decreased carbohydrate oxidation, and kept protein use constant. These results demonstrate a complex dynamic in fuel utilization with both temperature and metabolic demand, and both studies suggest that at low temperatures fish may preferentially use carbohydrates as fuels. 33 Oxygen consumption and the role of the mitochondria As introduced previously, the OLTT hypothesis predicts that oxygen limitations at high and low temperatures could result in performance declines at high and low temperatures setting the limits of an organism's thermal niche (Pdrtner, 2002). One of the main predictions of the hypothesis is that in order to maintain function in the cold, cold-adapted organisms should have greater mitochondrial content and/or function than closely related, warm-adapted organisms. However, this increase in mitochondrial activity would be predicted to come at the cost of a reduction in high temperature tolerance in cold-adapted organisms, thus setting up a trade-off between cold and warm temperature tolerance. Several pieces of information suggest that there might be intraspecific variation at the level of the mitochondria in killifish. Work by Whitehead and Crawford (2006) using cDNA microarrays demonstrated that populations of Fundulus heteroclitus differ in the mRNA levels for a variety of mitochondrial genes including cytochrome c oxidase (COX) and ATP synthase (ATP Syn) (Whitehead and Crawford, 2006). Intraspecific variation in these genes was correlated with habitat temperatures after correction for non-independence due to phylogeny suggesting that killifish populations may differ in mitochondrial densities. The genetic differences between killifish populations in mtDNA sequences have revealed an extremely steep cline in mtDNA compared to other loci (Gonzalez-Villasenor and Powers, 1990; Beraardi et al , 1993; Adams et al., 2006), consistent with strong natural selection on mtDNA (Powers et al., 1993). Killifish populations have also been shown to differ in isolated tissue metabolic rates when acclimated to and compared at a common temperature such that northern fish have a higher metabolic rate than southern fish (DiMichele and Westerman, 1997 (embryos); Podrabsky et al., 2000 (heart)). Whole-organism measures of routine oxygen consumption of northern and southern killifish have also shown that northern killifish have 1.7 fold higher rates of oxygen consumption than southern fish at 5, 15, and 25°C acclimation temperatures (Appendix A; Fangue et al., unpublished). In addition, I have shown 34 that northern fish are more cold tolerant and less heat tolerant than their southern counterparts (Chapter Two; Fangue et al., 2006). OLTT predicts that higher metabolic rates should be associated with differences in thermal tolerance, as appears to be the case for killifish. What is not known, however, is what role adjustments at the level of mitochondrial content and/or function play in setting the boundaries of killifish thermal performance niches. Mitochondrial content cannot be simply defined as the number of mitochondria per cell. Rather, mitochondria exist as a dynamic syncytium where cristae surface areas as well as mitochondrial volume can change under various conditions (Bereiter-Hahn, 1990; reviewed in Gazaryan and Brown, 2007). Cristae density obtained from measurements of mitochondrial ultrastructure is a good correlate of mitochondrial capacity (Suarez, 1996), but these analyses are time consuming and prone to sampling artefacts. Consequently, most studies use the specific activity (V m a x ) of marker enzymes to assess mitochondrial content. In particular, cytochrome c oxidase (ETC component) and citrate synthase (TCA cycle enzyme) are the most commonly measured (Dalziel et al., 2005; Lucassen et al., 2006). Studies of isolated mitochondria have greatly enhanced our understanding of mitochondrial function. Intact mitochondria can be studied using respirometry techniques in which isolated mitochondria are supplied with oxidative substrates and ADP, and oxygen consumption measured (reviewed in Ballantyne, 1994). The determination of maximum oxidative capacity (State III) involves providing the isolated mitochondria with an oxidative substrate, a carbon intermediate to spark the T C A cycle, and saturating concentrations of ADP. As ATP is produced, an increase in oxygen consumption is measured and when all the ADP is converted to ATP, State IV is achieved. To estimate proton leak (State IV0i), oligomycin (an inhibitor of the ATP synthase) can be added to the preparation. Uncoupled rates of respiration (an index of ETC activity) can be obtained by administering the proton ionophore carbonyl-cyanide p-trifluoro-methoxyphenyl hydrazone (FCCP) as an uncoupler of oxidative 35 phosphorylation. Quantitative predictions of maximum oxygen consumption rates in vivo by the mitochondria are difficult because they are influenced by many factors including intracellular pH (Moyes et al, 1988), availability and affinity for ADP and N A D H (Brand and Murphy, 1987; Guderley and St. Pierre, 1999), membrane properties (Kraffe et al., 2007) and delivery of oxygen and fuels by the circulation (Mathieu-Costello et al., 1992). Isolated mitochondrial measurements, however, are a valuable tool and widely used proxy to estimate the maximal potential oxidative capacity of the mitochondria (Guderley et al., 1997; Johnston et al., 1998; Portner, 2002; Guderley, 2004; Sommer and Pdrtner, 2004). OBJECTIVE THREE: LOCATION OF THE THERMAL OPTIMA USING THERMAL PREFERENCE DATA In principle, all of the performance traits I have chosen to measure have a thermal optimum, but the location of the thermal optimum may vary among physiological traits within a species. For example, the thermal optimum measured for swim performance may not be reflective of the thermal optimum for growth or vice versa (for other examples, see Magnuson and Destasio, 1997). One approach to determine an organism's thermal optimum integrated across multiple performance parameters is to quantify the organism's preferred temperature. Temperature preference in fishes is a reflection of the behavioral control of body temperature by the selection of appropriate water temperatures (Houston, 1982) and a large body of literature suggests that fish have a finely tuned mechanism to discriminate between small differences in environmental temperatures (Steffel et al., 1976). Fry (1947) specifically defined the final preferendum as 'the temperature at which individuals will ultimately congregate regardless of their thermal experience before being placed in the gradient, and at which acclimation temperature and acute preferred temperatures are equal'. The underlying implications of this paradigm are that all other factors being equal, fishes in nature will tend to occur at their preferred temperature when available, and that the final preferenda will coincide with the 36 temperature at which key physiological, biochemical, and life-history processes are optimized. It is presumed, although debatable, that fish select 'thermal niches' to satisfy these criteria. There is an abundance of data supporting these tenets (reviewed by Reynolds and Casterlin, 1979); however, there are also many examples to confuse the issue. For example, centrarchids select temperatures in the lab well above those available in their natural habitat (Magnuson and Beitinger, 1978), while bluegill sunfish acclimated to low temperatures voluntarily select high temperatures resulting in 100% mortality (Beitinger and Magnuson, 1976). These examples suggest that thermal preference data must be carefully interpreted to resolve the adaptive value, if any, of a precise thermal preferendum. There is, however, general agreement between performance optima of many physiological correlates such as feeding, growth, metabolic rates, swimming performance, and reproduction (Beitinger and Fitzpatrick, 1979; Coutant, 1987) with preferred temperatures (Jobling, 1981; McCauley and Casselman, 1981). If the rate function optima coincide with thermal preference, the adaptive value will be much more clear than simply reporting preferred temperatures alone. Preferred temperatures in fishes are quantified in the laboratory using a variety of thermal gradient devices (reviewed in Fry, 1947; McCauley, 1977; Crawshaw and O'Connor, 1997). Vertical and horizontal thermal gradients are the most commonly used, but other designs such as rosette, cross-gradient, and electronic shuttlebox techniques have been successfully used in fish (reviewed in McCauley, 1977). Comparisons of thermal preference data from different studies are often confounded by differences in techniques or statistical treatment of the data, but overall there is good agreement in the preferred temperatures for a particular species across studies (McCauley, 1977; Crawshaw and O'Connor, 1997). Temperature preference typically incorporates both acute preference measured within 2 hours of a fish's exposure to a temperature selection gradient, which is reflective of the fish's thermal acclimation history, and the final preferendum, which occurs when the fish has been exposed to the gradient for a prolonged time 37 period (Fry, 1947; Schurmann et al., 1991). Temperature selection data consist of a range of occupied temperatures bounded by upper and lower avoidance temperatures. The preferred temperature is actually a statistical measure of central tendency (mode) of the temperature distribution (Reynolds and Casterlin, 1979) and is reflective of the temperature most often occupied by the fish. THESIS ORGANIZATION Following this introductory chapter, I have organized my thesis into four data chapters presented in manuscript form. The thesis concludes with a general discussion of my salient findings and future research directions (Chapter Six). CHAPTER TWO In Chapter Two, I quantified the thermal tolerance limits of several killifish populations, and investigated the role of heat shock protein as a mechanistic correlate of thermal tolerance testing the hypothesis that thermal tolerance limits and hsp mRNA expression patterns in killifish are shaped by adaptation to local environmental temperature. This hypothesis generated the following predictions: • Southern killifish should have higher CTMax values consistent with their warmer thermal habitats. • Northern killifish should have lower CTMin values than southern fish consistent with their colder thermal habitats. • Critical thermal limits should respond to thermal acclimation. • There is a tradeoff between high temperature tolerance and low temperature tolerance such that an improvement in heat tolerance should come at the expense of cold tolerance, and vice versa. 38 • Heat shock protein threshold induction temperature (T o n) has been shown to increase with increasing environmental temperatures among closely related organisms such that organisms from warmer environments have higher T o n 's. Therefore, I predicted that the T o n of hsp expression should be higher in more thermally tolerant southern killifish. CHAPTER THREE In Chapter Three, I tested the hypothesis that thermal preference is correlated with thermal tolerance limits. I quantified the acute and final thermal preferenda of northern and southern killifish acclimated to 5, 15, and 25°C. Secondly, I tested whether killifish preferred temperatures reflect habitat temperatures during the summer, active period for each population. The specific predictions were: • Southern killifish with higher C T M limits (Chapter Two) should prefer warmer temperatures than northern fish. • The preferred temperature for southern fish would be ~25°C compared to ~20°C for northern fish, reflective of summer habitat temperatures. CHAPTER FOUR In Chapter Four, I assessed the effects of temperature on critical swimming speed (UCn't) in killifish populations acclimated to a wide range of temperatures. Swimming trials were conducted at acclimation and acute challenge temperatures to determine the thermal sensitivity of Ucnt measures. Standardized exercise challenges as well as metabolic measurements were investigated as possible mechanistic correlates underlying intraspecific variation in swimming performance. The first hypothesis tested in this experiment was that the position of the thermal reaction norm for swimming performance in killifish populations would shift in the direction consistent with adaptation to local thermal regimes (see Figure 1.6 for predictions). 39 Northern killifish Southern killifish -5 0 5 10 15 20 25 30 35 40 Temperature (°C) Figure 1.6 Predicted patterns of swim performance in killifish populations acclimated to a wide temperature range. Relationships between northern and southern killifish populations at 10 and 25°C derived from DiMichele and Powers (1982b). The prediction for southern fish maintaining swim performance at higher temperatures relative to northern fish is suggested because of the higher thermal tolerance limits I have shown for southern killifish (Fangue et al., 2006). A second hypothesis tested in this chapter was whether or not killifish populations known to differ in L D H properties would show differences in the patterns or types of metabolic fuels (particularly carbohydrates) supporting exercise. Because northern fish have higher LDH-B activity in liver (Segal and Crawford, 1994), they may have a higher capacity to generate pyruvate and rely more heavily on gluconeogenic pathways to support exercise compared to southern fish that may rely more heavily on glycolysis. CHAPTER FIVE In this chapter, I tested the hypothesis that the differences in thermal limits of killifish populations are related to aerobic capacity adjustments at the level of the mitochondria, as suggested by the OLTT hypothesis (Pdrtner, 2002). In this experiment, I assessed mitochondrial density and function adjustments with thermal acclimation in eurythermal killifish populations. I quantified mRNA expression of several genes coding for mitochondrial proteins, assessed mitochondrial tissue content using mitochondrial enzyme activities, and measured the functional 40 properties of isolated mitochondria from northern and southern killifish acclimated to 5, 15, and 25°C. The OLTT hypothesis generated the following predictions: • Low temperature acclimation should increase mitochondrial density and/or function per mitochondria. • If northern killifish demonstrate patterns of cold-adaptation at the level of the mitochondria, northern killifish populations should show more pronounced increases in mitochondrial density and/or function than southern killifish. 41 REFERENCES Ackerman, P.A., and G.K. Iwama. 2001. Physiological and cellular stress responses of juvenile rainbow trout to Vibriosis. Journal of Aquatic Animal Health. 13: 173-180. Adams, S.M., Lindmeier, J.B., and D.D. Duvernell. 2006. 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Canadian Journal of Fisheries and Aquatic Sciences. 50: 703-707. 59 C H A P T E R T W O : I N T R A S P E C I F I C V A R I A T I O N I N T H E R M A L T O L E R A N C E A N D H E A T S H O C K P R O T E I N G E N E E X P R E S S I O N I N C O M M O N K I L L I F I S H , FUNDULUS HETEROCLITUS1 INTRODUCTION Temperature's pervasive effects on biochemical and physiological processes are thought to play a fundamental role in shaping the distribution and abundance of organisms. This is particularly true for ectotherms such as fish. Although studies comparing species that are widely divergent phylogenetically have revealed major patterns of thermal adaptation, understanding the role of temperature in establishing fine-scale patterns of thermal tolerance in closely related species or among populations within a species can provide additional insights into the nature of adaptive variation in thermal tolerance (Somero, 2002). The relationship between environmental conditions and variation in thermal tolerance, both intraspecifically and between closely related species, has been extensively studied in Drosophila (reviewed in Hoffmann et al., 2003). Comparisons of thermal tolerance of lab-bred flies derived from distinct geographic locations, however, have produced conflicting results. In some cases, geographic comparisons of thermal tolerance traits indicate that differences among populations exist consistent with what is predicted by local environmental thermal regimes (Krebs and Loeschcke, 1995; Guerra et al., 1997; Sorensen et al., 2001). Other comparisons where latitudinal variation in thermal tolerance is expected have failed to reveal such differences (Davidson, 1990; Kimura et al., 1994). Similarly, in fish, evidence for intraspecific variation in thermal tolerance is mixed. Many studies have shown that populations of a species sampled from differing thermal environments and acclimated to common temperatures have thermal tolerance limits such that fish from cooler latitudes exhibit lower tolerance limits than their warm-water counterparts (Hart, 1952; ' A version of this chapter has been previously published. Fangue, N.A., Hofmeister, M., and P.M. Schulte. 2006. Journal of Experimental Biology. 209: 2859-2872. 60 McCauley, 1958; Otto, 1973; Fields et al., 1987; Lohr et al., 1996; Strange et al., 2002). On the other hand, some studies have failed to show thermal tolerance differences in populations from thermally contrasting environments (Brown and Feldmeth, 1971; Elliott et al., 1994; Smale and Rabeni, 1995). Common killifish {Fundulus heteroclitus Linnaeus), inhabit estuaries and salt marshes along the east coast of North America through a latitudinal temperature gradient, and thus have been studied extensively as a model to investigate mechanisms of thermal adaptation. It has been shown that substantial variation exists within the species in morphological, molecular, genetic, and physiological traits (reviewed in Powers et al., 1993; Powers and Schulte, 1998; Schulte, 2001). This variation shows significant directional change with temperature/coastal latitude such that two distinct regional subspecies have been suggested - the northern form, Fundulus heteroclitus macrolepidotus, occurring from the Gulf of St. Lawrence, Canada to New Jersey, USA, and the southern form, Fundulus heteroclitus heteroclitus, distributed from Virginia, USA to the North-eastern coast of Florida, USA (Morin and Able, 1983). At the extremes of the species' range, northern fish experience temperatures ranging from -1.4 to 21°C, while southern fish encounter temperatures ranging from 7 to 31°C, and monthly mean temperatures are on average, 13°C higher in the south than in the north at any given time of year (calculated from N O A A NERRS Data, Sapelo Island, G A and Wells Inlet, ME). Clearly, the ability to acclimate to seasonal and daily temperature fluctuations is critical to all killifish populations, but the temperature range over which they must make adjustments is very different between populations and seasons. Early work on the thermal tolerance limits of killifish confirmed that these fish do in fact possess the ability to acclimate and tolerate a wide range of temperatures (Bulger, 1984; Bulger and Tremaine, 1985). This work, however, was performed on a single killifish population from Virginia (southern subspecies) acclimatized to seasonal photoperiod and temperature combinations, and only upper thermal tolerance limits were 61 quantified. These data, although useful in describing patterns of acclimation/acclimatization and tolerance for a single killifish population, provide no information about the nature of, or mechanisms involved in, intraspecific variation in thermal performance. A number of physiological and biochemical traits that are influenced by temperature and may play important roles in thermal performance of organisms have been proposed (Hochachka and Somero, 2002). At the molecular level, many candidate genes have been identified as potential targets of adaptive evolution to temperature (reviewed in Hoffmann et al., 2003; Somero, 2005). In particular, heat shock proteins (Hsps) are thought to play an ecologically and evolutionarily important role in thermal adaptation (Parsell and Lindquist, 1993; Feder and Hofmann, 1999). As molecular chaperones, Hsps interact with proteins that are in their non-native conformation (stress-denatured) in such a way that they prevent these proteins from interacting inappropriately with one another (for a review see Lindquist, 1986; Hightower, 1991; Morimoto, 1998). It has been clearly shown that a species' threshold for Hsp expression is correlated with the levels of thermal stress they naturally experience, and that natural fluctuations in environmental temperatures are sufficient to elicit the heat shock response (Roberts et al., 1997; Tomanek and Somero, 1999; Buckley et al., 2001). Taken together, these findings suggest that Hsps are good candidates as ecologically relevant mechanisms used by animals to ameliorate thermal stress and likely have important roles in thermal adaptation. It has long been known that heat shock proteins are encoded by multiple genes that are assigned to families based on sequence similarity and molecular weight. Two important Hsp families are Hsp90 and Hsp70, each containing several members, some of which are expressed constitutively under normal physiological conditions (i.e., Hscs) and some of which are induced in response to protein-denaturing stress (i.e., Hsps) (Gething, 1997). Members of both the Hsp90 and Hsp70 families are known to be important in folding of nascent polypeptides as well as renaturation of heat damaged proteins (Morimoto and Santoro, 1998). However, our 62 understanding of the true biochemical diversity of the heat shock response in an ecologically relevant context remains limited, because much of the early work addressing this question was performed using 1-dimensional gel electrophoresis, which often fails to discriminate among related heat shock proteins within a family. As a result of these technical limitations most of the work that has comprehensively addressed the relationship between the heat shock response, whole-organism thermal tolerance and population distribution and abundance has been performed on a few well-characterized model species (Feder and Hofmann, 1999; Sorensen et al., 1999; 2001; 2005; Michalak et al, 2001; although see also White et al., 1994; Norris et al., 1995; Tomanek, 2005). In this study, we quantified thermal tolerance and investigated the mRNA expression patterns of a variety of isoforms of heat shock proteins (Hsps) in killifish populations in order to address several questions: 1) Are there differences in thermal tolerance between killifish populations that correlate with latitudinal temperature ranges? 2) Is intraspecific variation in thermal tolerance related to differences in the sequence of hsp genes between populations? 3) Is intraspecific variation in thermal tolerance related to differences in the expression patterns of hsp genes? 4) Are differences in hsp expression patterns between populations consistent across multiple hsp genes? By addressing these questions we are able to provide valuable insight into how local adaptation can occur between fish populations from two different environments even when the local environments are themselves highly variable. MATERIALS AND METHODS EXPERIMENTAL ANIMALS Adult killifish of the northern subspecies (Fundulus heteroclitus macrolepidotus) were collected from three locations: Hampton, New Hampshire (NH; 42° 54' 46" N), USA, Salsbury 63 Cove, Maine (ME; 44° 25' 54" N), USA, and Antigonish, Nova Scotia (NS; 45° 37' 0" N), Canada. Fish of the southern subspecies (Fundulus heteroclitus heteroclitus) were also collected from three sites: Brunswick, Georgia (GA; 31° 7' 31" N), USA, Whitney Island, Florida (WI; 29° 39' 34" N), USA, and Fernandina Beach (FB; 30° 40' 51"N), Florida, USA. All collections were made in late spring of 2002 (northern (NH) and southern (GA) thermal tolerance experiments) or 2004 (interpopulation thermal tolerance and heat shock experiments). Fish were held in 75 L glass aquaria with biological filtration at 20 ppt salinity, 20 ± 2°C, and 12h:12h (L:D) photoperiod for a minimum of 3 weeks before the experimental acclimations described below. Fish were fed TetraMin® fish flakes supplemented with commercial trout chow (PMI Nutrition International, Brentwood, MO, USA) daily to satiation, but were not fed for 24 h prior to experimental trials. Treatment of all experimental animals was in accordance with the University of British Columbia animal care protocol #A01-0180. UPPER AND LOWER LETHAL LIMITS Upper and lower thermal acclimation limits were quantified for northern (NH) and southern (GA) killifish populations using chronic thermal tolerance methodology. Following a two-week holding period at 20 + 0.5°C, 30 fish from each population were subjected to either increasing or decreasing water temperatures of 0.5°C per day. Bennett et al. (1997) suggest this rate as it is slow enough to allow the fish's thermal acclimation to keep pace with the temperature change but yet is ecologically realistic. The respective chronic thermal maximum or minimum value is taken as the high or low temperature at which 50% morbidity is observed (Fields et al., 1987; Bennett and Beitinger, 1997). 64 THERMAL TOLERANCE METHODOLOGY Temperature tolerance in killifish populations was determined using the critical thermal methodology (CTM). The critical thermal maximum (CTMax) and critical thermal minimum (CTMin) are typically defined as the upper and lower temperatures, respectively, at which fish lose the ability to escape conditions that will ultimately lead to death (Cox, 1974; Becker and Genoway, 1979; Beitinger et al., 2000). The C T M test chamber consisted of a plastic rectangular water bath (50 x 35 x 15 cm) containing 10 individual 1-liter plastic test beakers. The water bath was filled with dilute ethylene glycol that could be heated or cooled with an immersion coil connected to a Lauda RM6 benchtop unit, and circulated with a Mag-Drive model 1.5 pump to insure complete mixing. Each beaker was filled with seawater and individually aerated to maintain oxygen concentrations at saturation and prevent thermal stratification during the trials. Beaker temperatures were monitored with Fisherbrand® NIST certified mercury thermometers (Fisher Scientific, Nepean, ON, Canada) and heating/cooling rates were between 0.28 and 0.33°C • min"1 for all trials. Loss of equilibrium (LOE) was chosen as our experimental endpoint, and critical thermal maxima and minima were calculated by taking the arithmetic mean of the L O E temperatures for each acclimation group (Cox, 1974; Beitinger et a l , 2000). At the end of each trial, fish were weighed (wet mass ± 0.1 g), measured (total length ± 0 . 1 cm), and returned to their acclimation conditions for recovery. We achieved >95% post-trial survival in all acclimation groups. EFFECTS OF A CCLIMA TION We assessed the relationship between acclimation temperature and upper or lower thermal tolerance of northern (NH) and southern (GA) killifish by estimating CTMax and CTMin of fish acclimated to one of seven constant temperature treatments ranging from 2.3 to 34.0°C. Acclimation temperatures were controlled with Fisherbrand® NIST traceable 65 temperature controllers and Ebo Jager 250 W submersible heaters. Killifish were acclimated for a minimum of 21 days to each treatment temperature under a 12h:12h (L:D) photoperiod and 20 ppt salinity. Three replicate 75 L acclimation tanks per temperature treatment were divided to house 10 northern fish on one side and 10 southern fish on the other. Five fish from each population per acclimation tank were randomly chosen to be in either a CTMax or CTMin trial. In total, 30 fish from each population (n=15, CTMax and n=15, CTMin) were tested from each acclimation group. All C T M trials were run between 10am and 2pm to minimize any effects of daily rhythms in thermal tolerance. V INTRASPECIFIC VARIATION In a second experiment, we explored intraspecific variation in thermal tolerance between replicate northern and southern killifish populations. Three northern populations (NH, M E , and NS) and three southern populations (GA, WI, and FB) were acclimated to 22 (± 0.25)°C under identical experimental conditions to those described above. Thermal tolerance trials and calculations were performed as previously described. IDENTIFICA TIONAND SEQUENCING OF HSP GENES Isolation of genomic DNA. total RNA extraction, and reverse-transcriptase PCR amplification Several genes of interest were cloned from killifish tissues including hsc70, hsp70-l, and hsp70-2 (gill, liver, and/or spleen), hsp90a (liver), and hsp90(3 (gill). The intronless hsp70 genes were cloned from both cDNA and genomic DNA, while all other genes were cloned from cDNA only. Genomic DNA was isolated from killifish spleens either by proteinase K digestion followed by phenolxhloroform extraction essentially as in Sambrook et al. (1989), or by the salting out method of Medrano et al. (1990). 66 Total RNA was extracted from either heat shocked or control tissues using the guanidine isothiocyanate method outlined by Chomczynski and Sacchi (1987) using TRIzol® Reagent (Invitrogen Life Technologies, Burlington, ON, Canada). Following isolation, RNA was quantified spectrophotometrically and electrophoresed on an agarose-formaldehyde gel (1% w/v agarose, 16% formaldehyde) to verify RNA integrity. RNA was stored at -80°C. First strand cDNA was synthesized from 5 u.g total RNA using oligo (dTi8) primer and Revert Aid™ H Minus M-MuLV reverse transcriptase as per the manufacturer's instructions (MBI Fermentas Inc., Burlington, ON, Canada). cDNA was stored at -30°C for up to 1 month or at -80°C for longer-term storage. Partial hsp90 sequence was obtained using primers determined from conserved regions of European sea bass, Dicentrarchus labrax (Accession No. AY395632), Atlantic salmon, Salmo salar (Accession No. AF135117), and zebrafish, Danio rerio (Accession No. AF042108). The forward primer was 5'-GGA CC(A/C) G(G/C/A)A A C C C(C/T)G A(C/T)G A C A T-3' and the reverse primer was 5'-CCT G(G/T)G C(C/T)T T C A TGA TCC (T/G)CT CC-3'. All sequences were aligned with ClustalW and primers were designed with the assistance of GeneTool Lite software (www.biotool.com'). Complete sequences of two isoforms of the inducible hsp70 and one isoform of the constitutive hsc70 were obtained from northern killifish (NH). Degenerate primers were initially used to obtain a 1500 bp fragment in the central region of each gene. These primers were designed based on conserved regions of zebrafish hsc70 (Accession No. Y l 1413), Ictalurus punctatus hsp70 (Accession No. U22460), Oncorhynchus tschawytscha hsp70 (Accession No.U35064), Xenopus laevis hsc70 (Accession No. X01102), and Gallus gallus hsp70 (Accession No. J02579). The sequence of the forward primer (Hsp701F) was 5'-GGA C C A C A C C(C/A)A GCT TGG-3' and the reverse primer (Hsp701R) was 5'-CGT TIG T G A TIG T G A T C T T G T T C - 3 ' . 67 Polymerase chain reactions (PCRs) were carried out in a PTC-200 MJ Research thermocycler using Taq DNA polymerase (MBI Fermentas Inc., Burlington, ON, Canada) and either cDNA (for all genes) or genomic DNA (for hsp70-l and hsp70-2) isolated as described above. Each PCR consisted of 40 cycles of 30 s at 94°C, 30 s at the primer specific annealing temperature, and lminute for every 1,000 bp of expected product at 72°C. PCR products were electrophoresed on 1.5% agarose gels containing ethidium bromide and bands of appropriate size were extracted from the gels using the QIAEXII gel extraction kit (Qiagen Inc., Mississauga, ON, Canada). Extracted PCR products were ligated into a T-vector (pGEM-T easy; Promega; Fisher Scientific, Nepean, ON, Canada), transformed into heat shock competent Escherichia coli (strain JM109; Promega; Fisher Scientific, Nepean, ON, Canada) and colonies were grown on ampicillin Luria-Bertani (LB) agar plates. Several colonies containing the ligated PCR product were selected and plasmids were isolated from liquid culture using GenElute Plasmid Miniprep kit (Sigma-Aldrich, Oakville, ON, Canada) and sequenced using an ABI 377 automated fluorescent sequencer at York University Molecular Biology core facility (Toronto, ON, Canada), or at the NAPS core facility at the University of British Columbia (Vancouver, BC, Canada). At least three clones of each fragment were sequenced bidirectionally. Consensus sequences for the hsp90 fragments were submitted to GenBank (hsp90a, Accession No. DQ202281; hsp90f3, Accession No. DQ202282). To determine the complete cDNA sequences for hsc70, hsp70-l, and hsp70-2 genes, isoform-specific nested PCR primers were designed based on the central fragment sequences obtained above, and used for 5'and 3' rapid amplification of cDNA ends (Smart R A C E cDNA amplification kit; BD Bioscience Clontech, Mississauga, ON, Canada). Primer sequences for 5' R A C E of hsp70 were as follows: external primer (for both isoforms) 5' T T C A C C T C A AA(C/T) A T G C C G T C C 3'; internal primer for hsP70-l 5' C A T TGC GCT C T C C T C TTT TGC 3'; internal primer for hsp70-2 5' TTT CTC TCT CCC GTC T T G CC 3'. Primer 68 sequences for 3' R A C E of hsp70 were as follows: external primer (for both isoforms) 5' A G C C A T G A C C A A G G A C A A C A A 3'; internal primer for hsp70-l 5' C C A G A G G A G T G C C A C A G A T A G A G 3', internal primer for hsp70-2 5' A G G TTT G A G C T G A C G G G A A T C 3'. Primer sequences for 5' R A C E of hsc70 were as follows: external primer 5' TTT GGC C G G G T G C T G T C A T T G A 3 ' , internal primer 5' A A A C C G C C G GCC A A T C A A CC 3'. Primer sequences for 3' R A C E of hsc70 were external primer 5'-CAC TGC T G G A G A T A C T C A TCT T G G T G G G-3\ internal primer 5' G C G G T G T T C C A C A G A T T G A G G T G A CCT T 3'. Each PCR consisted of 3 min at 94°C followed by 35 cycles of 30 s at 94°C, 30 s at 60°C and 1 min at 72°C, followed by 7 min at 72°C. PCR products were then electrophoresed and cloned as described above. At least 3 clones per fragment were sequenced in both directions at least twice, and a majority-rule consensus for the full-length cDNA transcript was developed for each isoform. Sequence assembly and analysis were performed using GeneTool Lite and DNAstar (Lasergene) software. Comparison with published sequences in GenBank was made with the BLAST algorithm (Altschul et al., 1997) and multiple alignments were produced using ClustalW (Thompson et al., 1994). Complete cDNA sequences have been deposited into GenBank (hsc70, Accession No. DQ202278; hsp70-l, Accession No. DQ202279; hsp70-2, Accession No. DQ202280). Sequence variation in hsc70 and hsp70 isoforms To determine whether there are any fixed differences in the sequences of hsc70 or hsp70 isoforms between northern and southern populations of killifish that might affect the function of these proteins, a series of isoform-specific PCR primers were developed to amplify the complete coding region of each isoform: hsp70-l forward 5' CTC A G A TCT TTT C C A CGT A C T C A 3', hsP70-l reverse 5'CTC C A G T A G T G A A A T GAT G C A GT 3'; hsp70-2 forward 5' C T G 69 A A A G G A A A G T G A GCC A A G A T G 3', hsp70-2 reverse 5' T A A A C A GTC C A G G A G A T G A G A GT 3', hsc70 forward 5' CCC G G A G A G GTC T G C TGT GT 3', hsc70 reverse 5' G G A GGT C T G A G G A T G G A A T G G T 3'. These primers were used to obtain the complete coding regions of these genes from at least three individuals from both northern (NH) and southern (GA) killifish populations. The sequences of a central fragment of the coding region of each gene (1,409 bp) were also determined for an additional five individuals from each population using the following primers: for hsp70-l forward 5' C A T G A A C C C C A C C A A C A C A A T C 3', reverse 5' C G A C A G C A G A C A CGT T T A G G A 3'; for hsp70-2 forward 5' CGC GTA C G G TCT GGA C A A A G G C 3', reverse 5' GCC CTT C A A GCT CTC GTC GTC C A 3'; for hsc70 the original degenerate primers (HSP701F and HSP701R) were used on cDNA from control fish. Phylogenetic analysis Amino acid sequences were deduced from the nucleotide sequence of each isoform for both northern and southern fish using GeneTool Lite Software. Because there were few fixed differences between populations, only northern killifish sequences were used in the phylogenetic analysis. Protein sequences or deduced amino acid sequences were obtained from GenBank for all the available complete fish hsp/hsc70 genes, and Bos and Homo sequences were used as representative mammalian species. Sequences were aligned using ClustalW and phylogenetic analysis was performed using the neighbour-joining method with pairwise deletion of gaps using MEGA2 software (Kumar et al., 2001). The support for each node was assessed using 1000 bootstrap replicates, and isoforms were named according to their position on the phylogenetic tree. 70 RELA TIONSHIP BETWEEN THERMAL TOLERANCE AND HEA T SHOCK PROTEINS Heat shock experiment To determine the threshold induction temperature of heat shock proteins in killifish, we acclimated northern (NH) and southern (GA) killifish to a common temperature of 20°C for 8 weeks as previously described. Groups of six fish per population were sampled directly from the acclimation tank (control) or transferred to one of several acute thermal challenge groups: 30, 31, 32, 33, 34, 35, and 36°C (GA only) or to 20°C (handling control) for 2 hours. Preliminary experiments with northern (NH) killifish acutely transferred from 20 to 36°C resulted in 100% mortality within 1 hour. Fish were then transferred back into 20°C recovery tanks for 1 hour. We chose a 1-hour recovery period based on a synthesis of literature values for eurythermal fish and several pilot experiments indicating that these exposure and recovery times were sufficient to induce changes in gene expression. Following the recovery period, fish were sacrificed by rapid decapitation and the gills were dissected and immediately frozen in liquid nitrogen. All tissues were stored at - 8 0 ° C until analysis. We elected to examine expression in the gill because preliminary experiments indicated that inter-individual variation in hsp gene expression was lowest in this tissue, thus maximizing our ability to detect inter-population differences in gene expression. Quantitative Real-time PCR analysis of hsc70, hsp70 and hsp90 gene expression Total RNA was extracted using TRIzol® Reagent, quantified spectrophotometrically, and cDNA was synthesized using 5 pg total RNA per sample (as previously described). Gene expression data were obtained using quantitative real-time PCR (qRT-PCR) on an ABI Prism 7000 sequence analysis system (Applied Biosystems Inc., Foster City, CA, USA). Gene specific primers were designed using Primer Express software (version 2.0.0; Applied Biosystems Inc., 71 Foster City, CA, USA) and are reported in Table 2.1. qRT-PCR reactions were performed using 2u.L cDNA, 4pmoles of each primer and 2X SYBR Green Master Mix (Applied Biosystems Inc., Foster City, CA, USA) to a total volume of 22 u.L under the following conditions: 1 cycle of 50°C for 2 min, 1 cycle of 94°C for 10 min, 40 cycles of 95°C for 15 sec, 60°C for 1 min. At the end of each qRT-PCR reaction, PCR products were subjected to a melt curve analysis to confirm the presence of a single amplicon. In addition, representative samples were sequenced to verify that the appropriate gene fragments were amplified. Samples of RNA that had not been reverse transcribed were also subjected to qRT-PCR to detect the possible presence of genomic DNA contamination. For the constitutively expressed genes as well as the control gene elongation factor-la, genomic DNA contamination was below 1:1024 starting cDNA copies for hsc70, 1:4096 for hsp90fi, and 1:2048 for EF-1 a. For the inducible genes (hsp70-l, hsp70-2, and hsp90ct), we considered a sample to be induced when mRNA levels were at least 32-fold greater than background genomic contamination. One highly induced sample was used to develop a standard curve relating threshold cycle to cDNA amount for each primer set. Results were then normalized using elongation factor-la {EF-1 a; Accession No. AY430091) as mRNA levels of this gene do not change with heat shock in killifish gills (data not shown). STA TISTICAL ANALYSES Thermal tolerance data sets were analyzed by analysis of co-variance (ANCOVA) with length or mass as co-variates. Corrected C T M values differed by no more than 0.1 °C to actual values; therefore, data sets from both the thermal tolerance and heat shock experiments were analyzed by multiple analysis of variance (ANOVA) with population, acclimation group, and/or heat shock temperature as factors without statistical adjustment for body size. Simple linear 72 regression (SLR) and polynomial regressions were used to explore and model the statistical relationship between CTMax or CTMin of killifish and acclimation temperature. All data met the assumptions of normality, and data were log transformed where necessary to meet assumptions of homogeneity of variance. When interactiomterms were not significant, post-hoc comparisons were performed among the groups with the Student-Newman-Keuls multiple range test (SNK MRT). If the interaction terms were significant, the data were separated and analyzed independently using one-way ANOVA. For all statistical analyses, a was set at 0.05. RESULTS THERMAL TOLERANCE IN KILLIFISH POPULATIONS Using laboratory acclimation studies, we identified acclimation ranges and thermal tolerance scopes for killifish from northern (NH) and southern (GA) populations. Chronic thermal maximum and minimum experiments revealed pronounced differences in survival between northern and southern killifish populations (Figure 2.1). Survival data were fit to a third order regression for each population, which predicted the chronic thermal maxima to be 36.4°C for northern fish and 38.2°C for southern fish. Chronic thermal minima experiments, however, revealed no difference between northern and southern populations with both populations surviving until the water ultimately froze at -1 .1°C. Acclimation temperature had a substantial effect on both CTMax and CTMin in northern (NH) and southern (GA) killifish populations such that C T M values increased with increasing acclimation temperature in both killifish populations (Figure 2.2). Simple linear regressions of CTMax and CTMin on acclimation temperature for northern and southern killifish populations were highly significant (SLR, PO.001, r 2 = 0.958 (CTMax) and 0.858 (CTMin), for northern fish and SLR, PO.0001, r 2 = 0.935 (CTMax) and 0.910 (CTMin), for southern fish) (Table 2.2). 73 Critical thermal maxima increased by 0.41° (northern fish) and 0.36° (southern fish) for every 1.0°C increase in acclimation temperature, while CTMin increased by 0.28° (northern fish) and 0.35° (southern fish) for every 1.0°C increase in acclimation temperature. Although simple linear regressions, as reported above, are traditionally used to model the relationship between acclimation temperature and thermal tolerance, we also used multiple regression techniques to determine the best-fit relationship for our data. Second order regression models (Figure 2.2) increased the coefficients of determination (r2) for each regression with the greatest improvements made in the CTMin models. By using these second order regression models, we were able to explain 95% or more of the variation in critical thermal limit by variation in acclimation temperature in all four models. EFFECTS OF BODY SIZE ON THERMAL TOLERANCE There were significant differences within northern (NH) and southern (GA) killifish populations in mean length and mass between acclimation groups (Table 2.3). When C T M values were adjusted by analysis of covariance (ANCOVA) using either total length (cm) or wet mass (g) as covariates, the corrected C T M values differed by no more than 0.1 °C from the actual values (Table 2.3). Therefore, no statistical adjustments of thermal tolerance values were necessary for either length or mass, and we used only the actual measured critical thermal tolerance values for all subsequent data interpretation and comparisons. INTRA- AND INTERPOPULATION VARIATION IN THERMAL TOLERANCE CTMax and CTMin were analyzed using two-way A N O V A with population and acclimation group as factors. Two-way ANOVA's revealed a significant effect of population and acclimation group, as well as a significant interaction term for both the CTMax and CTMin data sets (P<0.001 for all comparisons). One-way ANOVA's followed by post-hoc tests 74 revealed that within a killifish population, CTMax were significantly different at each acclimation temperature with two exceptions; CTMax for southern fish acclimated to 7.2 and 12.4, and CTMax for southern fish acclimated to 32.1 and 34.0°C were not statistically significantly different from one another (SNK MRT, P=0.242 and P=0.709, respectively) (Figure 2.2A). CTMin responses within a population of killifish were also significantly different at each acclimation temperature with the exception of the three lowest acclimation temperature groups. There were no significant differences in the CTMin of northern fish acclimated to 2.3, 7.2, and 12.4°C (P=1.000 for all comparisons). Similarly, no differences in CTMin within the southern population acclimated to 2.3, 7.2, and 12.4°C were found (P-values between 0.544 to 1.000 for all comparisons) (Figure 2.2B). Between-population comparisons revealed that the CTMax of southern fish within a temperature acclimation group was always significantly higher than the CTMax of northern fish (P<0.001 for all comparisons) (Figure 2.2A). The CTMin of southern fish within an acclimation group were also significantly higher than that of northern fish, except at acclimation temperatures of 12.4°C or lower where the CTMin's converged at the freezing point of brackish water (P-values between 0.544 and 1.000 for all comparisons) (Figure 2.2B). In order to determine if thermal tolerance differences between northern and southern killifish were consistent across multiple populations within a subspecies, we quantified CTMax and CTMin in samples of killifish from six different populations (3 of the northern subspecies and 3 of the southern subspecies) acclimated to a common temperature of 22°C (Figure 2.3). Within a subspecies, killifish populations did not differ in CTMax (P-values ranged from 0.060 to 0.928) or in CTMin (P-values ranged from 0.092 to 0.921). Between subspecies, however, southern killifish always had significantly higher CTMax (P<0.001 for all comparisons) and CTMin (PO.001 for all comparisons) than the northern forms (Figures 2.3A and 2.3B). 75 Northern (NH) and southern (GA) killifish were sampled from the same geographic location in two separate years and acclimated to 22°C (2002-thermal tolerance experiment, Figure 2.2; 2004-intraspecific experiment, Figure 2.3). There were no significant differences in C T M values within a population sampled in either year (compare Table 2.3 and Figure 2.3). SEQUENCE VARIATION IN FUNDULUS HSPS The degenerate primers used in this study allowed us to obtain three distinct heat shock protein 70-related transcripts in killifish gills. Phylogenetic analysis of the complete amino acid sequences of these transcripts (Figure 2.4) revealed that one was highly similar to other fish hsc70 sequences, whereas the remaining two transcripts were similar to other fish hsp70 sequences. The putative hsc70 from killifish was cloned from both control and heat shock samples, whereas the putative hsp70 transcripts were found only in cDNA isolated from fish exposed to heat shock, confirming their identification as constitutive and inducible transcripts, respectively. The two hsp70 transcripts did not group together phylogenetically, but instead each grouped with a distinct sequence from Xiphophorus maculatus. We tentatively named the hsp70 transcripts hsp70-l and hsp70-2, following the convention established for X. maculatus. In order to determine whether there were any fixed differences in hsp sequences between northern and southern killifish populations that might affect their function, we obtained the complete coding sequences of hsp70-1, hsp70-2, and hsc70 from a sample of individuals from each population (-10 individuals from each of the NH and GA populations). There were no fixed differences between populations in hsp70-2, and only a single fixed difference between populations in hsp70-l, which did not result in a change in the amino acid sequence. There were two fixed differences between populations in hsc70, one of which was silent, and one of which resulted in a change from serine in the southern population to threonine in the northern population at amino acid position 98. 76 Despite the high conservation of these sequences at the amino acid level, there was substantial silent polymorphism in these genes, most of which was found in the southern population, consistent with the pattern observed for other genes (e.g. Bernardi et al., 1993). There were 14 polymorphic sites in hsp70-l, all of which were found in the southern population, whereas the sample from the northern populations exhibited no variation among individuals. Similarly, hsp70-2 had 5 polymorphic sites, only one of which was found in the northern population. In contrast, there were three silent polymorphic sites in hsc 70 (in addition to the two fixed differences), two of which were present in both populations, and one of which was found only in the northern population. VARIA TION IN HSP EXPRESSION Prior to heat shock treatment, there were no differences in control and handling control mRNA levels in the gill between northern (NH) and southern (GA) killifish for any of the three hsp70 genes measured. The mRNA levels for hsc70 (constitutive isoform) in northern fish did not change with heat shock (Figure 2.5A). Southern fish, however, had elevated hsc70 mRNA levels in all heat shock groups, and this increase was significant in the 32°C group. As a result, southern fish had significantly higher hsc70 mRNA at all heat shock temperatures when compared to northern fish (P< 0.019 for all comparisons). The patterns of mRNA expression differed substantially between hsp70-l and hsp70-2 (inducible isoforms). Levels ofhsp70-l increased gradually with increasing heat shock temperatures in both populations and there was no difference between populations in the magnitude of this progressive induction (Figure 2.5B). The induction profile for hsp70-2 was more typical of an inducible gene with a clear onset temperature of expression (Figure 2.5C). Both northern and southern killifish demonstrated a significant elevation above control values in 77 mRNA levels (T o n) for hsp70-2 at 33°C. However, northern fish had significantly higher levels of hsp70-2 at 33, 34 and 35°C than southern fish (p<0.05 for all comparisons). Both northern and southern killifish populations showed a gradual increase in hsp90a expression (inducible isoform) with increasing heat shock temperature, and the overall magnitude of induction did not differ between populations (Figure 2.6A). T o n for hsp90a, however, did differ between populations, with significant induction occurring at 30°C in southern fish (P=0.021), and at 32°C in northern fish (P=0.012). Levels of hsp90B (constitutive isoform) did not change with heat shock in southern killifish. Northern killifish had slightly elevated hsp90B levels at 31-33°C. Two-way A N O V A for hsp90B mRNA levels with population and heat shock temperature as factors revealed a significant effect of heat shock temperature (P=0.001) and population (P=0.037) and no significant interaction (P=0.675) with southern individuals having overall higher hsp90B mRNA levels than northern fish. Post-hoc tests, however, revealed a significant difference in hsp90B mRNA levels only in control northern fish compared to southern fish. DISCUSSION KILLIFISH THERMAL TOLERANCE From the data presented here, it is clear that killifish are impressive eurytherms and can be acclimated to an exceedingly wide range of environmental temperatures. Both northern (NH) and southern (GA) killifish were able to survive temperatures approaching the freezing point of brackish water for several days and had chronic upper lethal limits of 36.4 and 38.2°C, respectively (Figure 2.1). As a result, fish from a southern population (GA) had a chronic scope (chronic maximum - minimum) of 39.3°C (Figure 2.1), which is larger than any previously 78 published value for fishes (reviewed in Beitinger et al., 2000). Furthermore, both northern and southern killifish had CTMax that approached or exceeded 42°C and CTMin of approximately -1.1 °C (Table 2.3), which are among the highest and lowest values, respectively, measured in fishes (reviewed in Beitinger et al., 2000). Of the available thermal tolerance scopes (CTMax -CTMin), only three of the most eurythermal fish have scopes that meet or exceed those of the killifish: the Amargosa pup fish, Cyprinodon navadensis (Feldmeth, 1981), the sheepshead minnow, C. variegates (Bennett and Beitinger, 1997), and the Atlantic stingray, Dasyatis sabina (Fangue and Bennett, 2003). The combined thermal acclimation and tolerance attributes of both northern and southern killifish populations encompass the entire range of daily and seasonal temperatures they naturally experience and may allow killifish to move freely between dynamic thermal microhabitats. Consistent with previous suggestions of local thermal adaptation, there were differences in thermal tolerance between killifish populations from different geographic locations. On average, southern (GA) killifish had critical thermal maxima that were 1.5°C higher than northern (NH) fish across all acclimation temperatures (Figure 2.2A). This 1.5°C difference between populations was also maintained for critical thermal minima except at the three lowest acclimation temperatures (Figure 2.2B). These differences in thermal tolerance between killifish populations are small relative to their large thermal acclimation ability, but comparisons within species for many physiological traits often show small overall intraspecific variation compared to the variation seem among species or higher taxa (Feder et al., 2000). The difference in C T M values between northern and southern subspecies was consistent across multiple sampling sites: southern (GA, WI and FB) killifish populations had critical thermal tolerance values that were significantly higher than northern (NH, M E and NS) populations (Figure 2.3). These differences in thermal tolerance were also maintained across samples obtained from the same locations in N H and G A in different years (2002 and 2004). 79 Taken together, these data suggest that the critical thermal limits for each killifish subspecies are an intrinsic property of that subspecies and are constant from year to year. However, additional work on laboratory-reared killifish will be necessary to confirm this suggestion and to rule out the possibility of maternal or developmental effects. Killifish thermal tolerance limits vary in a direction consistent with that predicted for fish that have undergone localized adaptation to habitat temperatures. These functional differences between killifish subspecies are in agreement with a variety of work by others suggesting that the physiological specializations and genetic variation between subspecies are likely to be adaptive responses to temperature or some other factor correlated with latitude (reviewed in Powers and Schulte, 1998; Schulte, 2001). HSPS AND THERMAL TOLERANCE The heat shock response is thought to be important for adaptation of organisms to their thermal environment (Feder and Hofmann, 1999; Hoffmann et al., 2003; Somero, 2005). A number of characteristics of the heat shock response could, in principle, respond to thermal selection, including: 1) the functional efficiency of the heat shock proteins themselves, 2) the onset temperature (T o n) at which hsp expression is induced, and 3) the magnitude of hsp expression under either basal or induced conditions. Although there is some evidence for each of these mechanisms in natural populations or following laboratory selection (Tomanek and Somero, 1999; Michalak et al., 2001; Feder et al., 2002; Sorensen et al., 2005) little is known about whether similar patterns are observed across multiple isoforms within a species, and few studies have assessed the relative roles and generality of these mechanisms. We found no differences in the amino acid sequences of hsp70-l or hsp70-2 within or between populations of killifish, and there was only a single highly conservative substitution between populations in hsc70 (ser-thr at amino acid 98). These data strongly suggest that changes in the functional efficiency of the 70-kD heat shock proteins have not been involved in 80 the evolution of differences in thermal tolerance between northern and southern killifish populations. The onset temperature of hsp induction (T o n) and magnitude of hsp expression exhibited a variety of patterns among isoforms when compared between northern and southern killifish populations. Among the three inducible genes we examined (hsp90a, hsp70-l and hsp70-2), hsp90a had a lower T o n in southern populations, but populations did not differ in the magnitude of induction, while hsp70-2 did not differ in T o n between populations, but was induced to a greater magnitude by heat shock in northern fish than in southern fish. In contrast, neither T o n nor the magnitude of expression differed between populations for hsp70-l. The observation that each isoform exhibited a different pattern of expression between populations suggests that differences in expression are not due to a global factor that affects all hsps, such as differences between populations in the stability of the total protein pool or overall rates of protein or mRNA turnover. Instead, this complex pattern of expression suggests that differences in mRNA levels between populations result from gene-specific mechanisms such as differences in transcription as a result of promoter sequence variation, or differences in mRNA stability as a result of sequence variation in the 5' or 3' untranslated region in a particular hsp gene (McGarry and Lindquist, 1986; Petersen and Lindquist, 1988). The lower T o n for hsp90a~we observed in southern killifish (Figure 2.6A) is in marked contrast to the results of most other studies, which have generally found that organisms from warmer environments induce Hsps at a higher temperature than closely related organisms from colder environments (Huey and Bennett, 1990; Dietz and Somero, 1992; Fader et al., 1994; Gehring and Werner, 1995; Hofmann and Somero, 1995; 1996; Tomanek and Somero, 1999; 2000). This discrepancy might be explained by the fact that our study examined mRNA levels, while most previous studies have focused on protein levels (Feder and Hofmann, 1999; Tomanek and Somero, 1999; 2000; Buckley et al., 2001). However, if this were the case, we would have 81 to postulate a decoupling of transcription and translation for hsp90ct Alternatively, the lower Ton for hsp90a could reflect an anticipatory response in southern killifish. Southern fish frequently experience peak water temperatures greater than 30°C, whereas water temperatures exceeding 30°C are rare in northern habitats. There is some support from experiments in Drosophila for maximum rather than mean environmental temperature as the important environmental thermal feature structuring adaptive thermal responses (Davidson, 1988; Anderson et al., 2003). An anticipatory upregulation ofhsp90a could allow southern fish to protect critical components of their protein-pool in the face of high environmental temperatures. The greater magnitude of hsp70-2 upregulation in northern killifish (Figure 2.5C) is consistent with the hypothesis that these fish are more sensitive to thermal stress. The magnitude of the heat shock protein response is typically proportional to the severity of the heat shock and associated protein damage (e.g. see DiDomenico et al., 1982). Similar to the results presented here, lines of Drosophila selected for high-temperature resistance have decreased expression of Hsp70 in response to heat shock compared to control lines suggesting that a sub-lethal heat exposure is less stressful for heat adapted populations thus leading to less cell damage and an overall smaller magnitude of stress protein induction (Sorensen et al., 1999; 2001). However, the hypothesis of differential thermal sensitivity is not supported by the results with hsp70-l mRNA levels, which exhibited no differences between populations in either T o n or the magnitude of induction (Figure 2.5B). Possible explanations for the differences in response between the two isoforms include differential sensitivity of these two isoforms to the denatured protein pool or differences in specificity between the hsp70-2 response and the hsp70-l response. Southern killifish had generally higher basal levels of the two constitutive mRNAs (hsc70 and hsp90jJ) than northern killifish. If these mRNA levels are indicative of differences in the standing protein pool, these differences could be protective and are thus consistent with the 82 greater thermal tolerance of southern fish. Work in poeciliid fishes (dilorio et al., 1996) suggests that higher constitutive levels of Hsc70 protein may be as or more important for thermal tolerance than changes in the inducible genes. However, there is some evidence in Drosophila and Arabidopsis that enhanced levels of Hsps under basal conditions can be deleterious (Krebs and Feder, 1997; 1998; Zatsepina et al., 2001; Sung and Guy, 2003), and thus increased levels of the constitutive Hsps in killifish in unstressed fish could have a negative effect. Basal levels of heat shock proteins, particularly Hsp90, can also affect the induction of heat shock protein genes (for review see Voellmy, 2004). Heat shock protein induction occurs via the binding of a transcription factor, the heat shock factor (HSF). Under unstressed conditions, HSF is present as a protein complex that includes Hsp90, Hsp70 and other chaperone molecules. This complex does not bind DNA, and thus inducible heat shock genes are transcribed almost undetectably under unstressed conditions. During thermal stress, the chaperone proteins dissociate from the HSF protein complex and bind to unfolded proteins within the cell, releasing HSF from inhibition (Wu, 1995). Based on this mechanism, we would predict that southern killifish populations, which have a higher level of hsp90BmRNA under unstressed conditions, would require a higher level of thermal stress to remove the repression of HSF by Hsp90, and thus would have a higher T o n and lower magnitude of expression at a given temperature for all inducible heat shock proteins. This was not the case. In fact, the only difference we observed in T o n between populations was a lower T o n for hsp90a in southern fish, in direct contrast to the predictions of this model. However, northern fish had a greater magnitude of hsp70-2 induction, which is consistent with model predictions. Although hsc70 and hsp90Bave considered to be constitutively expressed, we observed statistically significant increases in the expression of both of these genes in response to heat shock. It has been shown that some fish have the ability to upregulate hsc70 levels with increasing acclimation temperatures as well as with heat shock (Deane and Woo, 2005), while 83 other research has shown a pattern more typical of a constitutive isoform with no change in hsc70 levels with heat shock (Yamashita et al., 2004; Ojima et al., 2005). The pattern of hsc70 and hsp90B induction differed between killifish populations. Southern killifish exhibited a substantial increase hsc70 levels in response to heat shock while no change was observed in northern fish. The upregulation of hsc70 expression by southern killifish in response to heat shock may suggest an important role for Hsc70 in handling protein damage associated with daily fluctuations in environmental temperatures, and is consistent with the greater thermal tolerance exhibited by killifish from southern populations. In contrast, there was a small but statistically significant elevation of hsp90B in northern fish but not southern fish following heat shock, which is not obviously consistent with a hypothesis of thermal adaptation. The modulation of hsp mRNA expression patterns involving multiple isoforms from several Hsp families has been suggested as one mechanism ectotherms use to maintain flexibility in thermal phenotype in response to changing thermal environments (Hightower, 1991; Hochachka and Somero, 2002). The combinations of Hsps expressed, however, vary widely between organisms and the reason for this variation in protein expression is unknown. Often, this variation reflects both the evolutionary histories of the species and the recent thermal acclimation conditions encountered by that organism (White et al., 1994; Hofmann and Somero, 1995; Roberts et al., 1997; Tomanek and Somero, 2002; Tomanek, 2005) suggesting that substantial adaptive variation exists in the heat shock response. To our knowledge, only a single study has addressed the mRNA expression profiles of multiple hsp genes from several gene families in fish (Ojima et al., 2005). This work was performed in an immortalized rainbow trout gonadal fibroblast cell line and only a single acclimation temperature and heat shock temperature treatment was evaluated. Even with this simple experimental design, however, the authors demonstrate gene-specific variation in hsp mRNA levels. Results from microarray studies in fish exposed to constant or cycling environmental temperatures (Podrabsky and Somero, 2004) or 84 during cold acclimation (Gracey et al., 2004) show complex gene expression signatures that involve many gene classes known to be associated with thermal tolerance. However, these studies, and the current report, have examined these processes at the mRNA level, and mRNA levels are not necessarily predictive of the behaviour of the protein pool. Recently, Tomanek (2005) used 35S-labelling of newly translated proteins followed by two dimensional gel electrophoresis in turban snails exposed to heat shock showed the induction of over 30 proteins from several Hsp families with varying patterns among isoforms. While it is not yet known whether these proteins are coded by different genes or represent post-transcriptional modifications, it is clear that these protein variants are important and could contribute to the phenotypic plasticity seen in eurythermal organisms. Although correlative studies such as those of Tomanek (2005) and the current study cannot directly establish a causal link between the patterns of hsp isoform expression and whole organism thermal tolerance, these studies provide critical evidence of the under-appreciated diversity of the patterns of hsp expression in natural populations, their relationship to differences in whole-organism thermal tolerance, and their possible role in the establishment of biogeographical patterns. 85 REFERENCES Altschul, S.F., Madden, T.L., Schaffer, A.A. , Zhang, J., Zhang, Z., Miller, W. and D J . Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research. 25: 3389 -3402. 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Zatsepina, O.G., Velikodvorskaia, V.V. , Molodtsov, V.B. , Garbuz, D., Lerman, D.N., Bettencourt, B.R., Feder, M.E. , and M.B. Evgenev. 2001. A Drosophila melanogaster strain from sub-equatorial Africa has exceptional fhermotolerance but decreased Hsp70 expression. Journal of Experimental Biology. 204:1869-1881. 92 Table 2.1 Primers used for qRT-PCR of heat shock protein genes. Gene Sequence (5'-3') HSC70 F: A C A C C A C C A TCC C G A C A A A R: C A C A C C A G G CTG GTT A T C A G A GT HSP70-1 F: C G G A A T A A A TGT CCT G C G GAT R: C A A A A G TGC CTC C A C C A A GAT C HSP70-2 F: C T G A T C A A A C G C A A C A C C A C C R: CTC CCC TTC GTA G A C CTG GAT HSP90a F: C A G A T C TGC TGC GCT TCT A C A R: C G A G A A A C A T A G TCT TTG A G G G A A A C HSP90b F: T G A GCT GCT G C G CTA C C A R: CAT A C G GGT G A G GTA CTC TGT C A A EF-1 a F: G G G A A A G G G CTC CTT C A A GT R: A C G CTC GGC CTT C A G CTT Table 2.2 Simple linear regression equations and model comparisons of r2. Second order regressions are given in Fig. 2.2. Population Simple Linear Regression Models n P-value r2 (SLR) r 2 (2nd order) r 2 difference (2nd order - SLR) Northern (NH) CTMax N = 28.525 + (0.410 * Acc Temp) 103 O.001 0.958 0.972 0.014 Southern (GA) CTMaxs = 30.874 + (0.362 * Acc Temp) 102 <0.001 0.935 0.945 0.010 Northern (NH) C T M i n N = -3.190 + (0.279 * Acc Temp) 100 <0.001 0.858 0.963 0.105 Southern (GA) C T M i n s = -3.573 + (0.349 * Acc Temp) 103 <0.001 0.910 0,973 0.063 Table 2.3 Critical thermal maxima and minima (°C), total length (cm), and mass (g) for northern (NH) and southern (GA) killifish acclimated to a 12:12 L:D photoperiod and constant temperatures (Acc Temp) for 21 days. Acc Temp Pop Total Length Weight CTMax (°C) CTMin (°C) CTMax corrected for: CTMin corrected for: m e a n ± S D m e a n ± S D m e a n ± S D # of Fish m e a n ± S D # of Fish m e a n ± S D Length Mass Length Mass (°C) (cm) (g) (n) (°C) (n) (°C) (°C) (°C) (°C) (°C) 2.3 ± 0.47 N 6.21 ±0.54 2.88 ±0.79 15 28.6 ± 1.05 15 -1.1 ± 0 28.6 28.6 -1.1 -1.1 S 7.79 ±0.54 7.25 ± 0.79 15 30.8 ± 1.44 15 -1.1 ± 0 30.8 30.8 -1.1 -1.1 7.2 ±0.11 N 6.18 ±0.54 2.76 ±0.77 14 32.0 ±0.73 15 -1.1 ± o 32.0 32.0 -1.1 -1.1 S 7.38 ±0.95 5.13 ± 1.92 14 34.6 ±0.65 15 -1.1 ± 0 34.6 34.6 -1.1 -1.1 12.4 ±0.24 N 6.21 ±0.69 2.91 ± 1.10 15 33.6 ±0.87 15 -1.1 ± 0 33.6 33.6 -1.1 -1.1 S 7.40 ±0.83 5.83 ±2.04 15 34.9 ± 0.93 15 -1.0 ±0.41 35.0 35.0 -1.0 -1.0 22.4 ±0.35 N 6.44 ±0.73 3.24 ± 1.29 15 38.2 ± 0.45 14 1.7 ±0.90 38.2 38.2 1.8 1.8 S 7.76 ± 0.92 7.08 ± 2.73 15 39.4 ±0.20 15 2.9 ±0.73 39.4 39.4 2.9 2.9 26.5 ± 0.28 N 5.11 ±0.91 3.04 ± 1.28 15 40.4 ±0.33 15 3.8 ±0.36 40.5 40.4 3.6 3.8 S 6.17 ± 1.11 6.26 ± 2.73 13 41.4 ±0.33 14 5.8 ±0.41 41.3 41.4 5.8 5.8 32.1 ±0.44 N 6.57 ±0.75 3.33 ± 1.33 14 41.8 ±0.29 14 5.5 ±0.49 41.7 41.8 5.5 5.5 S 7.82 ±0.87 7.13 ±2.23 15 42.4 ±0.84 15 7.7 ±0.47 42.4 42.4 7.7 7.7 34.0 ±0.32 N 6.63 ± 0.60 3.43 ± 1.14 15 41.3 ±0.71 12 8.6 ±0.49 41.2 41.2 8.6 8.6 S 8.27 ±3.26 8.32 ±3.26 15 42.5 ± 0.96 14 9.6 ±0.90 42.6 42.6 9.6 9.6 F i g u r e 2.1 C h r o n i c thermal m a x i m a for northern ( N H , triangles) and southern ( G A , circles) k i l l i f i s h . C h r o n i c thermal m i n i m a (data not shown) were estimated to be -1.1 ° C for b o t h populat ions. D a t a were fit w i t h a th i rd order regression for calculat ions o f m a x i m a and m i n i m a . 96 Figure 2.2 Critical thermal max (A) and min (B) for northern (NH, triangles) and southern (GA, circles) killifish acclimated to temperatures between 2.3 and 34.0°C and a 12:12 L:D photoperiod. Second order regression models of CTMax or CTMin within a population are shown. Significant differences in CTMax or CTMin within a population are indicated with different letters. An asterisk (*) indicates a significant difference in critical thermal limit between populations at a given acclimation temperature. Data are expressed as mean ± SD and PO.001 for all significant comparisons. 97 42 41 O '1 40 • "ro E o 39 ro o 38 O 37 V o "E ro E .C ro o O 3 4. 2 4 A. CTMax P771 Northern Fh D Southern Fh V B. CTMin [777] Northern I H Southern Fh X X X y JL y _ j _ N-NS N-ME N-NH S-GA Population S-FL (FB) S-FL (Wl) Figure 2.3 Critical thermal max (A) and min (B) for three northern (Nova Scotia (NS), Maine (ME) and New Hampshire (NH); hatched) and three southern (Georgia (GA), Fernandina Beach, F L (FB) and Whitney Island, FL (WI); grey) populations of killifish acclimated to 22°C and 12:12 L:D photoperiod. Data are expressed as mean ± SD, and significant differences between populations within each panel are indicated by different letters (P<0.001 for all significant comparisons). 98 100 100 34 27 64 58 87 100 Xiphophorus hsp70-1 (AB062113) Fundulus hsp70-l (DQ202279) Paralichthys hsp70 (ABO 10871) Rhabdosargus hsp70 (AY436787) Damo hsp70a (AB062116) Danio hsp70b (AF210640) 95 100 1—o. i 97 O. mykiss hsp70A (AB 176854) O. tshawytscha hsp70 (U35064) /»>*«• hsp70B (AB 176855) Fueu hsD70-4 fY0858n Fugw hsp70-2 (Y08577) Oreochromis hsp70 (AJ001312) Oryz/cw hsp70-l (AF286875) Xiphophorus hsp70-2 (AB062114) Fundulus hsp70-2 (DQ202280) Dicentrarchus hsp70 (AY423555) Homo hsp70-AlL (NM_005527) 5oshsp70-l (NM-174550) Bos hsp70-2 (NM-203322)) Homo hsp70-Al A (NM-005345) Homo hsp70-AlB (NM_005346) 16 51 > i 100 26 30 29 Xiphophorus hsc70 (AB062115) O. mykiss hsc70A (AB 196460) Gallus hsc70 (AJ00940) Bos hsc70-8 ( N M J 74345) Ictalurus hsc70 (U22460) Carrassius hsc70 (AY 195744) Dan/o hsc70-8 (BC045841) Pimephales hsc70 (AY538777) I Fundulus hsc70 (DQ202278) Oryzias hsc70 (D13669) Paralichthys hsc70 (AB006814) Rhabdosargus hsc70 (AY436786) Tetraodon hsc70 (CR656379) TetraqdonhsclO^ _(CR7_34665)_ _ 100 100 100 Homo hsp70-4 (LI 2723) Cyprinus hsp70-4 (AU279339) Danio hsc70-4 (BC65970) Figure 2.4 Phylogenetic relationships among vertebrate hsc/hsp70 amino acid sequences. The tree was constructed using the neighbor-joining method and bootstrap values (percentage of 1000 replicates) are shown at each branch point. Bold font indicates the sequences identified in this study. GenBank accession numbers of all sequences are shown in brackets following the gene name. 99 3 o < z a. 1/ / i Northern Fh (no induction) F ^ - | Southern Fh (T^FC) * * hi hi tl a * / / / / 1 / / 1 / / / / hi I hi I CH 30°C 31 °C 32°C 33°C 34°C 35°C 36°C 1.5 0) < z a. 8-•c rr-7\ Northern Fh (Jm-30°C) | Southern Fh (Tm-30°C) 32°C 33°C .°C 36°' 0.75 Uj < z a: E 6 a 0.50 0.00 y-p, Northern Fh (T^33°C) H 1 Southern Fh (T=33°C) 0.25 H ah ah T.ra n , F l ,h hi a " ajj be CH 30°C 31 °C 32°C 33°C 34°C 35°C 36°C Heat Shock Temperature Figure 2.5 Branchial hsc70 (A), hsp70-l (B), and hsp70-2 (C) mRNA levels in northern (NH, hatched bars) and southern (GA, grey bars) killifish in response to heat shock. Control (C) samples were taken prior to experimentation, and handling controls (CH) were treated the same as the experimental temperature groups but were transferred back to the acclimation temperature of 20°C. All mRNA data are normalized to the control gene EF-1 a (mean ± SE). A difference in letters indicates significant differences between treatments but within a population, and an asterisk (*) indicates a significant difference between northern and southern fish within a treatment. 100 1.5 UJ O ^—< Q) > ro CD a: E o •c ro !c o c co A r7771 Northern Fh f^m Southern Fh 1.0 0.5 0.0 a h a h = yk c _ ! kl kl I CH 30°C 31 °C 32°C 33°C 34°C 35°C 36°C UJ o CD > ro P 2.5 2.0 S 1-5 H E « L 1.0 O •c 1 0.5 H o c ro 0.0 B [7771 Northern Fh i r ~ ^ Southern Fh I h I b i b h I I h .1 1 h I CH 30°C 31°C 32°C 33°C 34°C 35°C 36°C Treatment Groups Figure 2.6 Branchial hsp90a (A) and hsp90B (B) mRNA levels for northern (NH, hatched bars) and southern (GA, grey bars) killifish exposed to heat shock. Control (C) samples were taken prior to experimentation, and handling controls (CH) were treated identically to the heat shock groups but exposed only to the acclimation temperature of 20°C. A l l mRNA expression data are normalized to the control gene EF-1 a (mean ± SE). A difference in letters indicates significant differences between treatments but within a population, and an asterisk (*) indicates a significant difference between northern and southern fish within a treatment. 101 C H A P T E R T H R E E : C O U N T E R G R A D I E N T V A R I A T I O N I N T E M P E R A T U R E P R E F E R E N C E I N P O P U L A T I O N S O F K I L L I F I S H , FUND UL US HETEROCLITUS2 INTRODUCTION The preferred temperature of fishes is a reflection of the behavioral control of body temperature by the selection of appropriate water temperatures (Houston, 1982). Fishes have been shown to discriminate temperature changes of as little as 0.03°C (Bardach and Bjorklund, 1957; Steffel et al., 1976), and goldfish have been successfully trained to perform a task (lever pushing) to receive an injection of cold water that transiently lowered the temperature of a 41°C tank by 0.3°C (Rozin and Mayer, 1961). These and other studies on the thermoregulatory centers of the central nervous system of fishes suggest that fish are capable of very precise temperature selection and regulation (reviewed in Crawshaw and O'Connor, 1997). In experimental studies, fish typically express two different temperature preferenda: an acutely selected temperature (T°C a C ute) measured within 2 hours of a fish's exposure to a temperature selection gradient and a final thermal preferendum (T°Cf, n ai). The T°C acute typically reflects the fish's thermal acclimation history, while Fry (1947) defined T°Cf i n a i as 'the temperature at which individuals will ultimately congregate regardless of their thermal experience before being placed in a thermal gradient'. Regardless of acclimation temperature, fish generally achieve T°Cf i n a i within 1-3 days of being placed in a thermal gradient (Fry, 1947; Crawshaw, 1975). The final thermal preferendum paradigm implies three ecologically relevant hypotheses: 1) the final temperature preferendum is species-specific and independent of acclimation temperature, 2) all other factors being equal, fishes in nature will tend to occur at their preferred temperature when available, and 3) the final thermal preferendum will coincide with the temperature at which key physiological, biochemical, and life-history processes are optimized. 2 A version of this chapter will be submitted to Ecology Letters. 102 There are many examples in fishes demonstrating the selection of thermal niches to maintain body temperature at or near the final thermal preferendum (reviewed by Reynolds and Casterlin, 1979). Largemoufh bass in natural reservoirs, for example, survive near lethal summer water temperatures (found throughout the majority of the reservoir) by congregating near cool, spring-fed areas where fish maintain their body temperatures 5-10°C lower than reservoir temperatures and at a temperature near the laboratory-determined final preferendum (Van Den Avyle and Evans, 1990). In the cooler months of the year, bass distributed themselves much more widely throughout the reservoir. However, there are also many examples to confuse ecologically relevant interpretations of preferred temperature data. Some centrarchids, for example, select temperatures in the laboratory well above those available in their natural habitat (Magnuson and Beitinger, 1978). Bluegill sunfish acclimated to low temperatures voluntarily select high temperatures resulting in 100% mortality (Beitinger and Magnuson, 1976). Thermal preference data, therefore, must be carefully interpreted to resolve the adaptive value of a particular thermal preferendum. One approach to better understand the significance of preferred temperatures has been to compare these temperatures to ecological and physiological thermal optima. In many fish species, there is general agreement between performance optima of physiological correlates such as feeding, growth, metabolic rates, swimming performance, and reproduction with preferred temperatures (Beitinger and Fitzpatrick, 1979; Jobling, 1981; McCauley and Casselman, 1981; Coutant, 1987). As well, a positive correlation between thermal tolerance thresholds (critical thermal maxima (CTMax) and lethal temperatures) and the final thermal preferendum has been shown across fish species with correlation coefficients greater than 0.85 suggesting that these two parameters are closely connected (Jobling, 1981; Tsuchida 1995). In the current study, we assessed the potential for linkage between thermal tolerance thresholds and thermal preferenda using populations of the common killifish (Fundulus 103 heteroclitus), which have been shown to differ in thermal tolerance (Fangue et al., 2006). Killifish populations inhabit estuaries and salt marshes along the east coast of North America. Mean annual temperature across this latitudinal gradient increases by 1°C with every degree decrease in latitude such that at the northern and southern extremes of the killifish's range temperatures are, on average, 13°C higher in the south than in the north at any given time of year (calculated from N O A A NERRS Data). These killifish populations are thought to have undergone adaptation to their local thermal environments (Powers et al., 1993; Powers and Schulte, 1998; Schulte, 2001), and population-specific differences in thermal tolerance have been shown, consistent with predictions for local adaptation to environmental temperature (Fangue et al., 2006). The objective of this experiment was to compare the acute and final thermal preferenda of northern and southern killifish populations acclimated to 5, 15, and 25°C. Because the thermal habitats of northern and southern killifish differ substantially and these populations differ in their thermal tolerance limits, we predicted that fish from cooler northern environments would prefer lower temperatures than fish from southern populations. MATERIALS AND METHODS EXPERIMENTAL ANIMALS Killifish are divided into regional subspecies with a northern subspecies (Fundulus heteroclitus macrolepidotus) distributed from the Gulf of St. Lawrence, Canada to New Jersey, USA and a southern subspecies (Fundulus heteroclitus heteroclitus) distributed from Virginia, USA to the North-eastern coast of Florida, USA (Morin and Able, 1983). Adult killifish of the northern subspecies were collected from Hampton, New Hampshire (NH; 42° 54' 46" N), USA, and fish of the southern subspecies were collected from Brunswick, Georgia (GA; 31° 7' 31" 104 N), USA. All collections were made in late spring of 2002. Fish were held in 75 L glass aquaria with biological filtration at 3ppt salinity, 20 ± 2°C, and 12h:12h (L:D) photoperiod for a minimum of 3 weeks before experimental acclimation. Fish were then acclimated to 5, 15, or 25 ± 0.5°C. Temperature was changed at a rate of 1°C per day until each acclimation temperature was reached and fish were maintained at their acclimation temperature for a minimum of 4 weeks prior to experimentation. Three replicate 75 L acclimation tanks per temperature treatment were divided to house 4 northern fish on one side and 4 southern fish on the other for a total of 12 fish per population and acclimation temperature. Acclimation temperatures were controlled with Fisherbrand® NIST traceable temperature controllers and Ebo Jager 250 W submersible heaters. Fish were fed TetraMin® fish flakes and commercial trout chow (PMI Nutrition International, Brentwood, MO, USA) daily to satiation. All fish were fasted for 24 hours prior to temperature preference trials as selected temperatures in some fish species have been shown to change in response to fed state (Magee et al, 1999; Despatie et al, 2001). The initial 4-week acclimation period was carried out at the University of British Columbia (UBC). Fish were then transported at their acclimation temperatures to Portland State University (PSU), and thermal acclimation was continued as described above for a minimum of 5 days before temperature preference determinations were conducted. Following temperature preference trials, fish were removed from the thermal gradient, weighed (wet mass ± 0 . 1 g), and measured (total length ± 0 . 1 cm). Fulton's condition factor (k) was calculated according to the formula: Total mass(g) k = Total length1, (cm) x 100 THERMAL GRADIENT APPARATUS Temperature preference measurements were conducted at PSU using a 9 lane thermal gradient apparatus (Wollmuth et al., 1987). Each lane was 2.5 m in length, 28 cm wide, and 105 water depth was 8 cm. Temperature in each lane of the longitudinal gradient was linear and a gradient spanning up to 30°C could be achieved. Water temperature was measured with thermocouples placed every 12.5 cm in each lane. The position of a fish was recorded every 6 seconds using a wide-angle camera mounted above the gradient. This image was digitized using frame grabber software (Data Translation Co., Marlboro, MW, USA), and the fish's position was converted to the water temperature measured by the thermocouples using customized software. A single fish occupied each lane and their selected temperature was recorded every 6 seconds for 11.5 hours. Four or five fish per population were run per day, and lane assignments were randomized between populations. All trials began at 8 am. At the start of the trial, individual killifish were introduced to the thermal gradient in a partitioned section that corresponded to the fish's acclimation temperature and the fish remained in this section for 30 min. The partition was then removed and the fish could freely explore the gradient. Some fish jumped between thermal gradient lanes or were poorly detected by the camera and were eliminated from the analysis. Thus, a total of 9 to 12 fish were analyzed per population and acclimation temperature treatment. ANAL YSIS OF SELECTED TEMPERA TURES Preliminary temperature gradient trials demonstrated that even 5°C acclimated fish never entered temperatures below 5°C, and thus, experimental trials were conducted using gradient temperatures from 5-35°C. Preliminary temperature gradient trials also allowed the determination of the maximum trial length killifish needed to reach their final thermal preferenda. In these preliminary experiments, northern and southern killifish were exposed to the temperature gradient for 2 consecutive days. All fish, regardless of their acclimation temperature or population of origin, reached their final thermal preferenda in 8 hours or less. 106 Based on these preliminary results, we carried out all of the experimental temperature selection trials for a minimum of 11 hours. Temperature recordings for each fish were analyzed individually by averaging the modal (± SE) and mean (± SE) selected temperatures across 10 min intervals for the duration of each preference trial. Data exploration revealed that there were no differences in the results obtained using 5 and 10 min bins, but information was lost if longer bin intervals were used. A previous thermal selection study using this thermal gradient system also showed that 10 min bin intervals were optimal for data analysis (Clutterham et al., 2003). The acute thermal preferendum (T°C a cute) , the selected temperature (T°C s ei), and the time to T°C s e i (Timesei) were obtained individually for each fish and expressed as the mode (± SE) as well as the mean (± SE). Acute thermal preference, by definition, occurs within the first 2 hours of exposure to the temperature gradient (Fry, 1947). We defined T°C a c u t e as the average temperature chosen by each fish for 3 consecutive 10 min bin intervals that fell within 1°C of one another during the first 120 min of the trial. The final selected temperature is the temperature that the fish ultimately chooses by the end of a prolonged trial. This temperature was determined as the temperature occupied by the fish for a minimum of 3 consecutive 10 min bins within 1°C of each other, but was often much longer. Once the final selected temperature was determined, the T°C s e i and the time to T°C s ei (Timesei) was taken as the time where the fish first spent 3 consecutive 10 min bins at a temperature ±0.5°C of the final selected temperature. In addition to the analysis of preferred temperatures of individual fish, the mean modal selected temperature at each 10 min bin interval for all fish from each population and acclimation temperature group was plotted for the duration of the trial. The final thermal preferendum is statistically defined as the central tendency (mode) of the temperature distribution (Reynolds and Casterlin, 1979) and is reflective of the temperature 107 most often occupied by the fish. By definition, the final thermal preferendum ( T ° C f , n a i ) is independent of acclimation temperature, and thus was calculated for each population by taking the average of each fish's modal T ° C s e ] values from all three acclimation groups. As well, we also report the average of the mean T ° C s e i values. As a complementary approach to the analysis of the final thermal preferenda for individual fish, we also plotted the mean modal selected temperatures for each 10 min bin for all northern or southern fish from each experimental treatment. The T ° C f i n a i for each population and acclimation group was then calculated as the grand mean of the modal selected temperatures from the T ° C s e i to the end of the thermal gradient trial. STATISTICS To determine whether temperature preference varied with body mass, condition factor, or sex, correlation analysis was performed within each killifish population as well as for the pooled data for all populations. Selected modal and mean temperatures and times were analyzed by multiple analysis of variance (ANOVA) with population, and/or acclimation temperature as independent categorical variables. All data met the assumptions of normality, and data were log transformed where necessary to meet assumptions of homogeneity of variance. When interaction terms were not significant, post-hoc comparisons were performed among the groups with the Student-Newman-Keuls multiple range test (SNK MRT). If the interaction terms were significant, the data were separated and analyzed independently using one-way A N O V A . For all statistical analyses, a was set at 0.05. 108 RESULTS SIZE, CONDITION, AND SEX Fish used in this experiment were all of similar size, and there were no significant differences in body mass between populations or between thermal acclimation groups. The mean mass of northern killifish was 6.26 ± 0.55g (mean ± SE), and the mass of southern killifish was 6.16 ± 0.63 g. Condition factor values ranged from 1.41 to 1.64 for all killifish groups. Roughly equal numbers of males and females per population and acclimation temperature were used in the analysis. There were no significant correlations between body mass, condition factor or sex with any of the measured temperature selection parameters. FISHBEHA VIOR IN THE THERMAL GRADIENT Northern fish acclimated to 5°C and allowed to select from temperatures ranging from 5-35°C immediately selected temperatures that were equivalent to their upper thermal tolerance limits (Critical Thermal Maxima, CTMax = 30.3°C for northern killifish acclimated to 5°C; Fangue et al., 2006). These fish lost equilibrium and the selected water temperatures were lethal if the fish were not excluded from them. This response was never seen in southern fish acclimated to 5°C (CTMax = 32.5°C). Fish acclimated to warmer temperatures did not have access to temperatures greater than their CTMax in the gradient. Selected temperatures measured every 6 seconds and plotted over time showed substantial variability between individuals, reflecting differences in the temporal patterns of the exploratory bouts between individual fish in the thermal gradient. As an example, Figure 3.1 A shows the selected temperature over time for one representative 5°C acclimated northern fish. These exploratory swims over the length of the gradient were brief, however, and resulted in 109 each fish spending very little time at temperatures other than those at or near the mode and mean temperatures calculated in 10 min bins. The modal (Figure 3. IB) and mean (Figure 3.1C) selected temperatures are shown for the same 5°C acclimated northern fish in Figure 3.1 A. Figure 3. IB also shows an example of how T 0 C a C u t e , T°C s ei, Time s ei, and the final selected temperature were obtained for each fish. Values for both the mode and the mean gave very similar trends over time as the temperatures selected by the fish most frequently (mode) approximated the average temperature (mean) indicating just how little time was actually spent at temperatures away from either the mode or mean temperatures. Therefore, we report modal preferred temperatures as the measure of preferred temperatures in killifish. Values of the modal T°C a c u te, T°C s ei, and Time s ei were calculated for each fish, and the means of these modal values were computed for each population and acclimation temperature group (Figure 3.2). ACUTE THERMAL PREFERENDA (T°CACUTS) A two-way A N O V A for the modal T°C a c u te with population and acclimation temperature as factors detected a significant effect of acclimation temperature (p=0.008; Figure 3.2A). Both killifish populations increased their modal T°C a c u te with increasing acclimation temperature, and post-hoc comparisons revealed that 25°C acclimated northern fish had significantly higher T°C a Cute values than did the 5 and 15°C acclimated northern fish (p<0.022 for both comparisons). Post-hoc tests also revealed a significant difference between populations such that northern killifish acclimated to 25°C had a significantly higher T°C a c utethan southern fish acclimated to 25°C (p=0.032). 110 TIME (TIMESEO AND SELECTED TEMPERATURES (T°CSEI) Two-way A N O V A for modal T°C s e i values revealed a significant effect of population (p=0.035; Figure 3.2B) and no significant affect of acclimation temperature or a significant interaction term. While acclimation temperature did not have a significant effect on the selected temperatures (T°C s e i ) of killifish populations, the two-way A N O V A for modal time to the selected temperature (Time s ei) detected a significant effect of acclimation temperature (p=0.002; Figure 3.2C), and no significant effect of population or a significant interaction term. Post-hoc tests revealed that for modal Time s ei, northern fish acclimated to 5°C took significantly longer than northern fish acclimated to 15 and 25°C to reach their final selected temperature T°C s e i (p<0.004 for both comparisons). In southern fish, there were no significant differences between acclimation groups in modal Time s ei. SELECTED TEMPERA TURE AS A FUNCTION OF TIME In order to evaluate selected temperature as a function of time, we also computed the mean for all fish in each population and temperature acclimation group of the modal selected temperature for each fish for each 10 min bin (Figure 3.3). Mean modal preferred temperature increased with increasing exposure time in the thermal gradient, and this was particularly apparent in 5 and 15°C acclimated fish. The selected temperatures for 5°C acclimated fish were less variable overall in the early parts of the trial (100-350 min) compared to the 15 and 25°C acclimation groups. In 15°C acclimated fish, both northern and southern killifish explored the gradient during both the early and late portions of the trial with a more stable period at -200-400 min. For 25°C acclimated fish, both northern and southern killifish tended to explore the gradient more consistently for the entire duration of the preference trial. I l l As an alternate method to compute preferred temperature for each population, we calculated the grand mean of the mean modal selected temperatures for each 10 min bin for each fish from T°C s ei to the end of the trial, and then used these values to calculate the mean selected temperature for each population and acclimation group (Table 3.1). A two-way A N O V A comparing these values revealed a significant effect of population (p=0.002) and no significant effect of acclimation or a significant interaction term. At all three acclimation temperatures, northern killifish had a higher mean modal selected temperature than southern fish, and post-hoc tests showed a significant difference between populations for 25°C acclimated fish (30.7°C, north; 25.6°C south; p=0.006). This difference can also be seen in Figure 3.3 as southern killifish acclimated to 25°C spent significantly higher proportion of their time at cooler temperatures than northern fish. FINAL THERMAL PREFERENDA (T°CFINAI) The final thermal preferendum (T°Cfinai) is defined as the temperature at which individuals will ultimately congregate regardless of their thermal experience (Fry, 1947). As acclimation temperature had no significant effect on T°C s ei in either killifish population, T°Cfinai was calculated for each population from the data reported in Figure 3.2B across 5, 15, and 25°C acclimated fish (Figure 3.4). The modal T°C s e i for northern killifish was 1.6°C higher than that for southern fish, and a t-test revealed that this difference was significant (p=0.026). Similarly, the mean T°C s ei was significantly different between killifish populations with northern fish having a T°C s ei that is 2°C higher than southern fish (p=0.002). We also calculated the T°Cfjnai from the data reported in Table 3.1. The mean modal preferred temperature for northern fish was 29.1 ± 0.51°C compared to 26.5 ± 0.65°C for southern fish, resulting in a 2.6°C difference between populations that was highly significant (t-test, p=0.002). 112 DISCUSSION THE FINAL THERMAL PREFERENDA (T°CFINAI) DIFFER BETWEEN KILLIFISH POPULATIONS Contrary to our initial hypothesis that northern killifish from cooler thermal habitats would prefer lower temperatures, we found that northern killifish had a significantly higher final thermal preferenda (T°Cfm ai) than southern fish (Figure 3.4). One possible explanation for these differences could be related to the salinity we chose for our experiments (3 ppt). Garside and Morrison (1977) found that northern killifish (Nova Scotia population) acclimated to 0.5 ppt preferred temperatures of ~24°C while seawater acclimated fish preferred temperatures 3-6°C higher, suggesting that low salinity is an osmotically challenging situation for killifish and the selection of lower temperatures is a strategy to limit the metabolic costs associated with this osmoregulatory stress. Experiments in our laboratory on northern and southern killifish populations have revealed profound differences in freshwater tolerance with northern fish being much more tolerant of freshwater than southern fish (Scott et al., 2004) possibly due to the poor ability of southern fish to minimize CI" loss and maintain CI" balance in freshwater (Scott et al , 2004; Scott et al, 2005). If southern fish are more strongly affected by low salinity, this would account for the lower T 0Cfi n ai detected in this experiment. However, our T°Cf i nai values agree closely with those for seawater acclimated Nova Scotia northern killifish previously described by Garside and Morrison (T°Cf, n a i= 30°C, Garside and Morison, 1977 and this study) suggesting that our fish were not deleteriously affected by low salinity. As well, northern and southern fish in our laboratory do equally well at salinities of 3ppt, and southern fish only begin to suffer mortality at salinities lower than 0.3ppt (G.R. Scott, pers comm). It has also been shown in several killifish populations that seawater gill morphology persists until salinities drop below 113 ~0.3ppt suggesting that these fish only become stressed at these very low salinities (Lacy, 1983; Karnaky, 1986; Katoh et al., 2001). COUNTERGRADIENT VARIATION IN FINAL THERMAL PREFERENDA While our results do not support the prediction that preferred temperature would be positively correlated with habitat temperatures, these results are consistent with countergradient variation in this trait. Countergradient variation predicts an inverse relationship between habitat temperature and the value of traits such as growth rate, and is thought to have evolved as a compensatory response to shorter growing seasons in high-latitude, cold-adapted populations (Conover and Schultz, 1995; Yamahira and Conover, 2002). The prediction for countergradient variation in thermal preference is that fish from colder environments would select higher temperatures when presented with a thermal gradient than those fish from warmer environments (Conover and Schultz, 1995; Yamahira and Conover, 2002; Freidenburg and Skelly, 2004). While countergradient variation in thermal preference has not been previously shown in fishes, it has been demonstrated in larval wood frogs, Rana sylvatica (Freidenburg and Skelly, 2004). As well, there are a number of examples of countergradient variation in early development, growth and metabolic rates, and body size of fishes (Alvarez et al , 2006; Marcil et al., 2006) including newly hatched killifish populations (Schultz et al., 1996), lending support for this phenomenon as a potential explanation for the higher temperatures preferred by northern killifish. Low THERMAL RESPONSIVENESS IN NORTHERN KILLIFISH Northern fish acclimated to cold temperatures (5°C) voluntarily exposed themselves to lethal temperatures upon entering the thermal gradient. This response, however, was not seen in cold-acclimated southern fish. This peculiar behavior and mortality has been seen before in several warm-water species acclimated to low temperatures and has been termed 'low thermal 114 responsiveness' (Meldrim and Gift, 1971; Barans and Tubb, 1973; Beitinger and Magnuson, 1976). The slower time to T°C s e i (Figure 3.2B) as well as the voluntary exposure to lethal temperatures seen in 5°C northern killifish suggest an offset in the temporal response between behavioral acclimation mechanisms and physiological and biochemical tolerance acclimation in cold-acclimated northern killifish. To our knowledge, this is the only demonstration of population-specific differences in the phenomenon of low thermal responsiveness. One hypothesis to explain this difference between populations is that even in winter, when mean temperature is low, southern fish may encounter microhabitats approaching lethal temperatures in their natural environment whereas cold-acclimated northern fish rarely if ever see temperatures that approach their maximum thermal tolerance values. As such, southern fish may maintain a well-developed avoidance strategy for lethal habitat temperatures that has been lost in northern populations. Differences between populations in their ability to counterbalance preferred temperatures and lethal temperatures could be one contributing factor keeping northern killifish from expanding their range southward. RELATIONSHIP BETWEEN T°CFINALAND THERMAL HABITAT Evolutionary ecologists and physiologists have speculated that preferred temperatures should be under strong selection (Angilletta et al., 2002; Angilletta et al., 2006), but there is little evidence to support or refute this claim (Freidenburg and Skelly, 2004). There have been a handful of studies using geographic clines to evaluate hypotheses related to the evolutionary responses of thermal preferences within a species, but these studies have generated contradictory results (Ellner and Karasov, 1993; Diaz, 1997; Freidenburg and Skelly, 2004). For example, bluegill sunfish, largemouth bass, and redband trout exhibit constant laboratory final thermal preferenda regardless of geographic origin or thermal history (Beitinger and Fitzpatrick, 1979; Gamperl et al., 2002). In contrast, white perch, Morone americana, (Hall et al., 1978), and coho 115 salmon, Oncorhynchus kisutch, (Konecki et al., 1995) preferred temperatures that differed significantly according to geographic location with populations from warmer habitats selecting higher temperatures than those from cooler habitats. In this experiment, we have shown that killifish populations differ in their final thermal preferenda in direct contrast to the predictions derived from their thermal habitat characteristics. Southern killifish experience mean monthly temperatures near 30°C during the summer months, and temperatures above 25°C are experienced in at least 8 of the 12 months of the year, suggesting a good match between habitat temperatures and T ° C f j n a i in southern killifish. Interestingly, however, the preferred temperature of ~30°C for northern fish would rarely, if ever, be available in northern thermal habitats. 30°C is substantially higher than the warmest mean summer temperature of most northern habitats (~20°C) and greater than the maximum habitat temperatures these fish are likely to experience acutely (NOAA NERRS Data, Wells Inlet, ME). The data for northern killifish are consistent with a number of other examples of fishes selecting warmer temperatures than those typically available in their natural environments (reviewed in Magnuson and Beitinger, 1978; Crawshaw and O'Connor, 1997). It is possible that the high T ° C f j n a i in both killifish populations is not under strong selection to closely approximate mean habitat temperature but rather preferred temperatures in these populations are more related to some other environmental factor beyond simply mean thermal habitat temperature, such as selection of high local microhabitat temperatures to maximize growth and fecundity. RELA TIONSHIP BETWEEN CRITICAL THERMAL LIMITS AND FINAL THERMAL PREFERENDA A positive correlation between critical thermal maxima and final thermal preferenda has been shown across many fish species (reviewed in Coutant, 1987; Tsuchida, 1995). However, this relationship has rarely been studied at the intraspecific level, despite the fact that population-116 specific differences in upper temperature tolerance have frequently been demonstrated (reviewed in Beitinger et al., 2000; Fangue et al., 2006), as have differences in thermal preference (Hall et a l , 1978; Konecki et al., 1995) albeit in a different subset of species. One of the few intraspecific examples directly comparing CTMax and thermal preference comes from genetically distinct populations of largemouth bass, Micropterus salmoides, in which significant differences in thermal tolerance were found with no differences in final thermal preferenda between populations (Fields et al., 1987; Koppelman et al., 1988). Northern and southern killifish populations have been shown to differ in CTMax by ~1.5°C with southern fish tolerating warmer temperatures and northern fish tolerating colder temperatures, consistent with local thermal adaptation predictions (Fangue et al., 2006). In the current experiment, however, thermal preferences for killifish populations revealed that northern fish pick slightly warmer temperatures than southern fish (T°Cf, nai~1.5 0C higher in northern fish; Figure 3.4), contrary to predictions generated from thermal tolerance measures. Taken together, these experiments indicate a disconnect between thermal tolerance and final thermal preferenda in killifish. In fact, our data for these two measures in killifish are contradictory and may suggest that the positive correlation seen in CTMax and selected temperatures across species may not hold true within a species. CONCLUSIONS The preferred temperature of a fish is often assumed to be optimal for physiological and biochemical processes such as growth, swimming performance and reproduction, as well as being well-matched to a fish's thermal habitat (Fry, 1947). In fact, many fish have physiological and life-history correlates that are optimized to temperatures very near their final thermal preferenda and these temperatures occur frequently in their natural environments (Brett, 1971; Webb, 1978; Jobling, 1981, Kelsch, 1996; although see also Larsson, 2005). However, some 117 fish species including the killifish populations used in this experiment have a perplexing mismatch between habitat and preferred temperatures. While we did not specifically address the relationship between physiological and ecological correlates and thermoregulatory behavior in this experiment, experiments in our laboratory on thermal tolerance (Fangue et al., 2006; Chapter Two), and swim performance assessed using critical swim speed methodology (Ucrit) (Chapter Four) quantified at a wide range of temperatures show that both killifish populations perform very well at acclimation and test temperatures equivalent to the T°Cfm ai of = 30°C measured in this experiment. Taken together, these studies suggest that overall, both populations are broadly tolerant to a wide range of temperatures, but differ subtly in thermal tolerance in a direction consistent with local thermal adaptation, but that thermal preference is instead consistent with countergradient variation in this trait. 118 REFERENCES Alvarez, D., Cano, J.M., and A.G. Nicieza. 2006. 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Ecology. 83:1252-1262. 123 Table 3.1 Mean modal selected temperatures (± SE) calculated from T°C s ei to the end of the temperature preference trial for northern and southern killifish acclimated to 5, 15, and 25°C. Acclimation Temperature (°C) Population 5 15 25 Northern Southern 2 8 . 8 ± 2 . 4 1 a 2 6 . 2 ± 3 . 2 5 x 27.5 ± 1.99x 28.9 ± 1.64! a 3 0 . 7 ± 3 . 8 6 ; 25.6 ± 4.77x Values are calculated as the grand mean of the mean modal selected temperatures for all fish per population and acclimation temperature group (n=9-12 fish per population and acclimation temperature). Significant differences within a population between acclimation temperatures are indicated with different letters. An asterisk (*) indicates a significant difference between populations at a given acclimation temperature. 124 35 30 A B 25 H 20 15 5°C North 60 120 180 240 300 360 420 Time (minutes) 480 540 600 660 720 O 60 120 180 240 300 360 420 480 540 600 660 720 Time (minutes - 10 minute bins) O Time (minutes - 10 minute bins) Figure 3.1 Selected temperature as a function of time for one representative 5°C acclimated northern fish (A) shown as an example. The modal (B) or mean (C) selected temperatures averaged for consecutive 10 min bin intervals are shown as well as an example of how T°C a Cute, T°C s e i , Time to T°C s e i , and the final selected temperature were obtained from each fish's selected temperature trace (B). 125 u o • o c a 10 H -o o I I Northern killifish r~7~a Southern killifish CL E u B Northern killifish Southern killifish E c_f o H o u E P I Northern killifish 7~7~i Southern killifish Figure 3.2 Modal acute thermal preference (T°C a Cute, A) , modal final selected temperature ( T ° C s e i , B) and modal time to final selected temperature (Time s ei, C) (± SE) for northern (open bars) and southern (hatched bars) killifish acclimated to 5, 15, and 2 5 ° C (n=9-12 fish per population and acclimation temperature). Significant differences within a population between acclimation temperatures are indicated with different letters. A n asterisk (*) indicates a significant difference between populations at a given acclimation temperature. 126 Time (minutes -10 minute bins) T i m e ( m i n u t e s . 1 0 minute bins) Figure 3.3 Mean modal selected temperatures (± SE) as a function of time in northern and southern killifish acclimated to 5, 15, and 25°C. Each point represents the mean of the modal temperatures for each 10 min bin interval for the fish in each experimental treatment (n = 9-12 fish per population and acclimation temperature). 127 Figure 3.4 Modal and mean final thermal preferenda ( T ° C n n a i ) (± SE) for northern (open bars) and southern (hatched bars) killifish. The final selected temperatures ( T ° C s e i ) for all three temperature acclimation groups for each population were pooled to obtain T°Cf , n ai (n=28 for north, and n=27 for south). An asterisk (*) indicates a significant difference between populations. 128 C H A P T E R F O U R : I N T R A S P E C I F I C V A R I A T I O N I N S W I M M I N G P E R F O R M A N C E A N D E N E R G E T I C S A S A F U N C T I O N O F T E M P E R A T U R E I N K I L L I F I S H , FUNDULUS HETEROCLITUS3 INTRODUCTION Common killifish (Fundulus heteroclitus) are found in estuaries and salt marshes along the east coast of North America from Newfoundland to central Florida. Killifish populations encounter a steep latitudinal temperature gradient across the species' range such that monthly mean water temperatures are, on average, 13°C higher in the south than in the north at any given time of year. However, there is substantial seasonal variation within localities such that monthly mean temperatures range from 3°C to 31.6°C, depending on the location and the time of year (calculated from N O A A NERRS Data, Sapelo Island, G A and Wells Inlet, ME). At the extremes of the species' range, northern fish are exposed to mean monthly temperatures ranging from 3 to 21°C, while southern fish encounter mean monthly temperatures ranging from 7 to 31.6°C resulting in only a relatively narrow temperature range between 7 and 21°C that is common to both northern and southern killifish populations. In addition to seasonal temperature variation, daily temperature fluctuations of 5-10°C are common in most months and across the entire distribution range (Sidell et al., 1983 and calculated from N O A A NERRS Data). Clearly, the ability to cope with seasonal and daily temperature fluctuations is critical to all killifish populations, but the temperature range over which they must make adjustments differs between populations and seasons. Consistent with their highly variable thermal environment, we have demonstrated that killifish from either extreme of the species' distribution can be acclimated to temperatures from 2-35°C and have thermal tolerance values amongst the highest ever recorded for fish (Fangue et al., 2006). 3 A version of this manuscript is accepted pending revision at Physiological and Biochemical Zoology. 129 Correlated with differences in their thermal environment, substantial variation between northern and southern populations of killifish has been shown in morphological, molecular, genetic, and physiological traits (reviewed in Powers et al. 1993; Powers and Schulte 1998; Schulte 2001; Fangue et al. 2006) and this variation is consistent with adaptation to different environmental temperatures. Biochemical studies of glycolytic enzymes indicate that metabolic organization differs between killifish populations. Extensive work on the lactate dehydrogenase-B (Ldh-B) locus has shown that Ldh-B genotype is tightly correlated with latitude/temperature, with the Ldh-Bb genotype found in northern populations, and the Ldh-B" genotype being dominant in southern fish (Powers et al. 1991). LDH-B isozymes are known to differ in their kinetic properties such that L D H - B b (northern genotype) has greater catalytic efficiency than LDH-B a (southern geneotype) at low temperatures and vice versa at warmer temperatures (reviewed in Powers et al. 1993). Northern killifish also have higher lactate dehydrogenase (LDH) activity in heart (Pierce and Crawford 1997; Podrabsky et al. 2000), and liver (Segal and Crawford 1994) when compared to southern fish across a variety of acclimation temperatures. There is also a correlation between Ldh-B genotype and swimming performance in killifish (DiMichele and Powers 1982).. These authors found that killifish sampled from a Delaware population (near the center of the hybrid zone) differed in critical swimming speed (Ucrit) such that the fish bearing the northern Ldh-B genotype (Ldh-Bb) outperformed fish bearing the southern Ldh-B genotype (Ldh-Ba) when tested at 10°C. However, the performance of the two genotypes converged at warmer temperatures (25°C). DiMichele and Powers (1982) ascribed this pattern to differences in Ldh-B genotype causing differences in glycolytic flux in erythrocytes, which cause differences in ATP levels and thus blood hemoglobin oxygen affinities between populations, resulting in northern fish having a greater ability to deliver oxygen to tissues at low temperatures. 130 A l t h o u g h D i M i c h e l e and P o w e r s (1982) s h o w e d that temperature di f ferentia l ly affects s w i m m i n g performance i n k i l l i f i s h bearing alternate Ldh-B genotypes, the magnitude o f the temperature effect was modest (Qio =1.2 for Ldh-Bb and 1.3 for Ldh-Ba) at a c c l i m a t i o n temperatures between 10 and 25°C. These f indings are consistent w i t h w o r k b y Targett (1978), w h o s h o w e d that k i l l i f i s h have a large zone o f temperature-independent metabol ic rate f r o m -13-30°C over w h i c h the Qjo o f respiration rate was just over 1. A t very l o w temperatures, however , o x y g e n c o n s u m p t i o n dec l ined steeply and showed a strong effect o f temperature (Qio = 4.4 f r o m 13 to 5°C) (Targett 1978). T h i s steep decl ine i n resting o x y g e n c o n s u m p t i o n at temperatures b e l o w 13°C suggests that a largely aerobic process such as sustained s w i m m i n g performance might be strongly affected at l o w temperatures. These l o w temperatures are environmenta l ly relevant for northern populat ions o f k i l l i f i s h , but are s e l d o m experienced b y f ish i n southern populat ions, and the effects o f Ldh-B genotype at these temperatures are u n k n o w n . In this study, w e assessed the effects o f temperature o n c r i t i c a l s w i m m i n g speed (Ucrit) i n k i l l i f i s h populat ions acc l imated to temperatures between 5 and 32°C. A d d i t i o n a l l y , w e tested whether the relative thermal independence o f s w i m m i n g performance observed b y D i M i c h e l e and P o w e r s (1982) is a result o f temperature a c c l i m a t i o n or due to an intr insic insensi t iv i ty to temperature b y c o m p a r i n g U c r j t values o f f i s h tested at their a c c l i m a t i o n temperature to those acutely chal lenged at var ious temperatures. W e also examined metabol ic differences that c o u l d underl ie differential s w i m m i n g performance between populat ions b y us ing a standardized exercise challenge to determine the patterns o f metabol ic fuel use and energy reserves supporting exercise m e t a b o l i s m across a c c l i m a t i o n temperatures i n a variety o f tissues p r i o r to and f o l l o w i n g exercise. 131 MATERIAL AND METHODS EXPERIMENTAL ANIMALS Adult killifish of the northern subspecies (Fundulus heteroclitus macrolepidotus) were collected from Hampton, New Hampshire (NH; 42° 54' 46" N), USA. Individuals of the southern subspecies (Fundulus heteroclitus heteroclitus) were collected from Brunswick, Georgia (GA; 31° 7' 31" N), USA. Collections were made in late spring of 2002. A second collection was conducted in spring of 2004 that again included the original NH and G A killifish populations, as well as two additional northern populations from Salsbury Cove, Maine (ME; 44° 25' 54" N), USA, and Antigonish, Nova Scotia (NS; 45° 37' 0" N), Canada, as well as two additional southern populations from Whitney Island, Florida (WI; 29° 39' 34" N), USA, and Fernandina Beach, Florida (FB; 30° 40' 51"N), USA. All fish were collected using baited minnow traps. Fish were held in a 6,000 L recirculating aquarium system with biological filtration at 20 ppt salinity, 20 ± 2°C, and 12h:12h (L:D) photoperiod for a minimum of 3 weeks before transfer to 75 L experimental acclimation tanks. Acclimation temperatures were controlled with NIST traceable temperature controllers and 250 W submersible heaters (Fisherbrand®, Fisher Scientific, Nepean, ON, Canada). Water temperatures were adjusted at a rate of 0.5°C per day until the desired acclimation temperature was reached. Killifish were acclimated for a minimum of 21 days to each treatment temperature (ranging from ~5-30°C) under a 12h:12h (L:D) photoperiod and 20 ppt salinity before all experiments. We elected to utilize a common photoperiod for all comparisons, despite the possibility of photoperiod influencing swimming performance (Kolok, 1991, Smiley and Parsons, 1997, Day and Butler, 2005) in order to allow the direct determination of the effects of temperature independent of other factors. The northern 132 and southern source populations that we used only experience a relatively narrow temperature band in common and are exposed to these temperatures in different seasons. However, when all localities along the coast with resident populations of killifish are considered, mean monthly temperatures ranging from 6-28°C are found in combination with a 12:12 photoperiod (calculated from N O A A NERRS Data), suggesting that essentially all of the temperatures that we tested could, in principle, be encountered by killifish acclimatized to a 12:12 photoperiod in the natural habitat. Fish were fed TetraMin® fish flakes supplemented with commercial trout chow (PMI Nutrition International, Brentwood, MO, USA) daily to satiation, but were not fed for 24 hours prior to experimental trials. Treatment of all experimental animals was in accordance with the University of British Columbia animal care protocol #A01-0180. CRITICAL VELOCITY MEASURES To establish the swimming capacity of killifish, critical or maximum prolonged swimming speed (U c rjt) was determined using the methodology described by Brett (1964). Individual fish were introduced into a Beamish-style swim tunnel (4000 mL volume) and allowed to acclimatize at a linear water velocity of 3 cm-s"1 for 30 min. During this acclimatization period, fish oriented to the water current but maintained a stationary position on the bottom of the swim tube with little body movement. After the acclimatization period, fish were given a pre-trial swim that entailed an initial 10 min swim at 1 body length per second (BL-s"1) followed by water velocity increases of 0.3 BLs" 1 every 2 min until the fish fell against the back screen. This pre-trial swim was performed in order to allow the fish to become familiarized with the tunnel (Jain et al , 1997; MacNutt et al., 2004). All fish completed the pre-trial swim, and the U c r i t determined in the pre-trial was qualitatively similar to that determined in the test protocol. Following the pre-trial, each fish was given a 3 hour recovery period, which has been shown to be sufficient for the recovery of U c r i t (Jain et al., 1998). For the determination of Ucrit, water velocity was increased stepwise by 0.3 BL-s"1 every 10 min, from a starting velocity of 1 BL-s' 1, until the fish fatigued. A swimming interval of 10 min was chosen based on a synthesis of literature showing that 10 min is an appropriate interval for U c r i t determination (Jones, 1971; Beamish, 1980; Hammer, 1995). When fish failed to maintain swim performance, water velocity was decreased to zero to allow the fish to come off of the screen and resume swimming. Velocity was then increased to the velocity of failure within 10 s. Final fatigue was established when the fish fell against the back screen 3 consecutive times. U C r i t was then calculated using the following formula from Brett (1964): Ucrit = U, + where Uj is the highest speed fish swam for the full time period (cm-s"1), Uu is the incremental speed increase (cm-s"1), tj is the time the fish swam at the final speed (minutes), and tjj is the prescribed period of swimming per speed (10 min). The cross sectional areas of the fish were between 8 and 12% of the area of the swimming chamber; therefore, the measured swimming speeds were corrected for solid blocking effects according to the calculations described by Bell and Terhune (1970). The absolute value of Ucritwas converted to relative swimming speed in BL-s"1 by taking the Ucrit value (cm-s"1) and dividing it by the total body length (cm) of each fish (Kolok, 1999). The water current in the tunnel was produced by a submersible pump (Little Giant Pump Co., Oklahoma City, OK, USA) and water velocity was controlled with a rheostat. The tunnel water velocities were calibrated with an inline flowmeter (Onicon Inc., Clearwater, Fl, USA). We conducted two kinds of swimming performance trials. For the first type of Ucrit determination which involved determination of performance at the fish's acclimation 134 temperature, the swimming chambers were submerged in thermostated water baths and water temperatures were maintained within ±0.5°C of the specified acclimation temperature for each experiment using NIST traceable temperature controllers and 250 W submersible heaters (Fisherbrand®, Fisher Scientific, Nepean, ON, Canada). The second type of swimming performance measure involved an acute temperature challenge in which fish acclimated to 18°C were introduced to the swimming chamber at one of four challenge temperatures (5, 18, 25, or 34°C). After the 30-minute tunnel acclimatization at the challenge temperature, the pre-trial swim and Ucr,t were conducted at the challenge temperature, and temperatures were maintained as described above. In all experiments, Ucrit was determined for six fish per population and experimental treatment. Following exercise trials, fish were removed from the tunnel, weighed (wet mass ± 0 . 1 g), and measured (total length ± 0 . 1 cm). Fulton's condition factor (k) was calculated according to the formula: f Total mass(g) 1 ^ m Total length2,(cm) STANDARDIZED EXERCISE CHALLENGE Temperature-dependent metabolic fuel availability and mobilization patterns were measured in populations of killifish exposed to a standardized exercise challenge protocol. Killifish were given a pre-trial swim (as described for Ucrjt), and a 3 hour recovery, followed by a 2 hour exercise challenge. During the exercise challenge, velocity was increased over a 30 min period to a swimming speed that corresponded to 80% of the Ucrit determined for that temperature, population, and fish length. Fish were then swum at this speed for 1.5 hours. Control fish were also kept in a swim tunnel with circulating aerated water for 3 hours at a velocity of 3 cm-s"1. At the end of the swimming period, 8 mL of an ethyl p-amino-benzoate solution (benzocaine, Sigma-Aldrich, Oakville, ON, Canada; stock solution made by dissolving 135 62.5 g of benzocaine in 500 mL ethanol) was introduced to the tunnel. Fish continued to swim for ~1 min and then fell against the back screen at complete anaesthesia. Fish were quickly sampled for blood plasma, white muscle (lateral portion posterior to the dorsal fin), and liver. Tissues were immediately frozen with aluminum blocks pre-cooled in liquid nitrogen, and sampling time never exceeded 1 min per fish. ANALYTICAL TECHNIQUES Frozen white muscle and liver samples were ground into a fine powder under liquid nitrogen in an insulated mortar and pestle. Metabolites were extracted by adding ~100 mg of powdered tissue to 1.0 mL of ice-cold HCIO4 (1 molT 1), and homogenizing at 0°C for 20 s with a PowerGen homogenizer (Fisher Scientific, Nepean, ON, Canada). 200 fil of each homogenate was removed and frozen at - 8 0 ° C for glycogen determination. The remaining homogenate was centrifuged at 10,000 g for 10 min at 4°C, and the supernatant was neutralized with 3 molT 1 K2CO3. The neutralized extract was then centrifuged at 10,000 g for 10 min at 4°C. The resulting supernatant was immediately assayed spectrophotometrically for ATP and phosphocreatine (PCr) according to the methods of Bergmeyer (1983) and stored at - 8 0 ° C for later lactate and glucose determination. Samples for glycogen determination were enzymatically digested according to the methods of Hassid and Abraham (1957), and all samples were analyzed for glucose following the methods of Bergmeyer (1983) modified for spectrophotometry. Plasma was analyzed directly for lactate and glucose as above. Muscle total lipid was determined by the sulphophosphovanilun method described in Barnes and Blackstock (1973). Intramuscular triacylglycerol (IMTG) was determined using the methodology outlined by Denton and Randle (1967). Glycerol was assayed following the methods of Bergmeyer (1983) modified for spectrophotometry. 136 STA TISTICAL ANAL YSIS To determine whether U c r j t (expressed as either cm-s"1 or BL-s"1) varied with body mass or condition factor, correlation analysis was performed within each killifish population as well as for the pooled data for all populations. Critical swimming speed and exercise challenge metabolite data sets were analyzed by multiple analysis of variance (ANOVA) with population, temperature, and/or rest/exercise as independent categorical variables. All experiments were conducted independently and fish were never used in more than one experiment or treatment group. Thus all data points can be considered as independent. All data met the assumptions of normality, and data were log transformed where necessary to meet assumptions of homogeneity of variance. When interaction terms were not significant, post-hoc comparisons were performed among the groups with the Student-Newman-Keuls multiple range test (SNK MRT). If the interaction terms were significant, the data were separated and analyzed independently using one-way ANOVA. For all statistical analyses, a was set at 0.05. RESULTS BODY SIZE Fish used in this experiment were all of similar size, and there were no significant differences in body mass between populations or treatment groups. The mean mass of northern killifish was 5.39 ± 0.60g (mean ± SE), and the mass of southern killifish was 5.94 ± 0.50 g. Condition factors ranged from 1.44 to 1.68 for all killifish groups, and there were no significant correlations between body mass or condition factor with Ucrit (expressed as either cm-s"1 or BL-s"1) in any of our experimental treatments. 137 EXPERIMENT 1: EFFECTS OF A CCLIMA TION Critical swimming speed (U c n t) for northern (NH) and southern (GA) killifish from the 2002 collection year acclimated to temperatures of 5.2, 10.4, 14.7, 21.5, 28.7, and 32.4°C were analyzed using two-way A N O V A with population and acclimation temperature as factors (Figure 4.1). Two-way ANOVAs revealed a significant effect of population and acclimation temperature (P<0.001 for both comparisons) with no significant interaction (P=0.379). Post-hoc tests revealed that, at all acclimation temperatures, northern killifish had significantly higher U c rit values than southern killifish (SNK MRT, P<0.001 for all comparisons). Within the northern population, there were no differences in U c r j t values between acclimation temperatures of 10.4 and 32.4°C (P-values ranging from 0.217 to 0.858), while the U c r i t S o f both northern and southern fish acclimated to 5.2°C were significantly lower than all other acclimation temperatures (SNK MRT, P<0.001 for all comparisons). The patterns in the southern population were qualitatively similar, although the 28.7°C group had very slightly, but significantly, higher performance than the 21.5°C acclimation group. EXPERIMENT 2: PERFORMANCE IN ACUTE CHALLENGE Figure 4.2 presents the effect of acute temperature challenge on the U c rit of fish acclimated to 17.8°C. Two-way A N O V A revealed a significant effect on U c r it of population (P=0.011), temperature (PO.001), and a significant interaction between the two (P=0.012). As in the previous experiment, northern fish acclimated to ~18°C swam significantly better than southern fish (SNK MRT, PO.001 )(Figure 4.2). When 17.8°C acclimated fish were acutely challenged to swimming temperatures of 5.0, 25.1, or 34.0°C, however, there were no differences between populations at any of the acute challenge temperatures (SNK MRT, P-values ranging from 0.212 to 0.875). For northern fish, performance was independent of temperature 138 from 18-35°C, but declined significantly at 5°C (SNK MRT, P<0.001). In contrast, southern fish significantly increased performance approximately linearly with temperature from 5-25°C (SNK MRT, P-values ranging from <0.001 to 0.03) and performance was temperature independent from 25-35°C (SNK MRT, P=0.594). EXPERIMENT3: STANDARDIZED EXERCISE CHALLENGE All fish successfully performed at 80% Ucrit for the entire 2 hour swimming period. Muscle glycogen and lactate Three-way A N O V A for muscle [lactate] (Figure 4.3C and D) revealed a significant effect of acclimation temperature (P=0.003) and exercise state (P<0.001), and a significant interaction between acclimation temperature and population (P=0.034) as well as between population and exercise state (P=0.004). There was no significant difference in resting muscle [lactate] between populations except at 29°C where southern fish had significantly elevated resting [lactate]. In northern fish, muscle [lactate] increased with exercise at all temperatures (Figure 4.3C; SNK MRT, PO.005 for all comparisons). In southern killifish, [lactate] significantly increased with exercise only in the 15°C group (Figure 4.3D). Other muscle metabolites Most muscle metabolites ([glucose], [ATP], intramuscular triacylglycerol [EVITG]) changed only modestly or inconsistently with acclimation temperature, population, and exercise state (Table 4.1). Three-way A N O V A for muscle [PCr] (Table 4.1) revealed a significant effect of acclimation temperature (PO.001), population (P<0.001), and rest/exercise (P=0.018), and a significant interaction between temperature and population (P=0.010). At 5°C, southern fish had higher [PCr] than northern fish at rest and following exercise, but there were no differences between populations at higher temperatures. In contrast, northern fish had significantly higher 139 [total muscle lipid] than southern fish as indicated by the results of the 3-way A N O V A analysis (Pop, P<0.001; no significant effect of temperature, exercise state, or any significant interactions). Although this effect was consistent in all groups it was detected as significant in post-hoc tests only for the 15°C exercise and 29°C rest groups. Liver glucose and glycogen Liver [glucose] changed only modestly or inconsistently with acclimation temperature, population, and exercise state (Table 4.1). In contrast, 3-way A N O V A revealed a significant effect of acclimation temperature (P<0.001) and population (P=0.012) and a significant interaction between them (P=0.020) for liver glycogen (Table 4.1). Northern fish had higher liver [glycogen] at 5 and 15°C than at 29°C, and as a result northern fish had generally higher liver [glycogen] than southern fish at lower temperatures, while the reverse was the case at 29°C, although this effect was not consistently detected in post-hoc tests. When liver [glycogen] values from all acclimation temperatures and exercise states were pooled by population, a t-test revealed that overall, northern killifish had significantly higher mean liver glycogen than southern fish (P =0.029). Plasma glucose and lactate With the exception of resting southern fish at 29°C, plasma [glucose] was very consistent across populations, acclimation temperatures, and exercise states (Table 4.1). Plasma [lactate] increased significantly with exercise in northern fish at all acclimation temperatures, but consistent with the data for muscle [lactate] this effect was only significant for southern fish at 15°C (Table 4.1). 140 EXPERIMENT 4: MUL TIPLE POPULA TIONS In order to determine if U c r i t differences between northern and southern killifish were consistent across multiple populations within a subspecies, we quantified U c r i t in samples of killifish from six different populations (3 of the northern subspecies and 3 of the southern subspecies; collection year 2004) acclimated to a common temperature of 23°C (Figure 4.4A). One-way A N O V A comparing killifish populations revealed no differences in U c r i t among any of the populations tested (P=0.345). The U c r i t values of all six killifish populations were consistent with those of northern fish from the first experiment (compare Figure 4.1 and 4.4A). Resting levels of muscle [glycogen] (Figure 4.4B) and muscle [glucose], liver [glycogen], and liver [glucose] (Table 4.2) were also determined for all killifish populations. With the exception of a single southern killifish population (WI) having significantly higher liver [glucose] than the other populations, there were no significant differences in metabolite levels between populations in any of the metabolites measured (Table 4.2). T-test's comparing metabolite levels of all northern fish combined to all southern fish combined, however, revealed a significant difference between subspecies for muscle [glycogen] (higher in northern fish, P=0.0019) as well as for liver [glucose] (higher in southern fish, P=0.008). However, there was no significant difference in liver [glycogen] (P=0.262) EXPERIMENT 5: STANDARDIZED EXERCISE CHALLENGE To determine the patterns of fuel utilization for northern and southern killifish subspecies that had both high U c r i t s and high levels of resting muscle and liver [glycogen], northern (NH) and southern (GA) fish from the experimental groups in experiment 4 were exposed to a standardized exercise challenge. With respect to resting fish, the NH population had significantly higher muscle [glycogen] than did the G A population (P=0.002; Table 4.3) similar to the situation in experiment 4, which revealed a significant difference when all populations 141 within a subspecies were pooled. However, the values for southern fish in both experiment 4 and 5 were much higher than those observed in experiment 3 (compare Figures 4.3, 4.4, and Table 4.3). Liver [glycogen], however, was similar across all experiments (compare Tables 4.1, 4.2, and 4.3). Exercise resulted in an increase in muscle and plasma lactate levels in both killifish populations, and this effect was significant for southern fish (SNK MRT, P=0.018 and 0.034, respectively). There was no significant decline in muscle and liver [glycogen] with exercise in either population. EXPERIMENT 6: LOW TEMPERA TURE A ecu MA TION Because previously published work (DiMichele and Powers, 1982) and experiment 1 of the current study indicated significant differences in swimming performance of northern and southern killifish at 5°C, we also determined the effect of low temperature (7°C) acclimation on Ucnt and metabolite levels in killifish with high starting levels of muscle [glycogen] (2004 collection year, Table 4.4). The U c r i t values for northern (NH) and southern (GA) killifish acclimated to 7°C were 6.2 and 6.3 (BL-s"1), respectively, and there was no significant difference between populations (P=0.832) (Table 4.4). Additionally, these values were not significantly different from the values of northern fish in the first experiment at 10°C (compare Figure 4.1 and Table 4.4). There was no difference in muscle [glycogen] levels of northern fish before or after acclimation to 7°C. There was, however, a significant decline in muscle glycogen levels of southern fish after acclimation to 7°C for 21 days. No other metabolites showed any significant differences between populations or during acclimation. 142 DISCUSSION Four important observations emerge from the data presented here. (1) Regardless of population, F. heteroclitus has an extremely broad zone of essentially temperature-independent swimming performance, as assessed using U c r j t . (2) Much of this zone can be attributed to an intrinsic temperature-insensitivity of swimming performance, rather than any beneficial effects of acclimation, particularly for northern fish. (3) Observed differences between populations in swimming performance varied from year-to-year. (4) This variation appeared to be related to metabolic factors such as muscle glycogen content that differed from year-to-year in southern fish. TEMPERA TURE INDEPENDENT SWIMMING PERFORMANCE Both northern and southern populations of killifish showed little change in swimming performance, as assessed using U c r j t , at acclimation temperatures from 10 to 33°C. The extremely large zone of temperature-independent swimming performance for killifish is consistent with the observations of Targett (1978), who showed that killifish metabolic rate is independent of temperature at acclimation temperatures from 13-30°C, a range of more than 15°C. Temperature independence has also been shown in burst performance in northern killifish acclimated to 10 and 35°C (Johnson and Bennett 1995). This extremely broad range of temperature-independent performance is very unusual among fishes. For example, experiments on carp (Heap and Goldspink 1986), tinfoil barbs, river barbels (O'Steen and Bennett 2003), and several salmonid species (reviewed in MacNutt et al. 2004) have all shown a positive relationship between temperature and swimming performance up to a thermal optimum, with a plateau in performance where maximum performance is maintained only across a few degrees, followed by declines in performance at higher temperatures. For example, in salmonids this plateau extends 2 to 5°C beyond the thermal optimum and is reflective of habitat temperature 143 (Randall and Brauner 1991; Beaumont et al. 1995; Day and Butler 1996; Swanson et al. 1998; Altamiras et al. 2002; Lee et al. 2003; MacNutt et al. 2004). However, the much wider zone of temperature insensitivity of performance for killifish may also be reflective of their habitat temperatures, because killifish habitat temperature is extremely variable. Mean monthly temperatures in killifish habitat range from ~3°C to 32°C, depending on the location and the time of year. Thus, just as in other fishes, the thermal sensitivity of performance in killifish is well-matched to their habitat temperatures. The broad zone of temperature-independent swimming performance that we observed in both northern and southern killifish extends almost to their thermal tolerance limits. Killifish cannot survive long-term exposure to temperatures greater than 36.4°C for northern killifish, or 38.2°C for southern killifish (Fangue et al. 2006), whereas optimal performance is maintained up to ~33°C. Thus there is a very narrow window between optimum performance and death in this species (3.4 or 5.2°C, depending on the population). For killifish in the extreme southern parts of the species distribution, mean monthly temperature ranges from 12-29°C, with temperatures dropping below 10°C for only a few days in December and January (calculated from N O A A NERRS Data, Matanzas, FL). This suggests that fish in southern habitats operate within the zone of temperature independence of swimming performance most of the year. In contrast, killifish from the extreme northern parts of the species distribution experience mean monthly temperatures from 2.5-18°C, and mean monthly temperatures are below 10°C for 6 months of the year in some localities. Indeed, mean monthly temperatures below 3°C are common in winter, with temperatures routinely dropping below 1°C (calculated from N O A A NERRS Data, Wells, ME). This suggests that northern fish operate for much of the year at temperatures below their thermal optimum. It is possible that reduced performance at low temperatures in northern fish was a consequence of the relatively long-day photoperiod to which these fish were acclimated in the laboratory, compared with those they might experience in natural habitats. Photoperiod has been 144 demonstrated to have an effect on swimming performance in fish, with some but not all studies demonstrating improved performance at seasonally appropriate photoperiods (Kolok, 1991; Smiley and Parsons, 1997; Day and Butler, 2005). Our northern source population experiences mean monthly temperatures ranging from 6-15°C in combination with a photoperiod of approximately 12:12, suggesting that the photoperiod we selected was indeed seasonally appropriate for the lower acclimation temperatures. Shorter-day photoperiods in the northern habitat are associated with even lower temperatures than those tested here. As a result, it is unlikely that the decrease in performance at low temperatures that we observed is a result of the effects of inappropriate photoperiod. A drop in performance at low temperatures has been observed in other eurythermal species (reviewed in Johnston and Temple 2002), and it is thought that these fish may not maintain the potential for high activity at very low temperatures and enter a state of winter dormancy where activity is greatly reduced. This strategy of reducing activity has the benefit of conserving energy during the winter season when food availability is low as well as keeping fish hidden from homeothermic predators such as birds (Crawshaw 1984). During the summer, daily water temperatures in northern habitats typically range between 12 and 24°C, indicating that during the summer season when they are most active, northern killifish operate within their zone of temperature independent swimming performance. INTRINSIC TEMPERATUREINSENSITIVITYDURING ACUTE THERMAL CHALLENGE The wide zone of temperature-independent swimming performance that we observed was not solely due to the effects of acclimation, and this was particularly apparent in northern fish. For northern killifish acclimated to 18°C, swimming performance was independent of acute test temperature from 18-34°C. This response curve is atypical of those reported for other species where a positive correlation between acute swimming temperature and U c r j t is observed (Beamish 1978, 1981; Hammer 1995). Our data for northern fish are, however, consistent with the work 145 of Johnson and Bennett (1995) who quantified burst performance in northern killifish during acute temperature exposure. Their work showed that at the whole organism level, groups of killifish acclimated to either 10 or 35°C and acutely challenged with a series of swimming temperatures between 5 and 30°C (10°C acclimation group) and 10 and 35°C (35°C acclimation group), burst performance declined only slightly when 10°C acclimated fish were tested at 30°C and 35°C acclimated fish were tested at 10°C. In contrast, southern fish did not demonstrate such a large zone of temperature independent performance. Swimming performance of southern fish acclimated to 18°C increased significantly (and approximately linearly) with temperature between 5 and 25°C. However, above 25°C, these fish also showed a plateau in performance, with a zone of temperature insensitivity of ~10°C. Although this is small in comparison to the thermal insensitivity of the northern populations, it is still impressive in comparison with the data available for other fish species (reviewed in O'Steen and Bennett 2003). One possible explanation of these results is that our acclimation temperature o f 1 8 ° C represents a very different seasonal temperature for northern and southern killifish. Summer mean monthly temperatures are close to 18°C in northern climates, whereas 18°C for southern habitats is much more representative of mean temperatures in early spring (estimated from N O A A NERRS data). Northern fish experience the greatest daily temperature fluctuations in June and July when daily excursions to greater than 30°C from the mean of ~18°C are not uncommon. Therefore, the ability of northern fish to be thermally independent within this temperature range could be an ecologically relevant mechanism used by northern populations to maintain locomotory performance in the summer months. Southern fish, however, are most likely to experience a mean temperature of 18°C in March and April when the magnitude of the variation between the monthly mean and daily temperature extremes is small (<5°C) suggesting that when acclimated to 18°C in the wild 146 southern fish do not need to perform at a wide range of temperatures. Daily temperatures exceeding 25°C do not occur until May, when the mean temperature has already reached 23°C. These observations emphasize that simple metrics such as mean habitat temperature may oversimplify the coastal temperature regime experienced by killifish, and may obscure important differences such as temperature extremes relative to mean habitat temperatures. PLASTICITY IN SWIMMING PERFORMANCE As was previously observed by DiMichele and Powers (1982), we found that sustained swimming performance differed between northern and southern killifish at low acclimation temperatures, with northern fish outperforming southern fish in our first experiment on fish collected in 2002. Indeed, our study detected a larger performance difference between populations than was found in the previous work. This is not unexpected, as the study by DiMichele and Powers (1982) was conducted on fish collected from the Delaware Bay hybrid population and genotyped for Ldh-B. These fish were not genotyped at any other loci making the overall genetic context in which the LDH-B isoforms were functioning unknown. Northern and southern killifish populations, however, differ at a number of loci (reviewed in Powers et al. 1993; Powers and Schulte 1998) that might also influence swimming performance. In addition, hybrid fish from the same collection location would be assumed to have been exposed to more similar environmental/developmental conditions compared to fish that were wild-caught from different locations reducing potential irreversible effects of differing developmental conditions in hybrid fish. However, in our first experiment (on fish collected in 2002) we also detected differences in swimming performance between northern and southern fish acclimated at high temperatures, unlike the studies of DiMichele and Powers (1982), who observed no differences in performance at high temperatures. 147 In our subsequent study on fish collected in 2004, however, these differences in swimming performance were not repeatable, and we detected no differences in swimming performance between northern and southern fish acclimated at either low or high temperatures. In this second study, the performance of southern fish improved to values equivalent to northern fish from both the 2002 and 2004 collection years. There was generally good agreement between our studies and those of DiMichele and Powers (1982) for northern fish acclimated to 25°C (6.3 BL-s"1 and 6.2 BL-s"1 current study and 5.8 BL-s"1, DiMichele and Powers (1982)). However, agreement was poor both within and between studies for southern fish acclimated to 25°C (4.0 BL-s"1 and 7.9 BL.s"1 current study and 5.6 BL-s"1, DiMichele and Powers (1982)), and similar patterns were observed at low temperatures. Taken together, these data suggest that the swimming performance of southern fish differed between collection years, as a result of some unidentified factor that varied from year-to-year and between studies, either in the field or in the laboratory. Critical swimming speed is often used to assess the swimming capabilities of fishes and is thought to be an ecologically relevant measure of a fish's ability to survive ecological challenges (reviewed in Plaut 2001), but the long-term repeatability of U c r j t has only been investigated in a handful of studies. Critical swimming speeds of samples of wild-caught blacknose dace from the same location in different years (Nelson et al. 2003), and wild-caught European sea bass held in mesocosms with natural fluctuations in biotic and abiotic parameters and tested periodically over 6 months (Claireaux et al. 2007) were highly repeatable from year to year. This is consistent with our results for northern fish, but is in contrast to our findings for southern fish, suggesting that U c r j t assessed in laboratory-acclimated fish may not be a repeatable indicator of ecologically relevant performance in all fish species. Although we saw substantial variability in the swimming performance of southern fish from year-to-year, we observed little variation in swimming performance from year-to-year or among populations of northern fish. This variability in southern but not northern populations may be the result of differences between populations in the effects of acclimation on swimming performance. By comparing Figures 4.1 and 4.2, it is clear that acclimation has little effect on the swimming performance of northern killifish. This is not the case for southern fish, in which performance declined with acclimation to temperatures above 20°C. It is possible that the poor performance of southern fish in our first experiment (compared to that in our subsequent experiment or that of DiMichele and Powers 1982) could be a result of generally poor condition of these fish following acclimation. Although a positive correlation between condition and U c r i t has been shown in some species (Lapointe et al. 2006), the condition factor of our fish as well as biochemical measurements of mobilizable stores (e.g. hepatic lipids and hepatic glycogen) routinely used to assess a fish's condition did not reveal any differences between populations under any of the acclimation conditions; therefore, generally poor condition is likely not the reason underlying the lower performance of the southern fish in 2002. There were also no significant differences between the condition factors of the fish used in the DiMichelle and Powers study (1982) and the fish in our experiment. All northern and southern fish were acclimated under identical conditions and were housed together in split tanks. Thus, if the difference in the swimming performance of southern fish as assessed in experiments conducted in different years was a result of some factor due to housing conditions, northern fish must not be susceptible to these effects. BIOCHEMICAL CORRELATES OF SWIMMING PERFORMANCE Critical swimming speed in fish has been shown to be influenced by a variety of biological factors (e.g. body size, nutrition, training) and environmental perturbations (e.g. temperature, oxygen, pH, salinity) (reviewed in Keiffer 2000). Our biochemical data suggest that low white muscle glycogen stores are associated with poor swimming performance. In all 149 cases where northern fish outperformed southern fish, northern fish had 2-4 fold higher muscle glycogen (Figure 4.3) suggesting that muscle glycogen stores are an important determinant of performance. In the second set of U c r it experiments, which found high swim performance values that were not significantly different between killifish populations (Figure 4.4, Table 4.4), all populations had muscle glycogen values that were higher than those of southern fish in our first series of experiments (Figure 4.3), again suggesting the importance of muscle glycogen as an exercise fuel. Unfortunately, measurements of muscle glycogen were not made in the DiMichele and Powers (1982) study for comparison. There were no differences in [IMTG], [liver glycogen], or condition factor between populations or collection years. It is well known from mammalian studies that muscle glycogen levels are positively correlated with exercise endurance (Widrick et al. 1993; Johnson et al. 2004; Rauch et al. 2005). The relationship between endurance and white muscle glycogen has been investigated to some extent in fish, and high levels of muscle glycogen have been linked with elevated lactate during exercise (Pearson et al. 1990; Schulte et al. 1992), faster recovery from exhaustion (Scarabello et al. 1991), greater survivability upon stream release (Hochachka and Sinclair 1962), and overall higher endurance performance (McFarlane and McDonald 2002). At this point, we have no specific hypotheses to advance that would account for the low muscle glycogen in southern fish collected in 2002, compared to those collected in 2004. Laboratory housing and feeding conditions were similar between years, and northern and southern fish were maintained in split tanks, eliminating the possibility of a tank effect causing the low muscle glycogen in southern fish. At higher temperatures, southern fish tend to be in reproductive condition, and we have observed greater degrees of agonistic interactions among southern fish than among northern fish (personal observations), which might cause higher activity levels, and thus lower muscle glycogen, and reduced swimming performance. Fish that were obviously gravid were not utilized in this experiment, but reproductively active males were not excluded, which could have influenced the 150 results. However, we also observed low muscle glycogen in southern fish from the 2 0 0 2 collection year after long-term acclimation to 5 and 1 5 ° C , temperatures at which reproductive activity is absent or very minimal in southern fish. As a result, we consider it unlikely that differences in reproductive condition could account for the differences in swimming performance between northern and southern fish. Similarly, we saw no obvious evidence of disease or parasitism in the southern fish. In addition, because of the split-tank design of these ' experiments, any diseases would likely have been transmitted to northern fish as well. However, it is still possible that some uncontrolled factor in the laboratory that differed from year-to-year, to which southern fish were susceptible, but which did not affect northern fish, might have resulted in the low muscle glycogen and poor swimming performance of southern fish from the 2 0 0 2 collection year. Although the causes for the differences in muscle glycogen and swimming performance from year-to-year are difficult to determine, it is clear that both of these parameters are plastic, at least in southern fish, and that previously observed differences in swimming performance between killifish genotypes are not repeatable under our experimental conditions. CONCLUSIONS The results presented here demonstrate that killifish are impressive eurytherms with the ability to maintain essentially equivalent U c r i t across acclimation temperatures from 1 0 - 3 3 ° C . In fish from a northern population, much of this zone of temperature independent performance could be attributed to an intrinsic temperature insensitivity of swimming performance, since performance did not vary when fish acclimated to 1 8 ° C were tested at temperatures from 1 8 -3 3 ° C . Southern fish, in contrast, were somewhat more sensitive to acute temperature change, such that Ucrit of fish acclimated to 1 8 ° C increased when the fish were exposed to higher temperatures. However, even in this population, performance was not affected by temperature at 151 temperatures from 25-33°C. In fact, both populations achieved their highest UCnt values at their respective summer habitat temperatures. 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Journal of Applied Physiology. 74: 2998-3000. 157 Table 4.1 Muscle, liver, and plasma metabolites for northern (N) and southern (S) killifish at rest or after a standardized exercise challenge (experiment 3). 5^  15° 29° Metabolite Pop Rest Exercise Rest Exercise Rest Exercise Muscle Glucose N 1.11 ± 0 . 0 6 a 1 . 3 4 ± 0 . 0 7 x 1 . 4 8 ± 0 . 1 4 a 1 .93±0 .04 y ' * 1 . 3 5 ± 0 . 2 2 a A § 2.20 ± 0.3 l y S 1 . 4 6 ± 0 . 3 5 a 0 . 9 7 ± 0 . 0 7 x 1.32 ± 0.10 a 1 . 3 7 ± 0 . 0 9 y 2 . 2 7 ± 0 . 1 6 b 1 . 8 9 ± 0 . 0 8 z Muscle A T P N 1.14 ± 0.17 a 1.38 ± 0.10 x 1 .38±0 .20 a 1 . 7 2 ± 0 . 1 8 x 1.44 ± 0.15 a 1.58 ± 0.18 x S 1 . 6 0 ± 0 . 1 9 a 1 . 2 5 ± 0 . 0 5 x 1.62 ± 0.21 a 1.61 ± 0 . 2 0 x 2.22 ± 0.22 a 1 . 8 4 ± 0 . 2 6 x Muscle P C r N 10.48 ± 1.15a* 8.04 ± 1.89x'* 9.62 ± 2.50 a 8.08 ± 1.22x 8.54 ± 1.38a 4 . 5 7 ± 0 . 9 8 x S 17.30 ± 1.31a 16.53 ± 2 . 6 5 x 10.25 ± 1.77b 7 . 2 3 ± 2 . 1 0 y 11.12 ± 1.85b 8.18 ± 1.03y Muscle Total L i p i d N 30.04 ± 2 . 5 7 a 27.38 ± 0 . 6 1 x 29.68 ± 4.40 a 33.68 ± 5.44"-' 26.27 ± 2.46"'* 25.79 ± 2 . 6 7 x S 24.58 ± 2.32 a 21.05 ± 1.16" 23.54 ± 1.79a 20.76 ± 1.74x 18.14 ± 2 . 0 8 a 23.83 ± 1.81x I M T G N 4.72 ± 1.02a 5 . 5 4 ± 0 . 3 5 x 4.25 ± 0.50 a 5 . 6 2 ± 0 . 3 3 x 4.72 ± 0.68 a 6.80 ± 1.40x S 4.77 ± 0.27 a 5.79 ± 1.29x 6.78 ± 0.72 a 5.85 ± 0 . 5 3 " 6.57 ± 1.55a 5.95 ± 0.85 x Liver Glucose N 3 . 3 2 ± 0 . 4 1 a 5.93 ± 0.65 x 4.90 ± 0.57 a b 5.75 ± 1.01x 7.80 ± 1.77b 10.98 ± 2.47 y'* S 4.41 ± 0 . 1 8 a b 4.92 ± 0.44 x 2.72 ± 0.37 a 4.30 ± 1.24x 7.11 ± 0 . 8 7 b 7.49 ± 1.87x Liver Glycogen N 647.73 ± 138.39 3 703.46 ± 116.59x'* 793.94 ± 92.65 a '* 761.98 ± 5 7 . 9 1 x 250.16 ± 6 4 . 2 1 b 463.85 ± 177.66 x S 410.56 ± 115.09 3 369.44 ± 58.74 x 445.43 ± 113.83 3 701.21 ± 117.48 x 371.81 ± 2 9 . 3 5 a 493.33 ± 4 5 . 7 5 x Plasma Glucose N 4.35 ± 0.32 a 6.45 ± 0.80 x 5 . 7 2 ± 0 . 6 2 a 7 . 1 2 ± 0 . 7 2 x 6.72 ± 1.973'* 10.17 ± 2 . 2 8 x S 4 . 8 0 ± 0 . 7 0 a 5 . 1 1 ± 0 . 5 2 x 4.31 ± 0 . 6 4 a 5 . 2 6 ± 0 . 5 3 x 12.60 ± 2 . 0 8 b , § 8.74 ± 0.92 y Plasma Lactate N 3.58 ± 1.46a'§ 8.56 ± 1.44x 4.22 ± 0 . 9 l a ' § 11.68 ± 1.88x 1.85 ±0.46^ 1 3 . 5 0 ± 3 . 1 6 x S 3 . 5 8 ± 0 . 9 7 a 5.50 ± 1.63x 3.52 ± 1.09a'§ 10.88 ± 3 . 3 2 y 7.38 ± 1.65a 1 1 . 3 0 ± 2 . 4 8 x y Collect ion sites: Hampton, N H ( N H ) and Brunswick, G A ( G A ) in 2002. Values are mean ± SE , n=6 for all groups PCr , creatine phosphate; I M T G , intramuscular triacylglycerol Muscle glucose, muscle A T P , muscle P C r , plasma glucose, plasma lactate, liver glucose, liver glycogen are /unol-g"1 wet tissue Muscle total l ip id expressed as /jg-mg"1 tissue; I M T G expressed as glycerol /unol-g"1 tissue Different letters indicate a significant difference between temperatures within a population and metabolic state * indicates a significant difference between populations at a given acclimation temperature and metabolic state (indicated on northern values) § indicates a significant difference between rest and exercise within a population and acclimation temperature (indicated on rest values) Table 4.2 Resting metabolites for multiple killifish populations acclimated to 23°C (experiment 4). Northern killifish Southern killifish Metabolite Nova Scotia-NS Maine-ME New Hampshire-NH Georgia-GA Florida-FB Florida-WI Muscle glucose 0 . 9 9 ± 0 . 3 3 a 1.51 ± 0.28a 1 . 4 5 ± 0 . 2 3 a 2.06 ± 0.37a 0 . 8 2 ± 0 . 4 3 a 0 . 7 6 ± 0 . 1 4 a Liver glycogen 549.31 ± 15.383 471.69 ±30 .51 a 482.89 ± 26.62a 414.23 ± 102.703 5 3 6.14 ± 25.69a 323.43 ± 5 2 . 1 1 Liver glucose 4.40 ± 0.433'* 4.41 ± 0.713'* 4.74 ± 0.463'* 6 . 9 4 ± 1 . 5 2 a b 5.25 ± 0.673 9 . 0 6 ± 1 . 3 7 b a Metabolites are expressed as /Ltmol-g-1 wet tissue. Values are mean ± SE, n=6 for all groups. Different letters indicate a significant difference in metabolite level between each population Collection sites: Antigonish, NS (NS); Salsbury Cove, M E (ME); Hampton, NH (NH); Brunswick, G A (GA); Femandina Beach, Fl (FB); Whitney Island, Fl (WI) in 2004 * indicates an overall significant difference in metabolite level between northern (ME, NH, NH) and southern (FB, GA, and WI) killifish (indicated on northern values only) Table 4.3 The effects of a standardized exercise challenge on metabolites in northern (NH) and southern (GA) killifish (experiment 5). Muscle Liver glycogen glucose lactate glycogen glucose lactate North (NH) - Rest 54.59 ± 3 . 3 6 a 2.32 ± 0.76a 1 . 3 7 ± 0 . 4 0 a 602.21 ± 3 1 . 0 5 a 6.18 ± 1.08a 2 . 8 7 ± 0 . 8 0 a North (NH) - 80%Ucr i t 44.04 ± 7.70a 2.01 ± 0.24a 4.87 ± 1.67a 613.66 ± 5 8 . 6 6 a 5 . 5 4 ± 0 . 6 1 a 9 . 0 7 ± 2 . 3 7 a South (GA) - Rest 25.10 ± 3 . 9 6 b 1 . 6 3 ± 0 . 2 9 a 0.75 ± 0.38a'* 467.56 ± 80.763 2 . 7 2 ± 0 . 3 6 b 2.52 ± 1.193'* South (GA) - 80% U c r i t 31.02 ± 6 . 1 3 a 2.05 ± 0.36a 6.41 ± 2 . 4 5 a 413.76 ± 4 8 . 8 4 b 5.11 ± 1.06a 10.83 ± 3.96a Metabolites are expressed as /miol-g"1 wet tissue. Values are mean ± S.E., n=6 for all groups. Different letters indicate a significant difference in resting or exercising metabolite levels between populations Collection sites: Hampton, NH (NH) and Brunswick, G A (GA) in 2004 * indicates a significant difference in metabolite level within a population between rest and exercise (indicated on resting values only) Table 4.4 The effects of low temperature acclimation (7°C) on U c r j t and metabolites in northern (NH) and southern (GA) killifish (experiment 6). U c r i t (BLsec1) Metabolite Muscle glycogen Muscle glucose Liver glycogen Liver glucose NH - Pre-acc NH - Post-acc GA - Pre-acc N.D. 6.16 ± 1.00 N.D. GA - Post-acc 6.31 ± 1.3.7 35.64 ± 8 . 2 1 a 0.13 ± 0.13a 487.68 ± 61.69a 1 . 3 8 ± 0 . 3 0 a 36.43 ± 6 . 6 1 a 0.02 ± 0.02a 423.42 ± 69.36a 1 . 4 3 ± 0 . 3 7 a 28.21 ± 8.30a 0.30 ± 0.24a 299.29 ± 92.40a 3.02 ± 1.80a 9 . 0 6 ± 2 . 5 6 b 0.00 ± 0.00a 397.09 ± 78.18a 0.49 ± 0.20a Metabolites are expressed as /imol-g"1 wet tissue. N.D. = not determined. Values are mean ± S.E., n=6 for all groups. Different letters indicate a significant difference in metabolite level among acclimation groups Collection sites: Hampton, NH (NH) and Brunswick, G A (GA) in 2004 10 8 - * * b b 6 - I •C 4 A 0 A Northern (NH) killifish • Southern (GA) killifish * a - 1 — 5 yz b y b I * b 1 I yz 10 15 20 25 30 Acclimation Temperature (°C) 35 Figure 4.1 Critical swimming speed (Ucrit; BL-sec"1) for northern (NH, triangles) and southern (GA, circles) killifish collected in 2002 and acclimated to temperatures between 5.2 and 32.4°C (experiment 1). Significant differences in Ucrit within a population are indicated with different letters. An asterisk (*) indicates a significant difference in U c r j t between populations at a given acclimation temperature. Data are expressed as mean ± SE (n=6) and P<0.001 for all significant comparisons. 162 o 10 0 b yz A Northern (NH) killifish • Southern (GA) killifish "i ' r ~i ' r 0 10 15 2 0 25 30 35 Swim Temperature (°C) 4 0 Figure 4.2 Critical swimming speed ( U c r i t ; BL-sec" 1) for northern ( N H , triangles) and southern ( G A , circles) kil l if ish collected in 2002 and acclimated to 18°C and swum at the acclimation temperature or at 5, 25, or 34°C (experiment 2). Significant differences in U c r i t within a population are indicated with different letters. A n asterisk (*) indicates a significant difference in Ucrit between populations at a given temperature. Data are expressed as mean ± SE (n=6) and P<0.05 for all significant comparisons. 163 Figure 4.3 (following page) Northern (NH) and southern (GA) killifish muscle glycogen (NH, panel A; GA, panel B) concentrations (umol-g"1 wet tissue) and muscle lactate (NH, panel C; GA, panel D) concentrations (umol-g"1 wet tissue) for resting (open columns) and exercising fish (hatched columns) collected in 2002 and acclimated to 5, 15 and 29°C (experiment 3). An asterisk (*) indicates a significant difference (P<0.05) between populations at a given acclimation temperature and exercise state (indicated on NH panels only). Different letters indicate a significant difference (P<0.05) between temperatures within a population and exercise state. An (§) indicates a significant difference between rest and exercise within a population and acclimation temperature (indicated on the rest values). Values are means ± SE (n=6). 164 165 12 N - N S N - M E N - N H S - G A S - FB S - WI Population 60 -i N - N S N - M E N - N H S - G A S - F B S - WI Population Figure 4.4 Critical swimming speed (U c ri t; BL-sec"1; panel A) and resting muscle glycogen levels (umol-g"1 wet tissue; panel B) for multiple northern (Nova Scotia, NS; Maine, M E ; New Hampshire, NH; open columns) and southern (Brunswick, GA; Fernandina Beach, Fl, FB; Whitney Island, Fl, WI; hatched columns) killifish populations collected in 2004 and acclimated to 23°C (experiment 4). Different letters indicate a significant difference (P<0.05) in U c r j t values between populations (panel A) or in muscle glycogen between populations (panel B). The asterisk (*) indicates that the three northern populations had statistically significant higher glycogen levels than the three southern populations. U c rjt data are expressed as mean ± SE (n=6) and muscle glycogen as mean ± SE (n=6). 166 C H A P T E R F I V E : I N T E R - P O P U L A T I O N V A R I A T I O N I N T H E E F F E C T S O F T H E R M A L A C C L I M A T I O N O N M I T O C H O N D R I A L P R O P E R T I E S I N K I L L I F I S H {FUNDULUS HETEROCLITUS)4 INTRODUCTION Temperature's pervasive effects on biochemical and physiological processes are thought to be fundamental in shaping the distribution and abundance of aquatic ectotherms such as fish (Hochachka and Somero, 2002), but the precise mechanisms involved in establishing both the upper and lower thermal limits of organisms are still poorly understood (Angilletta et al., 2006; Pdrtner et al., 2006). Recently, the concept of oxygen limited thermal tolerance (OLTT) has been proposed as a unifying theory of the effects of temperature on organismal performance limits (Pdrtner, 2002; Pdrtner et al., 2007). The OLTT hypothesis proposes that at both low and high temperatures, organismal performance is limited by the inability to supply oxygen to the respiring mitochondria, i.e. there is a mismatch between oxygen supply and demand (Pdrtner, 2001; 2002; Pdrtner et al., 2004). Increasing temperatures result in increased oxygen demand by the mitochondria, and at some critical temperature threshold (Tp, pejus temperature=getting worse, Shelford, 1931), the mitochondrial oxygen demand outstrips the ability of the circulatory and ventilatory systems to supply oxygen (reviewed in Pdrtner et al., 2004). As a result, aerobic scope declines, causing a reduction in performance. As temperature (and thus metabolic rate) continues to increase beyond T p , a critical temperature (Tc) is eventually reached at which even standard metabolic rate cannot be maintained and system failure results. Decreasing temperatures, in contrast, cause declines in the ability of the mitochondria to produce ATP, compromising an organism's ability to perform normal physiological functions including the function of the ventilatory muscles and the circulatory pumps that are needed in order to supply oxygen to the working tissues at low temperatures. Thus, acclimation or adaptation to the cold 4 A version of this chapter will be submitted for publication. 167 must involve increases in either mitochondrial density or changes in their functional properties to improve ATP production in the cold. Under the OLTT framework, however, this increase in overall mitochondrial capacity in the cold comes at a cost under warming conditions. At warmer temperatures, not only is oxygen demand/metabolic rate increased, but there is also an increase in oxygen radical production associated with the greater mitochondrial capacity in cold-adapted organisms. According to the OLTT hypothesis, this increased standard metabolic rate at warm temperatures would be expected to decrease the upper thermal limits of the organism. Thus, Portner (2001) has suggested that " adjustments of mitochondrial densities and their functional properties appear as a critical process in defining and shifting thermal tolerance windows". The predictions derived from the OLTT framework at the level of the mitochondria are the following: 1) low temperature acclimation should increase mitochondrial density and/or function per mitochondria, 2) low temperature adaptation should result in more pronounced changes in mitochondrial density and/or function in 'low temperature adapted' populations, and 3) differences in upper and lower thermal tolerance between populations should be related to differences in mitochondrial properties between populations. In support of these predictions, acclimatory changes have been shown for a number of fish species with mitochondrial function, mitochondrial densities, and/or mitochondrial enzyme activities increasing in response to cold acclimation'(reviewed in Guderley, 2004). As well, cold-adapted populations show elevations in mitochondrial densities and increases in the activities of mitochondrial enzymes (Tschischka et al , 2000; Guderley, 2004; Sommer and Portner, 2004; Lannig et al, 2003). Evidence in stenothermal species suggests that no evolutionary modification of mitochondrial function has occurred with cold adaptation (Johnston et al, 1998), but more recent evidence in eurytherms suggests that function may be upregulated in cold-adapted eurythermal populations (Tschischka et al., 2000; Sommer and Pdrtner, 2004). Taken together, these data suggest a possible distinction between the strategies that stenofherms and eurytherms use to modulate mitochondrial 168 function with cold adaptation. The upper and lower thermal tolerance limits of an organism are known to shift unidirectionally during thermal acclimation. This suggests that the mechanism(s) involved in setting an organism's thermal niche should be investigated at both the high and low temperatures because of the tradeoff between high and low thermal tolerance. Mitochondrial density and function adjustments appear to be one of the key mechanisms contributing to these unidirectional shifts in both low and high pejus and critical temperatures with thermal acclimation (Pdrtner, 2002). The purpose of the current study was to test the predictions of the OLTT hypothesis in a temperate-zone eurythermal fish species that experiences a wide range of environmental temperatures in its natural habitat. Common killifish (Fundulus heteroclitus Linnaeus) have been studied extensively as a model to investigate mechanisms of thermal adaptation (Schulte, 2001). These fish inhabit estuaries and salt marshes along the East Coast of North America from Newfoundland to central Florida and experience substantial variation in environmental temperatures such that killifish in the northernmost populations experience temperatures that are on average, ~12°C cooler than those experienced by their southern relatives (NOAA NERRS data). Killifish have a broad capacity to tolerate and acclimate to environmental temperatures and are thought to have undergone local adaptation to differences in habitat temperatures (Schulte, 2001). Within the species, substantial variation exists in morphological, molecular, genetic, and physiological traits (reviewed in Powers et al., 1993; Powers and Schulte, 1998; Schulte, 2001; Fangue et al., 2006) such that two distinct regional subspecies have been suggested - a northern form, Fundulus heteroclitus macrolepidotus, occurring from the Gulf of St. Lawrence, Canada to New Jersey, USA, and a southern form, Fundulus heteroclitus heteroclitus, distributed from Virginia, USA to the North-eastern coast of Florida, USA (Morin and Able, 1983). 169 Recent cDNA microarray studies have revealed substantial variation between populations of killifish in mRNA expression of mitochondrial genes, particularly in several subunits of cytochrome c oxidase (Cox) (Whitehead and Crawford, 2006), while proteomics techniques have shown differences between populations in ATP Synthase levels (B. Rees, pers comm), suggesting that northern and southern killifish populations may differ in mitochondrial density. As well, analysis of killifish mtDNA sequences have revealed a steep cline with two distinct lineages of killifish with 'northern' and 'southern' haplotypes thought to have diverged ~1 million years ago (Gonzalez-Villasenor and Powers, 1990; Bernardi et al., 1993; Adams et al., 2006). The cline in mtDNA is much steeper than that for other genetic loci, suggesting the possibility of strong selection on mtDNA (Powers et al., 1993). Killifish populations also differ in isolated tissue and whole organism metabolic rates when acclimated to and compared at a common temperature such that northern fish have a higher metabolic rate than southern fish (DiMichele and Westerman, 1997 (embryos); Podrabsky et al., 2000 (heart); Fangue et al., Appendix A). In addition, northern fish are also more cold tolerant and less heat tolerant than their southern counterparts (Fangue et al., 2006). Higher metabolic rates and lower thermal tolerance limits in northern fish are consistent with OLTT predictions for cold adaptation in northern killifish populations. Together, these lines of evidence suggest that killifish may be an interesting model in which to test the predictions of OLTT. MATERIALS AND METHODS EXPERIMENTAL ANIMALS Adult killifish of the northern subspecies (Fundulus heteroclitus macrolepidotus) were collected from Hampton, New Hampshire (42° 54' 46" N), USA, and fish of the southern subspecies (Fundulus heteroclitus heteroclitus) were collected from Brunswick, Georgia (31° 7' 170 31" N), USA. All collections were made in late spring of 2004. Fish were held in 75 L glass aquaria with biological filtration at 15 ppt salinity, 20 ± 2°C, and 12 h:12 h (L:D) photoperiod for a minimum of 3 weeks before experimental acclimation. Fish were then acclimated to 5, 15, or 25 ± 0.5°C. Temperature was changed at a rate of 1°C per day until each acclimation temperature was reached and fish were maintained at their acclimation temperature for a minimum of 4 weeks prior to experimentation. Six replicate 75 L acclimation tanks per temperature treatment were divided to house 10 northern fish on one side and 10 southern fish on the other. Acclimation temperatures were controlled with Fisherbrand® NIST traceable temperature controllers and Ebo Jager 250 W submersible heaters. Fish were fed TetraMin® fish flakes and commercial trout chow (PMI Nutrition International, Brentwood, MO, USA) daily to satiation. TISSUE SAMPLING Fish were stunned by a blow to the head and rapidly killed by spinal cord transection. Sex, body mass, total length, and wet weight measurements were taken, and muscle and liver tissues quickly dissected and frozen in liquid nitrogen for later analysis of enzyme activities and mRNA levels. Eight fish from each population and acclimation group were sampled for these analyses. Additional individuals were sacrificed and fresh liver tissue was sampled for the isolation of mitochondria (see below). The mean mass of northern killifish was 7.39 + 0.72 g (mean ± SE), and the mass of southern killifish was 7.94 ± 0.56 g, and no significant differences in mass were found between acclimation groups, populations, or sex. Treatment of all experimental animals was in accordance with the University of British Columbia animal care protocol #A01-0180. 171 TOTAL RNA EXTRACTION Total R N A was extracted from muscle and liver tissues using the guanidine isothiocyanate method outlined by Chomczynski and Sacchi (1987) using TRIzol® Reagent (Invitrogen Life Technologies, Burlington, ON, Canada). Following isolation, RNA was quantified spectrophotometrically and electrophoresed on an agarose-formaldehyde gel (1% w/v agarose, 16% formaldehyde) to verify RNA integrity. RNA was stored at -80°C. First strand cDNA was synthesized from 5 jag total RNA using oligo (dT]g) primer and RevertAid™ H Minus M-MuLV reverse transcriptase as per the manufacturer's instructions (MBI Fermentas Inc., Burlington, ON, Canada). cDNA was stored at -80°C. QUANTITATIVE REAL-TIME PCR ANALYSIS OF GENE EXPRESSION Gene expression data for citrate synthase (CS), cytochrome c oxidase subunit two (COXII), and ATP synthase /3-chain (ATPSyn) in liver and muscle tissue were obtained using quantitative real-time PCR (qRT-PCR) on an ABI Prism 7000 sequence analysis system (Applied Biosystems Inc., Foster City, CA, USA). Gene specific primers were designed from published sequences for Fundulus heteroclitus (Accession No. CN983139 (CS); AY735182 (COXII); CV822026 (ATPSyn)) using Primer Express software (version 2.0.0; Applied Biosystems Inc., Foster City, CA, USA) and are reported in Table 5.1. qRT-PCR reactions were performed using 2 pX cDNA, 4 pmoles of each primer and 2X SYBR Green Master Mix (Applied Biosystems Inc., Foster City, CA, USA) to a total volume of 22 u.L under the following conditions: 1 cycle of 50°C for 2 min, 1 cycle of 94°C for 10 min, 40 cycles of 95°C for 15 sec, 60°C for 1 min. At the end of each qRT-PCR reaction, PCR products were subjected to a melt curve analysis to confirm the presence of a single amplicon. Samples of RNA that had not been reverse transcribed were also subjected to qRT-PCR to detect the possible presence of genomic of mitochondrial DNA contamination. For citrate 172 synthase and ATP synthase /3-chain, genomic DNA contamination was below 1:625 starting cDNA copies. For cytochrome c oxidase (subunit II), mitochondrial DNA contamination was 1:25 starting cDNA copies. One liver and muscle tissue sample was used to develop a standard curve relating threshold cycle to cDNA amount for each primer set. Results are expressed relative to total RNA used in the reverse transcription reaction. PROTEIN ISOLA TION AND ENZYME ASSA YS Frozen liver or muscle tissue was homogenized in 9 volumes of ice-cold buffer (100 mM Hepes, 5 mM EDTA, 1 mM DTT, and 0.05% Triton T-100, pH=7.4 at 20°C) using 2 low-speed passes of 10 seconds each with a Polytron homogenizer (Fisher Scientific, Nepean, ON, Canada). Cellular debris was removed by a 5 min centrification at 2,500 g and 4°C, and preliminary tests ensured the complete release of enzymes from the tissues using this procedure. The remaining supernatant was further diluted with buffer containing only 100 mM Hepes and 5 mM EDTA, pH=7.4 at 20°C as appropriate for each assay. Cytochrome c oxidase (COX) activity was determined as described in Moyes et al. (1997) in 100 mM Hepes and 0.05 mM reduced cytochrome c, pH=7.4 at 20°C. Cytochrome c was reduced with ascorbate, dialyzed exhaustively in 100 mM Hepes buffer (pH=7.4 at 20°C) and stored at -80°C. The decrease in the absorbance at X^SO nm was monitored using a spectrophotometer thermostated to 25.0°C. Citrate synthase (CS) activity was determined according to Moyes et al. (1997) in 100 mM Hepes, 0.2 mM 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB), 0.3 mM acetyl CoA, and 1 mM oxaloacetate, pH=7.4 at 20°C. The increase in the absorbance at A.=412 was measured spectrophotometrically at 25°C. The protein concentration of each sample was determined using the bicinchoninic acid method (BCA) (Smith et al., 1985) and enzyme activity in Units per mg 173 protein was calculated using an extinction coefficient (e550) for cytochrome c of 29.5 mM and for DTNB (e412) of 13.6 mM. ISOLA TION OF MITOCHONDRIA Liver tissue was finely diced on cooled glass plates and introduced to 9 volumes of ice-cold isolation media. Approximately 4-6 fresh livers were pooled for each preparation to obtain sufficient material that could be used for 5-7 assays. Isolation media contained 250 mM sucrose, 50 mM KC1, 25 mM K H 2 P 0 4 , 10 mM Hepes, 0.5 mM EGTA, and 1.5% bovine serum albumin (BSA, fraction V "fatty acid free"), pH=7.4 at 20°C (Bagarinao and Vetter, 1990). Liver tissue was then homogenized on ice by three passes with a motorized Teflon tissue grinder. The resulting homogenate was centrifuged at 600 g for 10 min at 4°C to pellet cellular debris. The supernatant was transferred to a new, pre-cooled tube by pouring through glass wool to remove the majority of the fat. The defatted supernatant was then centrifuged at 6,000 g for 10 min at 4°C to pellet the mitochondria, and any remaining fat was carefully removed from the mitochondrial pellet with tissue. The pellet was washed twice with isolation media, gently resuspended, and kept on ice until all mitochondrial respiration measurements were completed. MITOCHONDRIAL RESPIRA TION MEASUREMENTS Oxygen consumption of isolated mitochondria was measured with an Oroboros Oxygraph 2-k high-resolution respirometry system (Oroboros Instruments, Innsbruck, Austria). The system was calibrated daily with air saturated assay medium at each experimental temperature and zero oxygen measures were made by the addition of sodium dithionite. Oxygen solubility in the assay media at different temperatures was calculated as described in Gnaiger and Forstner (1983). The assay medium contained 150 mM KC1, 25 mM K H 2 P 0 4 , and 20 mM Hepes, pH=7.4 at 20°C (Bagarinao and Vetter, 1990). Approximately 0.3 mg mitochondrial protein was added to 1.8 ml 174 air saturated assay media at the assay temperature. Oxygen consumption was measured following the addition of 0.25 mM malate to spark the Kreb's cycle, and 5 mM pyruvate was added as the carbon substrate. Pyruvate was selected in order to obtain maximum rates of State III respiration in fish mitochondria (Guderley and St. Pierre, 1996). State III rates of oxygen consumption were obtained by adding saturating ADP to a concentration of 0.625 mM. When all ADP had been phosphorylated, the rate of State IV respiration was measured for 5 min before oligomycin, an inhibitor of mitochondrial FoFi-ATPase, was added at 0.625 mM (State IV 0i respiration reflecting proton leak). Respiratory control ratios (as indices of mitochondrial coupling) were determined by dividing State III by State IV (RCR; Estabrook, 1967) or State III by State IV 0, (RCRoi; Pdrtner et al, 1999). Mitochondrial preparations were first assayed at the fish's acclimation temperature (5, 15, or 25°C), and the RCR was calculated to determine the coupling of the mitochondrial preparation. Preparations with RCR values less than 4 were not used for further analysis. Following this initial test, samples of the mitochondrial preparation were assayed at temperatures between 2 and 37°C. The order of assay temperature was randomized, and the final assay of the day was repeated at the fish's acclimation temperature to ensure that the mitochondrial preparation had not lost capacity or become progressively uncoupled over the duration of the experiment. All assays for each mitochondrial preparation were completed within 8 hours from the start of the mitochondrial isolation, but preparations were often stable for more than 12 hours. Within each acclimation temperature group and killifish population, mitochondrial respiration measurements were conducted at each experimental assay temperature for 4-6 independent mitochondrial preparations. Protein concentrations (mg/ml) were determined for each sample used for a mitochondrial respiration assay using the B C A method (Smith et al., 1985). Briefly, each sample 175 was introduced to 1 % triton to solubilize mitochondrial membranes and protein concentrations were corrected for BSA present in the mitochondrial suspension from the isolation medium. S T A T I S T I C A L A N A L Y S E S Mitochondrial respiration, enzyme activity, and mRNA expression data sets were analyzed by multiple analysis of variance (ANOVA) with population, acclimation group, and/or assay temperature as factors. All data met the assumptions of normality, and data were log transformed where necessary to meet assumptions of homogeneity of variance. When interaction terms were not significant, post-hoc comparisons were performed among the groups with the Student-Newman-Keuls multiple range test (SNK MRT). If the interaction terms were significant, the data were separated and analyzed independently using one-way A N O V A followed by SNK MRT. For all statistical analyses, a was set at 0.05. For the analysis of proton leak ( F V o i X slope discontinuities were determined using the Regress® program developed by Yeager and Ultsch (1989) for statistical determination of critical points in continuous data sets by determining the intersection of two best fit lines. RESULTS G E N E R A L M I T O C H O N D R I A L C H A R A C T E R I S T I C S Liver mitochondria from both northern and southern fish were highly coupled at all but the highest assay temperatures. Respiratory control ratios, RCR ( I I L I V ) as well as R C R o i ( I I L I V o i ) , were difficult to estimate accurately at low temperatures due to the extremely low State I V and I V o i rates at temperatures below 10°C, but the estimates were always greater than 5.37 (RCR) and 13.0 ( R C R o i ) . RCR and R C R o i values (mean ± SE) at assay temperatures between 10 and 35-37°C were always greater than 5.35±0.93 North, 4.19 ± 0.66 South (RCR) and 7.23 ± 176 1.35 North, 6.65 ± 1 . 5 1 South (RCRoi). Thermal acclimation affected the upper temperature sensitivity of mitochondrial oxidative phosphorylation. State III measurements for mitochondria prepared from both northern and southern fish acclimated to 5°C and assayed at 37°C could not be made, as these mitochondria were insensitive to ADP at this assay temperature, whereas mitochondria from 15- and 25°C-acclimated fish remained coupled and responsive to ADP up to 37°C, uncoupling only at higher temperatures. THERMAL SENSITIVITY OF MITOCHONDRIAL RESPIRA TION Maximum oxidative capacity (State III) Acclimation had different effects on the response of mitochondria to acute temperature challenge in each killifish population (Figure 5.1). Northern mitochondria from the 25°C acclimation group had significantly higher State III rates when assayed at 2 and 5°C compared to southern mitochondria (Figure 5.1, Panel C). At all other assay temperatures, State III rates of 25°C acclimated mitochondria were very similar between populations, with both northern and southern fish showing essentially linear increases in State III activity with acute increases in temperature. There were also significant differences between populations in the 15°C acclimation group. Southern mitochondria acclimated to 15°C showed a linear response to acute temperature challenge, whereas mitochondria from northern fish had a depression in State III rates at acute challenge temperatures of 15-25°C (Figure 5.1, Panel B), resulting in significant differences between populations when assayed at 25 and 30°C. For 5°C acclimated mitochondria, northern fish had a plateau in State III rates across acute temperature challenges of 10-30°C. Southern fish acclimated to 5°C showed a triphasic response where State III was linear from 2-15°C, steeply increased at temperatures above 15°C and plateaued at 25-35°C. As a result of these differences in the shapes of the acute temperature response curves between populations, 177 there were significant differences in activity between 5°C acclimated northern and southern fish at both low and high assay temperatures (2-10°C and 25-30°C; Figure 5.1, Panel A). Figure 5.2 shows State III rates compared between temperature acclimation groups assayed at 5, 15, and 25°C. Warm-acclimated mitochondria (25°C) from both northern and southern fish exhibited State III rates one-third lower than rates for 5- or 15°C-acclimated mitochondria in both populations assayed at both 2 and 5°C (Figure 5.2, Panel A) demonstrating that cold-acclimated mitochondria perform better than warm-acclimated mitochondria at low temperatures. For example, northern mitochondria acclimated to 5°C and assayed at 2 and 5°C had State III rates of 118 and 167.5 pmol O mg"1 mitochondrial protein s"1, respectively, while 25°C-acclimated northern mitochondria had State III rates of 77 and 116.6 pmol O mg"1 mitochondrial protein sec"1 when assayed at 2 and 5°C. This trend for warm-acclimated mitochondria showing reduced State III rates compared to cold-acclimated mitochondria remained for southern mitochondria assayed at 15 and 25°C. Northern mitochondria, regardless of acclimation temperature, had equivalent State III rates between acclimation groups when assayed at 15 and 25°C (Figure 5.2, Panels B and C). • In contrast to cold acclimation improving State III rates when assayed in the cold (Figure 5.1, Panel A), we did not see evidence for warm acclimation improving performance at warmer assay temperatures as there were no examples where 25°C-acclimated mitochondria performed better than the other acclimation groups at a given assay temperature (Figure 5.2). We did, however, show that warm acclimation shifted the upper performance temperature of mitochondria such that State III rates could be obtained for 15 and 25°C acclimated mitochondria at an assay temperature of 37°C whereas 5°C-acclimated mitochondria were uncoupled at this temperature (Figure 5.1). 178 Proton leak (State I V n Q Rates of oxygen consumption for mitochondria inhibited by oligomycin were determined as an index of proton leak (State I V 0 i ) . Assay temperature had a significant effect on State I V 0 i with rates increasing exponentially with assay temperature in all acclimation groups and for both populations (Figure 5.3). However, the assay temperature at which these curves reached their inflection point (the largest change in slope between two assay temperatures) differed among acclimation temperature groups such that mitochondria from fish acclimated to 5°C showed an inflection at assay temperatures of 21.8°C for northern and 21.4°C for southern fish (Figure 5.3, Panel A). In the 15°C acclimated groups, the temperature of inflection shifted to 27.5°C for northern mitochondria and 27.1°C for southern mitochondria (Figure 5.3, Panel B). When acclimated to 25°C, the inflection point was 29.8°C for northern mitochondria and 29.7°C for southern mitochondria (Figure 5.3, Panel C). The only significant difference between populations in proton leak rates was found for 25°C acclimated southern mitochondria. These mitochondria had I V 0 i rates at assay temperatures of 35 and 37°C that were significantly higher than those of all northern fish as well as southern fish acclimated to 5 and 15°C tested at 35 and 37°C assay temperatures (Figure 5.3, Panel C). Mitochondrial efficiency (State I H - I V n i ) The efficiency of mitochondrial function (State III-rV0i) was calculated and these data showed similar trends as seen for State III (data not shown). This suggests that in fish acclimated to 5°C northern mitochondria are significantly more efficient than southern mitochondria when assayed at temperatures of 2, 5, and 10°C, while their efficiency is significantly lower when assayed at 25 and 30°C. For 15°C-acclimated fish, mitochondria from southern fish are significantly more efficient at assay temperatures of 25 and 30°C than those of 179 northern fish, but for 25°C-acclimated fish, there were no significant differences in efficiency between populations. VA RIA TION IN ENZYME A CTIVITY . Cytochrome c oxidase (COX) activity (Units per mg protein) measured in the liver and analyzed by two-way A N O V A with acclimation temperature and population of origin as factors revealed a significant effect of population, but no significant effect of acclimation temperature or any significant interaction. Liver COX activity in northern fish was higher than southern fish at all acclimation temperatures (40%, 20%, and 10%o higher in northern fish acclimated to 5, 15, and 25°C, respectively), and post-hoc tests revealed a significant difference between populations at 5°C (Figure 5.4, Panel A). For COX activity measured in killifish muscle, the two-way A N O V A revealed no significant differences with acclimation or between populations (Figure 5.4, Panel B). For liver citrate synthase (CS) activity (Units per mg protein), two-way A N O V A detected a significant effect of acclimation temperature and population with no interaction. Post-hoc tests revealed that both populations increased CS activity with cold acclimation. Northern killifish had significantly higher CS activity compared to southern fish, and this difference was detected as significant in post-hoc tests in fish acclimated to 5°C (Figure 5.4, Panel C). Citrate synthase activity in muscle showed a significant effect of acclimation temperature and population with no interaction. Post-hoc tests revealed that 5°C-acclimated northern fish had CS activities that were significantly higher than activities in fish acclimated at 15 and 25°C, and 5°C acclimated northern fish also had significantly higher CS activity than all three southern groups. There was no effect of acclimation temperature on CS activity levels in southern fish (Figure 5.4, Panel D). 180 VARIA TION IN GENE EXPRESSION A two-way A N O V A with acclimation temperature and population of origin for COXII mRNA expression in liver revealed a significant effect of acclimation temperature and no significant effect of population or a significant interaction. Liver COXII mRNA levels were significantly higher in 5°C-acclimated northern and southern fish relative to 15- and 25°C-acclimated fish (Table 5.2). In muscle, there was a significant effect of population such that northern fish had higher COXII mRNA levels at all acclimation temperatures, and northern fish had significantly higher (~2 fold) muscle COXII mRNA than southern fish at 5°C acclimation temperatures. Two-way A N O V A for liver CS mRNA levels showed a significant effect of acclimation temperature and population such that northern fish had significantly higher CS mRNA expression (2-3 fold) than southern fish at all three acclimation temperatures. Secondly, northern fish increased liver CS mRNA expression with decreasing acclimation temperature, and this increase was detected as significant in post-hoc tests in northern fish acclimated to 5°C relative to 15 and 25°C acclimated fish. In muscle, northern fish had overall higher mRNA expression levels for CS than southern fish, and post-hoc tests revealed significant differences between populations at 5 and 25°C acclimation temperatures. Northern fish had significantly higher mRNA expression in muscle CS at 5°C acclimation relative to 15 and 25°C acclimation groups. The results of the two-way A N O V A indicated that muscle CS mRNA levels in southern fish increased with decreasing acclimation temperatures, although these differences were not detected as statistically significant in post-hoc tests. Two-way A N O V A for liver ATPSyn mRNA expression revealed no significant effect of population or acclimation temperature, but there was a significant interaction. Post-hoc tests revealed that northern fish had greater ATPSyn mRNA expression at 25°C relative to 5 and 15°C 181 acclimation groups. In contrast, southern fish liver ATPSyn mRNA expression increased with decreasing acclimation temperature and this increase was significant at 5°C. Populations differed at 5 and at 25°C acclimation such that ATPSyn mRNA expression was significantly higher at 5°C and lower at 25°C in southern fish. In muscle, two-way A N O V A for ATPSyn mRNA expression revealed a significant effect of population with northern fish having higher ATPSyn mRNA levels at all acclimation temperatures and post-hoc tests showed a statistically significant difference between populations at the 15°C acclimation temperature. DISCUSSION We have shown that both mitochondrial function and content change with thermal acclimation in killifish, and that these responses differ between populations, suggesting the possibility of evolutionary modulation in these complementary mechanisms of enhancing oxidative capacity in the face of temperature challenges. We have also shown that cold acclimation temperatures dramatically alter the response of northern and southern killifish mitochondria to acute temperature challenge, while warm-acclimated mitochondria have very similar function between killifish populations. A CCLIMA TION AFFECTS MITOCHONDRIAL CONTENT Acclimation to cold temperatures has been shown to result in increases in mitochondrial content and cristae surface area in a variety offish species in both muscle and liver tissue (Johnston et al., 1998, Pdrtner et al., 1998; Guderley, 2004). Our data for citrate synthase activity in liver suggest that both killifish populations increase CS activity in the cold, but northern fish do so to a greater extent than southern fish (Figure 5.4). In muscle, northern fish again upregulated CS activity with cold acclimation whereas CS levels in southern fish remained 182 unchanged with temperature acclimation and were significantly lower than those of northern fish acclimated to 5°C. Cytochrome c oxidase activity showed no change with acclimation temperature in either muscle or liver in both killifish populations. However, in liver, northern fish had higher COX levels than southern fish at all acclimation temperatures (Figure 5.4). Taken together, these data suggest that the modulation of aerobic capacity differs between killifish populations as well as between tissue types, with northern fish having a greater ability to modulate aerobic capacity during cold acclimation relative to southern fish, which is consistent with the predictions of OLTT. Population specific differences in the activity levels of CS and COX have been seen in other fish species. In muscle of cod, upregulation of CS and COX activity was observed with cold acclimation, and cod populations experiencing colder environmental temperatures had higher enzyme activity levels than populations experiencing more moderate temperatures (Lannig et al., 2003; Lucassen et al., 2006). In cod liver, however, CS was upregulated with cold acclimation, but no change was seen in COX suggesting that in liver, the relationships between matrix enzymes and membrane bound components may be different than those in muscle (Lannig et al, 2003; Lucassen et al., 2006). Similarly, Lucassen et al. (2003) showed an increase in CS activity but no change to COX activity in the liver of eelpout during a time-course of cold acclimation. Our results for liver and muscle CS activity and liver COX activity are in general agreement with these studies and suggest that cold-adapted northern killifish populations increase enzyme capacities in the cold to a greater degree than warmer-adapted southern populations. We did not, however, see evidence for any changes in muscle COX activity with acclimation temperature or population. 183 RELA TIONSHIP BETWEEN MRNA AND PROTEIN LEVELS The nature of the relationship between mRNA levels and mitochondrial content is currently unclear from the available literature making predictions for this relationship difficult in the context of the OLTT. To gain a better understanding of the sites and levels of regulation of mitochondrial proliferation and differentiation between killifish populations, we compared mRNA levels to enzyme activities for a number of different mitochondrial proteins. Citrate synthase enzyme activity values in killifish liver and muscle were loosely correlated to mRNA levels, and both tended to increase following long-term acclimation to cold in both killifish populations. Northern fish had higher CS mRNA expression in liver and muscle than southern fish, and this was consistent with the significantly higher enzyme activity values of northern fish at the coldest acclimation temperature. Similarly, Lucassen et al. (2003) showed a loose relationship in citrate synthase activity and mRNA expression with cold acclimation in liver of North Sea eelpout. In contrast, more recent data from cod populations have shown that CS activity and mRNA levels are well correlated in muscle (Lucassen et al., 2006) suggesting transcriptional control of enzyme activity of this gene, but no such strong correlation was seen in cod liver. Work in the tunas and billfish, however, suggests that differences in CS content in muscle are largely due to post-transcriptional regulation rather than mRNA transcript abundance (Dalziel et al., 2005). Taken together these data suggest that the relationship between CS mRNA levels and enzyme activities is unpredictable, and may vary among species, tissues, or acclimation conditions. Cytochrome c oxidase activity and COXII mRNA levels were not well correlated in either liver or muscle in either killifish population. This may not be surprising as a number of complicating factors influence COX regulation. COX is made up of 13 subunits (3 composing the catalytic core and encoded by the mitochondria, and 10 nuclear encoded subunits involved in subunit assembly and regulation), and COX assembly requires several accessory proteins and 184 chaperones (Capaldi, 1990). COX is regulated allosterically by ATP and numerous isoforms of the nuclear encoded C O X subunits are present (Kadenbach et al , 1997). In trout muscle, COX activity has been shown to increase in cold acclimation with no increase in C6lY7mRNA and several other messengers of mitochondrial encoded genes (Battersby and Moyes, 1998). Hardewig et al. (1999) showed that COXI, COXII, and COXIVmRNA levels were overcompensated relative to the enzyme capacities in muscle and liver of eelpout from the North Sea, whereas a strong correlation in muscle and liver enzyme activities and COXmRNAs was found for Antarctic eelpout although the overall levels were significantly lower in this cold-adapted species compared to the related North Sea species. Lucassen et al., (2006) did not find a strong correlation between COX activity and COXII mRNA expression with temperature acclimation. While COXII mRNA abundance may not be a good correlate of COX activity, it may be a more useful measure of the transcription rate of the mitochondrial genome as the entire mitochondrial genome is transcribed together (Fernandez-Silva et al, 2007). Our results show that both northern and southern fish have significantly higher liver COXII mRNA expression at cold acclimation temperatures suggesting higher transcription rates of mitochondrial genes in these groups. In muscle, however, we saw significantly higher COXII expression in northern fish, but no effect of thermal acclimation. A CCLIMA TION AFFECTS MITOCHONDRIAL FUNCTION Our data show that mitochondria from cold-acclimated (5 and 15°C) northern and southern killifish populations have significantly higher State III rates than mitochondria from warm-acclimated fish when assayed at low temperatures (2 and 5°C) (Figure 5.1). This is consistent with the first prediction of OLTT, which suggests that mitochondrial function should increase with low temperature acclimation. As well, cold-acclimated northern mitochondria perform better than southern mitochondria at cold assay temperatures (Figure 5.1), except in the 185 15°C acclimation group. Therefore, the second OLTT prediction that cold adapted organisms should have higher mitochondrial function than warm adapted organisms is upheld when mitochondria are assayed in the cold. Similar increases in State III rates with cold acclimation have been seen in muscle mitochondria from shorthorn sculpins (Guderley and Johnston, 1996), rainbow trout (Guderley et al., 1997; Bouchard and Guderley, 2003; Kraffe et al., 2007), and lugworms (Sommer and Portner, 2004), but to our knowledge, this is the first demonstration of intraspecific variation in State III rates in fishes with cold acclimation. Acclimation to warmer temperatures, however, did not result in any differences in maximum oxidation rates at warm assay temperatures of 35 or 37°C. Al l acclimation groups and both populations achieved similar State III rates at these warm assay temperatures. These observations are not consistent with the third pre'diction of the OLTT hypothesis, which suggests that the increase in mitochondrial function in the cold (as a result of either acclimation or adaptation) should come at a cost of increased mitochondrial respiration in response to acute warming. Warm acclimation did, however, result in a shift in the upper thermal sensitivity of mitochondria to acute temperature challenge such that mitochondria from northern and southern fish acclimated to 15 and 25°C remained responsive to ADP at assay temperatures of 37°C (Figure 5.1, Panels 2 and 3) whereas 5°C acclimated mitochondria could not be measured at temperatures greater than 35°C (Figure 5.1, Panel A). The most dramatic differences we observed in mitochondrial function between killifish populations were in the response of maximum oxidation rates (State III) to acute temperature challenge. As a result, mitochondrial acute performance curves differed greatly in shape both between populations and acclimation temperature treatments (Figure 5.1). The maximum oxidation rates of southern fish increased roughly linearly with increasing assay temperatures in 15 and 25°C acclimation groups and only had a slightly less than linear relationship in the 5°C 186 acclimated mitochondria (Qio's ranging from 2-2.5 for all three acclimation groups). In contrast, the State III acute performance curves differed greatly in shape for northern fish at each acclimation temperature. At the coldest acclimation temperature of this study (5°C), northern killifish demonstrated a significantly higher maximum State III rate when assayed at cold temperatures (Figure 5.1), but dramatically lower rates when acutely challenged with warm assay temperatures relative to southern fish (Figure 5.1, Panel A). This depression in State III rates in cold-acclimated mitochondria from northern fish assayed at warmer temperatures resulted in a large thermally insensitive zone of mitochondrial respiration (Qi 0=l.l, 20-30°C assay temperatures) whereas southern mitochondria increased State III rates with increasing assay temperature across this range (Qio=2.1, 20-30°C assay temperatures). Acclimation to 15°C revealed a similar, but less pronounced plateau in State III rates in northern fish relative to southern fish (Figure 5.1, Panel B). Mitochondria from 25°C acclimated fish, however, did not differ in capacity between populations, and both populations demonstrated similar Qio's of 2.2 (north) and 2.5 (south) across the entire assay temperature range (Figure 5.1, Panel C). While there are no data sets for a single fish species acclimated to several temperatures and tested across such a broad range of assay temperatures, Johnston et al. (1998) tested the maximum oxidative capacities of mitochondria from multiple fish species sampled from very different thermal environments and found Qio's ranging from 1.8-2.7 for the species and temperature ranges tested. While our overall Q j 0 values calculated across the entire range of assay temperatures are similar to those described by Johnston et al. (1998), our data show intraspecific variation as well as acclimation-dependent patterns in the shape of mitochondrial acute response curves. In contrast to the non-linear patterns of thermal sensitivity of mitochondria to acute temperature challenge described in this study, essentially linear increases in State III rates with increasing assay temperatures were demonstrated in a study of marine lugworms (Sommer and Pdrtner, 2004) that compared State III oxidation rates from three 187 acclimation temperature groups and across a full range of acute assay temperatures in two populations. These data showed that State III rates increased with increasing assay temperatures in all acclimation groups. As well, mitochondria from cold-acclimated worms always had higher State III rates than the warm acclimation groups at all assay temperatures tested (0-32°C) resulting in State III acute performance lines that remained parallel to one another across all assay temperatures. These data did not show any evidence that State III performance at warm assay temperatures converged between acclimation groups or between populations, nor did the upper performance temperature shift with thermal acclimation. Intraspecific variation in State III rates were shown such that the cold-adapted lugworm population had higher State III rates at all assay temperatures than the worms from warmer environments when acclimated to a common temperature and assayed across the entire thermal assay range (Sommer and Pdrtner, 2004). Studies comparing acute effects of temperature on mitochondrial respiration with thermal acclimation and adaptation are rare, and to our knowledge the only other comparable study was conducted on various species of abalone (Dahlhoff and Somero, 1993), but only the Arrhenius break temperatures, and not the shape of the acute mitochondrial performance curves were reported. As well, this study utilized uncoupled respiration rates and made interspecific rather than an intraspecific comparisons. While the lugworm data set as well as what we have shown in killifish suggest that mitochondrial function is upregulated in populations of cold adapted eurytherms, our data for mitochondrial performance at intermediate acute challenge temperatures showed dramatically different patterns than what was shown in lugworms. Our data are the first demonstration of intraspecific variation in the modulation of mitochondrial function in response to thermal acclimation and acute temperature challenge, suggesting that there is an underappreciated complexity in the biochemical responses of the mitochondria to thermal acclimation and acute exposure. 188 PROTON LEAK (IVOL) Acclimation temperature had no significant effect on State I V 0 i rates, and State I V 0 i rates did not differ between populations except for 25°C acclimated southern mitochondria assayed at 35 and 37°C, which had significantly higher leak rates than northern mitochondria (Figure 5.3). This finding was surprising because previous studies have shown that State I V as well as State I V o i both increase with cold acclimation compared to warm-acclimated organisms assayed at a common temperature (Guderley et al, 1997; Bouchard and Guderley, 2003; Sommer and Pdrtner, 2004). Increases in proton leak go hand in hand with the increases seen in State III rates with cold acclimation in these species. Proton leak decreases the potential across the inner mitochondrial membrane and leads to oxygen uptake without ATP synthesis, thus a considerable proportion of standard metabolic rate is devoted to proton leak (Rolfe and Brand, 1996; Rolfe et al., 1999). An increase in proton leak (as well as in State III) with cold acclimation is thought to be adaptive because it limits ROS production (Skulachev, 1998). Increased leak, however, is one of the features thought to lead to the loss in aerobic scope at a lower temperature with warming in cold-acclimated organisms relative to warm-acclimated organisms (Pdrtner, 2001; 2002). Our data suggest that killifish from both populations maintain very similar proton leak rates at all acclimation temperatures. Our analysis did, however, reveal a minor shift in the inflection point of the State I V 0 i respiration to warmer assay temperatures with increasing acclimation temperature (Figure 5.3), which might be related to changes in mitochondrial membrane unsaturation with thermal acclimation (Brand et al., 1991). PA TTERNS OF A CCLIMA TION AND ADAPTA TION IN EUR YTHERMS There is considerable debate surrounding whether or not cold-adapted eurytherms and cold-adapted stenotherms modulate their overall mitochondrial capacities in the same ways (Pdrtner, 2002; Guderley, 2004). Eurytherms are faced with the challenge of maintaining 189 activity across a broad temperature range. In contrast, stenotherms living in the permanent cold are faced with the challenge of ATP production in the cold, but have a much more limited temperature range over which they must perform. The consensus in the literature is that eurythermal fish species show higher activity levels, increased metabolic rates, as well as increased metabolic scopes relative to stenothermal species (reviewed in Pdrtner, 2002; Pdrtner et al., 2007). These data have been used to argue for a distinction between the strategies for adaptation to the cold in eurytherms compared to stenotherms at the level of mitochondrial proliferation and function (Pdrtner et al., 2000; 2002; Pdrtner, 2004). Mitochondrial proliferation has occurred during adaptation for life at permanently cold temperatures, particularly in the oxidative muscles of demersal, moderately active stenothermal fish species (Johnston et al., 1998). Increases in mitochondrial content have also been shown to occur in eurythermal species in response to cold acclimation, but to a lesser degree compared to content adjustments seen in stenotherms (Johnston et al., 1998; Pdrtner, 2002). One argument for why this mitochondrial proliferation has occurred at low temperatures is for the facilitation of intracellular distribution of oxygen and metabolites (Egginton and Sidell, 1989; Sidell, 1998). It has also been suggested that these increases in mitochondrial content (at least in muscle) in response to the cold are less dramatic in eurytherms relative to stenotherms because this increase in content could compromise contractile force production and associated activity by sacrificing cellular space to accommodate more mitochondria. At the level of mitochondrial function, however, studies comparing species from different thermal habitats have shown no evidence for enhanced maximum oxidation rates in cold-adapted fish species (Johnston et al, 1998). While these studies have been mostly conducted on stenotherms, it is thought that oxidative capacity is not enhanced over evolutionary time scales because of the costs associated with maintaining highly active mitochondria (e.g. increased baseline metabolism, proton leak, and ROS production) (reviewed in Pdrtner, 2004; Guderley, 190 2004). It is thought that mitochondria with low oxidative capacities, but that are distributed across a large proportion of the cell can provide a compromise between the benefits of an extensive mitochondrial network for oxygen and metabolite uptake, the negative impacts of highly active mitochondria, coming at a cost of the sacrifice of intracellular space potentially compromising contraction. It is possible, however, that eurytherms and stenotherms may make different mitochondrial function and content compromises such that eurytherms may adopt a strategy to increase ATP synthesis capacities by the mitochondria so as not to solely depend on increases in mitochondrial content, which could limit muscle contraction and activity. Stenotherms may make little to no mitochondrial functional adjustments, but instead increase mitochondrial densities as they can afford to sacrifice cellular space because of their less active lifestyles. Our data suggest a role for intraspecific modulation in both mitochondrial content and function in a northern hemisphere, eurythermal fish species in response to temperature acclimation and adaptation. IMPL1CA TIONS FOR WHOLE-ORGANISM PERFORMANCE Unifying principles relating the mechanisms of aerobic capacity modulation to seasonal acclimatization and latitudinal thermal adaptation are still elusive. The data presented here suggest that northern killifish demonstrate enhanced oxidative capacity (i.e. upregulating mitochondrial content and function) relative to southern killifish when acclimated to and assayed at cold temperatures. With warming, however, mitochondria from cold-acclimated northern killifish have lower State III rates relative to those from southern fish. We suggest that this strategy in northern killifish may represent a mechanism to avoid the deleterious affects of high mitochondrial oxidative capacity with warming temperatures, which could reduce whole-organism thermal tolerance. Linking isolated mitochondrial function and content measures to whole organism function, however, is difficult. Quantitative predictions of maximum oxygen 191 consumption rates in vivo by the mitochondria are complicated by the influences of many factors including intracellular pH (Moyes et al, 1988), availability and affinity for ADP and N A D H (Brand and Murphy, 1987; Guderley and St. Pierre, 1999), membrane properties (Kraffe et al., 2007) and delivery of oxygen and fuels by the circulation (Mathieu-Costello et al., 1992). It is possible that the oxidative performance of killifish mitochondria is regulated at one or several of these levels, but this remains to be tested. However, our data clearly suggest the use of contrasting strategies between populations and between acclimation temperatures, suggesting that mitochondrial function and content modulation may differ between populations of eurythermal killifish, and adaptive patterns in the modulation of aerobic capacity may be more complex than previously thought. 192 REFERENCES Adams, S.M., Lindmeier, J.B., and D D : Duvernell. 2006. 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Journal of Experimental Biology. 203: 3355-3368. Whitehead, A. and D.L. Crawford. 2006. Neutral and adaptive variation in gene expression. Proceedings of the National Academy of Sciences. 103: 5425-5430. Yeager, D.P, and G.R. Ultsch. 1989. Physiological regulation and conformation: A BASIC program for the determination of critical points. Physiological Zoology. 62: 888-907. 197 Figure 5.1 (following page) Temperature dependence of State III (pmol O mg"1 mitochondrial protein min"1) respiration rates, determined in mitochondria from 5°C (panel A), 15°C (Panel B) and 25 °C (Panel C) acclimated northern (filled triangles) and southern (open circles) killifish. Significant differences within a population and acclimation temperature are indicated with different letters. An asterisk (*) indicates a significant difference between populations within an acclimation temperature for a given assay temperature. A § indicates a significant difference between the 25 °C acclimation group and the 5 and 15°C acclimation groups when assayed at the same temperature (indicated on 25°C acclimation data). Data are expressed as mean + SE (n = 4-6), and P < 0.05 for all significant comparisons. 198 0 1800 -. o d) u> c 1600 -'a> 2 1400 -C L rial 1200 --a c o .c 1000 -o o E 800 -CO E 600 -O o 400 -E 200 -0 0 -s C ~-1800 o <U 1600 -c B 2 1400 -Q . rial 1200 -T3 C o .c 1000 -o o 1 800 -E 600 -O o 400 -E Q . 200 -£ 0 -3 w 10 15 20 25 30 35 40 Assay Temperature f C ) B 15°C Acclimation e e T - - A r - North - • e - South C 25°C Acclimation — North - - 6 - South * § * § rs r r ab a a 5 l 10 rs ab st be 4 de 10 15 20 25 30 35 40 Assay temperature (°C) Assay temperature (°C) Figure 5.2 (following page) State III (pmol O mg"1 mitochondrial protein min"1) respiration rates determined in mitochondria from northern and southern killifish acclimated to 5 (black bars), 15 (light grey bars), and 25°C (dark grey bars) and assayed at 5°C (panel A), 15°C (Panel B) and 25°C (Panel C). Significant differences within a population at a given assay temperature are indicated with different letters. An asterisk (*) indicates a significant difference between populations within an acclimation temperature. Data are expressed as mean ± SE (n = 4-6), and P < 0.05 for all significant comparisons. 200 250 • S 200 A o 150 A 100 E O o E 500 • 300 200 E O Q, 100 • B S 400 A-Assayed at 5°C * mm 5°C acc a 15°Cacc a 25°C acc North South Population Assayed at 15°C mm 5°C acc 15°C acc 25°C acc North South Population 1200 • E O o E 400 • ~ 200 • C Assayed at 25°C mm 5°C acc 15°C acc 25°C acc * a a North South Population 201 Figure 5.3 (following page) Temperature dependence of State rv 0i (pmol O mg"1 mitochondrial protein min"1) respiration rates, determined in mitochondria from 5°C (panel A), 15°C (Panel B) and 25°C (Panel C) acclimated northern (filled triangles) and southern (open circles) killifish. Significant differences within a population and acclimation temperature are indicated with different letters. An asterisk (*) indicates a significant difference between the 25°C acclimation group and the 5 and 15°C acclimation groups when assayed at the same temperature (indicated on 25°C data). An arrow (i) indicates the temperature of inflection (the assay temperature where the largest change in slope was observed) within each acclimation temperature group. Data are expressed as mean ± SE (n = 4-6), and P < 0.05 for all significant comparisons. 202 300 C 250 £ 2: 200 A 5°C Acclimation - A — North - - e - South E O 150 A 100 • 50 • 10 15 20 25 30 35 Assay Temperature (°C) 40 300 • 250 • £ o Q. ro • D c o - C o o 'I 'c» E 2 50-o E Q. 200 • 150 A 100 • OH B 15°C Acclimation — A — North - -e- South 10 15 20 25 30 35 Assay temperature (°C) 40 300 • £ o Q. ro •o c o Ui E 25°C Acclimation 250 200 A 150 A 100 A _ 50 • o E Q. 0 1 - ± — North - •e- South 10 15 20 25 30 35 Assay Temperature (°C) 40 203 Liver Acclimation Temperature (°C) ro E 3 s i > o 00 O Liver 0.07 0.06 0.05 0.04 -0.03 • 0.02 -0.01 • 0 a x b xy • North H South b y 15 25 Acclimation Temperature (°C) B Muscle Acclimation Temperature (°C) D Muscle 0.07 0.06 -0.05 -_ „ 0.04 -£ o 0.03 < a 0.02 « 0.01 0 E 3 i f • North 0 South b x b x 1 I P 1 s, 15 25 Acclimation Temperature (°C) Figure 5.4 Cytochrome c oxidase (COX) and citrate synthase (CS) activity in northern (open bars) and southern (hatched bars) killifish acclimated to 5, 15, or 25°C. Results for liver are given in panels A and C, and for muscle in panels B and D. Significant differences within a population between acclimation temperatures are indicated with different letters. An asterisk (*) indicates a significant difference between populations at a given acclimation temperature (indicated on northern bars). Data are expressed as mean ± SE (n = 6-8), and P < 0.05 for all significant comparisons. 204 Table 5.1 Primers used for qRT-PCR. Gene Sequence (5'-3') CS F:CGG CAT GAC GGA GAT GAA CT R:GAG GGC CCG GGA CAC A CoxII F:AGT TTA GGA ATC AAA ATA GAC GCA GTT R:CGG GAG GTA ATG AAG GCT GTT ATPSyn F:TGG TGC CCC TCA AGG AAA R: TCA TAC TCG CCT CCC AGG AT qRT-PCR, quantitative real-time PCR; F, forward; R, reverse Table 5.2 Cytochrome c oxidase, subunit two (COXII), citrate synthase (CS), and ATP synthase /3-chain (ATPSyn) mRNA levels (expressed relative to total RNA). Gene Pop 5°C 15°C 25°C mean ± SE mean ± SE mean ± SE COXII (liver) N 0.80" 0.06 0.61b 0.05 0.57b 0.05 S 0.9X 0.09 0.56y 0.05 0.66y 0.06 COXII (muscle) N 1.19a* 0.22 0.70b 0.11 1.00ab 0.15 S 0.65" 0.09 0.51x 0.07 0.71x 0.08 CS (liver) N 1.89a* 0.35 1.09b'* 0.15 1.25b'* 0.09 S 0.85" 0.14 0.43x 0.11 0.35x 0.06 CS (muscle) N 0.84a'* 0.07 0.39b 0.06 0.52b'* 0.09 S 0.26" 0.05 0.21x 0.06 0.16x 0.02 ATPSyn (liver) N 0.72a'* 0.05 0.71a 0.05 0.93b'* 0.03 S 1.04x 0.11 0.76y 0.04 0.70y 0.04 ATPSyn (muscle) N 0.82" 0.10 0.693'* 0.06 0.753 0.09 S 0.64x 0.05 0.49x 0.06 0.66x 0.05 Different letters indicate a significant difference between temperature acclimation groups within a population. An asterisk (*) indicates a significant difference between populations at a given acclimation temperature. * indicated on northern values. 206 C H A P T E R S I X : G E N E R A L D I S C U S S I O N A N D C O N C L U S I O N S OVERVIEW The goal of the research presented in this thesis was to use killifish as a model to study mechanisms of local thermal adaptation and acclimation. Overall, my work has allowed me to assess the thermal niche of killifish and to investigate candidate mechanisms involved in defining this niche. Figure 6.1 graphically demonstrates some of the candidate mechanisms, from molecule to organism, that respond to thermal heterogeneity mapped onto each of my thesis chapters. An additional layer of complexity involves the temporal scales over which these mechanistic adjustments occur, which is shown on the x-axis of Figure 6.1. Adjustments to any one or several of these mechanisms across various time scales can alter the functional performance of the organism. © Q o Organism. Tissue Celt T£J Organelle Molecule Killifish Intraspecific Variation Behavior 7fter/^tofe>^<r^&preference, swimperformance. SecondsrMinutes Organ/System function: Metabolic fuels 'Muscle-Physiology Mitochondrial junction Enzyme activity Gene sequence^..expression . Heritable" variation Ch. 2,3, & 5 ^ Ch. 2 Ch. 4 Ch.3 & 4 Hours-Days,' Timescale Weeks-Years Figure 6.1 The interplay between timescale, physiological and biochemical mechanisms, and biological levels of organization with killifish performance (modified from Angilletta et al., 2006). The types of measures made in this thesis are italicized and the arrows indicate the thesis chapters utilizing each of these measures. 207 SUMMARY OF SALIENT FINDINGS As discussed in Chapter One, organisms occupy a thermal niche, and this niche can be altered by both thermal acclimation and adaptation. In this thesis, I have quantified the thermal performance niche optimum, niche limits, and niche shape in killifish. Figure 6.2, below, illustrates a thermal reaction norm, showing the critical temperature thresholds identified in the oxygen-limited thermal tolerance framework (Portner, 2001; 2002), as this conceptual framework will be the basis for the integrated discussion of my thesis chapter findings. The pejus temperatures (Tp) are the upper and lower temperatures where performance declines, the critical temperatures (Tc) are thought to represent the temperatures where organismal thermal tolerance becomes time-limited and dependent on the organism's ability to use anaerobic metabolism and to upregulate protective mechanisms such as antioxidants and heat shock proteins, and the failure temperatures (Tf) are the organismal death temperatures (modified from Pdrtner et al., 2007). o u Temperature Figure 6.2 Conceptual scheme for critical temperatures at which an organism's performance is altered. In the context of the OLTT hypothesis, T p = temperatures where performance declines, T c = temperatures of time-limited tolerance, and Tf = temperatures of whole organism failure (modified from Portner et al., 2007). 208 LOCATION OF THE THERMAL OPTIMUM As an integrated measure of whole-organism thermal optimum, I assessed the thermal preference (Chapter Three) of northern and southern killifish with the prediction that the more thermally tolerant southern killifish (Chapter Two) would prefer warmer temperatures than northern fish. In contrast to this prediction, I showed that northern killifish had a modal final thermal preferenda that was significantly higher than that of southern fish (T°C f i n a i = 30.6 versus 29.0°C, respectively). These data were inconsistent with my original predictions, but instead suggest countergradient variation in thermal preference in killifish populations. Countergradient variation in high-latitude populations has been suggested to evolve as a compensatory response to a shorter growing season (Conover and Schultz, 1995; Yamahira and Conover, 2002; Freidenburg and Skelly, 2004). Northern fish may select higher temperatures than might be expected based on their cooler northern habitat as a strategy to maximize their metabolic rates when given the opportunity to exploit warm waters. An increase in metabolic rate could translate into a rise in activity, feeding efficiency, and growth rates to compensate for the shorter growing season in the north, if these temperatures do not exceed the maximum tolerated temperatures. For example, northern killifish have been shown to occupy warm pools for the winter rather than cooler creeks typically occupied in the summer (Smith and Able, 1994) and these authors suggested that one explanation for this behavior was to maximize metabolic rates in the winter. Cold-acclimated northern fish do not, however, appear to avoid potentially lethal warm temperatures (demonstrating a phenomenon termed low thermal responsiveness; Chapter Three). The opportunity to select warm temperatures approaching the lethal limits of northern killifish may be quite rare in northern habitats in winter. Indeed, water temperatures above 10°C are rarely recorded in most northern habitats in the winter months (NOAA NERRS Data). In contrast to northern fish, cold-acclimated southern fish do not immediately pick lethal temperatures suggesting a well-developed avoidance response for upper lethal temperatures 209 (Chapter Three), which are certainly available in southern habitats. As well, persistent periods where water temperatures remain near 5°C for more than a day or two are rare in southern habitats potentially decreasing the need to maximize metabolic rates over these very short timescales. Over an entire growing season in southern habitats, it may not be possible to support a metabolism dictated by the selection of the highest water temperatures available as energetic resources may be limiting over such long time scales. Countergradient variation in early development and metabolic rate has been shown in killifish populations lending support for this phenomenon in killifish (Schultz et al., 1996). To my knowledge, my finding of countergradient variation in temperature preference is the first demonstration of this phenomenon in fishes. QUANTIFICA TION OF THERMAL NlCHE LIMITS In order to assess the critical temperatures (T c, see Figure 6.2) of killifish, I quantified the upper and lower chronic thermal maxima and minima as well as CTMax and CTMin (Chapter Two). Chronic thermal maximum (i.e. the theoretical maximal acclimation temperature) was 1.8°C greater in southern than in northern killifish, but there were no differences in chronic minima (Chapter Two). As well, northern and southern killifish differed in their thermal tolerance limits (as assessed by CTM) with southern fish having higher critical thermal maxima than northern fish by ~1.5°C at each acclimation temperature (from 2-34°C) although the difference was slightly larger at low acclimation temperatures (~2.2°C) than at warm acclimation temperatures (~1.0°C). Critical thermal minima were significantly lower in northern killifish by ~1.5°C at acclimation temperatures above 22°C, but the CTMin of the two populations converged at freezing point of brackish water at acclimation temperatures below 22°C. These data are consistent with predictions that local thermal adaptation to the warmer temperatures found in southern killifish habitats would result in these populations showing higher thermal tolerance limits. 210 Critical thermal limits in northern and southern killifish populations are plastic (Chapter Two). Thermal acclimation resulted in changes to the C T M limits in both populations by more than 10°C across their entire thermal acclimation range. Despite this plasticity, however, differences in thermal tolerance between killifish populations persisted, and were highly repeatable among multiple northern and southern killifish populations across collection years. This impressive plasticity in acclimation as well as the repeatable differences in thermal tolerance between killifish populations within a species makes F. heteroclitus an attractive model for studying the mechanistic basis of thermal acclimation as well as thermal adaptation. For example, in this system, it is possible to address whether the processes involved in thermal acclimation are similar or different to those involved in thermal adaptation. The results presented in this thesis also provide some insight into 'pejus' temperatures (Tp= temperatures where performance declines, see Figure 6.2) in killifish. When killifish are acclimated to 34°C and swim performance is tested at this temperature, U c r i t values are equivalent to those obtained from fish from all thermal acclimation groups between 7 and 34°C suggesting that Ucnt is independent of acclimation temperature across this wide temperature range (Chapter Four). Indeed, U c r i t values for northern and southern killifish acclimated to 18°C and acutely plunged to 18, 25, and 35°C vary little across this range of acute plunge temperatures and do not differ between populations at 25 and 35°C. Taken together, these data suggest that thermal acclimation does not improve performance at 34°C, and that U c r i t performance for both northern and southern killifish is thermally insensitive across a broad temperature range. As well, these data suggest that killifish can maintain aerobic scope at swimming temperatures of at least 35°C as there is no decline in swimming performance, suggesting that the upper T p is greater than 35°C in northern and southern killifish. Preliminary acute thermal challenge swim trials using 18°C acclimated killifish were initially attempted at 36°C, and these trials showed that killifish could not consistently swim 211 when acutely challenged with this temperature (Fangue, pers obs). Unfortunately, the sample size was limited and collected without particular attention to population. It is clear, however, that both northern and southern fish swim to maximal U c rit values at 35°C and failed at temperatures near 36°C. 35°C, however, closely approaches the critical temperatures (Tc) for killifish. Using the regression equations reported in Chapter Two, CTMax values for 18°C acclimated fish would be 36.7°C for northern fish and 38.0°C for southern fish. Therefore, killifish are able to perform maximally at temperatures only 2-3°C lower than their CTMax values. Taken together, these data suggest that the T p for both killifish populations (acclimated to 18-34°C) is somewhere between 35 and 36°C, and that T p and T c are separated by no more than 3°C. Because killifish differ in their thermal niche limits (defined by CTM) such that southern fish are more heat tolerant than northern fish, the finding that northern killifish selected a higher final thermal preferenda than southern fish (T°C f m a i = 29.0°C (south) to 30.6°C (north)) was initially surprising (Chapter Three). However, when I compared the final thermal preferenda of ~30°C to the performance of killifish acclimated to 30°C, my data show that 30°C is well within the thermal acclimation limits for both northern and southern killifish (Chapter Two), and is also well within the optimal temperature range for swimming performance (Ucrit) for both populations (Chapter Four). In fact, my swim performance data do not give any indication that swim performance declined at temperatures of 30°C in either killifish population or under acclimatory or acute thermal plunge conditions. These data suggest that for both northern and southern killifish, temperatures near 30°C do not inhibit physiological performance for the traits measured in this thesis. At swimming temperatures below 7°C, both the acclimation and acute thermal challenge data sets (Chapter Four) suggest that swim performance declines in both killifish populations, with UCrit values at 5°C approximately half those of the values found at swimming temperatures 212 between 7 and 35°C. At an acclimation temperature of 18°C, the calculated CTMin for northern fish is 0.2°C and for southern fish is 1.2°C suggesting that the window in the cold between T p and T c (estimated to be ~5-7°C) is substantially larger than that in the warm. This is not surprising, however, as performance in the warm often plateaus across several temperatures before drastically dropping, whereas a much more gradual decline in performance is often seen with decreasing temperatures. Asymmetric performance curves are typical for many performance measures including mitochondrial oxidation rates and proton leak, cardiac and ventilatory outputs, and whole organism rates of aerobic performance (Farrell et al., 1997; Angilletta et al., 2002; Portner et al., 2005). Very careful measurements, however, at the extreme low and high swimming temperatures would be necessary to fully describe the relationship between performance failure and critical thermal tolerance. As well, measurements of aerobic scope under thermal acclimation and acute thermal challenge conditions would be invaluable to help elucidate critical transition temperatures defining northern and southern killifish performance limits. The quantitative temperature difference between T p and T c in the warm (~3°C) versus the cold (~5-7°C) could have important implications for killifish in their natural environments. Setting the maximum or optimum performance temperature very close to the upper thermal limit may allow killifish to maximize metabolic rates and thereby enjoy maximum growth and reproductive rates in nature. A number of terrestrial and aquatic ectotherms have been shown to behaviorally select temperatures that satisfy these criteria (reviewed in Angilletta, 2002; Angilletta et al., 2006). The countergradient arguments previously discussed also support the selection of warm temperatures when available for the maximization of physiological performance. As well, the ability of killifish to maintain high levels of activity at very warm temperatures may give killifish a performance advantage allowing them to exploit thermal microhabitats that are potentially more thermally stressful for other competing, less tolerant, 213 organisms. In the cold, however, reductions in performance and metabolic rate for winter dormancy strategies might be more important than maximizing physiological rates in the winter. Allowing your metabolic rate to fall during times when productivity in the environment is low may be the most appropriate strategy. There is some suggestion in my data that northern and southern fish may take a slightly different approach to cold-acclimation. Based on resting metabolic rate measures (Appendix A; Fangue et al., unpublished), northern fish acclimated to 5°C have a 1.7 fold higher metabolic rate, more mitochondria (Chapter Five), and higher functioning mitochondria in the cold (Chapter Five) relative to southern fish, suggesting the retention of higher activity in northern fish that could translate into higher physiological performance in the extreme cold. Taken as a whole, my thesis data suggest that northern and southern fish are capable of performing very similarly at warm temperatures, but that northern fish may have enhanced performance in the cold resulting in an overall larger functional temperature range. This is in contrast to predictions that cold-adapted populations should be specialized to function best in the cold and warm-adapted populations should be specialized for the warm implying a trade-off between function in the warm and cold. Instead, it may be that northern fish are more broadly tolerant of temperatures than southern fish such that northern fish do not make the tradeoff for reduced performance in the warm in order to maintain performance in the cold. This lack of specialization to the cold has been seen in high latitude ectothermic species, and it is thought that the more thermally variable nature of higher latitudes in comparison to southern latitudes may select for the maintenance of plasticity and broader tolerances in northern populations (Angilletta et al., 2006). Specialization for increasing metabolic rate in the cold by northern fish might result in the consistently higher metabolic rate seen in northern fish at warmer temperatures as well (25°C acclimated northern fish have 1.7 fold higher routine oxygen consumption than do southern fish) (Appendix A; Fangue et al., unpublished). This high metabolic rate, however, may 214 be too costly for a northern fish to maintain for long periods in the warmer southern waters. In combination with their lower maximum thermal tolerance, high metabolic rates may partially explain why northern fish have not colonized southern habitats. MECHANISTIC BASIS OF NICHE LIMITS One possible mechanism setting thermal niche limits is mitochondrial failure. When acutely challenged to warm acute assay temperatures, mitochondria from the 5 and 15°C acclimation groups from both killifish populations remained coupled at 35 and 37°C assay temperatures, respectively (Chapter Five). These temperatures are beyond the calculated CTMax values for 5 and 15°C acclimated fish and very near to the theoretical maximum swimming temperatures described above (Table 6.1). Acclimation Temperature (°C) Population CTMax (°C) Mitochondrial uncoupling temperature (°C) 5 N 30.3 . 35 5 S 32.5 35 15 N 35.4 37 15 S 36.9 37 25 N 39.3 37 25 S 40.3 37 Table 6.1 The calculated CTMax temperatures for 5, 15, and 25°C acclimated northern (N) and southern (S) killifish compared to the upper thermal limits for isolated mitochondrial State III rates (Chapter Five). The CTMax values are calculated from the regression equations generated in Chapter Two. Somewhat surprisingly, isolated mitochondria from the 25°C acclimation groups only remained well coupled up to 37°C. In general, most studies have suggested that isolated mitochondria are capable of functioning at temperatures several degrees higher than those tolerated by the more thermally sensitive intact organism (reviewed in Hochachka and Somero, 2002; Pdrtner et al., 2005; 2007). Calculated CTMax values for 25°C acclimated fish were 39.3 and 40.3°C (north and south, respectively; Table 6.1), higher than the highest temperature at which mitochondria 215 could be reliably assayed. The highest temperatures to which killifish could be acclimated, however, were 36.4 and 38.2°C for northern and southern killifish suggesting mitochondrial failure temperatures may be very close to whole organism maximal thermal acclimation temperature. The lowest isolated mitochondria assay temperature was 2°C, and there is no evidence that mitochondria from any group or population lost function at this assay temperature. In principle, there are two possible explanations for why the failure limits for isolated mitochondrial function and whole-organism function are so close together, in marked contrast to the results of other studies: 1) technical differences or limitations of our mitochondrial preparations relative to those of other studies, and 2) the extreme eurythermicity of killifish compared to most other fish examined to date. One possible technical explanation for the failure of warm-acclimated isolated killifish mitochondria at temperatures lower than expected could be that isolated mitochondria lose function while being kept on ice during isolation and before assay (preparations from 25°C acclimated fish were, in fact, stable for a shorter time on ice than those from the 5 and 15°C groups). Another confounding technical factor could be the stringent limits for measuring maximum oxidation rates I set in my experiment. All the preparations I utilized for these experiments involved highly coupled mitochondria (RCR values greater than 4 at all assay temperatures), and stable State II rates were achieved before stimulation by ADP to obtain an increase in oxygen consumption (State III). Preparations assayed at temperatures where ADP stimulation did not increase oxygen consumption were not included in my analysis of failure temperatures. Other studies on temperate eurytherms have reported State III rates at temperatures greater than 40°C even though the RCR's were ~2 at these warm assay temperatures (Sommer and Pdrtner, 2004). Uncoupled respiration rates have been used in congeneric abalone species and hydrofhermal vent organisms to estimate the failure temperature rather than classic State III rates due to the inconsistent coupling of preparations assayed at high temperatures (Dahlhoff et al., 1991; Dahlhoff and Somero, 1993). As a result, comparisons of 216 absolute maximum functional temperatures between studies may be difficult because of these differences in methodologies. Another difficulty in comparing my mitochondrial failure temperature data to other studies is that a large majority of isolated mitochondria studies in fishes comes from Antarctic, sub-Antarctic, and temperate fish species where often the highest assay temperature reported for State III rates is ~20°C (Guderley and Johnston, 1996; Weinstein and Somero, 1998; Johnston et al., 1998; Blier and Lemieux, 2001; Bouchard and Guderley, 2003; Lannig et al., 2005). One explanation to this pattern is assay of mitochondria from the hydrothermal vent worm, Riftia pachyptila, in which the upper failure temperature for succinate-stimulated, coupled respiration was reported to be 35°C (O'Brien et al., 1988), similar to the failure temperatures reported here. To my knowledge, however, there are no studies that have applied the same stringent conditions as were used in my experiments for obtaining State III rates from fish species as eurythermal as killifish acclimated to and assayed at very warm temperatures. It is possible that once other eurythermal species are examined, the pattern of mitochondrial performance continuing at temperatures substantially greater than the maximum temperatures for whole organism performance may also be violated. In addition, several studies have shown that there is a positive relationship between acclimation/adaptation temperature and the temperature at which even the uncoupled rates of mitochondrial oxidation cannot be maintained (Dahlhoff et al , 1991; Dahlhoff and Somero, 1993; Weinstein and Somero, 1998). My data are broadly consistent with these studies in that increased acclimation temperatures did result in an increase in the upper failure temperature for coupled killifish mitochondria (Table 6.1). A second mechanism that could play a role in setting the thermal niche of an organism is that of heat shock proteins (Hsps). The heat shock response is thought to be important for adaptation of organisms to their thermal environments, and Hsps play a protective role for 217 organisms approaching their critical thermal limits (reviewed in Feder and Hofmann, 1999; Hoffmann et al., 2003; and see Chapter One). In theory, thermal selection could target the functional efficiency of the Hsps themselves, the onset temperature for hsp induction (T o n) and/or the magnitude of basal or inducible hsp expression. In Chapter Two, I showed that there were no fixed differences in amino acid sequence between populations in either hsp70-l or hsp70-2, and only a single conservative substitution between populations in hsc70 strongly suggesting that the functional efficiency of the 70kDa Hsps does not differ between killifish populations. Northern and southern killifish did, however, differ in their patterns of mRNA expression for a number of hsp genes (Chapter Two). When killifish from both populations were acclimated to 20°C and acutely challenged to temperatures between 30-35°C (north) or 30-36°C (south), hsp70-l mRNA levels increased gradually and to the same extent in response to heat shock in both killifish populations. In contrast, both northern and southern killifish significantly increased hsp70-2 levels above control values (T o n) at a heat shock temperature of 33°C, but the magnitude of this induction was greater in northern fish, suggesting that northern fish may be more susceptible to thermal damage than are southern fish. The constitutive isoform (hsc70) was significantly elevated by heat shock in southern fish, but not in northern fish, while the 90kDa constitutive isoform (hsp90fJ) was not significantly induced with heat shock in either killifish population. The more thermally tolerant southern killifish had a T o n for hsp90a, the inducible isoform, of 30°C, 2°C lower than that of northern fish. This observation combined with the ability of southern killifish to upregulate hsc70 in response to heat shock suggests a possible role for these hsps in whole-organism differences in thermal tolerance. The finding that each hsp isoform exhibited a different pattern of mRNA expression with heat shock suggests that the expression patterns of all hsp genes are not regulated by a global factor such as differences in stability of the protein pool or overall rates of protein or mRNA turnover. Instead, this complex pattern of expression suggests that differences in mRNA levels 218 between populations result from gene-specific mechanisms such as differences in transcription as a result of promoter sequence variation, differences in mRNA stability as a result of sequence variation in the 5' or 3' untranslated region in a particular hsp gene (McGarry and Lindquist, 1986; Petersen and Lindquist, 1988), differences in HSF levels (Wu, 1995), or differences in the number of copies of these genes (Krebs and Feder, 1998). Experiments are currently underway to try to distinguish between these possibilities to explain the differential expression patterns shown for hsp70-2 between killifish populations. Taken together, these data suggest that considering the complexity of the heat shock response across many genes and gene families may be critical to understanding their role in thermal tolerance. ESTIMA TING THE SHAPE OF THE NICHE The data presented in Chapter Four demonstrate that killifish from both populations could maintain some level of swimming performance at all temperatures investigated in this thesis (5-35°C). In fact, within a population, killifish maintained equivalent performance at all acclimation/test temperatures between 7 and 34°C, and performance only declined at swimming temperatures of 5°C in both populations. These data show a broad zone of relative thermal independence in swimming performance in both northern and southern killifish populations. To fully determine the shape of the thermal niche, a careful analysis of several acclimation temperature groups acutely challenged to temperatures across their tolerance range would be necessary to generate comparative reaction norms for multiple thermal acclimation groups and populations. While this full data set is not available, my U c r i t data set for 18°C acclimated killifish acutely challenged to 5, 18, 25, and 35°C suggests that the shape of the thermal performance niche is very flat from 18-35°C, with performance declining at 5°C (Chapter Four). Unfortunately, I do not have acute thermal challenge swim performance data between 5 and 18°C that would allow for the determination of the shape of the curve between 5 219 and 18°C. The existing data, however, suggest a broad zone of thermal independence in the thermal niche shape for swimming performance, but information from fish acclimated to other temperatures and acutely challenged would be helpful in order to fully define the thermal niche shapes. Up to this point in the discussion, I have suggested that northern and southern killifish acclimated to 18°C are insensitive to thermal effects on U c r i t up to 35°C, suffering no performance decline at this temperature, which suggests that aerobic scope is maintained up to at least 35°C. However, the temperatures for hsp induction for northern and southern fish acclimated to 20°C (Chapter Two), for several hsp isoforms were between 30 and 33°C suggesting that Hsps are induced at a temperature where swimming performance is still maintained at its maximum. This point is important because the literature to date shows no evidence that molecular damage (indicated by the induction of Hsps) occurs prior to the onset of a temperature-induced hypoxemia and a reduction in aerobic scope (discussed in Pdrtner, et al., 2007). It is important to note, however, that U c r i t trials are somewhat insensitive measures of aerobic performance as anaerobic metabolism is often recruited at swimming speeds of -50-70% of U C r i t values. In fact, experimental manipulations of functional hemoglobin concentrations in salmon have shown that hemoglobin concentrations can be reduced by 50%> before a reduction in Ucrit is detected (Brauner et al, 1993). If we accept the contention that Hsp induction occurs only after the onset of hypoxemia, my Hsp data suggest that aerobic scope has already declined in killifish at swimming temperatures above 30°C, although U c r i t has not yet declined. This would suggest an increased reliance on anaerobiosis to support swimming at higher temperatures. In fact, the muscle and plasma lactate values obtained for northern and southern fish subjected to a standardized exercise challenge at 5, 15, and 25°C showed a trend to increase with increasing swimming temperatures in both killifish populations (Chapter Four), consistent with the prediction that the anaerobic contribution to maintaining a high U c r i t increases with 220 increasing swimming temperatures. Jain and Farrell (2003) also found that post-exercise lactate levels in rainbow trout were greater in warm-acclimated fish, but in this case the warm-acclimated fish had higher U c r i t -In the case of killifish, the question then arises as to why fish at lower acclimation temperatures do not increase U c r j t by increasing the anaerobic component of metabolism. One possibility is that there is a volitional component to swimming such that increases in temperature 'encourage' the fish to swim harder and increase the use'of anaerobic pathways to achieve high Ucrit values. ACUTE THERMAL PERFORMANCE OF MITOCHONDRIA IN RESPONSE TO THERMAL ACCLIMATION In Chapter Five of my thesis, I provided evidence that acclimation temperature dramatically altered the shape of the isolated mitochondria acute performance curves. Both northern and southern killifish mitochondria had acute State III curves that differed in shape between 5 and 25°C acclimation groups (with 15°C acclimated mitochondria having curves with intermediate shapes). Both populations had approximately linear curves for 25°C acclimated mitochondria with very few differences between populations, but 5°C acclimated mitochondria from northern and southern fish differed in response to acute thermal challenge. Cold-acclimated northern mitochondria had increased State III rates in the cold and reduced State III rates in the warm relative to southern fish (Chapter Five) demonstrating that thermal acclimation clearly influenced the function of the mitochondria in response to acute thermal challenge in very different ways between northern and southern killifish populations. While interpreting the adaptive value of these differential acute curve shapes is difficult as in vivo mitochondrial function is regulated at a number of different levels (reviewed in Guderley, 2004), if these functional differences in isolated mitochondria translate into differences in whole organism performance, they could have a dramatic affect on the shape of the thermal performance niche of 221 each killifish population. As well, I have shown that northern killifish had higher mitochondrial content (Chapter Five), particularly at low acclimation temperatures, which could also influence whole-organism aerobic capacity. To try to understand the relationship between isolated mitochondrial function and content measurements versus whole-animal responses, future experiments involving whole-organism metabolic rate measurements under thermal acclimation and acute thermal challenge conditions will be necessary. CONCLUSIONS From the information presented in this thesis, it is clear that numerous measures of performance respond to thermal acclimation and differ between killifish populations. I have shown that southern fish had higher thermal tolerance limits than northern fish, that both killifish populations demonstrated a high degree of plasticity in thermal tolerance, that both populations differed in hsp mRNA expression patterns, and that both populations differed in mitochondrial amount and function, particularly when fish are acclimated to cold temperatures. While these findings were broadly consistent with predictions based on the thermal habitats of killifish as well as predictions developed from the thermal biology literature including the OLTT hypothesis, this was not always the case. For example, the thermal optimum (as assessed by preferred temperatures) was higher in northern killifish, and the estimated pejus temperatures where swimming performance declined were very similar between populations with both populations showing a very large zone of essentially thermally independent swimming performance (~7-35°C). As well, differences in hsp mRNA expression were shown between populations, but the patterns of expression were not broadly consistent with the thermal tolerance differences between populations or consistent between hsp genes, suggesting inherent complexity in the regulation of the Hsp response. 222 In summary, I have shown that there are differences between the northern and southern subspecies of F. heteroclitus at the physiological, biochemical, and molecular levels, some of which are consistent with local adaptation to their thermal habitats. My thesis highlights the importance of considering multiple potential mechanisms that could underlie differences in whole organism performance when investigating the interplay between thermal acclimation and local thermal adaptation. Despite the intensive use of killifish as a model for local thermal adaptation in fishes (Powers et al., 1993; Powers and Schulte, 1998; Schulte, 2001; Cossins and Crawford, 2005), much of the work that has been done to date has been carried out at a single or across very few thermal acclimation temperatures. By limiting comparisons to only a few temperature treatments, one killifish population could be favored over the other making the interpretation of the results difficult. My thesis has clearly shown that thermal acclimation temperature can have a profound effect on the way killifish populations perform in the face of thermal challenge, and these responses may differ between populations suggesting that comparisons across multiple temperature treatments are critical to fully understand the mechanisms involved in thermal acclimation and adaptation in killifish. FUTURE DIRECTIONS The findings from my thesis have provided the backbone for developing an integrated view of thermal performance niches in fishes, and I suggest that the OLTT framework may be a useful tool to further describe the thermal reaction norm for each killifish population (Pdrtner et al., 2007). The data set from my thesis can be expanded to include additional measures of whole organism performance, for example growth and aerobic scope. Measures of the oxygen delivery cascade such as cardiac output, tissue perfusion and oxygenation parameters would provide an excellent complement to measures of oxygen consumption of the whole organism or at the level of isolated mitochondria. While some of these measurements are possible in killifish, others will 223 be difficult to obtain. To further test the OLTT hypothesis, defining the upper and lower T p , T c , and Tf for each of these measurements from killifish acclimated to and acutely tested to a broad range of temperatures will be required. Because the temperature thresholds of T p , T c , and Tf are influenced by the type of measurement technique utilized as well as how these thresholds are defined, careful consideration and interpretation of the values obtained will be crucial to our understanding of the thermal niche of killifish. While the suite of mechanisms required to explain the performance differences between killifish are not fully described, future experiments should be focused on trying to understand the mechanisms that co-define the critical temperature thresholds of an organism's thermal performance niche. 224 REFERENCES Angilletta, M J . Jr., Bennett, A.F. Guderley, H. Navas, C A . Seebacher, F., and R.S. Wilson. 2006. Coadaptation: A unifying principle in evolutionary thermal biology. Physiological and Biochemical Zoology. 79: 282-294. Angilletta, M.J. Jr., Niewiarowski, P.H., and C A . Navas. 2002. The evolution of thermal physiology in ectotherms. Journal of Thermal Biology. 27:249-268. Brauner, C.J., Val, A.L. , and D.J. Randall. 1993. The effect of graded methemoglobin levels on the swimming performance of Chinook salmon (Oncorhynchus-Tshawytscha). Journal of Experimental Biology. 185: 121-135. Blier, P.U., and H. Lemieux. 2001. The impact of the thermal sensitivity of cytochrome c oxidase on the respiratory rate of Arctic charr red muscle mitochondria. Journal of Comparative Physiology. 171: 247-253. Bouchard, P., and H. Guderley. 2003. Time course of the response of mitochondria from oxidative muscle during thermal acclimation of rainbow trout, Oncorhynchus mykiss. Journal of Experimental Biology. 206: 3455-3465. Conover, D.O., and E.T. Schultz. 1995. Phenotypic similarity and the evolutionary significance of countergradient variation. Trends in Ecology and Evolution. 10: 248-252. Cossins, A.R., and D.L. Crawford. 2005. Fish as models for environmental genomics. Nature Reviews Genetics. 6: 324-340. Dahlhoff, E. Obrien, J., Somero, G.N. and R.D. Vetter. 1991. Temperature effects on mitochondria from hydrothermal vent invertebrates - evidence for adaptation to elevated and variable habitat temperatures. Physiological Zoology. 64: 1490-1508. Dahlhoff, E. , and G.N. Somero. 1993. Effects of temperature on mitochondria from abalone (Genus Haliotis): adaptive plasticity and its limits. Journal of Experimental Biology. 185:151-168. Farrell, A.P. 1997. Effects of temperature on cardiovascular performance. In Global warming: Implications for freshwater and marine fish. (ed. C M . Wood and D.G. McDonald), pp. 135-158. Cambridge University Press, Cambridge. Feder, M.E. , and G.E. Hofmann. 1999. Heat shock proteins, molecular chaperones, and the stress response: Evolutionary and ecological physiology. Annual Reviews in Physiology. 61: 243-282. Freidenburg, L.K. , and D.K. Skelly. 2004. Micro geographical variation in thermal preference by an amphibian. Ecology Letters. 7:369-373. Guderley, H. 2004. Locomotor performance and muscle metabolic capacities: impact of temperature and energetic status. Comparative Biochemistry and Physiology. 139: 371-382. 225 Guderley, H., and I.I. Johnston. 1996. Plasticity of fish muscle mitochondria with thermal acclimation. Journal of Experimental Biology. 199: 1311-1317. Hoffmann, A.A. , Sorensen, J. G., and V. Loeschcke. 2003. Adaptation of Drosophila to temperature extremes: bringing together quantitative and molecular approaches. Journal of Thermal Biology. 28: 175-216. Jain, K.E. , and A.P. Farrell. 2003. Influence of seasonal temperature on the repeat swimming performance of tainbow trout, Oncorhynchus mykiss. Journal of Experimental Biology. 206: 3569-3579. Johnston, I.A., Calvo, J., Guderley, H., Fernandez, D., and L. Palmer. 1998. Latitudinal variation in the abundance and oxidative capacities of muscle mitochondria in perciform fishes. Journal of Experimental Biology. 201:1-12. Krebs, R.A., and M.E. Feder. 1998. Hsp70 and larval thermotolerance in Drosophila melanogaster. how much is enough and when is more too much? Journal of Insect Physiology. 44: 1091-1101. Lannig, G., Storch, D., and H.O. Pdrtner. 2005. Aerobic mitochondrial capacities in Antarctic and temperate eelpout (Zoarcidae) subjected to warm versus cold acclimation. Polar Biology. 28: 575-584. McGarry, T., and S. Lindquist. 1986. The preferential translation of hsp70 mRNA requires sequences in the untranslated leader. Cell. 42: 903-911. National Oceanic and Atmospheric Administration, Office of Ocean and Coastal Resource Management, National Estuarine Research Reserve System-wide Monitoring Program. 2004. Centralized Data Management Office, Baruch Marine Field Lab, University of South Carolina http://cdmo.baruch.sc.edu. O'Brien, J., Parks, E .M. , and G.N. Somero. 1988. Factors that influence the upper temperature limit of mitochondrial respiration. American Zoologist. 28: A44. Petersen, R., and S. Lindquist. 1988. The Drosophila hsp70 message is rapidly degraded at normal temperatures and stabilized by heat shock. Gene. 72: 161-168. Pdrtner, H.O. 2001. Climate change and temperature-dependent biogeography: oxygen limitation of thermal tolerance in animals. Naturwissenschaften. 88: 137-146. Pdrtner, H.O. 2002. Climate variations and the physiological basis of temperature dependent biogeography: systemic to molecular hierarchy of thermal tolerance in animals. Comparative Biochemistry and Physiology. 132: 739-761. Pdrtner, H.O., Lucassen, M . , and D. Storch. 2005. Metabolic biochemistry: its role in thermal tolerance and in the capacities of physiological and ecological function. In The Physiology of Polar Fishes, (ed. A.P. Farrell and J.F. Steffensen). pp. 79-154. Academic Press, San Diego. 226 Portner, H.O., Peck, L. , and G.N. Somero. 2007. Thermal limits and adaptation in marine Antarctic ectotherms: an integrative view. Philosophical Transactions of the Royal Society. Powers, D.A., Smith, M . Gonzalez-Villasenor, I. DiMichele, L. Crawford, D. Bernardi, G., and T. Lauerman. 1993. A multidisciplinary approach to the selectionist/neutralist controversy using the model teleost, F. heteroclitus. In Oxford Survey of Evolutionary Biology, (ed. D. Futuyuma and J. Antonovics). pp. 43-107. Oxford University Press, Oxford. Powers, D.A., and P.M. Schulte. 1998. Evolutionary adaptations of gene structure and expression in natural populations in relation to a changing environment: A multidisciplinary approach to address the million-year saga of a small fish. Journal of Experimental Zoology. 282: 71-94. Schulte, P.M. 2001. Environmental adaptations as windows on molecular evolution. Comparative Biochemistry and Physiology. 128: 597-611. Schultz, E.T., Reynolds, K.E. , and D.O. Conover. 1996. Countergradient variation in growth among newly hatched Fundulus heteroclitus: Geographic differences revealed by common-environment experiments. Functional Ecology. 10:366-374. Smith, K.J., and K.W. Able. 1994. Salt-marsh tide pools as winter refuges for the mummichog, Fundulus heteroclitus, in New Jersey. Estuaries. 17: 226-234. Sommer, A . M . and H.O. Pdrtner. 2004. Mitochondrial function in seasonal acclimatized versus latitudinal adaptation to cold in the lugworm Arenicola marina (L.) Physiological and Biochemical Zoology. 77:174-186. Weinstein, R.B., and G.N. Somero. 1998. Effects of temperature on mitochondrial function in the Antarctic fish Trematomus bernacchii. Journal of Comparative Physiology. 168: 190-196. Wu, C. 1995. Heat shock transcription factors: Structure and regulation. Annual Review of Cell and Developmental Biology. 11: 441-469. Yamahira, K., and D.O. Conover. 2002. Intra- vs. interspecific latitudinal variation in growth: adaptation to temperature or seasonality? Ecology. 83:1252-1262. 227 APPENDIX A o E CM O 25 20 15 10 5 0 • Northern ^Southern a x 5°C 15°C Acclimation Temperature (°C) 25°C Metabolic rates (u.mol/g/hr) for northern (NH, white) and southern (GA, hatched) killifish in response to acclimation temperatures (5°C, 15°C, and 25°C). A l l M 0 2 values are the mean ± SE. N=7-8 per population and temperature treatment. Different letters indicate significant differences (PO.05) between treatments within a population, while an asterisk indicates a significant difference (P<0.05) between the populations of a single treatment (Fangue, Quan, and Schulte, unpublished). These data were collected by Michelle Quan, an undergraduate project student under my direct supervision, and will be incorporated into Chapter Five for publication. 228 APPENDIX B THE UNIVERSITY OF BRITISH COLUMBIA A N I M A L C A R E C E R T I F I C A T E Application Number: A04-0238 Investigator or Course Director: Patricia M. Schulte Department: Zoology Animals: Fish 2050 Start Date: May 1, 2002 Funding Sources: Approval Date: July 12,2006 Grant Agency: Grant title: Grant Number: Grant Agency: Grant Title: Unfunded title: Natural Science Engineering Research Council Environmental regulation of gene expression: responses and mechanisms 01-3976 Natural Science Engineering Research Council Environmental regulation of gene expression N/A The Animal Care Committee has examined and approved the use of animals for the above experimental project. This certificate is valid for one year from the above start or approval date (whichever is later) provided there is no change in the experimental procedures. Annual review is required by the CCAC and some granting agencies. A copy of this certificate must be displayed in your animal facility. Office of Research Services and Administration 102,6190 Agronomy Road, Vancouver, BC V6T 1Z3 Phone: 604-827-5111 Fax: 604-822-5093 229 

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