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Maximum heart rate as a means of rapidly estimating optimal temperature for aerobic scope in salmon :… Casselman, Matthew Thomas 2012

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MAXIMUM HEART RATE AS A MEANS OF RAPIDLY ESTIMATING OPTIMAL TEMPERATURE FOR AEROBIC SCOPE IN SALMON: ITS POTENTIAL FOR APPLICATION  by  Matthew Thomas Casselman B.Sc., Simon Fraser University, 2006   A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in The Faculty of Graduate Studies (Zoology)   THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)   March 2012  © Matthew Thomas Casselman 2012  ii ABSTRACT  Knowing the optimal temperature (Topt) for aerobic scope of fishes may be useful for predicting responses to warming environmental temperature because Topt is when fish can allocate the most oxygen to activity. However, the broad application of Topt measurements is hampered by the time required to determine Topt of a species. This thesis sought to develop a rapid method of estimating Topt of Pacific salmon species (genus Oncorhynchus) based on evidence that suggests the decline in aerobic scope above Topt is triggered by a limitation on maximum heart rate (fH). Thus, maximum fH at elevated temperature is thought to limit oxygen convection through the circulatory system, and hence limits both maximum metabolic rate and aerobic scope. Measurements of metabolic rate and fH were taken over a range of temperatures at rest and following exhaustive exercise in juvenile coho salmon (O. kisutch) to confirm the association between Topt and maximum fH. In vivo measurements revealed a maximum fH limitation at the Topt for aerobic scope, supporting the use of fH to estimate Topt. The potential for expediting measurements of maximum fH during acute warming was investigated using anaesthetized coho salmon and pharmacological stimulation of fH. In coho salmon sedated with low doses of anaesthetic, pharmacologically stimulated fH was equivalent to the maximum fH measured in vivo. Breakpoint analysis of the relationship between maximum fH and temperature demonstrated a limitation on maximum fH that corresponded closely with the Topt for aerobic scope. Further, while Topt measurements took three weeks to complete, maximum fH measurements were completed in three days. Therefore, the novel maximum fH technique considerably reduced the time  iii needed to estimate Topt and may be broadly suited to estimating Topt both within and outside of the Oncorhynchus genus. Potential application of this rapid Topt estimation method is discussed in relation to temperature data collected from two local coho salmon-bearing streams. Temperature data also allowed for the examination of stream warming and cooling dynamics and identification of habitat critical to buffering anthropogenic disturbances to stream temperature. These data highlight the importance of riparian areas for maintaining the thermal integrity of waterways.  iv PREFACE  A version of Chapter 2 has been published. M.T. Casselman, K. Anntila, and A.P. Farrell. 2012. Using maximum heart rate as a rapid screening tool to determine optimum temperature for aerobic scope in Pacific salmon Oncorhynchus spp. Journal of Fish Biology. 80(2): 358-377. M.T. Casselman carried out the vast majority of experimental design, data collection, and manuscript preparation. A.P. Farrell provided valuable supervision on experimental design, data analysis, and manuscript preparation. K. Anttila provided assistance with data collection and manuscript preparation. Members of the Seymour Salmonid Society and West Vancouver Streamkeepers assisted with data collection for Chapter 3 and Chapter 4. All procedures were approved by the University of British Columbia Animal Care Committee in accordance with guidelines set out by the Canadian Council on Animal Care (UBC Animal Care protocols A10-0002 and A10-0236).  v TABLE OF CONTENTS ABSTRACT....................................................................................................................... ii! PREFACE......................................................................................................................... iv! TABLE OF CONTENTS ................................................................................................. v! LIST OF TABLES .......................................................................................................... vii! LIST OF FIGURES ....................................................................................................... viii! LIST OF ABBREVIATIONS .......................................................................................... x! ACKNOWLEDGEMENTS ............................................................................................ xi! CHAPTER 1: INTRODUCTION.................................................................................... 1! 1.1! The Influence of Temperature ....................................................................... 2! 1.2! The Importance of Topt ................................................................................... 4! 1.3! Limits to Maximum Oxygen Delivery........................................................... 6! 1.3.1! Oxygen Delivery and Uptake at the Gills .......................................... 10! 1.3.2! Oxygen Convection by the Circulatory System.................................. 11! 1.3.3! Oxygen Delivery at the Tissues .......................................................... 12! 1.3.4! Topt and Maximum fH .......................................................................... 13! 1.4! Expediting Topt Estimations ......................................................................... 14! 1.4.1! Estimating Topt for Aerobic Scope...................................................... 15! 1.4.2! The fH Method for Determining Topt ................................................... 16! 1.5! Study Species ............................................................................................... 18! 1.6! Chapter 2 Objectives.................................................................................... 19! 1.7! Applying Topt Measurements ....................................................................... 20! 1.8! Chapter 3 and Chapter 4 Objectives ............................................................ 22! CHAPTER 2: USING MAXMIMUM HEART RATE AS A RAPID SCREENING TOOL TO ESTIMATE OPTIMUM TEMPERATURE FOR AEROBIC SCOPE IN SALMON......................................................................................................................... 25! 2.1! Introduction.................................................................................................. 25! 2.2! Materials and Methods................................................................................. 28! 2.2.1! Experimental Animals and Care ........................................................ 28!  vi 2.2.2! Aerobic Scope and fH Measurements in Unanaesthetized Fish ......... 28! 2.2.3! ! ˙ M O2 Measurements........................................................................... 30! 2.2.4! fH Measurements ................................................................................ 31! 2.2.5! Maximum fH in Anaesthetized Fish .................................................... 32! 2.2.6! Experimental Apparatus .................................................................... 33! 2.2.7! Data and Statistical Analysis ............................................................. 34! 2.3! Results.......................................................................................................... 35! 2.3.1! Effect of Temperature on ! ˙ M O2  and fH in Unanaesthetized Fish ....... 35! 2.3.2! Effect of Temperature on fH in Anaesthetized Fish ............................ 37! 2.3.3! Comparison of Topt Estimates............................................................. 39! 2.4! Discussion .................................................................................................... 39! 2.4.1! Accuracy of Topt and fH Measurements for Unanaesthetized Fish ..... 40! 2.4.2! Measuring Maximum fH in Anaesthetized Fish.................................. 42! CHAPTER 3: TEMPERATURE CONDITIONS AND DYNAMICS IN TWO COHO SALMON-BEARING STREAMS ................................................................... 55! 3.1! Introduction.................................................................................................. 55! 3.2! Materials and Methods................................................................................. 58! 3.2.1! Study Watersheds ............................................................................... 58! 3.2.2! Temperature Monitoring and Data Analysis ..................................... 59! 3.3! Results.......................................................................................................... 60! 3.4! Discussion .................................................................................................... 63! 3.4.1! Resolution of Temperature Monitoring ............................................. 64! 3.4.2! Stream Temperature Dynamics.......................................................... 67! CHAPTER 4: DISCUSSION AND CONCLUSIONS ................................................. 87! 4.1! Combining Rapid Topt Determinations with Temperature Data................... 89! 4.2! Comparative Assessments using the fH Protocol ......................................... 92! 4.3! Extending the Applicability of the fH Protocol ............................................ 94! 4.4! Adapting the fH Protocol to Species and Conditions ................................... 95! 4.5! Conclusions and Perspectives ...................................................................... 97! REFERENCES.............................................................................................................. 102!  vii LIST OF TABLES  Table 2.1. Changes in heart rate following pharmacological treatments...........................47  Table 2.2. Physical and blood characteristics, cardiac collapse, and Arrhenius break temperature analysis of heart rate during an acute temperature change ..................................................................................48  Table 3.1. Temperature logger locations and basic stream temperature parameters from the study watersheds ..............................................................73  Table 3.2. Differences in temperature between the upper and lower loggers for each stream reach within the study watersheds ...........................................74  Table 4.1. Habitat thermal quality assessment of the Seymour River and Brothers Creek watersheds................................................................................99  Table 4.2. Results from the field application of the fH protocol to wild juvenile coho salmon from the Seymour River and Brothers Creek watersheds.............................................................................................100  viii LIST OF FIGURES  Figure 1.1. The temperature-dependent response of reaction rates, Arrhenius plot transformation, and Arrhenius break temperature analysis...............................23  Figure 1.2. Changes in resting metabolic rate, maximum metabolic rate, and aerobic scope with temperature.........................................................................24  Figure 2.1. Changes in water bath temperature, internal body temperature, and heart rate during acute warming in anaesthetized coho salmon........................49  Figure 2.2. Oxygen consumption, heart rate, and aerobic scope over a range of temperatures in resting and post-chase coho salmon ........................................50  Figure 2.3. Arrhenius plot of resting and maximum heart rate..........................................51  Figure 2.4. Heart rate during acute warming of anaesthetized coho salmon.....................52  Figure 2.5. Arrhenius plots of pharmacologically stimulated maximum heart rate ..........53  Figure 2.6. Comparison of optimal temperature estimates ................................................54  Figure 3.1. Area map for the Seymour River, Brothers Creek, and Hadden Creek ..........75  Figure 3.2. Detail map for the Seymour River...................................................................76  Figure 3.3. Detail map for Brothers Creek and Hadden Creek..........................................77  Figure 3.4. Summer temperature frequencies on the Seymour River................................78  Figure 3.5. Summer temperature frequencies on Brothers Creek......................................79  Figure 3.6. Summer temperature frequencies on Hadden Creek .......................................80  ix  Figure 3.7. Summer temperature dynamics on the Seymour River...................................81  Figure 3.8. Temperature dynamics in Brothers Creek reach B1........................................82  Figure 3.9. Influence of air and input water temperature on warming in Brothers Creek reach B1 ..................................................................................................83  Figure 3.10. Temperature dynamics in Hadden Creek reach H2.......................................84  Figure 3.11. Influence of air and input water temperature on cooling in Hadden Creek reach H2..................................................................................................85  Figure 3.12. Regression of air versus water temperature for upper and lower loggers in reach H2............................................................................................86  Figure 4.1. Polynomial quadratic regression of aerobic scope for juvenile coho salmon and Fry curve to define the temperature ranges for stream thermal assessments.............................................................................101   x LIST OF ABBREVIATIONS  ABT  Arrhenius break temperature ATP  adenosine triphosphate Ca2+  calcium CaO2  oxygen content of arterial blood Ca-vO2  difference in oxygen content of arterial and venous blood CvO2  oxygen content of venous blood ECG  electrocardiogram ETC  electron transport chain fH  heart rate M  mass ! ˙ M O2   rate of oxygen consumption MS-222  tricaine methanesulfonate Na+  sodium NaCl  sodium chloride NaHCO3  sodium bicarbonate O2  oxygen PaO2  partial pressure of arterial blood PvO2  partial pressure of venous blood ! ˙ Q  cardiac output Q10  temperature coefficient Topt  optimal temperature; maximum aerobic scope available Tcrit   critical temperature; zero aerobic scope available Vs  stroke volume Ucrit  critical swimming speed   xi ACKNOWLEDGEMENTS  I would first like to thank Dr. Tony Farrell for the past two and a half years of supervision, inspiration and endless patience. Tony is an incredible supervisor who motivated and encouraged me throughout my degree. I greatly appreciate all his support, teachings and the frequent computer quandaries that were welcome diversions. I would also like to thank my committee members, Bill Milsom and Rick Taylor, for their valuable input on my thesis. The people I met during my time at UBC made my Masters degree a truly amazing experience. I would like to thank all members of the Farrell lab for their support. Big thanks to Katja for keeping me on a rigorous experiment schedule and keeping my inbox full; Georgina and Slabado MaGoo for being amazing office-mates (but probably extending my writing by a month); Erika for all her smarts and being the best beer fairy; and Linda for kicking ass (mine included) in the lab. I would also like to thank those in the Zoology and Forestry departments that have been terrific for both academic and non-academic support. In particular, Alison ‘gong show’ Collins, Ken Jeffries, Tim Clark, Mike Sackville, Sarah Fortune, and Trisha Atwood. The Coho Society of the North Shore, the Pacific Salmon Foundation, and the West Vancouver Streamkeepers provided support for my research. Thank you to the members of these organizations for their field assistance and making the community a part of my MSc. Finally I would like to thank my parents and family for all their love and support.  1 CHAPTER 1: INTRODUCTION  The rapid pace of climate change has created a pressing need to understand how animals will respond to the temperature regime shifts expected to occur in most ecosystems (IPCC 2007). Warming temperatures are of particular consequence for aquatic ectotherms such as fishes, where minor changes in temperature have implications for energetics, habitat availability, and ultimately distribution and abundance. For instance, increases in sea temperature of less than 2°C in the past three decades have resulted in significant northward shifts of numerous marine fish species (Dulvy et al. 2008; Perry et al. 2005). If meaningful predictions are to be made regarding ecosystem-level responses to warming, a measure of thermal tolerance is required that is not only applicable at the ecosystem level but can also provide such pertinent information in a timely fashion. My thesis aims to develop a methodology that has the potential to act as a rapid screening tool to assist with meaningful predictions of the tolerance of fishes to warming temperature. Pacific salmon (genus Oncorhynchus) are an ecologically, culturally, and economically important group of fishes that have been well studied in all aspects of their physiology and ecology. Using one species of salmon, this thesis investigated whether measurements of thermal tolerance using a proven, ecologically relevant matrix (aerobic scope) could be expedited using alternative measures based on known physiological limitations of salmon (heart rate) at their upper temperature limits. Further, this thesis sought to demonstrate how the thermal tolerance data generated by this investigation could be combined with water temperature measurements from two salmon-bearing streams to assess the thermal suitability of habitat within these watersheds. This information would be useful in assessing how salmon will respond to increasing environmental temperature due to climate change.  2 1.1 The Influence of Temperature  Temperature affects all levels of biological organization primarily by increasing the rate of biological reactions. Increases in temperature will raise the mean kinetic energy of molecules according to the Maxwell-Boltzmann distribution and exponentially increase the number of molecules with sufficient energy to participate in a reaction (Haynie 2008). In response, reaction rates also increase exponentially (Figure 1.1A). One of the earliest attempts to quantify the temperature dependence of chemical reaction rates was by van’t Hoff, who proposed several equations to quantify this relationship that Arrhenius later assimilated into the Arrhenius equation ( ! k = Ae-E" /RT ) (Logan 1982). The Arrhenius equation and the work underlying it showed that chemical reactions did indeed increase exponentially with temperature. Arrhenius (1915) later used the Arrhenius equation as well as its linear form ( ! lnk = "E# /RT + lnA ) in an Arrhenius plot (Figure 1.1B) to assess the temperature dependence of more complex biological reactions, such as the resting heart rate of a tortoise. Arrhenius found that not only was the temperature response of biological reactions also exponential, but the temperature dependence of these reactions was fairly consistent (Arrhenius 1915). Today, the temperature dependence of biological reactions is most commonly described using Q10 values (van’t Hoff 1898; as cited in Blackman 1905), the ratio of reaction rates across a 10°C temperature difference. Not surprisingly, and in keeping with the findings of Arrhenius (1915) that biological reactions increase exponentially, most biological reactions have a Q10 between 2 and 3, meaning they will double or triple with a 10°C increase in temperature. Thus, the inescapable laws of thermodynamics that drive increases in the  3 rates of chemical reactions also apply to biological reactions that similarly increase exponentially with temperature. The exponential increase in biological reaction rates has important consequences for the energy requirements of organisms. Energy produced during cellular respiration is primarily generated via oxidative phosphorylation and the transfer of electrons down the electron transport chain (ETC) to produce biochemical energy in the form of adenosine triphosphate (ATP). The ETC is located in the mitochondria and is an efficient energy-producing pathway that is dependent on oxygen as the terminal electron receptor (Hochachka and Somero 2002). Cells use the ATP generated in the ETC to power biological reactions that support whole- organism function. Increased rates of ATP-consuming reactions will therefore require increased ATP generation and increase the oxygen required by a cell. While ATP production in the absence of oxygen (anaerobic) is possible, it is far less efficient (Hochachka and Somero 2002). Alternative energy stores to ATP can also be utilized if oxygen is unavailable, but their supplies are finite and can only meet short-term energetic requirements (Pörtner 2001). Therefore, organisms ultimately rely on oxygen for energy production and this allows the use of whole-organism oxygen consumption ( ! ˙ M O2) as an indirect measure of metabolic rate – the combined rate of all energy being used by an organism. Measuring ! ˙ M O2 can be used to quantify changes in energy utilization by an organism with changing physiological state or environmental conditions.   The temperature-dependence of biological reaction rates is reflected at the whole- organism level as an exponential increase in resting ! ˙ M O2. Ege and Krogh (1914) were the first to characterize this response in an ectotherm using an acute temperature increase in the goldfish (Carassius auratus) (as cited in Clarke and Fraser 2004). This metabolic  4 response has since been characterized in fishes across taxa (Brett 1971; Gollock et al. 2006; Johnston et al. 1991; Lannig et al. 2004), demonstrating resting ! ˙ M O2increases with temperature according to thermodynamic principles (Gillooly et al. 2001). It is clear that to support such an increase in oxygen demand an organism must increase oxygen supply to the cells carrying out oxidative phosphorylation. Given that the early experiments of Ege and Krogh (1914) showed that goldfish could increase resting ! ˙ M O2 exponentially, it is apparent in this case at least, that oxygen supply was capable of meeting demand. However, in order to flourish in the environment, animals must increase their metabolic rate above resting in order to forage, grow, reproduce, avoid predators, and perform a host of other activities. Examining how resting ! ˙ M O2 changes with warming will only reveal the limits of thermal tolerance and the temperature at which an organism’s capacity to supply oxygen collapses. Resting ! ˙ M O2 alone does not provide sufficient insight as to how temperature effects on oxygen demand will influence whole-organism function.  1.2 The Importance of Topt   Fry (1947) was the first to recognize that temperature also influenced the activity of ectothermic animals as well as resting ! ˙ M O2. Like Ege and Krogh (1914), Fry used goldfish to demonstrate this relationship. However, a key distinction between Fry’s work and the earlier work of Ege and Krogh (1914) was that Fry incorporated measures of maximum ! ˙ M O2 and, quite importantly, realized that activity was intrinsically linked to ! ˙ M O2. Building on this, Fry recognized temperature controlled both the resting and the  5 maximum ! ˙ M O2 and that the capacity for an organism to perform activity lay in the difference between these two measurements, which Fry termed scope for activity. By measuring resting and maximum ! ˙ M O2 across a temperature range, Fry observed that resting ! ˙ M O2 increased exponentially across the temperature range, as expected, but maximum ! ˙ M O2 increased exponentially only at lower temperatures (Figure 1.2A). The increase in maximum ! ˙ M O2 then failed to keep pace with resting ! ˙ M O2 and plateaued at a temperature mid-way between the upper and lower temperatures measured for resting ! ˙ M O2. When Fry plotted the scope for activity over the temperature range, the curve was bell-shaped with a distinct maximum (Figure 1.2B). The maximum for this ‘Fry curve’ (Farrell 2009) defines the optimal temperature (Topt) where the capacity for extraneous activity should be maximal. As a result, Fry postulated that Topt would be a more ecologically relevant measure of thermal tolerance than previous measures that focused on upper and lower thermal limits (Brett 1944; Britton 1924).   Fry’s scope for activity framework is now known as aerobic scope and further studies of aquatic ectotherms and in particular salmonids, have confirmed the link between Topt for aerobic scope and activity. Gibson and Fry (1954) demonstrated maximal swimming speed coincided with Topt for aerobic scope of lake trout (Salvelinus namaycush) and Brett (1964) found equivalent results with juvenile sockeye salmon (Oncorhynchus nerka). Recent studies with adult coho salmon (Oncorhynchus kisutch) and sockeye salmon have not only reiterated this relationship, but also demonstrated that Topt is species- (Lee et al. 2003) and even population-specific (Eliason et al. 2011). The likely dependence of fitness on swimming ability (Plaut 2001) suggests that temperatures away from Topt that  6 reduce aerobic scope will have ecological relevancy. Further, growth may also decline at temperatures away from Topt (Brett et al. 1969; Elliott and Hurley 2000).  The ecological importance of Topt has been demonstrated with investigations into the impacts of climate change where warming trends in both freshwater and marine environments are already exposing fishes to adverse temperatures. In the marine environment, Pörtner and Knust (2007) found that temperatures exceeding the Topt of eelpout (Zoarces viviparus) in the North and Baltic Seas caused a northern distribution shift and reduced abundance. In freshwater, temperatures in the Fraser River, British Columbia, Canada have on occasions already exceed the Topt of migrating sockeye salmon and have been linked to upstream migratory failure (Farrell et al. 2008). Thus, measures of Topt are potentially useful in predicting when environmental temperature may negatively impact a species.  1.3 Limits to Maximum Oxygen Delivery   It is readily apparent when examining a Fry curve alongside maximum and resting ! ˙ M O2 that Topt for aerobic scope is defined by the plateau of maximum ! ˙ M O2 because resting ! ˙ M O2 continues to increase exponentially above Topt (Figure 1.1A). Fry and Hart (1948) commented that the plateau in maximum ! ˙ M O2  was likely due to a deficiency in oxygen transport. A modern synthesis of this hypothesis is the concept of Oxygen and Capacity-Limited Thermal Tolerance (Pörtner 2001, 2010), whereby thermal tolerance is ultimately set by the maximum capacity of the ventilatory and circulatory systems to deliver oxygen to the tissues. Given the likely fitness consequences of reduced aerobic  7 scope, there have been investigations (Eliason 2011; Steinhausen et al. 2008) to elucidate the mechanistic basis for the failure of maximum ! ˙ M O2 to continue increasing above Topt. Identifying potential mechanisms to explain this maximum ! ˙ M O2  limitation requires understanding how oxygen is moved through the cardiorespiratory oxygen cascade, how fish increase oxygen delivery to meet the increased oxygen requirements of activity, and how active fish support the additive oxygen demands of warming temperature.  Oxygenated water flows nearly continuously across the gills of the fish by the combined action of the buccal and opercular pumps. Oxygen diffuses from the water, across the secondary gill lamellae and into the lamellar capillaries where it binds to hemoglobin in red blood cells. Oxygen delivery to and across the gills is governed by ventilation rate and volume, diffusion distance, surface area, gill perfusion and the diffusion gradient for oxygen. Blood is then pumped by the heart from the gills, through the circulatory system, and delivered to the tissue capillaries. Convection of oxygen through the circulation is governed by cardiac output ( ! ˙ Q ) and hemoglobin concentration. At the tissues, the diffusion gradient between the blood and the tissues favors oxygen diffusion from hemoglobin to the cell’s mitochondria. Again, oxygen delivery to the tissues is governed by capillary perfusion, diffusion distance, surface area, and the partial pressure gradient. Partially deoxygenated venous blood then returns to the heart, which is the last organ in the circulatory system to receive oxygen before the venous blood is pumped to the gills and re-oxygenated.  During activity, the ! ˙ M O2 of active fish can be 15x that of resting fish (Brett 1964, 1965) with the swimming muscles accounting for nearly all of the increased oxygen consumption (Randall and Daxboeck 1982). To meet this increase in oxygen demand,  8 fish invoke a suite of physiological mechanisms to increase oxygen transport to the skeletal muscle mitochondria. Fish increase oxygen delivery to the gills by increasing ventilation rate and volume and may even ram ventilate if sufficient swimming velocities are obtained (Steffensen 1985). To increase oxygen uptake across the gills, fish increase lamellae perfusion (Booth 1979; Farrell et al. 1979). Oxygen diffusion from the water to the blood is also aided by a lower venous partial pressure of oxygen (PvO2) from increased tissue oxygen extraction that increases the diffusion gradient (Farrell and Clutterham 2003; Kiceniuk and Jones 1977). Oxygen convection in the circulatory system is increased via ! ˙ Q through a combination of increased heart rate (fH) and stroke volume (Vs) (Brett 1971; Farrell and Jones 1992; Kiceniuk and Jones 1977). For salmonids, ! ˙ Q is primarily increased via Vs during activity. Release of additional red blood cells from the spleen will also increase oxygen convection (Gallaugher et al. 1992). Finally, increased capillary perfusion at the tissues will increase the surface area for diffusion while potential Bohr and Root effects that decrease the oxygen-affinity of hemoglobin will increase tissue oxygen removal from the blood at the capillaries. Thus, at a fixed temperature, fish have the capacity to greatly increase oxygen supply when the demands of activity necessitate. For salmon at least, it is thought that the finite capacity for oxygen delivery is expressed during maximal aerobic swimming. Increased tissue oxygen demand with warming is supported by thermodynamic effects that increase the capacity of the oxygen delivery mechanisms and maximum ! ˙ M O2. For example, rates of muscle contraction and relaxation increase with temperature (Randall and Brauner 1991) allowing fish to further increase ventilation rate and ! ˙ Q (Heath and Hughes 1973; Steinhausen et al. 2008). Elevated temperature will also right-  9 shift the oxyhemoglobin dissociation curve (Jensen et al. 1998) enabling faster and greater oxygen unloading at the tissues. In addition, oxygen diffusion will occur exponentially faster at higher temperatures, although this effect is small for biologically relevant temperatures (Dejours 1981). As a result, the maximum ! ˙ M O2 that can be obtained increases with temperature. However, the plateau of maximum ! ˙ M O2 past Topt clearly demonstrates that temperature-induced increases in maximal oxygen delivery capacity eventually become limited. The temperature of this limitation is also well below the maximum temperature tolerable by resting fish. Thus, a weak link in the steps of the cardiorespiratory oxygen cascade develops when active fish are warmed beyond Topt. Data regarding the mechanistic underpinnings for this limitation at Topt are most readily available for salmon. Previous studies (Eliason 2011; Steinhausen et al. 2008) investigated the basis for this limitation at four of the five steps in the cardiorespiratory oxygen cascade: oxygen delivery (i) and uptake (ii) at the gills, oxygen convection by the circulatory system (iii), and oxygen diffusion at the tissues (iv). The role of mitochondrial respiration (v) in limiting maximum ! ˙ M O2  above Topt has not been investigated in salmon, although some studies have investigated changes in mitochondrial properties with temperature acclimation (Bouchard and Guderley 2003; St-Pierre et al. 1998). However, Pörtner (2001) suggested the thermal sensitivity of mitochondria is less than that of the more complex ventilatory and circulatory systems. Mitochondrial respiration rates may be maintained above Topt and would not contribute to a maximum ! ˙ M O2 limitation, although evidence to support this is lacking. The following sections will therefore outline the current knowledge regarding the limits to oxygen delivery at high temperatures in  10 three areas (gills, circulation, tissues) covering steps i-iv of oxygen cascade and discuss the contribution of each of these areas to the maximum ! ˙ M O2 limitation at Topt in salmon.  1.3.1 Oxygen Delivery and Uptake at the Gills   Early studies that observed a collapse in fish swimming performance at elevated temperatures speculated that the primary factor in this collapse was the reduced oxygen concentration in water (Brett 1971). This suggestion was based on the fact that the solubility of oxygen in water decreases by ~2% for every 1ºC increase in temperature (Dejours 1981). Decreased oxygen availability could then lead to a diffusion limitation for oxygen across the gill lamellae that would reduce arterial partial pressure (PaO2) and blood oxygen content (CaO2). In support of this hypothesis, Heath and Hughes (1973) observed decreased CaO2 with temperature in acutely warmed resting rainbow trout (Oncorhynchus mykiss). However, data from active fish obtained in recent studies support neither the speculations of Brett (1971) nor the findings of Heath and Hughes (1973). For example, Steinhausen et al. (2008) found that both PaO2 and CaO2 were unchanged during an acute temperature increase above Topt in sockeye salmon swimming at ~75% of maximal aerobic capacity. Further, investigation by Eliason (2011) with maximally swimming sockeye salmon also showed no decrease in PaO2 or CaO2 at temperatures above known Topt values. These more recent data suggest that the earlier findings on resting rainbow trout of Heath and Hughes (1973) were likely due to a ventilatory limitation. Thus in the absence of a ventilatory limitation, salmon appear to be able to supply sufficient oxygen delivery to the gills to maintain PaO2 and CaO2 above Topt.  11 1.3.2 Oxygen Convection by the Circulatory System  Convection of oxygen through the circulatory system is a product of the CaO2 and the ! ˙ Q of the heart, which itself is a product of Vs and fH. When fish are warmed, increases in ! ˙ Q occur entirely through increases in fH. This has been demonstrated in resting and swimming salmon (Brodeur et al. 2001; Clark et al. 2008; Gamperl et al. 2011; Sandblom and Axelsson 2007; Steinhausen et al. 2008) and also appears to be a general trend among non-salmonid fishes (Gollock et al. 2006; Mendonça and Gamperl 2010; Stevens et al. 1972). The reliance on fH for increases in ! ˙ Q is likely due to the temperature- dependent frequency of the heart’s pacemaker cells (Randall 1970), which will contribute to exponentially increasing both resting and maximum fH, at least at lower temperatures. Thermodynamic effects on fH should therefore maintain the scope for increasing fH during warming. However, fH has a finite capacity to increase (Farrell 1991) and would be expected to become limited as it approached this upper threshold, with maximum fH becoming limited at a lower temperature than resting fH. Maximum fH may increase slightly above this limitation, but fH scope will decline since resting fH continues to increase exponentially. Accordingly, a limitation on maximum fH would be expected to restrict oxygen convection through the circulatory system and limit maximum ! ˙ M O2. Indeed, the data of Steinhausen et al. (2008) and Eliason (2011) demonstrate that maximum fH in swimming sockeye salmon became limited and failed to increase exponentially once a temperature that coincided with the plateau in maximum ! ˙ M O2 had been reached. Further, Steinhausen et al. (2008) and Eliason (2011) found fH scope declined past Topt. Thus, there is solid evidence that a limitation on maximum fH plays a  12 central role in defining Topt by reducing fH scope, limiting oxygen convection through the circulatory system and causing a decline in aerobic scope.  1.3.3 Oxygen Delivery at the Tissues  Whole-organism tissue oxygen utilization is calculated by the Fick equation ( ! ˙ M O2 = ! ˙ Q x A-VO2), which is the product of the difference in oxygen content between arterial and venous blood (Ca-vO2) and ! ˙ Q . As previously discussed, blood is fully saturated with oxygen when leaving the gills and CaO2 does not decrease with temperature. Warming increases oxygen consumption at the tissues, decreasing CvO2 and increasing Ca-vO2. Thus, PvO2 should steadily decrease in the absence of a diffusion limitation. If an oxygen delivery limitation to the tissues did arise at Topt, for instance due to inadequate capillarization, venous oxygen parameters would remain constant and Ca-vO2 would plateau. Steinhausen et al. (2008) observed a general trend towards a steadily increasing Ca-vO2 and decreasing CvO2 with an acute temperature increase in swimming sockeye salmon, although neither changes in Ca-vO2 nor CvO2 reached statistical significance. These results suggest no diffusion limitation was present. However, Steinhausen et al. also found PvO2 did not change with warming, supporting the possibility of a diffusion limitation. The findings of Eliason (2011) with maximally swum sockeye salmon did not support a tissue limitation as both PvO2 and CvO2 decreased past Topt. Together, these studies demonstrate there is still a reasonable degree of uncertainty regarding the possibility of a diffusion limitation developing at the tissues of active salmon. However, the gradual onset of the observed changes suggests a diffusion  13 limitation would likely only develop at temperatures higher than Topt. Further, active salmon may still have some capacity to increase venous oxygen extraction above Topt, apparent as the more rapid decline of fH scope in comparison to aerobic scope.  1.3.4 Topt and Maximum fH  To summarize, the temperature at which maximum ! ˙ M O2 plateaus ultimately defines the Topt for aerobic scope because resting ! ˙ M O2 continues to increase exponentially above Topt. The current evidence from salmon indicates that the plateau of maximum ! ˙ M O2 at Topt is triggered by the failure of maximum fH to maintain an exponential increase with increasing temperature. Therefore, the temperature at which maximum fH becomes limited may be a useful predictor of the Topt for aerobic scope. The limitation on maximum fH has also been proposed to initiate a series of events that lead to the death of exercising salmon at high temperatures (Farrell et al. 2009). The maximum fH limitation at Topt, which causes a decline in fH scope and limits ! ˙ Q , places a perfusion limitation on the delivery of oxygen to the tissues and then eventually a diffusion limitation. Inadequately perfused tissues must switch to anaerobic metabolism, causing metabolic acidosis and an adverse venous environment for the cardiac muscle and the eventual collapse of cardiac function apparent as cardiac arrhythmia (Clark et al. 2008). The death of the salmon follows soon after.    14 1.4 Expediting Topt Estimations   Relatively few studies of salmon or other fishes have incorporated Topt measurements. The lack of Topt application is not so much because the results are uninformative, but rather because it is time consuming to determine Topt for aerobic scope with an exacting methodology. The length of time required to estimate Topt is therefore the most obvious shortcoming of the aerobic scope methodology. The association of Topt and a maximum fH limitation may provide an analytical method for expedited Topt estimates. Although an exponential increase is typical of biological rates, divergence from this relationship can occur if biological systems are observed over a wide enough temperature range. Crozier (1926) was one of the first to demonstrate a departure from the exponential increase in a biological rate function and interestingly, demonstrated this with the fH of larval fish. Crozier used Arrhenius plots (see Section 1.1) to describe a discontinuity or ‘break’ in the increase of fH at a certain temperature. Crozier observed that the normally linear increase in fH that occurs on an Arrhenius plot failed to maintain a constant slope throughout the measured temperature range (Figure 1.1C). The temperature at which a break in an Arrhenius plot occurs is known as the Arrhenius break temperature (ABT). These century-old observations highlight an analytical method that may allow a straightforward approach to estimating Topt of salmon. Given that Topt coincides with maximum fH failing to increase exponentially with temperature, the ABT for maximum fH could act as a surrogate measure of Topt. Further, this approach may allow Topt estimates to be expedited if the ABT for maximum fH can be  15 rapidly determined. Understanding why an expedited methodology is desirable, however, requires a familiarity with the current method for estimating Topt for aerobic scope.  1.4.1 Estimating Topt for Aerobic Scope  Estimating Topt for aerobic scope for a fish species requires measurements of resting and maximum ! ˙ M O2 across a range of tolerable temperatures in order to generate a Fry curve. Three approaches are used in order to obtain measurements across a suitable temperature range: fish can be acclimated to specific test temperatures over the long- (weeks) (Zeng et al. 2010) or short-term (days) (Eliason et al. 2011); be tested at ambient temperatures (Lee et al. 2003); or receive an acute (hours) temperature increase to the test temperature from a common holding temperature (Clark et al. 2008; Eliason et al. 2011). Once a fish is at the appropriate temperature, resting ! ˙ M O2 can be obtained using intermittent flow respirometry (Ege and Krogh 1914; Steffensen 1989). Inevitably, handling of the fish is required prior to any resting measurements and fish must be allowed to recover from handling stress and the associated metabolic perturbations (Davis and Schreck 1997). Approximately 12 h has emerged as a standard minimum recovery time, although slightly shorter times are permissible (Clark et al. 2008). Further, if diurnal fluctuations in activity are to be considered, resting ! ˙ M O2 should be continuously monitored over a minimum 24 h period (Steffensen 1989). Generating maximum ! ˙ M O2 requires forcing a fish to actively swim and can be accomplished with exhaustive exercise (Reidy et al. 1995; Scarabello et al. 1991, 1992) or the use of a swim tunnel (Brett 1964; Farrell et al. 2003) and a standardized exercise  16 protocol, such as the critical swimming speed (Ucrit) test (Jain et al. 1997). Both of these techniques must also be combined with respirometry. Exhaustive exercise protocols are the most rapid exercise method and can generate maximum ! ˙ M O2 with a brief period (minutes) of manual chasing (Scarabello et al. 1991, 1992). Minimizing the time required for this protocol by allowing the minimum handling recovery time and using an acute temperature increase combined with exhaustive exercise, would permit resting and maximum ! ˙ M O2 values for a single fish to be obtained in ~24 h. However, resting and maximum ! ˙ M O2 measurements must be repeated on a sufficient number of fish at a minimum of five temperatures to generate a suitable Fry curve and permit a Topt estimation. Taken together, generating a complete Fry curve necessitates a minimum 3-4 week period of continuous experimentation.  1.4.2 The fH Method for Determining Topt   Using maximum fH as an alternative to ! ˙ M O2 measurements requires consideration of the methodology, given that the primary goal of any alternative technique should be to reduce experimental duration. For example, simply measuring fH in a maximally swimming fish during an acute temperature increase would provide no advantages in comparison to ! ˙ M O2 measurements. Each fish would have to be anaesthetized and undergo surgical procedures to either attach or implant (Clark and Farrell 2011) electrodes. Wireless fH monitoring is possible (Altimiras and Larsen 2000; Rommel 1973), but is highly susceptible to skeletal muscle interference during activity. In addition, a conservative rate of temperature change (2°C h-1; Steinhausen et al. 2008)  17 would be necessary to allow fish to reach a steady state at each test temperature while a number of temperatures would have to be tested for a wide temperature range. Variations of this protocol are possible, but the necessity of an exercise component to generate maximum fH and the moderate rates of temperature increase required for active fish prevent experimental duration from being significantly reduced.  In lieu of whole-organism activity to stimulate increases in fH, the cardiac control mechanisms in salmon present an opportunity to investigate whether pharmacological stimulation of maximum fH is possible. The salmon heart has an intrinsic rhythm set by the pacemaker cells located in the sinoatrial node. This cardiac rhythm is due to ‘funny’ Na+ channels that give pacemaker cells an unstable membrane potential that steadily depolarizes until an action potential threshold is reached. Modulation of the pacemaker rhythm occurs via two mechanisms: cholinergic inhibition and adrenergic excitation. The vagal nerve directly innervates the sinoatrial node and releases acetylcholine that interacts with muscarinic receptors on the pacemaker cells. The binding of acetylcholine triggers a signaling cascade that opens additional ion channels, hyperpolarizing the pacemaker cells, and decreasing action potential frequency and fH (bradycardia). In contrast, adrenaline and noradrenaline released from the chromaffin cells in the head kidney moves through the circulatory system, binds to !-adrenoreceptors on the heart, triggering a cascade that increases the rate and frequency of depolarization and elevates fH (tachycardia). In both cases, pharmacological agents are available that either block or stimulate these pathways. Atropine is a muscarinic antagonist that will block vagal tone and increase fH. Isoproterenol is a !-agonist that will stimulate !-receptors and further increase fH. Both atropine and isoproterenol have been used extensively to elucidate  18 cardiac control mechanisms in fishes (Altimiras et al. 1997; Axelsson and Farrell 1993; Randall et al. 1967; Stecyk and Farrell 2006; Wood et al. 1979). However, it is unknown whether pharmacological stimulation alone is sufficient to produce an increase in fH equivalent to that observed in exercising fish.  If maximum fH can be achieved with pharmacological stimulation, ABT analysis could be performed on individual fish. Each fish could be warmed and maximum fH data collected at numerous temperatures across a wide temperature range. This would permit sufficient fH data to be collected from an individual for ABT analysis (that requires a minimum number of temperatures on either side of the ‘break’). Pharmacological stimulation would also alleviate concerns regarding the effects of prolonged exercise on cardiac performance (e.g. Steinhausen et al. 2008). The absence of the required exercise might also allow the fish to be restrained or possibly anaesthetized to permit easier fH detection. Increased rates of temperature change may also be possible in anaesthetized fish. Chapter 2 of this thesis explored these possibilities with the overarching goal of expediting Topt measurements in salmon.  1.5 Study Species  Pacific salmon are the focus of this study because of the well-defined mechanistic basis for the collapse of aerobic scope above Topt. Of the five Pacific salmon species, I selected juvenile coho salmon because the fry life stage of coho salmon is characterized by a fluvial residency period of one to two years prior to seaward migration (Quinn 2005) and is one of the longest life history periods within Oncorhynchus. This is important,  19 given that thermal tolerance can vary with life stage (Pörtner and Farrell 2008). Using juvenile coho salmon would also allow for the potential application of Topt estimates to assess local coho salmon habitat (see Section 1.7). An important consideration with the use of juvenile coho salmon is that the evidence supporting maximum fH as the primary factor determining Topt has been derived entirely from studies of adult salmon. However, the adult life stage is not suited to the long-term freshwater holding necessary for this investigation due to the semelparous life cycle of salmon. In contrast, while juvenile coho salmon are amendable to prolonged holding there are no data from juvenile salmon to support the role of fH in setting Topt. Thus, the presence of a Topt-maximum fH relationship must be demonstrated prior to using juvenile coho salmon to investigate the possibility of expediting Topt estimates.  1.6 Chapter 2 Objectives  The first objective of Chapter 2 was to demonstrate that a limitation on maximum fH occurs at Topt for aerobic scope in juvenile coho salmon. This objective builds on the evidence from adult salmon that a limitation on maximum fH limits maximum ! ˙ M O2 and aerobic scope. I hypothesized that juvenile coho salmon will exhibit the same signs of cardiac impairment above Topt as adult salmon and that the Arrhenius break temperature for maximum fH will coincide with Topt for aerobic scope. The second objective in Chapter 2 was to develop a rapid assay to define Topt using pharmacological stimulation of fH in lieu of exercise. This possibility was investigated using anaesthetized juvenile coho salmon. First, I hypothesized that pharmacological  20 stimulation of maximum fH in anaesthetized coho salmon will produce an equivalent maximum fH to that seen in exercising salmon. Secondly, I hypothesized that during acute warming, the Arrhenius break temperature in maximum fH will occur at Topt.  1.7 Applying Topt Measurements  Application of Topt requires information on the temperatures encountered by a species. For fishes, this information is most easily obtained by measuring water temperature directly in a species’ habitat. Such data can then be combined with Topt estimates to assess, for instance, how current or future temperature regimes may impact fitness and survival. Examples of Topt application are most abundant in the Fraser River, British Columbia, Canada where over 60 years of historic temperature monitoring, detailed sockeye salmon escapement enumeration, and Topt estimates from several upstream migrating sockeye salmon populations have allowed complex Topt analyses. Farrell et al. (2008) correlated historically encountered river migration temperatures with Topt estimates to suggest population-level Topt adaptation in sockeye salmon based on the respirometry studies of Lee et al. (2003). Further, this association was used to retroactively explain the failed upstream migration of a sockeye salmon population during a year of uncharacteristically warm river temperatures. Eliason et al. (2011) expanded this analysis and confirmed local Topt adaptation and suggested climate warming would have population-specific effects on sockeye salmon. Hague et al. (2011) used climate change scenarios to model future Fraser River temperature and predicted  21 population-specific climate effects on upstream migratory success. However, outside of the upriver migration of Fraser River sockeye salmon, no other studies have applied Topt estimates to determine how thermal regimes may be affecting other salmon species. Upriver migration aside, the freshwater riverine environment still remains a priority for application of Topt given most salmon use freshwater habitat to some degree for juvenile rearing (Quinn 2005). The complex interaction of numerous abiotic and biotic factors at the fry life stage does not allow the same straightforward link between Topt and fitness as is possible with adult salmon migration, the dichotomous nature of which results in likely reproduction if migration is successful, or zero lifetime fitness if unsuccessful (Farrell et al. 2008). Regardless, juvenile salmon rearing at temperatures above Topt can be expected to have reduced fitness, given that sub-optimal temperatures limit swimming ability (e.g. Gibson and Fry 1954; see Section 1.3) and in turn affect foraging, growth, competitive ability, and predator avoidance. Consequently, species such as coho salmon, that are dependent on freshwater habitat as a nursery for emergent fry and are limited in their freshwater abundance by habitat availability (Quinn 2005), can see reduced overall abundance if temperatures exceed Topt in the freshwater environment. Therefore, even though a direct link to lifetime fitness cannot be made, there is still significant merit to combining Topt estimates with temperature data for rivers and streams where species such as coho salmon are present. Recording temperature in waterways has been made dramatically easier by the advent of inexpensive temperature loggers (Dunham et al. 2005). As a result, detailed measures of temperature can be used to record temperature regimes of individual streams and describe temperature dynamics. Combining such temperature information with a  22 rapid method to estimate Topt may provide species- and population- specific information on salmon habitat thermal quality. Further, temperature dynamics can be used to identify areas important to maintaining temperature conditions in streams. Such information would be useful to managers hoping to conserve salmon populations threatened by warming due to climate change or other anthropogenic disturbances.  1.8 Chapter 3 and Chapter 4 Objectives  The first objective of Chapter 3 was to describe the temperature conditions in two local, coho salmon-bearing waterways. To achieve this, I recorded high-resolution stream temperature data throughout the two study systems. A second objective of Chapter 3 was to examine the temperature dynamics within these streams to determine if particular stream reaches or habitat features acted to maintain or degrade the thermal integrity of waterways. Chapter 4 then discusses the potential for rapid Topt estimations from juvenile coho salmon to be combined with these stream temperature data and summarizes the findings of this thesis.   23   Figure 1.1. (A). The exponential increase in the rate of chemical and biological reactions with temperature as quantified by the Arrhenius equation ( ! k = Ae-E" /RT ). (B) Transformation of (A) into an Arrhenius plot using the linear version of the Arrhenius equation ( ! ln (k) = -E" /RT +  ln A). (C) Arrhenius plot showing the Arrhenius break temperature (*) as a discontinuity in the increase of the reaction rate with temperature.   24   Figure 1.2. (A) Increases in resting (green line) and maximum (blue line) oxygen consumption of the goldfish (Carassius auratus) with temperature. (B) The aerobic scope (maximum minus resting oxygen consumption) and optimal temperature for aerobic scope (Topt). Modified from Fry (1947).  25 CHAPTER 2: USING MAXMIMUM HEART RATE AS A RAPID SCREENING TOOL TO ESTIMATE OPTIMUM TEMPERATURE FOR AEROBIC SCOPE IN SALMON*  2.1 Introduction  It is well established that fishes have optimal temperatures for performance. Important rate functions such as ! ˙ M O2, growth and swimming ability are maximal at a species-specific Topt (Brett 1971; Elliott and Elliott 2010; Gibson and Fry 1954; Selong et al. 2001). The sharp decline in the capacity of fish to perform above Topt is proposed to be the result of an oxygen limitation brought about by the temperature dependence of aerobic scope (Pörtner 2001, 2010; Pörtner and Farrell 2008). Originally conceived by Fry (1947), aerobic scope is the difference between resting and maximum ! ˙ M O2 and measures the capacity of a fish to perform activity above basic requirements. A Fry curve for aerobic scope (Farrell 2009) describes the relationship between aerobic scope and temperature, with the greatest aerobic scope occurring at Topt. Above Topt, aerobic scope decreases because maximum ! ˙ M O2fails to keep pace with resting ! ˙ M O2 as temperature increases. With less available oxygen above Topt, reduced growth and abundance and increased mortality are possible (Farrell et al. 2008; Pörtner and Knust 2007). Measurements of Topt for aerobic scope are already proving to be an effective tool in predicting the affects of seasonal temperature change and future climate change  * A version of this Chapter has been published. M.T. Casselman, K. Anttila, and A.P. Farrell. 2012. Using maximum heart rate as a rapid screening tool to determine the optimal temperature for aerobic scope in Pacific salmon Oncorhynchus spp. Journal of Fish Biology. 80(2): 358-377.  26 scenarios on different fish species. Present applications of Topt estimates include the shifting distribution and abundance of eelpout and Atlantic cod (Gadus morhua) in the North Sea (Pörtner et al. 2001; Pörtner and Knust 2007), the impacts of climate change and ocean acidification on coral reef fishes from the Great Barrier Reef, Australia (Munday et al. 2009), the effects of warming on southern catfish (Silurus meridionalis) in the Yangtze and Jialing Rivers, China (Zeng et al. 2010), river migration of coho salmon and sockeye salmon in the Fraser River, Canada (Lee et al. 2003), and even local adaptation of sockeye salmon (Eliason et al. 2011). Despite Topt measurements being broadly applied to fish biology, the more than 25,000 fish species, along with the countless combinations of abiotic and biotic factors that can influence Topt (Pörtner 2010), create a need for a high-throughput method to estimate Topt. Generating a single Fry curve normally takes weeks, as ! ˙ M O2 measurements are required over a range of temperatures in resting and maximally swimming fish while ensuring fish are well recovered from handling (Steffensen 1989). Maximum ! ˙ M O2 can be achieved with a Ucrit test (Jain et al. 1997). However, somewhat faster Topt estimates are possible if maximum ! ˙ M O2 is measured following exhaustive exercise (Reidy et al. 1995; Zeng et al. 2010). An alternative and potentially even faster approach is to estimate Topt for maximum ! ˙ M O2 using maximum fH as a surrogate. The foundation for this idea is the original observation by Fry (1947) that maximum fH was limited around a purported Topt for brook trout (Salvelinus fontinalis) and a later suggestion by Brett (1964) that reduced performance above Topt could be due to a cardiac insufficiency in juvenile sockeye salmon, an idea recently confirmed for adults (Steinhausen et al. 2008). Steinhausen et al.  27 (2008) found that the upper limit for maximum ! ˙ M O2 and aerobic scope in exercising sockeye salmon was triggered by fH failing to increase significantly above Topt, thereby preventing further increases in ! ˙ Q with warming. Moreover, it is now clear that temperature-related increases in ! ˙ Q in both resting and active fishes are mediated entirely through increased fH (Brodeur et al. 2001; Clark et al. 2008; Cooke et al. 2003; Gamperl et al. 2011; Gollock et al. 2006; Mendonça and Gamperl 2010; Sandblom and Axelsson 2007; Steinhausen et al. 2008). Thus, fH is the primary variable that responds to a need for increased internal oxygen convection during warming. In view of the central role of fH in determining a fish’s response to warming, as particularly well demonstrated in salmon, the present study was carried out to assess the possibility that a limitation on maximum fH in coho salmon can be used to reliably estimate Topt in an expedited manner. This would eliminate the need for direct but more prolonged ! ˙ M O2 measurements necessary to generate a Fry curve. As a control, aerobic scope was measured by conventional means over a range of temperatures encompassing Topt for coho salmon (Brett et al. 1958; Edsall et al. 1999; Griffiths and Alderdice 1972) while additionally measuring fH as a confirmation that maximum fH was limited at the Topt for aerobic scope. To expedite Topt estimation and eliminate the problem of activity state altering fH, fish were anaesthetized during warming and maximum fH achieved through pharmacological means. Because anaesthetics can directly affect cardiac function (Cotter and Rodnick 2006; Fredricks et al. 1993; Hill et al. 2002; Hill and Forster 2004; Ryan et al. 1993), a comparison was made between tricaine methanesulfonate (MS-222), the most commonly used fish anaesthetic (Carter et al. 2011), and clove oil (Anderson et al. 1997).  28 2.2 Materials and Methods  2.2.1 Experimental Animals and Care  Juvenile coho salmon were obtained from the Seymour River Hatchery (North Vancouver, BC, Canada) and transported to the University of British Columbia (Vancouver, BC, Canada) in March 2010. Fish were held in a 1,000 l flow-through holding tank supplied with de-chlorinated municipal tap water and kept under a 12 h:12 h light:dark photoperiod while fed to satiation daily (BioClark’s Fry, Bio-Oregon Inc.; www.bio-oregon.com). Water temperature increased seasonally from 8ºC to 10°C at which point a chilling unit (BHL-1089-3, Frigid Units; www.frigidunits.com) maintained water temperature at 10.0ºC (range ± 0.5°C) for the duration of the experiment. All fish procedures were approved by the University of British Columbia Committee on Animal Care in accordance with the Canadian Council on Animal Care (A10-0236). Three weeks prior to experimentation fish were anaesthetized (100 mg l-1 MS-222 buffered with 100 mg l-1 NaHCO3, Sigma-Aldrich; www.sigmaaldrich.com) and individually tagged with a non-absorbable monofilament suture caudal to the dorsal fin. Tags showed no sign of rejection.  2.2.2 Aerobic Scope and fH Measurements in Unanaesthetized Fish  Resting and maximum values for ! ˙ M O2 and fH for a single group of fish (N = 12) were individually measured at each of five test temperatures (13, 15, 17, 19, and 21°C).  29 These measurements were completed over a 3-week period, with four fish measured daily as follows. After a 24 h fasting period, fish were individually transferred from the holding tank to one of four separate, custom-made respirometers (~450 ml) in two separate apparatus. Water temperature (10°C) in the respirometers was controlled with in-line, re- circulating chillers (F32-MD, Julabo Labortechnik GmbH; www.julabo.de; 3016D, Fisher Scientific; www.fishersci.ca). Fish were left overnight for a minimum of 12 h to adjust to the respirometer. Each experiment began with water temperature being increased at a rate of 2°C h-1 up to the test temperature, which was then held for a period of 1 h before making triplicate measurements of resting ! ˙ M O2 and fH over a 20 to 30 min period. Afterwards, one fish at a time was removed from its respirometer and placed in a 60 cm diameter circular tank containing aerated water at the test temperature, where it was chased to exhaustion over a 5 min period by combining hand chasing and tail pinches. Depending on the test temperature, fish generally displayed burst swimming for the first 30 s to 1 min, followed by 2 to 3 min of slower swimming with infrequent burst activity, and finally 1 to 2 min of only slow swimming until exhaustion, when the fish became refractory to touch regardless of temperature. The fish was immediately returned to the respirometer and the peak values for ! ˙ M O2 and fH that were recorded during the 45 to 60 min recovery period were taken as the maximum values for that test temperature. Aerobic scope and scope for fH were calculated as the difference between maximum and resting values. Individual Topt values were assigned to the test temperature at which aerobic scope was the greatest. After the recovery period, swimming activity had partially returned and the fish was placed in a recovery tank where temperature was slowly reduced to the acclimation temperature over a 30 to 80 min period, depending on the test  30 temperature. The fish was then weighed and returned to the holding tank. The above procedure was then performed on the remaining fish with the experimental start for two fish staggered by 2 to 5 h. Water temperature was matched to 0.1ºC between the two test systems using a Fisherbrand® Traceable® NIST-certified digital thermometer with a Type-K thermocouple (Fisher Scientific).  Each fish recovered for a minimum of 3 days in between temperature tests. The order of test temperatures was randomized, as was the order fish were tested on a given day/temperature.  2.2.3 ! ˙ M O2 Measurements  ! ˙ M O2 was measured using intermittent-flow respirometry, recording the rate of decrease in water oxygen saturation using a R-type oxygen probe connected to a NeoFox sensor system (Ocean Optics; www.oceanoptics.com) that was calibrated daily at the test temperature. The probe was inserted through the top of the respirometer and monitored changes in oxygen saturation directly in the chamber. Probe output was recorded on a laptop computer that ran the NeoFox Viewer software (Ocean Optics). Water was circulated in the respirometer during ! ˙ M O2 readings using a magnetic stir-bar, which was separated from the fish by a 20 gauge stainless-steel mesh. Each ! ˙ M O2 measurement involved at least a 10% decrease in oxygen saturation, which took 2 to 10 min depending on temperature as well as state of the fish. Water oxygen saturation never decreased below 80% and was quickly (~1 min) restored to !95% by water flow through the respirometer (~250 ml min-1) between recordings, during which time the stir-bar was turned off to reduce interference with the fH recording.  31 2.2.4 fH Measurements  Bioelectric signals from the heart propagate through water allowing the electrocardiogram (ECG) to be detected wirelessly using submerged electrodes (Altimiras and Larsen 2000). Thus, non-invasive fH recordings were obtained by using the stainless-steel mesh beneath the fish as an electrode grid, with a reference grid above the fish (Johnsson et al. 2001). Both electrodes extended to the interior edges of the respirometer and a small hole was made in the upper grid to allow the insertion of the oxygen probe. The electrodes were connected to a Grass P55 AC amplifier (Astro-Med Inc.; www.astro-med.ca) that amplified (1,000x to 10,000x) and filtered (60 Hz line filter; low-pass: 10 to 30 Hz; high-pass: 0.1 to 0.3 kHz) the ECG signal to reduce skeletal muscle activity and eliminate ambient electrical interference. Output from the amplifier was processed and recorded at a 1 kHz sampling frequency using a Powerlab ML870 data acquisition unit and LabChart software (AD Instruments; www.adinstruments.com). The R-peaks of the QRS complex were manually identified and fH calculated from the mean R-R interval of a continuous beats series (N = 15 heart beats per reading). Proper detection of the ECG required fish to maintain a vertical dorso-ventral orientation between the two electrodes. However, not all fish would necessarily maintain this orientation after exhaustive exercise. Thus, while resting fH values were obtained for all fish at each test temperature, some of the post-exercise fH values were lost at each temperature.    32 2.2.5 Maximum fH in Anaesthetized Fish  Anaesthetized juvenile coho salmon from the same population as those used for the aerobic scope trials were tested with pharmacological agents to elicit maximum fH. Individual fish were anaesthetized using either 30 ppm clove oil (1:10 clove oil:ethanol) or 75 ppm buffered MS-222 and transferred to the experimental apparatus (see below) where they were maintained in an anaesthetized state at 10ºC for 1 h prior to experimentation while monitoring fH. An anaesthetized state was maintained using either 30 ppm clove oil (N = 10), 15 ppm clove oil (N = 12), or 50 ppm MS-222 (N = 12). The 15 ppm clove oil and 50 ppm MS-222 represented the minimum concentrations for maintained anaesthesia for the duration of the temperature trial. A control group (N = 8) without pharmacological agents was tested using 30 ppm clove oil. Pharmacological stimulation of maximal fH was achieved via sequential intraperitoneal injections of 1.2 mg kg-1 atropine sulfate (Sigma-Aldrich) to block vagal tone and 4 µg kg-1 isoproterenol (Sigma-Aldrich) to stimulate cardiac adrenergic ß- receptors. Atropine was prepared daily and isoproterenol prepared immediately prior to use. Both agents were dissolved in 0.9% NaCl. Control fish received only saline injections. Each injection was followed by a 15 min equilibration period. Preliminary trials showed that doses used were sufficient to produce a maximal fH response as repeated injections of either atropine or isoproterenol did not further increase fH. In addition, these trials revealed the chronotropic and heart rate variability effects of atropine persisted >8 h post-injection, as reported in other studies (Altimiras et al. 1997).  33 Isoproterenol injections in the absence of atropine were also found to have a chronotropic effect that persisted >1.5 h. A step-wise temperature increase was applied in 1°C increments at a heating rate of 10°C h-1. A group of size-matched fish was tested to ensure temperature changes in the external bath were near synchronous with the internal body temperature and fH (Figure 2.1A). Preliminary experiments with heating rates of 2ºC h-1, 5ºC h-1, and 10ºC h-1 showed identical fH changes for 5ºC h-1 and 10ºC h-1 rates, and for 2ºC h-1 the only difference was at upper temperatures when fish had been anaesthetised for >6 h. Thus, the 10ºC h-1 heating rate was adopted for all experiments. Temperature and fH were allowed to briefly stabilize after each 1°C increment (Figure 2.1A), with the fH measurement being taken at the end of this period (N = 15 heart beats per reading). The experimental endpoint was a shift from rhythmic (Figure 2.1B) to arrhythmic fH (Figure 2.1C) at which point the fish was removed from the apparatus, a blood sample (0.2 to 0.5 ml) was withdrawn from the caudal vein into a heparinized syringe, and length and weight measurements were taken. Fish were then euthanized with a blow to the head, the heart excised to measure wet ventricular mass, and sex was noted.  Hematocrit was measured immediately in micro hematocrit capillary tubes spun for 5 min at 5900g.  2.2.6 Experimental Apparatus  The apparatus used to hold anaesthetized fish and measure fH consisted of a short section of PVC pipe cut lengthwise and capped on each end to form a 0.5 l, semicircular holding trough. Fish were orientated with a plastic mesh sling that suspended them in the  34 centre of the pipe and with weighted-foam pads that maintained the fish in a vertical dorso-ventral orientation. Fish were stationary and fully submerged throughout procedures. Custom-made chromel-A electrodes were positioned beneath the ventral surface of the fish underneath the plastic sling to permit non-invasive recording of the ECG. Duplicate troughs were each supplied with water inflow via a pressure-reduced diversion from the hose of a re-circulating chiller. Inflow was separated between a Y- shaped nozzle inserted in the mouth of the fish and outflowed into the troughs. The flow rate for each trough (~150 ml min-1) was sufficient to provide both gill irrigation and water temperature regulation (range ± 0.1ºC). Outflow from the holding troughs returned to the aerated reservoir of the re-circulating chiller. The water total volume of the chiller reservoir and the holding troughs was ~8 l.  2.2.7 Data and Statistical Analysis  Resting and maximum ! ˙ M O2 and fH values were recorded from all fish after an equal time period at a test temperature. There was no significant difference in ! ˙ M O2  or fH values between the first and second pair of fish tested daily (P >0.05; data not shown). Data are presented as mean ± S.E. For aerobic scope trials, combined resting and post- exercise data were not normally distributed and were separated by treatment (resting vs. post-exercise). Data that were normally distributed were tested with a one-way repeated- measures (RM) ANOVA followed by the Holm-Sidak post hoc test. Data that were not normally distributed were assessed with a one-way RM ANOVA on ranks with Dunn’s post hoc test. For the anaesthetized fish, the ABT was calculated for fH following Yeager  35 and Ultsch (1989). Arrhenius break temperature analysis of changes in fH with temperature was performed on: 1) the mean fH of all individuals at a test temperature to determine a single ABT for the group (mean analysis) and, 2) each individual separately to determine individual ABTs and calculate a mean group ABT (individual analysis). Regression slopes were compared with a paired t-test. Differences in fH between anaesthetics and pharmacological treatments were assessed with a two-way RM ANOVA and Holm-Sidak post hoc. Sampling and temperature response variables (arrhythmias, mean breakpoint) were compared with a one-way ANOVA. Statistical analysis was performed using SigmaPlot 11.0 (Systat Software Inc.; www.sigmaplot.com) with significance assessed as P <0.05.  2.3 Results  2.3.1 Effect of Temperature on ! ˙ M O2  and fH in Unanaesthetized Fish  All fish fully recovered from multiple temperature and chasing challenges, except for one fish that died while recovering after exhaustive exercise at 21°C (it also had the lowest aerobic scope of all fish tested). Post-exercise, fish resumed feeding soon after being returned to the holding tank and grew during the 3 week experimental period from an initial mass of 16.9 ± 0.2 g to 18.1 ± 0.3 g by the final exercise trial. There was no relationship (P >0.05; 1-way RM ANOVA) between the number of exercise challenges and resting or post-exercise ! ˙ M O2.  36 Resting ! ˙ M O2  increased significantly (P <0.001; 1-Way RM ANOVA; Holm- Sidak post hoc) with temperature from 3.7 ± 0.2 "mol h-1 g-1 at 13ºC to 7.0 ± 0.3 "mol h-1 g-1 at 21ºC, with a Q10 of 2.3 ± 0.1 (Figure 2.2A). Exhaustive exercise increased resting ! ˙ M O2 by 2.5 to 4.3 fold, depending on temperature. Between 13ºC and 17ºC, maximum post-exercise ! ˙ M O2 increased significantly (P = 0.004; 1-way ANOVA; Holm-Sidak post hoc) with a Q10 of 2.2 ± 0.3 and reached 20.3 ± 1.0 "mol h-1 g-1 at 17ºC. Post-exercise ! ˙ M O2 did not increase above 17ºC and showed signs of declining with increasing temperature since post-exercise ! ˙ M O2 was equivalent for 13ºC and 19ºC. Thus, based on these mean values, Topt for aerobic scope could be assigned to 17ºC (15.5 ± 1.1 "mol h-1 g-1; Figure 2.2C). Using individual Topt values for aerobic scope, Topt was 17.0 ± 0.7ºC (17.3 ± 1.0 "mol h-1 g-1). During the aerobic scope measurements, resting fH at 13ºC was 53.5 ± 2.7 beats min-1 and increased significantly (P <0.05; 1-way RM ANOVA on ranks; Dunn’s post hoc) with temperature to 110.8 ± 3.9 beats min-1 at 21ºC (Figure 2.2B), with a Q10 of 2.6 ± 0.2. Maximum post-exercise fH at 13ºC was 96.2 ± 1.3 beats min-1, a 1.8 fold increase over the resting fH. However, this factorial scope fH for was not maintained with increasing temperature, decreasing to a 1.1 fold increase at 21ºC when maximum fH was 125.8 ± 8.2 beats min-1. Thus, scope for fH was maximal at 17ºC (55.1 ± 6.7 beats min-1), and decreased precipitously at higher temperatures to only 10.0 ± 9.7 beats min-1 at 21ºC (Figure 2.2C) largely because maximum post-exercise fH increased little above 17ºC. Indeed, Q10 values for maximum fH were greatest (1.9) between 13ºC and 15ºC and lowest (1.1) between 19ºC and 21ºC. In contrast, the Q10 for resting fH was maintained near 2.6 between all test temperatures.  37 Arrhenius plots reveal discontinuities in temperature effects on rate functions. Such discontinuities were visible for both resting and maximum fH at ~19ºC and ~17ºC, respectively (Figure 2.3), but proper breakpoint analysis was impossible because of a limited number of test temperatures. However, a two-segment piecewise linear regression revealed a discontinuity in maximum fH values at 16.5ºC (P = 0.052) at the point where Q10 equivalents for regression slopes decreased from 1.8 in the lower segment (<16.5ºC) to 1.1 in the upper segment (>16.5ºC). This discontinuity for maximum fH fell within the previously defined range of Topt for aerobic scope. Matching statistical analysis of resting fH revealed a discontinuity at a higher temperature (18.9ºC; P = 0.073) at the point where Q10 values increased from 2.0 in the lower segment (<18.9ºC) to 5.2 in the upper segment (>18.9ºC).  2.3.2 Effect of Temperature on fH in Anaesthetized Fish  After 1 h of anaesthesia, initial fH was significantly (P <0.001) higher for 15 ppm clove oil and MS-222 than either 30 ppm clove oil treatments (Table 2.1). Atropine significantly increased fH by 6.8%, 9.7% and 11.5% in 30 ppm clove oil (P = 0.006), 15 ppm clove oil (P <0.001) and 50 ppm MS-222 (P <0.001), respectively. The subsequent isoproterenol injection did not significantly increase fH with any anaesthetic treatment (P >0.05), suggesting maximal sympathetic stimulation of the heart under these anaesthetic treatments at 10ºC. Saline injections had no affect on fH. The significantly higher initial fH with 15 ppm clove oil and MS-222 compared with 30 ppm clove oil treatments was maintained with warming up to 22ºC (P <0.05)  38 (Figure 2.4). Further, fH in the 15 ppm clove oil and MS-222 groups approximated the maximum fH values seen in unanaesthetized fish (Figure 2.4C,D). At temperatures from 20ºC to 23ºC, cardiac arrhythmias ensued and fH values above 23ºC were not different between anaesthetic treatments (P >0.05) likely as a result of reduced statistical power with fewer fish per temperature (Figure 2.4). Neither the temperature nor fH for the onset of cardiac arrhythmias was different among anaesthetic treatments (P >0.05) (Table 2.2). ABT analysis revealed significant (P <0.001) discontinuities for both individual (Table 2.2) and mean (Table 2.2; Figure 2.5) responses of fH to warming for each anaesthetic treatment. The range for individual ABTs was greatest (7.5ºC) for control fish and least (2.0ºC) for the MS-222 anaesthetic. For the individual fH responses, the ABT did not differ among treatments (P >0.05; Table 2.2) even though the fH corresponding to the ABT in each treatment was significantly higher (P <0.001) for the 15 ppm clove oil and MS-222 treatments than for either the 30 ppm clove oil treatment or the control fish. Therefore, the ABT was independent of an anaesthetic effect on fH. Comparison of regression slopes from individual fH responses revealed the initial Q10 for fH was significantly higher in control (P <0.001) and MS-222 (P = 0.001) treatments than the pharmacological 30 ppm clove oil treatment. Above the ABT, the Q10 for fH was reduced the greatest in the MS-222 treatment (Table 2.2). Thus, although different types and concentrations of anaesthesia affected the maximum fH corresponding to the ABT, the variability of the individual ABTs and the initial Q10 for maximum fH, none of these effects altered the ABT.  39 No correlations were found between individual physical characteristics (mass, relative ventricular mass, hematocrit; Table 2.2) and either ABT or the temperature when cardiac arrhythmias started (data not shown).  2.3.3 Comparison of Topt Estimates  The individual Topt estimate for aerobic scope (17.0 ± 0.7ºC) was not significantly different (P = 0.27) from the ABT estimated from any of the anaesthetic treatments using individual fH responses. The best agreement was for the individual values of ABT for 15 ppm clove oil treatment (17.1 ± 0.5°C) (Figure 2.6), but the least individual variability of ABT was for 50 ppm MS-222 (16.5 ± 0.2°C) (Table 2.2).  2.4 Discussion  The primary objective of this Chapter was to assess the potential for a high- throughput technique to estimate Topt using maximum fH as a surrogate measurement for aerobic scope. The aerobic scope protocol used here to generate maximum ! ˙ M O2 at each test temperature required 3 weeks to generate minimal data for a Fry curve (i.e. five temperatures at 2°C increments) using N = 12 fish. In contrast, a finer resolution ABT analysis of maximum fH in anaesthetised fish took only 3 days. Furthermore, Topt for aerobic scope was statistically indistinguishable from the ABT for maximum fH when individual comparisons were made to avoid rounding issues (see below). Therefore, my data strongly supports the idea that ABT analysis of maximum fH can be used a high-  40 throughput method for determining Topt that can act as a surrogate for aerobic scope measurements. Furthermore, using 50 ppm MS-222 as the anaesthetic could potential lead to less variability in the estimate of Topt.  2.4.1 Accuracy of Topt and fH Measurements for Unanaesthetized Fish  The accuracy of a Topt estimate is dependent upon the quality and quantity of the ! ˙ M O2 measurements used to generate the Fry curve. Resting ! ˙ M O2  measured here is comparable or slightly lower than previously reported values for juvenile coho salmon at similar or lower temperatures (3.7 to 4.4 "mol h-1 g-1; Janz et al. 1991; Davis and Schreck 1997) and far lower than resting values for juvenile rainbow trout (6.3 to 10.5 "mol h-1 g-1; Scarabello et al. 1991, 1992), indicating that the 12 h recovery period was sufficient. Likewise, post-exercise ! ˙ M O2  values closely match those for juvenile rainbow trout (20.1 to 22.1 "mol h-1 g-1; Scarabello et al. 1991, 1992). Thus, the factorial scope for ! ˙ M O2  (2.5 to 4.3) slightly exceeds that for previous chase studies with rainbow trout (2.1 to 3.2; Scarabello et al. 1991; Scarabello et al. 1992). The Topt for aerobic scope (17.0 ± 0.7ºC) is lower than the 19ºC to 20ºC Topt previously reported for swimming performance in 11ºC acclimated under-yearling (Griffiths and Alderdice 1972) and yearling coho salmon (Brett et al. 1958), but is greater than the 15ºC Topt for growth in coho salmon smolts (Edsall et al. 1999). The single fish mortality observed here at 21ºC likely would not have occurred if Topt of this particular group of fish was as high as that reported previously for under-yearling coho salmon.  41 Resting and maximum fH in non-anaesthetised juvenile coho salmon have not been previously reported for comparison. Resting fH for juvenile Atlantic salmon (Salmo salar) was reported as 25 to 40 beats min-1 at 10ºC (Knudsen et al. 1992). The present Q10 for resting fH of 2.6 ± 0.2 is higher than the 1.6 to 2.3 range previously reported for larger resting salmon (Clark et al. 2008; Steinhausen et al. 2008; Taylor et al. 1996), primarily because of a greater temperature sensitivity of resting fH above 19ºC. The absolute maximum post-exercise fH of 125.8 beats min-1 at 21ºC is near the 120 beats min-1 limit suggested for most fishes (Farrell 1991) and similar to that reported for adult sockeye salmon swimming near Topt (Steinhausen et al. 2008). Arrhenius break temperature analysis determined that maximum fH ceased to increase to any significant degree above the Topt for aerobic scope. This result supports the hypothesis that Topt coincides with a limitation on maximum fH in salmon. Increases in fH above the ABT were only modest (~6 beats min-1 between 17ºC and 21ºC) and are unlikely to provide any appreciable additional capacity for oxygen convection. This lack of additional capacity is supported by the decline in maximum ! ˙ M O2  above 17ºC, which corresponded to the decrease in aerobic scope. Thus, my data are consistent with the suggestion that, as fish are progressively warmed, a maximum fH limitation causes a decline in scope for fH that triggers a decline in scope for ! ˙ Q , which is followed by a collapse of aerobic scope (Farrell 2009). This supports the use of the ABT for maximum fH as a predictor of Topt.    42 2.4.2 Measuring Maximum fH in Anaesthetized Fish  Previous investigations have used Arrhenius plots to detect discontinuities in fH across species (Frederich and Pörtner 2000; Lannig et al. 2004; Stenseng et al. 2005). These studies performed analyses that were based on mean values, which does not incorporate individual variability and therefore tends to round the estimate. A better approach demonstrated here and used previously (Iwaya-Inoue et al. 1989) is to use individual rather than mean Arrhenius plots to determine ABTs. In fact, the very earliest work using Arrhenius analysis to detect discontinuities in fH was based on individual data across a wide variety of vertebrates (e.g. Crozier 1926), but these earliest studies preceded the development of analytical methods required for statistical analysis. My present experiments showed that fH discontinuities are revealed in anaesthetized juvenile coho salmon in a reliable and rapid manner.  This result raises the possibility of using this methodology as a tool to screen Topt in fishes. While numerous studies have considered using cardiovascular parameters as a predictor of ! ˙ M O2 in salmon (e.g. Brodeur et al. 2001), application has been limited by the high variability of fH and ! ˙ M O2 correlations (Priede and Tytler 1977). This is because different activity states and environmental factors alter the relationship of fH and ! ˙ M O2 (Thorarensen et al. 1996). By keeping the activity state of the fish constant using anaesthetics and achieving maximum fH by pharmacological means the confounding effects of activity state on fH could be circumvented. Therefore, the main concern then shifts to the reliability of measurements of fH in anaesthetised fish especially given the large amount of literature on the effects of fish anaesthetics on cardiac activity.  43 Consistent with the present observations, MS-222 anaesthesia brings about tachycardia by reducing parasympathetic tone (Cotter and Rodnick 2006; Ryan et al. 1993) by blocking neuronal Na+ channels (Burka et al. 1997; Carter et al. 2011). Clove oil also causes tachycardia (Cotter and Rodnick 2006), likely due to eugenol, the active ingredient in clove oil, blocking parasympathetic nerve action potentials (Moreira-Lobo et al. 2010). Blocking vagal tone in quiescent, unanaesthetized rainbow trout elevates fH by 30 to 50%, depending on temperature (Priede 1974; Wood et al. 1979). In the present study, the competitive muscarinic antagonist atropine increased fH by ~10% across treatments, indicating that vagal tone was reduced by all anaesthetic treatments, but not eliminated. Even so, the cardio-acceleratory effect of 30 ppm clove oil was attenuated in comparison with both 15 ppm clove oil and 50 ppm MS-222, possibly in part because eugenol blocks L-type Ca2+ channels (Sensch et al. 2000) and slows pacemaker depolarization. On the other hand, while MS-222 has been shown to negatively affect myocardial function in vitro (Hill et al. 2002; Ryan et al. 1993), in vivo studies have not reproduced these results (Fredricks et al. 1993; Hill and Forster 2004). Correspondingly, the highest fH values were recorded for the MS-222 trials and these were closest to those seen in unanaesthetized fish. The least amount of variability in ABT also occurred with the MS-222 trials. Therefore, the recommendation is to use 50 ppm MS-222 as the maintenance anaesthetic for ABT analysis of maximum fH. Stimulation of ß-adrenergic receptors following atropine treatment has previously been shown to increase fH of rainbow trout by 8 to 15% and in a temperature-dependent manner (Wood et al. 1979). Here, isoproterenol did not significantly increase maximum fH, suggesting cardiac ß-adrenergic receptors either were fully saturated or were inhibited  44 by the anaesthetic (Butterworth et al. 1997). Fish were sedated to stage 5 or 6 anaesthesia (Summerfelt and Smith 1990), which can temporarily elevate levels of circulating catecholamines (Iwama et al. 1989). No studies have examined plasma catecholamine levels in salmon under prolonged anaesthesia, so it is unclear if plasma catecholamine levels remain elevated. It seems unlikely that anaesthesia confounded the test results by decreasing maximum fH because maximum fH for fish anaesthetized with MS-222 exceeded that recorded post-exercise, possibly as a result of the membrane destabilizing effects of anaesthetics in general (Butterworth and Strichartz 1990). The maximum fH of fish sedated with MS-222 greatly exceeded that observed post-exercise at elevated temperatures, although a breakpoint was still observed in anaesthetized fish. A lower maximum fH in unanaesthetized fish could be due to increased vagal tone. Vagal tone was speculated to occur at high temperature in swimming sockeye salmon (Steinhausen et al. 2008) and could act as a cardio-protective mechanism by limiting fH and allowing adequate time for oxygen diffusion to the spongy myocardium as well as maintaining cardiac contractility. In anaesthetized fish, the above- mentioned membrane destabilizing affect of MS-222 also likely contributed to fH exceeding the ~120 beats min-1 maximum. However, maximum fH in atropinized, anaesthetized fish still failed to maintain an exponential increase with temperature and displayed a breakpoint. This limitation to increases in maximum fH may be an intrinsic property of the salmon heart resulting from the coincidence of a number of mechanistic limitations (contractility, oxygen delivery, Ca2+ exchange) (Lillywhite et al. 1999) at temperatures above Topt as the heart approaches its upper temperature and rate limit.  45 It may be possible to simplify the procedure developed in this thesis by eliminating the protracted recovery period following initial anaesthesia and by removing pharmacological treatments. In my experiment, the 1 h recovery period allowed sufficient time for fH to stabilize in order to demonstrate the possible effects of pharmacological stimulation on fH. In future studies aiming to achieve maximum fH of fish the pharmacological stimulation may be given immediately after fish are placed in the apparatus. Arguments can be made against the necessity of atropine and isoproterenol injections, given their relatively minor effects observed in this study. However, there is a distinct possibility of increased vagal tone at higher temperatures without the blocking effect of atropine. Moreover, the lack of response of coho salmon to isoproterenol does not preclude other species from responding differently. I recommend, therefore, that both atropine and isoproterenol injections are retained in future studies in order to further evaluate their effectiveness. In summary, Topt for aerobic scope in juvenile coho salmon was 17.0 ± 0.7ºC and was statistically indistinguishable from the ABT for maximum fH in anaesthetized fish (17.1 ± 0.5ºC for 15 ppm clove oil and 16.5 ± 0.2ºC for 50 ppm MS-222). Therefore, because the ABT analysis took 3 days versus 3 weeks for the Fry aerobic scope curve, I propose that ABT analysis of maximum fH in anaesthetized fish presents itself as a valuable, high-throughput screening tool to assess Topt in salmon. Further, the practicality and simplicity of this methodology lends it to field-based application. Field measurements, for instance, could be used to measure the Topt of rearing coho salmon in their natal streams. Recovery of fish following procedures may also be possible, potentially allowing this methodology to be applied to species of conservation concern.  46 Broader application of this technique may also be possible given that other fish species have been shown to similarly rely on fH to increase in ! ˙ Q during warming (Cooke et al. 2003; Gollock et al. 2006; Mendonça and Gamperl 2010). Interestingly, even though wireless field electrodes should theoretically be unable to detect the ECG in seawater, preliminary studies with Arctic cod (Arctogadus glacialis) have shown that an ECG can in fact be detected. This opens the possibility of rapid Topt determinations for fish species in both freshwater and marine environments.  47  Table 2.1. Changes in heart rate (fH) (beats min-1) following sequential pharmacological treatment with atropine and isoproterenol to stimulate maximum fH in juvenile coho salmon (Oncorhynchus kisutch) sedated with different anaesthetics at 10ºC. Injections were administered intraperitoneally and were given 15 min apart 1 h after fish were introduced to the anaesthetic. The 30 ppm sham-injected control group received saline injections only. Drugs Administered 30 ppm Clove Oil Control N = 8 30 ppm Clove Oil Stimulated N = 10 15 ppm Clove Oil Stimulated N = 12 50 ppm MS-222 Stimulated N = 12 1. Pre-injection 61.0 ± 1.3a 57.4 ± 1.1a 65.9 ± 1.5b 68.6 ± 0.9b 2. Atropine (1.2 mg kg-1) 59.6 ± 1.3a 61.3 ± 1.2a* 72.3 ± 1.2b* 76.5 ± 1.0b* 3. Isoproterenol (4 !g kg-1) 58.9 ± 1.4a 62.4 ± 1.1a 72.4 ± 1.1b 76.8 ± 1.3b All values are mean ± S.E. Different letters indicate significant differences (P <0.05) between anaesthetic treatments. A * denotes a significant difference from the previous fH.  48 Table 2.2. Physical and blood characteristics, cardiac collapse, and breakpoint analysis of heart rate (fH) during an acute temperature change (10ºC h-1) in sedated juvenile coho salmon (Oncorhynchus kisutch) under different anaesthetics with or without pharmacological stimulation of fH.  30 ppm Clove Oil Unstimulated N =8 30 ppm Clove Oil Stimulated N = 10 15 ppm Clove Oil Stimulated N = 12 50 ppm MS-222 Stimulated N = 12 Sampling Mass (M) (g) 22.7 ± 2.5a 18.6 ± 1.4a 15.8 ± 3.4a 18.4 ± 0.8a Relative ventricular mass (% M) - 0.102 ± 0.001a 0.099 ± 0.001a 0.099 ± 0.003a Hematocrit (%) - 27.7 ± 2.1a 31.0 ± 0.4ab 34.6 ± 1.6b Cardiac Collapse Pre-arrhythmia fH (beats min-1) 129.7 ± 5.0a 127.6 ± 4.5a 135.5 ± 7.2a 143.2 ± 5.7a Arrhythmia temperature (ºC) 22.6 ± 0.6a 22.5 ± 0.6a 23.3 ± 0.4a 22.9 ± 0.6a Mean Breakpoint Analysis Arrhenius break temperature (ºC) 17.4 19.4 16.8 16.8 Breakpoint fH (beats min-1) 103.1 116.7 118.7 128.4 Q10-equivalent slope (Low/High) 2.1/1.7 1.9/1.4 2.0/1.5 2.1/1.4 Individual Breakpoint Analysis Arrhenius break temperature (°C) 17.6 ± 0.7a 18.2 ± 0.6a 17.1 ± 0.5a 16.5 ± 0.2a Range (Min-Max) 12.4-19.9 14.5-21.3 15.2-20.4 15.5-17.5 Breakpoint fH (beats min-1) 105.1 ± 4.8a 106.0 ± 3.0a 121.1 ± 3.0b 125.5 ± 2.5b Q10-equivalent slope (Low) 2.1 ± 0.05a 1.9 ± 0.02b 2.0 ± 0.02ab 2.1 ± 0.02a Q10-equivalent slope (High) 1.6 ± 0.06a* 1.6 ± 0.06a* 1.5 ± 0.04a* 1.5 ± 0.04a* All values are mean ± S.E. Different letters indicate significant differences between anaesthetic treatments. A * indicates a significant difference (P <0.05) from previous Q10 value. Low and High refer to breakpoint slopes at temperatures less than/greater than the Arrhenius break temperature.  49  Figure 2.1. (A) Simultaneous recording of water bath temperature ( ), internal body temperature ( ), and heart rate (fH) ( ) in an anaesthetized juvenile coho salmon (Oncorhynchus kisutch) during a step-wise acute temperature increase (10ºC h-1). Insert in the main panel displays plateau of fH for ~1 min prior to subsequent temperature changes. Letters indicate where the electrocardiogram trace was sampled for lower panels. (B) Electrocardiogram trace prior to warming at 10ºC. (C) Electrocardiogram trace of arrhythmias and the collapse of cardiac function at ~23ºC.  50   Figure 2.2. (A) Resting ( ) and maximum post-exercise ( ) oxygen consumption ( ! ˙ M O2) and (B) resting ( ) and maximum ( ) heart rate (fH) of juvenile coho salmon (Oncorhynchus kisutch) over a range of temperatures (13ºC to 21ºC). (C) Mean individual differences between resting and maximum ! ˙ M O2 ( ) and fH ( ) at a given temperature are presented as aerobic scope and fH scope. Dissimilar letters indicate a significant difference (P <0.05) between temperatures within a group. For all samples N = 12 unless noted in brackets. Values are mean ± S.E.  51   Figure 2.3. Arrhenius plot of resting ( ) and maximum post-exercise ( ) heart rate (fH) of juvenile coho salmon (Oncorhynchus kisutch) repeatedly tested over a range of temperatures (13ºC to 21ºC). Discontinuities were approximated using a two segment piecewise linear regression (Region 1: y= b0 - b1 (x - T1), x < T1; Region 2: y = b0 + b2 (x - T1), x > T1) that was fitted to resting fH (b0 = 4.374, b1 = -13.524, b2 = -5.778, T1 = 3.424, P = 0.073, R2 = 0.997) and maximum fH (b0 = 4.784, b1 = -1.029, b2 = -5.148, T1 = 3.452, P = 0.052, R2 = 0.998). Resting and maximum fH showed discontinuities at 18.9ºC and 16.5ºC, respectively. Q10-equivalents for slopes are given for each segment of the regression. A * denotes the discontinuity.  52  Figure 2.4. Changes in heart rate (fH) of juvenile coho salmon (Oncorhynchus kisutch) during acute warming (10 ºC h-1) in each of four anaesthetic treatments. (A) 30 ppm clove oil with no pharmacological treatment ( ). (B) 30 ppm clove oil ( ), (C) 15 ppm clove oil ( ), and (D) 50 ppm MS-222 ( ) following sequential atropine and isoproterenol injections. Resting and maximum fH ( ) from aerobic scope trials are shown for reference. Symbols connected by solid lines represent the range of fH values used for Arrhenius break temperature analysis. n is in brackets for temperatures where cardiac arrhythmias reduced the number of individuals remaining at each temperature. * denotes a significant difference (P <0.05) from control and pharmacologically stimulated 30 ppm clove oil treatments. ** denotes a significant difference from all other treatments.   53   Figure 2.5. Arrhenius break temperature (ABT) analysis of mean heart rate (fH) during an acute temperature change for juvenile coho salmon (Oncorhynchus kisutch) under 30 ppm clove oil without pharmacological stimulation ( ) and 30 ppm clove oil ( ), 15 ppm clove oil ( ), and 50 ppm MS-222 ( ) with pharmacological stimulation of fH. Solid lines associated with symbols for each treatment show ABT regressions and * denotes the break point. Resting and maximum fH from aerobic scope trials ( ) are shown for reference.  54   Figure 2.6. Comparison of optimal temperature values for juvenile coho salmon (Oncorhynchus kisutch) estimated using measurements of aerobic scope or Arrhenius break temperature analysis of heart rate in acutely warmed, pharmacologically stimulated fish in two different anaesthetics (15 ppm clove oil or 50 ppm MS-222).  55 CHAPTER 3: TEMPERATURE CONDITIONS AND DYNAMICS IN TWO COHO SALMON-BEARING STREAMS  3.1 Introduction  The ecological relevance of Topt for aerobic scope in the life history of fishes has been suggested by linking changes in distribution and survival with environmental temperature (Farrell et al. 2008; Pörtner and Knust 2007). These previous studies examined this relationship in thermally homogeneous study systems and found associations between single-point environmental monitoring, Topt, and the observed ecosystem effects. For example, Farrell et al. (2008) combined sockeye salmon enumeration records with point source temperature data from the Fraser River to suggest temperatures in excess of Topt for sockeye salmon led to mortality during upstream migration. This particular correlation was facilitated by two factors. First, homogenous temperature conditions in the Fraser River are approached as a result of thorough mixing, limited tributary input, and a low surface area to volume ratio (Patterson et al. 2007). Thus, migrating sockeye salmon cannot behaviorally thermoregulate to any significant degree (Donaldson et al. 2009). Second, migration occurs over a narrow time frame with successful migration largely determined by swimming ability, which is directly influenced by the temperature dependence of aerobic scope. Failure of salmon to complete migration results in no lifetime fitness for this semelparous species (Farrell 2009) Thus, the temporally limited influence of temperature and the spatially invariable  56 conditions of the Fraser River required only straightforward temperature monitoring to extrapolate ecological consequences of temperatures exceeding Topt. A proposed use of the rapid Topt method presented in Chapter 2 is the investigation of the thermal tolerance of coho salmon living in low-order streams. Successful residence in freshwater is influenced by growth (Quinn and Peterson 1996), which is determined in part by the cumulative thermal history of a fish over several months during the summer (Holtby 1988). Temporal and spatial temperature variation is also high in low-order streams due to both natural and anthropogenic factors (Caissie 2006; Poole and Berman 2001). Small streams naturally have a high degree of diurnal temperature fluctuation due to their low thermal capacity (Caissie 2006). Anthropogenic disturbances such as deforestation (Holtby 1988), flow alterations (Sinokrot and Gulliver 2000) and reduced groundwater exchange (Story et al. 2003) will also increase diurnal temperature fluctuation. Temperature monitoring would therefore need to be of sufficient temporal resolution to capture the full range of temperatures occurring in a small stream. Spatially, groundwater input (Ebersole et al. 2003a), tributaries (Nielsen et al. 1994) and channel geomorphology (Matthews et al. 1994) create variability in stream temperature that would not be captured by monitoring at a single location (e.g. Farrell et al. 2008). This natural and anthropogenic variability will create a heterogeneous environment in which salmonids can behaviorally thermoregulate to avoid adverse temperatures (Nielsen et al. 1994). Accurately reconstructing the thermal history of fish in streams would be best performed with thermal biotelemetry tags (e.g. Matthews et al. 1994). A less complex method of estimating thermal history that does not require tagging fish is to use an array of water temperature loggers.  57 The objective of this chapter was to use water temperature monitoring to describe the temperature conditions of two local, coho salmon-bearing waterways. Temperature during the summer (1 June to 15 Sept) was the primary focus due to the importance of this period for coho salmon growth. Temperature was recorded in the streams for a portion of the summer of 2010 and all of summer 2011. Both study watersheds have significant human development (damming or urbanization), which is expected to create high stream temperature variation. To capture the spatial and temporal variation associated with these disturbances, temperature loggers were strategically deployed throughout both streams. Adequate characterization of the temperature variation occurring at each logger would allow the thermal history of fish within different stream reaches to be approximated. The potential for these temperature data to be incorporated with coho salmon Topt estimates is subsequently discussed in Chapter 4. The array of loggers also allowed for the examination of temperature dynamics across each watershed and within specific stream reaches. Temperature dynamics can provide important information on how human disturbances may have altered temperature conditions and the importance of habitat features in maintaining thermal conditions. Therefore, a second goal of this chapter was to assess stream temperature dynamics to determine the factors driving changes in temperature across each system and within specific stream reaches. Understanding the factors contributing to overall stream temperature may be important to mitigating thermal degradation due to habitat alternations or the impacts of climate change.     58 3.2 Materials and Methods  3.2.1 Study Watersheds  Water temperature was monitored in two coho salmon-bearing waterways: the Seymour River and Brothers Creek (Figure 3.1). The Seymour River watershed (Figure 3.2) covers 176 km2 in North Vancouver, BC, Canada. The Seymour River has naturally spawning populations of all Pacific salmon species with the exception of sockeye salmon. Coho salmon used in Chapter 2 were sourced from the Seymour River. From its headwaters the Seymour River flows south into Burrard Inlet. This flow is interrupted ~19 km from the mouth of the river by the Seymour Falls Dam that prevents upstream salmon migration and restricts adult and juvenile salmon to the lower portion of the watershed. The Seymour Falls Dam is a drinking water reservoir and regulated water release during summer months may create flow conditions that make the Seymour River susceptible to warming. Despite the dam construction, downstream reaches of the Seymour River support juvenile coho salmon. Habitat areas have been further enhanced with off-channel pools and in-stream structures. The lowest ~2 km of the Seymour River are characterized by urbanization and channelization. Juvenile coho salmon are present throughout the Seymour River (M. Casselman, personal observation) but are primarily found above the Spur 4 (Reaches S1-4; Figure 3.2).   59 Brothers Creek watershed (Figure 3.3) covers 6 km2 in West Vancouver, BC, Canada. Brothers Creek is a tributary of the Capilano River that drains into Burrard Inlet. Hadden Creek is a tributary of Brothers Creek. Although the headwaters of Brothers Creek are intact, significant urban development has occurred throughout the lower reaches. Development in the headwaters of Hadden Creek has resulted in the complete loss of riparian vegetation in some areas. Urbanization and the construction of impassable fish barriers have limited juvenile coho salmon rearing to just upstream of the Mathers Avenue logger on Brothers Creek (Reaches B2,B3; Figure 3.3). On Hadden Creek, however, hatchery-raised juvenile coho salmon are released above fish barriers into upper Hadden Creek (Reaches H1-H3; Figure 3.3).  3.2.2 Temperature Monitoring and Data Analysis  A total of 16 HOBO® Pro temperature loggers (± 0.2°C accuracy; Onset Computer Corporation; www.onsetcomp.com) were strategically deployed on the Seymour River (ten loggers; Figure 3.2; Table 3.1) and Brothers Creek (seven loggers; Figure 3.3; Table 3.1) in July 2010. Temperature loggers were calibrated and synchronized prior to deployment. Loggers were secured to in-stream structures, encased in protective sleeves to prevent direct solar input, and recorded water temperature every 10 min. Streams were divided into reaches according to logger placement with each reach defined by an upper and lower logger. Loggers recorded temperature year round. The July 2010 installation of loggers only allowed for a portion of 2010 summer temperatures  60 to be recorded. Temperature data from the Upper Seymour logger is only available for 2011 as the first logger placement was lost. For the summer (1 June to 15 Sept), the frequency of temperatures recorded at each logger was calculated in 0.5ºC increments. The frequency of temperatures was only calculated during the summer as this represents the time for maximum growth of coho salmon and when deleterious temperatures are most likely to be encountered. Daily maximum, mean, and minimum temperatures at each logger were used to calculate seasonal maximum temperature and diel variation. Summer temperature dynamics within stream reaches were evaluated using differences in daily maximum and mean temperatures between the upper and lower loggers. To determine what processes drive stream temperature, year-round stream temperature data was correlated with mean daily air temperature and rainfall data from weather stations located within or nearby the study watersheds (Environment Canada 2011; Figure 3.1). Water temperature correlations with environmental data were fit with linear regressions using Sigmaplot 11 (Systat Software Inc.; www.sigmaplot.com).  3.3 Results  Temperature frequencies recorded from the Seymour River, Brothers Creek and Hadden Creek indicated that there was a reduction in the frequency of high temperatures for 2011 versus 2010 in both the Seymour River and Brothers Creek watersheds (Figures 3.4-3.6). Reductions in the frequency of high temperatures were most apparent in the upper Seymour River (Figure 3.4B,C,D) and were least apparent in lower Hadden Creek  61 (Figure 3.6B,C). Summer maximum temperatures in 2011 were up to 4.2ºC lower in the Seymour River and 2.1ºC lower in Brothers Creek compared to 2010 (Table 3.1). Temperature dynamics in Seymour River were characterized by a gradual, downstream increase in temperature. In 2010, a progressive increase in seasonal maximum temperature occurred at each downstream logger (Table 3.1), with the exception of the Pool 88 logger where the seasonal maximum was 0.6ºC lower than the logger immediately upstream. This downstream warming trend was also apparent from the increased frequency of warmer temperatures (Figure 3.4) and overall temperature increase within reaches (Table 3.2) in both study years. Downstream warming in the Seymour River was less evident in 2011, likely due to colder overall conditions, although maximum temperatures in the lower Seymour remained warmer than in the upper Seymour (above Spur 4). The Seymour River did not display any significant warming or cooling trends within stream reaches, but unseasonable and abrupt temperature fluctuations were apparent in upper reaches of the river. The fluctuations can be attributed to flow alterations at Seymour Falls Dam. For example, in 2011, the seasonal warming beginning in early July was reversed in August when temperature in the upper river decreased from ~14ºC to ~10ºC, after which seasonal warming resumed (Figure 3.7A). In addition, a marked increase in temperature unassociated with a rain event occurred in the early summer, probably as dam flow switched from predominately bottom to surface water release. Substantial late-summer warming that coincided with high rainfall events (>90 mm) likely occurred during times of minimum dam discharge. Abrupt temperature  62 changes that occurred in upper Seymour River were generally not reflected in the lower river, although seasonal warming was curtailed in early August (Figure 3.7B). Water temperature within Brothers Creek and Hadden Creek displayed variable downstream dynamics. In Brothers Creek, both increases and decreases in seasonal maximum temperature occurred in the downstream direction, whereas only a decreasing maximum temperature occurred on Hadden Creek (Table 3.1). Changes in temperature frequency generally matched the downstream trends in seasonal maximum on both creeks, with an upward shift in the temperature frequency for Brothers Creek (Figure 3.5) and a downward shift for Hadden Creek (Figure 3.6). Warming of Brothers Creek occurred primarily within reach B1. Temperature at the lower logger in reach B1 was consistently higher than the upper logger (Figure 3.8A) resulting in stable summer warming in this reach (Figure 3.8B). The increase in mean daily temperature within reach B1 was the same in each study year (Table 3.2). Rain events had minimal effects on stream temperature in reach B1 and only temporarily reduced warming (Figure 3.8). The degree of warming was also independent of both air temperature (Figure 3.9A) and input water temperature (Figure 3.9B) during the summer, as well as the remainder of the year. Warming in reach B1 was partially reversed by downstream cooling in reach B2, although this trend was only apparent in 2010 (Table 3.2). Cooling on Hadden Creek was observed within reach H2. The mean difference in mean daily temperature between loggers was greater in 2010 than 2011 (Table 3.2), possibly due to the absence of data for early summer temperature differences in 2010. During 2011, the complete summer temperature record showed variable degrees of temperature reductions of input water temperature in reach H2. Minimal cooling occurred  63 during the first half of the summer (June to mid-July), as temperature at the upper and lower logger was approximately equal (Figure 3.10A). Temperatures diverged in the second half (mid-July onwards) (Figure 3.10B) and increasing air and input water temperature was associated with a progressively greater degree of cooling within reach H2 (Figure 3.11A,B). Closer examination of diel temperature fluctuations showed temperature concurrently increased at both loggers (Figure 3.10C) but diel variation was greater at the upper logger (Table 3.1). Maximum temperature differences thus occurred at peak daily water temperatures (Figure 3.10D) and this differential temperature gain possibly accounted for the gradual divergence in temperature during the summer. Differences in daily warming between the upper and lower logger may be partially explained by the reduced influence of air temperature on water temperature at the lower logger (slope = 0.71, R2 = 0.91, P < 0.0001) versus the upper logger (slope = 0.83, R2 = 0.89, P < 0.0001) (Figure 3.12A,B). Rain events dramatically reduced upstream temperature in reach H2 and this corresponded with a loss of the temperature differential between loggers (Figure 3.10). Cooling steadily returned during periods of no rainfall (Figure 3.10A).  3.4 Discussion  The temperature data I collected were largely able to describe the spatial and temporal water temperature variation within the Seymour River and Brothers Creek systems. As a result, the temperature data are of sufficient resolution to be incorporated with Topt estimates for resident coho salmon, although application would be limited to a  64 generalized thermal history of fish within different stream reaches. Analysis of temperature dynamics revealed different temperature trends in each study system, both largely influenced by human disturbances. Temperatures across the Seymour River demonstrated that the otherwise natural temperature regime of this river could be significantly perturbed by flow alterations at the upstream Seymour Falls Dam. In Brothers Creek and Hadden Creek, increased headwater temperatures due to riparian habitat loss in upper stream reaches could be partially buffered by cooling processes within intact stream reaches. One shortcoming of the present two-year data set is that both summer seasons were notably cooler and summer water temperatures greatly reduced from preceding years (B. Smith, Seymour River Hatchery, personal communication). Therefore, the possibility of higher temperatures in these watersheds remains to be fully assessed. Ongoing data collection will be important to assess how future temperature changes may impact thermal conditions in these streams. Overall, my study provides an assessment of both temperature conditions and the factors contributing to the temperature dynamics in each study system.  3.4.1 Resolution of Temperature Monitoring  The temporal resolution of temperature monitoring was able to capture the full extent of diel temperature fluctuation in each system. Infrequent temperature recording can lead to underestimation of both diel variation as well as daily maximum temperature (Dunham et al. 2005). Accurate measurement of diel variation is important if cumulative stream temperature data are used to calculate the frequency of temperatures encountered,  65 as summarized for the two study streams (Figures 3.4-3.6). Further, thermal habitat assessments for fishes that include a temporal component based on such data (e.g. Scruton et al. 1998) could underestimate the extent of adverse conditions if monitoring frequency is too low. Similar concerns would exist if stream temperature conditions were assessed with criteria based on daily maximum temperature (US EPA 2003). These issues were avoided by the high recording frequency of temperature loggers and aided by the relatively moderate diel temperature variation in these streams (Caissie 2006). For the purposes of Topt application, the temporal resolution of the present data is sufficient to estimate the thermal history of fishes, although spatial variability in streams must also be taken into account when determining how well temperature data from stationary loggers represents the thermal history of fish. Microhabitats in streams will increase spatial temperature variability. Cold-water patches can originate from emergent hyporheic (intragravel) flow (Ebersole et al. 2003a), groundwater or riparian shading (Clark et al. 1999), pool thermal stratification (Matthews et al. 1994) or tributary input (Nielsen et al. 1994) and have an area as small as a few cm. Such localized cold patches would not be captured in mean channel temperature monitoring, although the cumulative effect of such processes might be incorporated in overall cooling trends. As a result, the temperature monitoring on Brothers Creek and the Seymour River was not of sufficient spatial resolution to identify these areas. Microhabitat is an important consideration when applying Topt estimates in small streams because fish can behaviorally thermoregulate to exploit cooler habitats (Berman and Quinn 1991; Nielsen et al. 1994; Stevens and DuPont 2011; Tiffan et al. 2009; Torgersen et al. 1999), thus having a different thermal history from that estimated using water  66 temperature data. Application of Topt in small streams therefore will be dependent upon the spatial resolution of temperature data.  Identifying microhabitat areas could increase the spatial resolution of temperature monitoring. Handheld water temperature monitoring has been used to detect and determine the extent of microhabitat in streams (Ebersole et al. 2003b). Likewise, attempts have also been made to associate channel morphology with microhabitat abundance (Ebersole et al. 2003a) although definite linkages have yet to emerge. Alterations to streambed morphology have been suggested to reduce hyporheic exchange and microhabitat abundance (Poole and Berman 2001) while channelization has been shown to decrease microhabitat interconnectivity (Ebersole et al. 2003b). Channelization and other human disturbances in the Seymour River and Brothers Creek may have already reduced or eliminated microhabitats in these systems. Therefore, temperature data as recorded could be an accurate representation of temperature conditions, making Topt application more straightforward. Although small scale temperature variation was not a part of this work, future temperature monitoring on these systems could include identification of microhabitat and if present, the extent of cooling in these areas. Information on the availability and quality of cold-water refuge could be then incorporated with application of Topt estimates, under the assumption that fish can find and will use these cooler areas to behaviorally thermoregulate. A natural continuation of this temperature monitoring would be determining the extent of microhabitat use by fish.    67 3.4.2 Stream Temperature Dynamics  Temperature dynamics in the Seymour River and Brothers Creek were also examined. Stream temperature is determined by a suite of atmospheric and streambed heat exchange processes (Caissie 2006). For the most part, atmospheric processes make up the greatest proportion of heat flux in streams with solar radiation accounting for the majority of atmospheric heat gain whereas net long-wave radiation and evaporative cooling account for most of the heat loss (Johnson 2004; Webb and Zhang 1997). Streambed heat flux also contributes to the heat exchange in streams, although overall less so than atmospheric processes. Groundwater input can warm or cool streams depending on season (Shepherd et al. 1986) and conductive heat loss to the streambed can also play a role in streambed heat flux (Johnson 2004). Processes promoting heat loss, therefore, are important in moderating heat gain and can even lead to cooling in stream reaches with limited solar input (Scruton et al. 1998). The overall temperature regime of a stream is a product of the total heat flux by these factors and is dependent upon input water temperature and total stream discharge (Poole and Berman 2001). Typically, these atmospheric and streambed processes act to increase stream temperature in the downstream direction (Caissie 2006). Pronounced temperature dynamics can arise if the balance of these heat fluxes is altered by human disturbances to riparian habitat or the stream flow regime. As a result, normally gradual downstream warming can become much more acute.  Progressive downstream warming in the Seymour River is characteristic of streams with sufficient balance between natural warming and cooling processes that  68 buffer against significant heat gains (Caissie 2006). A lack of obvious temperature dynamics within the stream segments and the large scale of the Seymour River make it difficult to determine the processes driving stream temperature in this system. It is readily apparent from summer temperature dynamics, however, that releases from the reservoir on the Seymour River can profoundly alter water temperature throughout the system. The reversal of the seasonal warming trend in August likely resulted from a loss of warm, surface spillage and a change to primarily bottom reservoir release that reduced input water temperature by ~4ºC in the upper river. Sub-optimal temperatures will have negative consequences for coho salmon growth (Edsall et al. 1999), ultimately having the same effect as temperatures above Topt but without the possibility of approaching lethality. Application of Topt would therefore have to account for such cold temperature when determining the suitability of temperature conditions. Rapid increases in water temperature were also observed, likely due to rain events suddenly increasing reservoir levels and causing surface dam spillage, although the rate of increase (~1ºC h-1) would likely not cause undo stress in fish. Future temperature regimes of the Seymour River can be expected to be warmer than that observed in either study year. Trends towards earlier summer snowmelt (Stewart et al. 2005) will result in lower mid-summer flows and extended periods where reservoir discharge is minimal and at a higher temperature. Low flows not only reduce the thermal buffering capacity of streams, but also reduce the extent of hyporheic cooling (Poole and Berman 2001). Given the lack of significant cooling in any reach of the Seymour River, future years may be marked by high temperatures that would be of particular consequence for coho salmon residing in the lower reaches of the watershed.  69 Minimum summer discharge from the reservoir should therefore be of sufficient volume to allow natural heat exchange processes to occur and reduce the frequency of high temperatures (Sinokrot and Gulliver 2000). The temperature dynamics in Brothers Creek suggest streambed heat exchange processes may provide heat flux into some stream reaches, although the possibility of warming via surface water inputs cannot be eliminated. Reach B1 had the greatest absolute temperature gain of any stream reach within the studied watersheds. Such warming could not be attributed to atmospheric processes as riparian vegetation is generally intact and low solar input would be anticipated, although the extent of riparian shading in this reach was not quantified. Convective heat gain from surrounding air is also a relatively minor component of heat gain (Johnson 2004). Accordingly, warming was not influenced by air temperature during the summer or the remainder of the year (Figure 3.9A). Warming in reach B1 may therefore occur either through streambed heat exchange or tributary input. Streambed processes such as hyporheic exchange (Shepherd et al. 1986) and conductive heat exchange with the streambed (Caissie 2006; Johnson 2004) can dominate heat flux in the absence of solar input. Typically, these processes result in heat loss during the summer as warm surface water infiltrates streambed gravel, exchanges heat with the cooler stream substratum and hyporheic water, and reemerges at a lower temperature (Poole and Berman 2001). However, the snowmelt in 2011 that maintained low surface water temperatures could result in warming if subsurface water was warmer than in-stream water temperature. The possibility of streambed heat gain could be investigated by measuring intra-gravel temperature in this reach (Shepherd et al. 1986). Alternatively, warming could be due to surface water input from tributaries not  70 identified in this study. Further investigation is needed to determine the source of warming in reach B1, but the pronounced warming in this short length of stream suggest it is caused by human habitat alteration. Regardless of the source of heat gain, headwaters are an important area for maintaining source water temperatures (Scruton et al. 1998). If warming in Brothers Creek reach B1 persists at high temperatures, downstream thermal conditions could negatively affect coho salmon. Streambed heat exchange processes may also be responsible for moderating high input temperatures in Hadden Creek, although atmospheric processes likely play an important role. In contrast to the low input temperatures in Brothers Creek, input temperatures in Hadden Creek were the highest observed in this study. Loss of riparian vegetation in reach H1 likely caused the high input temperatures, as removal of canopy cover is well known to cause significant increases in solar input and stream temperature (Johnson and Jones 2000; Lynch et al. 1984; Rishel et al. 1982). In reach H2, the high input water temperatures from reach H1 were buffered by cooling processes that reduced daily maximum temperature up to 4.9ºC. Riparian vegetation present in reach H2 would significantly limit solar input as well as reduce wind near the water surface, thereby insulating against convective heat transfer (Poole and Berman 2001). Blocking of solar input can also allow for a net heat loss via long wave radiation and evaporation (Caissie 2006; Johnson 2004). Heat loss via atmospheric processes, however, may only confer part of the cooling in reach H2. Conductive heat loss to the streambed and hyporheic flow probably account for a portion of cooling in reach H2. Burton and Likens (1973) suggested conduction was responsible for the observed cooling when streams transitioned from clear-cut areas to  71 intact, forested reaches. Likewise, Story et al. (2003) found cooling via streambed conduction accounted for ~35% of an observed 2.3°C cooling in a small stream, with a further ~25% of cooling attributed to hyporheic flow. The influence of streambed processes in reach H2 is apparent from the different intercept and slope for the regressions between mean daily air and water temperature at the upper and lower loggers (Figure 3.12). Linear regressions of these variables will have slopes close to 1 and intercepts closer 0ºC if air temperature influence dominates (Erickson and Stefan 2000). In reach H2, the lower logger has both a higher intercept and lower slope than the upper logger, suggesting a moderating streambed component. Interestingly, cooling in reach H2 was only apparent at stream temperatures above 15ºC and increased in magnitude with higher input water temperatures and presumably, lower flow (Figure 3.11). Further, rain events and assumed high flow eliminating this cooling. Together, these observations suggest that the streambed processes present in reach H2 are only of sufficient magnitude to cool the stream during low flow periods. Given that high flows and warm input water temperatures are unlikely to occur at the same time, the effects of future climate warming on summer stream temperatures may be effectively moderated in this reach of the stream. This is critical given that snowmelt and rainfall dominated streams like Hadden Creek will be most affected by climate change (Mantua et al. 2010).  Together, the temperature data collected in this study demonstrate that anthropogenic disturbances have significantly altered the temperature regimes in these mountainous, high-gradient waterways. Managing for these disturbances and the additive affects of climate warming may be possible through flow compensation and the preservation of riparian areas and channel morphology. Such measures would maintain  72 water temperature-moderating processes and thermal heterogeneity in streams, both important for resident fishes. Salmon-bearing streams with contrasting channel morphology, such as low-gradient floodplain type streams, will also benefit from similar preservation strategies. Maintenance of channel complexity should be of particular focus on such streams given that hyporheic exchange has greater importance as channel width increases and the extent of riparian cover decreases (Poole and Berman 2001).  73 Table 3.1. Temperature logger locations in the Seymour River and Brother Creek watersheds and basic temperature parameters recorded from each logger during the summer (1 June  to 15 Sept). Logger Watershed Distance Upstream (m) Year Seasonal Maximum (ºC) Mean Diel Variation (ºC) Mean Diel Varation Range (ºC) Upper Seymour Seymour 18500 2010* - - -    2011 16.7 1.4 0.3 – 5.2 Hatchery Pool Seymour 17810 2010** 16.6 2.1 0.4 – 3.9    2011 16.4 1.7 0.4 – 3.3 Spur 7 Seymour 16230 2010** 16.7 2.7 0.6 – 4.4    2011 16.4 2.1 0.4 – 4.5 Pat’s Pool Seymour 15340 2010** 17.1 2.3 0.7 – 4.2    2011 16.5 2.0 0.4 – 4.4 Spur 4 Seymour 11650 2010** 19.5 3.6 0.7 – 5.3    2011 16.1 2.6 0.5 – 4.8 Cribbing Seymour 6970 2010** 20.7 4.1 1.0 – 6.2    2011 17.3 3.2 0.6 – 5.8 Twin Bridges Seymour 4860 2010** 20.7 3.5 1.1 – 5.6    2011 17.5 3.2 0.7 – 5.3 Pool 88 Seymour 3230 2010** 20.1 2.9 0.9 – 5.0    2011 17.4 3.0 0.7 – 5.1 Parkway Seymour 730 2010** 21.5 2.5 0.5 – 3.8    2011 17.3 2.7 0.6 – 4.9 Upper Brothers Brothers 4430 2010** 15.3 0.7 0.3 – 1.9    2011 13.7 0.9 0.3 – 2.3 Mathers Avenue Brothers 2020 2010** 19.2 1.6 0.2 – 3.0    2011 17.1 1.7 0.4 – 4.1 Newdale Avenue Brothers 1590 2010** 16.7 1.2 0.4 – 1.9    2011 16.8 1.5 0.3 – 3.4 Lower Brothers Brothers 130 2010** 17.9 1.6 0.4 – 2.9    2011 16.9 1.6 0.3 – 3.4 Upper Hadden Brothers 3030 2010** 22.3 1.7 0.2 – 3.6    2011 20.5 2.3 0.5 – 5.0 Stevens Drive Brothers 2270 2010** 18.8 1.4 0.4 – 2.6    2011 18.2 1.4 0.3 – 3.3 Upper Levels Brothers 1770 2010** 18.7 1.6 0.4 – 3.2    2011 17.9 1.4 0.3 – 3.1 * Data is unavailble due to the logger being lost. ** 2010 data are from 24 July to 15 Sept.  74 Table 3.2. Mean difference and difference in daily maximum temperatures between the upper and lower loggers in reaches of the Seymour River, Brothers Creek, and Hadden Creek during the summer (1 June  to 15 Sept). Difference in Daily Maximum Between Loggers Reach Reach Length (m) Year Mean Difference Between Loggers (ºC)  Mean Range S1 690 2010* - - -   2011 0.1 0.2 -2.5 – 1.2 S2 1580 2010** 0.0 0.3 -0.5 – 1.1   2011 0.1 0.3 -0.7 – 1.2 S3 890 2010** 0.3 0.0 -0.5 – 1.8   2011 0.1 0.0 -1.5 – 1.0 S4 3690 2010** 0.5 1.2 -1.0 – 3.2   2011 0.0 0.4 -0.8 – 3.1 S5 4680 2010** 0.8 0.9 -0.8 - 2.4   2011 0.6 1.0 0.3 – 2.5 S6 2110 2010** 0.3 0.0 -0.5 – 1.0   2011 0.2 0.1 -0.6 – 1.0 S7 1630 2010** 0.1 -0.1 -0.7 – 0.9   2011 0.3 0.1 -0.3 – 1.0 S8 2500 2010** 0.4 0.3 -1.0 – 1.6   2011 0.2 0.0 -0.6 – 0.7 B1 2410 2010** 3.2 3.6 2.4 - 4.4   2011 3.0 3.5 1.5 – 4.9 B2 430 2010** -1.1 -1.3 -2.5 – 0.2   2011 0.1 0.1 -2.1 – 2.3 B3 1460 2010** 0.5 0.7 -0.1 – 1.3   2011 0.6 0.6 -0.6 – 1.4 H2 760 2010** -2.1 -2.3 -4.9 – 0.3   2011 -1.1 -1.6 -3.4 – 0.1 H3 500 2010** -0.1 0.0 -0.4 – 0.2   2011 -0.1 -0.1 -0.6 – 0.2 Reaches are abbreviated; (S) Seymour River. (B) Brothers Creek. (H) Hadden Creek. * 2010 data are unavailable due to the upper logger being lost. ** 2010 data are from 24 July to 15 Sept.  75  Figure 3.1. Area map for the Seymour River, Brothers Creek, and Hadden Creek. The (!) indicates the location of Environment Canada weather stations that were the source for air temperature and rainfall data for the associated waterway.    76  Figure 3.2. Detail map of the Seymour River showing logger locations (") and the numbered stream reaches (S) between upper and lower loggers.   77  Figure 3.3. Detail map of Brothers Creek and Hadden Creek showing logger locations (") and the numbered stream reaches for Brothers Creek (B) and Hadden Creek (H) between upper and lower loggers.  78  Figure 3.4. Frequency distribution of temperatures for all logger locations on Seymour River for a portion of the summer in 2010 (24 July to 15 Sept) and all of the summer in 2011 (1 June to 15 Sept).   79  Figure 3.5. Frequency distribution of temperatures for all logger locations on Brothers Creek for a portion of the summer in 2010 (24 July to 15 Sept) and all of the summer in 2011 (1 June to 15 Sept).  80  Figure 3.6. Frequency distribution of temperatures for all logger locations on Hadden Creek for a portion of the summer in 2010 (24 July to 15 Sept) and all of the summer in 2011 (1 June to 15 Sept).  81  Figure 3.7. Summer temperature dynamics on the Seymour River. (A) Water temperature (thick line) at the Upper Seymour logger, mean air temperature (thin line), rain events >10 mm (#), and rain events >20 mm (stacked #) at the Upper Seymour logger from summer 2011. Gaps in air temperature indicate when data were unavailable. (B) Water temperature (dark line) at the Parkway temperature logger from summer 2011.  82  Figure 3.8. Temperature dynamics in Brothers Creek reach B1 (A) Water temperature at the Upper Brothers logger (thick black line), Mathers Avenue logger (thick grey line), mean air temperature (thin black line), rain events >10 mm (#), and rain events >20 mm (stacked #) from summer 2011. (B) Temperature difference between the Upper Brothers and Mathers Avenue loggers from summer 2011.  83  Figure 3.9. (A) Daily mean air temperature and (B) mean daily input water temperature versus the difference in temperature between the upper and lower loggers in reach B1 during the summer and the remainder of the year from 24 July 2011 to 15 Sept 2011. Days when mean daily air or water temperature was !0ºC are not shown.  84  Figure 3.10. Temperature dynamics in Hadden Creek reach H2. (A) Water temperature at the Upper Hadden logger (thick black line), Stevens Drive logger (thick grey line), mean air temperature (thin black line), rain events >10 mm (#), and rain events >20 mm (stacked #) from 1 June 2011 to 15 Sept 15 2011. (B) Temperature difference between the Upper Hadden and Stevens Drive loggers from 1 June 2011 to 15 Sept 2011. (C) Detailed view of daily temperature dynamics. (D) Detailed view of the difference in temperature between loggers.  85  Figure 3.11. (A) Mean daily air temperature and (B) mean daily input water temperature versus the difference in temperature between the upper and lower loggers in reach H2 during the summer and the remainder of the year from 24 July 2010 to 15 Sept 2011. Days when mean daily air or water temperature was recorded as ! 0ºC are not shown.   86   Figure 3.12. Regression (solid line) of mean daily air temperature versus mean daily water temperature on Hadden Creek at (A) the Upper Hadden logger and (B) the Stevens Drive logger from 24 July 2010 to 15 Sept 2011. Days when air or water temperature recorded as ! 0ºC were not included for analysis.  87 CHAPTER 4: DISCUSSION AND CONCLUSIONS  The primary objective of this thesis was to develop a rapid method for estimating Topt for aerobic scope that could be readily applied to fishes and thus provide an ecologically relevant measure of thermal tolerance in a rapid manner. Salmon were studied because the physiological basis for the decline in aerobic scope above Topt has been particularly well studied in this group of fishes. Moreover, recent evidence for adult salmon suggested the decline in aerobic scope above Topt is triggered by a limitation on maximum fH, thus providing a mechanism for the development of a rapid Topt estimation method. Chapter 2 presented a methodology using juvenile coho salmon where, after defining Topt for aerobic scope for this life stage and demonstrating Topt coincided with a limitation on maximum fH, this fH limitation was reproduced using pharmacological agents to stimulate maximum fH in anaesthetized fish while acutely warming them. The temperature of this limitation on maximum fH was demonstrated to occur at the same temperature as Topt for aerobic scope. Furthermore, the experimental time needed to estimate Topt with this novel protocol proved to be considerably less than with an abbreviated aerobic scope methodology (3 days versus 3 weeks). Therefore, the maximum fH protocol developed here provided an equivalent but much faster estimate of Topt than ! ˙ M O2 measurements.  While this novel Topt estimation method has potential for broad application, there are important tradeoffs with this method that must be recognized. Specifically, the fH protocol and corresponding Arrhenius break temperature analysis only generate a Topt estimate. In comparison, a Fry curve defines Topt and quantifies how aerobic scope  88 declines as temperatures increase above Topt and approach Tcrit, the temperature at which aerobic scope collapses (Pörtner 2001). Quantifying the rate of decline of aerobic scope for a species can be an important component in determining the extent to which variable environmental temperatures affect performance. Species that are eurythermal and maintain a high aerobic scope over a broad temperature range will not be as affected by elevated temperature as species that are stenothermal and can maintain aerobic scope only over a narrow temperature range. Thus, a Topt estimate generated with the fH protocol would indicate a temperature at which conditions in a fish’s habitat are beyond optimal, but more comprehensive measurement of Topt via ! ˙ M O2 may be necessary to fully understand the implications of elevated temperature. Section 4.1 discusses the incorporation of the fH protocol with temperature data from Chapter 3. Subsequent sections discuss the potential for further application of the fH protocol, how Tcrit data might be derived from the fH data, and how the protocol can be adapted to the various situations that might be encountered during application.  A second objective of this thesis was to comprehensively describe the temperature conditions and dynamics in coho salmon-bearing streams. Two local waterways were selected for water temperature monitoring. The temperature data revealed important but variable temperature dynamics within each stream that showed the potential for certain stream reaches to either help maintain or degrade habitat thermal quality. What emerged from this monitoring was that minimum flows and riparian areas on these streams are critical for moderating water temperature. Because the monitoring was of short duration, multi-year temperature trends could not be described. Local volunteers, however, were trained in the collection of temperature data and the expectation is that ongoing stream  89 temperature monitoring will be able to assess whether or not the factors that moderate water temperature will continue to do so as climate change progresses and stream temperature increases.  4.1 Combining Rapid Topt Determinations with Temperature Data   Numerous approaches have been used to assess the suitability of environmental temperatures for salmon and other species within the family Salmonidae (Flebbe et al. 2006; Isaak et al. 2010; Rieman et al. 2007; Scruton et al. 1998; Williams et al. 2009). Studies that have incorporated Topt estimates focused on temperatures during the narrow migration window of adult salmon on the Fraser River (Farrell et al. 2008; Hague et al. 2011). A more comprehensive approach may be necessary when applying Topt estimates to juvenile coho salmon since rearing success in streams is influenced by the cumulative thermal history of fish over several months (Holtby 1988; Quinn and Peterson 1996). One approach that may be appropriate for applying Topt estimates to juvenile coho salmon is that of Scruton et al. (1998), who used stream temperature data to calculate the frequency of suitable summer rearing temperatures. Originally, this method categorized temperatures using previous thermal tolerance data. Alternatively, Topt estimates from the fH protocol and Fry curve data can be used to define temperature categories. What follows is an example that illustrates how Topt and temperature data could be combined to assess temperature conditions, and how the results can be interpreted to determine the implications for coho salmon in these streams.  90 Two sets of categorical temperature criteria were generated based on the coho salmon Topt estimates in Chapter 2 and then used to assess the temperature data from Chapter 3. One set of criteria was based the fH protocol and defined temperature as either above or below Topt. The second set of criteria, based on ! ˙ M O2 measurements and the calculated Fry curve (Figure 4.1A), defined various temperature ranges for coho salmon, including an optimal range where aerobic scope is >80% of maximum (Figure 4.1B). This percentage was arbitrarily set, as it is unknown what level of aerobic scope is required by coho salmon in this environment. Temperature analysis using data collected in Chapter 3 and the fH protocol criteria shows these data can indicate the stream reaches where coho salmon would have potentially encountered adverse temperature conditions and where fish could seek refuge from high temperatures (Table 4.1). In 2010, the lower reaches of the Seymour River (S5-S8) and the upper reaches of Hadden Creek (H1) had a high frequency of temperatures exceeding Topt, suggesting sub-optimal conditions for performance and growth. Impacts of such temperatures to fish cannot be determined with this data, but speculation can be made regarding behavioral responses. Fish frequently encountering high temperatures would be expected to either seek out local thermal refuge (Ebersole et al. 2003b) or relocate to more thermally favorable habitat (Berman and Quinn 1991). Loss of microhabitat refugia in the Seymour River and Brothers Creek due to urbanization (Poole and Berman 2001) would likely force fish to thermoregulate via relocation. The fH protocol criteria results from 2010 also suggest coho salmon could seek thermal refuge in upstream reaches of the Seymour River (S1-S4) and downstream Brothers Creek reaches (B2-B3), assuming relocation is not impeded by thermal or  91 physical barriers. Thus, coho salmon could avoid high temperatures during the warmest of the two study years, although thermal refugia availability could also be limited by inter- and intra-specific competition (Hartman 1965; Mason and Chapman 1965). Together, the Topt estimate from the fH protocol and temperature monitoring provide a straightforward and useful overview of stream conditions as well as the potential for coho salmon to behaviorally thermoregulate and minimize exposure to temperatures exceeding Topt during the summer months. Stream temperature analysis with Fry curve data and the added designation of an optimal range allows for a broader assessment than that provided by the fH protocol, yielding an indication of the growth capacity for coho salmon in these streams. Growth is also maximal at an optimal temperature (Brett et al. 1969; Elliott and Hurley 2000) and the similarity of growth curves and Fry curves has led to juxtaposition of the two curves (Farrell 2009), suggesting interdependence. Coincidentally, growth of coho salmon with excess ration is maximal at 17ºC (Everson 1973; as cited in Sullivan et al. 2000), matching the Topt for aerobic scope as determined in Chapter 2. Coho salmon growth at this life stage is a determinate of survival (Quinn and Peterson 1996) and therefore has direct implications for fitness. Results of the temperature assessment therefore suggest the capacity for coho salmon growth would have been near maximal in 2010 in all stream reaches but restricted in 2011 in the Seymour River due to a high proportion of colder conditions. An important caveat in the application of aerobic scope as a surrogate for growth and fitness is that Topt for growth can decrease with reduced food availability (Brett et al. 1969; Elliott 1975; Elliott and Hurley 2000; Everson 1973). The daily energy intake of  92 naturally foraging coho salmon may be ~60% of maximum, reducing Topt for growth by 2-3ºC (Sullivan et al. 2000). Competition can also reduce the Topt for growth (McMahon et al. 2007). A lower threshold than the upper end of the optimal range temperature for aerobic scope, therefore, may be more appropriate for estimating growth potential. Further study is needed on the relationship between aerobic scope and growth, but a Topt estimate (and therefore the fH protocol) may be an index suitable for both a general assessment of conditions and an approximation of upper thermal thresholds for lost growth and fitness.  4.2 Comparative Assessments using the fH Protocol  The fH protocol may be best suited for screening for Topt differences among species or populations. Eliason et al. (2011) found population-level differences in Topt for aerobic scope amongst sockeye salmon that correlated with differences in the Topt for fH scope. Therefore, it would be expected that such population-level differences in cardiac performance would be reflected as an equivalent difference using the fH protocol, although this has yet to be tested. Such comparative assessments of populations would be particularly useful in prioritizing conservation efforts, as those populations with the lowest Topt and highest susceptibility to increasing temperature could be quickly identified and then perhaps more thoroughly tested. The Weaver Creek population of sockeye salmon had the lowest Topt of the six sockeye salmon populations considered by Eliason et al. (2011) and was therefore identified as the most vulnerable to climate change. It is unclear, however, whether or not the fH protocol can resolve between Topt  93 values for populations that are separated by only a few degrees. The Weaver Creek population of sockeye salmon with a Topt of 14.5ºC and the Chilko River population with a Topt that is 2.3ºC higher might be a good test of the methodology. Comparison of species within the family Salmonidae that have markedly different thermal tolerances would further test the resolving power of the fH protocol. For instance, redband rainbow trout Oncorhynchus mykiss gairdneri have one of the highest thermal tolerances of any trout species (Rodnick et al. 2004). On the other hand, bull trout Salvelinus confluentus are amongst the least thermally tolerant salmonids and show an optimal temperature below that of Weaver Creek sockeye salmon (~13ºC; Selong et al. 2001). Performing the fH protocol on each of these species would demonstrate the results for the extremes of thermal sensitivity and possibly provide enhanced analytical capacity to differentiate temperature tolerant and intolerant species. For instance, the ABT would obviously expected to be at a lower temperature for bull trout, but the reduction in the slope of the upper break regression might be more pronounced above the Topt for this species, implying a higher degree of cardiac impairment than in redband rainbow trout. Subsequent studies could use species with more similar thermal tolerances to determine the minimum resolution of the protocol. Initially, these studies would likely have to be paired with measures of aerobic scope (as in Chapter 2) in order to confirm that any differences observed with the fH protocol did indeed correspond to differences in aerobic scope. Population-level comparisons would then be a natural continuation of this investigation if the fH protocol did have sufficient resolution.    94 4.3 Extending the Applicability of the fH Protocol  It may be possible to use the arrhythmias that are triggered at extreme temperatures during the fH protocol to extend the applicability of the results from the fH protocol. Clark et al. (2008) observed arrhythmias near Tcrit and use of anaerobic metabolism (e.g. collapse of aerobic scope) for resting Chinook salmon (Oncorhynchus tshawytscha). In addition, Farrell et al. (2009) suggested that as a component of the ‘death spiral’ for maximally swimming salmon, cardiac arrhythmias would occur as result of the increasing reliance on anaerobic metabolism at high temperature, therefore marking the failure of fish to maintain adequate aerobic scope. In Chapter 2, arrhythmias were observed in fH trials at 22.9 ± 0.6°C (MS-222) and this temperature roughly corresponds with the estimated Tcrit in Figure 4.1. However, since unanaesthetized fish were only tested up to 21°C, the connection between the collapse of aerobic scope and arrhythmias in the fH protocol remains only a possibility. If future studies were able to draw a definitive link between arrhythmias in the fH protocol and Tcrit, the fH protocol could provide an indication of the breadth of a fish’s thermal tolerance. Although the rate of decline of aerobic scope away from Topt would still be unknown, the ‘optimal range’ for a fish could be estimated based on the temperature difference between the ABT and cardiac arrhythmias. Such information may be useful in the temperature assessment presented in Section 4.1. Interestingly, salmon are estimated to have a Topt ~7ºC below the collapse of aerobic scope (Farrell 2009) and the temperature difference between the ABT and the cardiac arrhythmias in the MS-222 trials was also ~7ºC.  95 Further confirmation of the relationship between the temperature difference of Topt, cardiac arrhythmias and the breadth of a fish’s thermal tolerance could be achieved by performing the fH protocol on markedly eurythermal and stenothermal species. Goldfish are a classic example of a highly eurythermal species that maintains high aerobic scope over a 15ºC range (Fry and Hart 1948). In contrast, coral reef fishes of the family Pomacentridae only maintain a high aerobic scope over 2-3ºC and display a rapid collapse of aerobic capacity (Johansen and Jones 2011; Nilsson et al. 2009). A corresponding temperature difference between Topt and arrhythmias and the breadth of the Fry curve in these species would lend further support to this difference defining the thermal tolerance of a fish as well as arrhythmias defining the collapse of aerobic scope.  4.4 Adapting the fH Protocol to Species and Conditions  This thesis restricted the investigation of an expedited Topt estimation method to one species within Oncorhynchus. However, the temperature dependence of fH (Randall 1970) and strong evidence that many fish species rely almost entirely on fH for increasing oxygen convection through the circulatory system when temperature is elevated (Brodeur et al. 2001; Clark et al. 2008; Gamperl et al. 2011; Gollock et al. 2006; Mendonça and Gamperl 2010; Sandblom and Axelsson 2007; Steinhausen et al. 2008; Stevens et al. 1972) suggest broad potential for this technique. Application of this technique to include other species and even potential field application will likely require modifications to certain aspects of the methodology.  96 Changing the method of fH detection, increasing the resolution of temperature intervals, and need to decrease as well as increase temperature are all potential modifications to the technique that may be required with other species and conditions. Simplification of fH detection to monitoring the ECG without directly wired electrodes is an appealing aspect of the anaesthetized methodology, but morphological limitations in some species may require alternate detection methods. From an analytical perspective, applying this technique to fishes where Topt and the collapse of aerobic scope are separated only by a few degrees (e.g. coral reef fishes) may not provide a sufficient number of temperature points to calculate the ABT. Therefore, it may be necessary to reduce the 1ºC temperature interval between fH measurements used in this study to 0.5ºC increments or smaller in order record sufficient temperatures below the ABT for an accurate ABT calculation. Further, a similar situation could arise if field application of this technique is undertaken to estimate Topt of wild fish in their native environment. Ambient environmental temperature could already be within a few degrees or even exceed the Topt for the species being studied. In this case, the temperature of the anaesthetized fish could be reduced to a common starting temperature prior to acute warming. Further methodological considerations not considered here would likely be necessary given the diversity in fish species and environmental conditions likely to be encountered during application. The methodology outlined in this thesis therefore should be considered a proof-of-content blueprint that other researchers applying this technique should verify and modify to suit their specific applications.   97 4.5 Conclusions and Perspectives   F.E.J. Fry originally conceived the scope for activity concept to provide an ecologically relevant measure of thermal tolerance. More than 50 years later, after both application and renaming, the significance of aerobic scope has finally been demonstrated for aquatic ectotherms with the work of Pörtner and Knust (2007) and Farrell et al. (2008). Both of these studies presented their findings in the context of climate change, a modern dilemma that is already strongly influencing and will continue to impact all types of biological phenomena (Rosenzweig et al. 2008). Conservation measures to preserve and protect species threatened by climate change are typified both by a sense of urgency and limited resources. Prioritizing conservation efforts requires quickly identifying the species that are most vulnerable to climate change while assessing if it is even possible to safeguard species given future climate scenarios. In this regard, this thesis outlined a method that has the potential to greatly assist in the identification of aquatic fish species susceptible to changing environmental temperature.  Contributions were made in this thesis to both the understanding of thermal tolerance in salmon and adaptation of the aerobic scope methodology to meet the challenges of climate change. While comprehensive aerobic scope measures from juvenile salmon were measured over 50 years ago, no previous studies had measured how fH in juvenile salmon responded to temperature. This study was not only the first to describe how both resting and maximum fH change with temperature in juvenile coho salmon, but also the first to relate the changes to aerobic scope and demonstrate that similar cardiorespiratory limits occur at high temperature in juvenile and adult salmon.  98 Building on this relationship, this thesis then developed a relatively simple methodology that accurately and rapidly predicts Topt for aerobic scope in Pacific salmon, making this method an important tool for screening different salmon species and populations, and determining those most vulnerable to warming environmental temperatures. Such a method will not only be important locally, but also has potential application to a much broader range of species. Finally, the simplicity and applicability of this technique may be best demonstrated by the ease with which field measurements were obtained from coho salmon in local waterways (Table 4.2) and proficiency with which local volunteers acquired the technique.  99  Table 4.1. Habitat thermal quality assessment of the Seymour River and Brothers Creek watersheds during the summer (1 June to 15 Sept). Data are presented as percentage of total time temperatures were within the designated range for different stream reaches.  Fry Curve  fH Protocol Reach Year Total Time (h)  Low (<13ºC) Optimal (13-20ºC) High (>20ºC)  <16.5ºC >16.5ºC S1 2010* 1296  24% 76% 0%  100% 0%  2011 2568  90% 10% 0%  100% 0% S2 2010* 1296  36% 64% 0%  99% 1%  2011 2568  89% 11% 0%  100% 0% S3 2010* 1296  17% 83% 0%  99% 1%  2011 2568  88% 12% 0%  100% 0% S4 2010* 1296  20% 80% 0%  88% 12%  2011 2568  86% 14% 0%  100% 0% S5 2010* 1296  13% 86% 1%  74% 26%  2011 2568  73% 27% 0%  99% 1% S6 2010* 1296  10% 89% 1%  71% 29%  2011 2568  69% 31% 0%  98% 2% S7 2010* 1296  6% 94% 0%  68% 32%  2011 2568  65% 35% 0%  98% 2% S8 2010* 1296  3% 96% 1%  59% 41%  2011 2568  57% 43% 0%  98% 2% B1 2010* 1296  7% 93% 0%  74% 26%  2011 2568  54% 46% 0%  99% 1% B2 2010* 1296  13% 87% 0%  99% 1%  2011 2568  55% 45% 0%  100% 0% B3 2010* 1296  11% 89% 0%  91% 9%  2011 2568  51% 49% 0%  100% 0% H1 2010* 1296  1% 85% 14%  46% 54%  2011 2568  12% 88% 0%  59% 41% H2 2010* 1296  2% 98% 0%  77% 23%  2011 2568  14% 86% 0%  92% 8% H3 2010* 1296  4% 96% 0%  78% 22%  2011 2568  15% 85% 0%  94% 6% Reaches are abbreviated; (S) Seymour River. (B) Brothers Creek. (H) Hadden Creek. * 2010 data is from 24 July to 15 Sept.  100 Table 4.2. Environmental conditions, physical parameters, cardiac collapse, and breakpoint analysis of heart rate (fH) during an acute temperature change (10ºC h-1) in wild juvenile coho salmon (Oncorhynchus kisutch) sedated with 50 ppm MS-222. Maximum fH was stimulated with atropine (1.2 mg kg-1) and isoproterenol (4 !g kg-1) injections. Fish were collected in 2011 from the Seymour River and Brothers Creek and field tested at the Seymour River Hatchery and Capilano Hatchery, respectively. Temperature adjustments to a lower initial temperature were performed in 0.5ºC step-wise changes during the 1 h equilibration period.  Seymour River N =12 Seymour River N = 8 Brothers Creek N = 9 Protocol Procedures Dates of Testing 17 July – 19 July 7 Sept – 10 Sept 29 Aug – 1 Sept Ambient Temperature (ºC) 8.1 – 8.5 13.8 – 14.2 14.7 – 15.3 Temperature Adjustment (ºC) - - -2.0ºC  Sampling Mass (g) 5.7 ± 0.3 7.1 ± 0.5 4.5 ± 0.3  Individual Breakpoint Analysis Arrhenius break temperature (°C) 14.3 ± 0.2 16.9 ± 0.4 18.2 ± 0.2 Range (Min-Max) 13.8 – 15.1 15.3 – 17.9 17.6 – 19.3 Breakpoint fH (beats min-1) 110.5 ± 1.6 114.8 ± 3.6 117.3 ± 2.6  Cardiac Collapse Pre-arrhythmia fH (beats min-1) 127.6 ± 10.4 138.1 ± 7.1 141.0 ± 4.3 Arrhythmia temperature (ºC) 23.2 ± 0.5 22.9 ± 0.9 23.0 ± 0.5 All values are mean ± S.E.  101  Figure 4.1. (A) Polynomial quadratic regression of aerobic scope for juvenile (Oncorhynchus kisutch) over the measured temperature range (solid line) and extrapolation to zero aerobic scope at high temperatures (dashed line). 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