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The relationship between thermal tolerance and hypoxia tolerance in Amazonian fishes Jung, Hyewon Ellen 2018

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THE RELATIONSHIP BETWEEN THERMAL TOLERANCE AND HYPOXIA TOLERANCE IN AMAZONIAN FISHES by  Hyewon Ellen Jung  B.Sc., The University of British Columbia, 2014  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Zoology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  March 2018   © Hyewon Ellen Jung, 2018  ii Abstract The Amazon contains 20% of the world’s freshwater fish species that are predicted to experience an increase in temperature by up to 2.2 to 7˚C within the next century. An increase in temperature will likely be associated with an increase in the frequency, duration, and magnitude of hypoxic bouts, creating an even greater challenge. Thermal tolerance may be limited by the ability to supply and deliver enough oxygen to tissues at critical temperatures, as is the case for hypoxia tolerance, thus both may be associated with similar mechanisms in fish. A direct relationship between thermal and hypoxia tolerance however, has not yet been investigated in a wide range of fish species. To address this, I conducted acute thermal tolerance (CTMax) and hypoxia tolerance (% air saturation at loss of equilibrium) assays in 20 species that spanned a broad phylogenetic range. In fish acclimated to the temperatures within the current temperature range of the Amazon River (28 or 31˚C), I found a positive relationship between CTMax and hypoxia tolerance. In fish acutely transferred to higher temperatures of 33 or 35˚C, there was a reduction in hypoxia tolerance relative to that at 28 or 31˚C. Acclimation (10 days or 4 weeks) to 33 or 35˚C did not increase hypoxia tolerance, and in some species there was a further reduction in hypoxia tolerance. Acclimation to 33 or 35˚C (10 days or 4 weeks) and exposure to hyperoxia (>200% air saturation) increased CTMax, although in most species only moderately. One of the most significant findings of my thesis was that most species failed to acclimate to the higher temperatures: of the 13 species investigated, 2 species did not survive 10 days or 4 weeks (chronic lethal maximum) at 31˚C, 9 species did not survive at 33˚C, and only 2 species survived 35˚C. Overall, acclimation to higher temperatures that are  iii predicted to occur within the next century had little or no effect on thermal tolerance and reduced hypoxia tolerance indicating that the high fish biodiversity of the Amazon may be at risk given the predicted changes in temperature and hypoxia associated with climate change.    iv Lay Summary Over the next century, the Amazon, home to 20% of the world’s fresh water fishes, is predicted to experience a 2.2 ~ 7˚C increase which will likely be associated with a reduction in aquatic oxygen levels (hypoxia). There is growing evidence that the physiological abilities to cope with changes in temperature and oxygen are associated in fish. To investigate this, I conducted thermal tolerance and hypoxia tolerance assays in 20 Amazonian species. In fish at the current thermal regime, I found a positive relationship between the two parameters. I also found that short-term exposure to higher temperatures reduced hypoxia tolerance. Long-term exposure to the same elevated temperatures further reduced hypoxia tolerance and had a minor effect on thermal tolerance. Ultimately, most species failed to survive long-term exposure to these temperatures. This thesis should be taken further to help protect one of the world’s fish biodiversity hotspots from the potential risks associated with climate change. v Preface Chapters 2 and 3 of this thesis are co-authored by Ellen Jung and Colin J. Brauner. I conducted all of the research in chapter 2 and 3 (research questions, experimental design, experimentation and data analysis) under the supervision of Dr. Colin J. Brauner, Chris M. Wood, and Adalberto L. Val. I wrote all 4 chapters of this thesis and received editorial feedback from my committee members, Drs. Colin J. Brauner, Jeffrey G. Richards, and Chris M. Wood. All experimental animals were treated according to the University of British Columbia Animal protocol #A15-0266 and Brazilian National and Instituto Nacional de Pesquisas da Amazônia (INPA) animal care regulations.  vi Table of Contents Abstract ........................................................................................................................... ii Lay Summary ................................................................................................................. iv Preface ............................................................................................................................ v Table of Contents .......................................................................................................... vi List of Tables ................................................................................................................. ix List of Figures ................................................................................................................. x List of Abbreviations ..................................................................................................... xi Acknowledgements ...................................................................................................... xii Chapter 1: General Introduction ................................................................................... 1 1.1 General Overview .......................................................................................................... 1 1.2 Thermal Tolerance ......................................................................................................... 3 1.2.1 Effect of acclimation to higher temperatures on thermal tolerance ........................... 5 1.2.2 Chronic lethal maximum ........................................................................................... 6 1.3 Hypoxia Tolerance ......................................................................................................... 7 1.3.1 Effect of an acute increase in temperature on hypoxia tolerance ............................. 9 1.3.2 Effect of acclimation to higher temperatures on hypoxia tolerance ........................ 11 1.4 Oxygen defining thermal limit: relationship between thermal tolerance and hypoxia tolerance ................................................................................................................. 12 1.4.1 Thermal tolerance in hyperoxia ............................................................................... 13 1.5 Application To Climate Change .................................................................................. 15 1.6 Overall objectives ........................................................................................................ 17  vii Chapter 2: The Relationship Between Thermal Tolerance and Hypoxia Tolerance in Amazonian Fishes at The University of British Columbia (UBC) ........................ 20 2.1 Introduction .................................................................................................................. 20 2.2 Material & Methods ...................................................................................................... 23 2.2.1 Experimental design ............................................................................................... 24 2.2.2 Thermal tolerance ................................................................................................... 24 2.2.3 Hypoxia tolerance ................................................................................................... 25 2.2.4 Statistical analysis ................................................................................................... 26 2.3 Result ............................................................................................................................ 28 2.3.1 Thermal tolerance and the effect of acclimation to higher temperatures ................ 28 2.3.2 Hypoxia tolerance and the effect of an increase in temperature (either acute or following acclimation) .......................................................................................................... 29 2.3.3 Relationship between thermal tolerance and hypoxia tolerance ............................. 30 2.4 Discussion .................................................................................................................... 30 2.4.1 Thermal tolerance ................................................................................................... 31 2.4.1.1 Effect of acclimation to higher temperatures on thermal tolerance ............................... 32 2.4.2 Hypoxia tolerance ................................................................................................... 34 2.4.2.1 Effect of an acute increase in temperature on hypoxia tolerance .................................. 36 2.4.2.2 Effect of acclimation to higher temperatures on hypoxia tolerance ............................... 37 2.4.3 Relationship between thermal tolerance and hypoxia tolerance ............................. 38 Chapter 3: The Relationship Between Thermal Tolerance and Hypoxia Tolerance in Amazonian Fishes at the Instituto Nacional de Pesquisas da Amazônia (INPA) ........................................................................................................................................ 43 3.1 Introduction .................................................................................................................. 43 3.2 Material & Methods ...................................................................................................... 44  viii 3.2.1 Experimental Design ............................................................................................... 45 3.2.2 Thermal tolerance ................................................................................................... 46 3.2.2.1 Thermal tolerance in hyperoxia ..................................................................................... 47 3.2.3 Hypoxia tolerance ................................................................................................... 47 3.2.4 Statistical analysis ................................................................................................... 48 3.3 Result ............................................................................................................................ 49 3.3.1 Thermal tolerance and the effect of acclimation to higher temperatures ................ 49 3.3.1.1 Thermal tolerance in hyperoxia ..................................................................................... 50 3.3.2 Hypoxia tolerance and the effect of an increase in temperature (either acute or following acclimation) .......................................................................................................... 51 3.3.3 Relationship between thermal tolerance and hypoxia tolerance ............................. 53 3.4 Discussion .................................................................................................................... 54 3.4.1 Thermal tolerance ................................................................................................... 55 3.4.1.1 Effect of acclimation to higher temperatures on thermal tolerance ............................... 56 3.4.1.2 Thermal tolerance in hyperoxia ..................................................................................... 59 3.4.2 Hypoxia tolerance ................................................................................................... 60 3.4.2.1 Effect of an acute increase in temperature on hypoxia tolerance .................................. 66 3.4.2.2 Effect of acclimation to higher temperatures on hypoxia tolerance ............................... 67 3.4.3 Relationship between thermal tolerance and hypoxia tolerance ............................. 70 Chapter 4: General Conclusion ................................................................................... 79 4.1 Future Research ........................................................................................................... 81 Bibliography .................................................................................................................. 86 Appendix ..................................................................................................................... 107  ix List of Tables Table 1.1. List of 20 species investigated in this thesis .................................................. 19  Table 3.1. Comparison of critical thermal maximum (CTMax, ˚C) measured at UBC and INPA ............................................................................................................................... 77  Table 3.1. Comparison of acute hypoxia tolerance (% air saturation at LOE) measured at UBC and INPA ........................................................................................................... 78    x List of Figures Figure 1.1 Schematic graphs of hypoxia and thermal tolerance following acclimation to higher temperatures ....................................................................................................... 15  Figure 2.1. Critical thermal maximum (CTMax) of 6 species following 4 weeks of acclimation to 31, 33 and 35˚C at UBC .......................................................................... 40  Figure 2.2. Hypoxia tolerance (% air saturation at LOE) of 6 species following acute exposure or acclimation (4 weeks) to 31, 33, and 35˚C at UBC. ................................... 41  Figure 2.3. Critical thermal maximum (CTMax) and hypoxia tolerance (% air saturation at LOE) of 6 species following 4 weeks of acclimation to 31, 33, and 35˚C at UBC ...... 42  Figure 3.1. Critical thermal maximum (CTMax) of 8 species following 10 days of acclimatio to 28, 31, 33, and 35˚C at INPA .................................................................... 72  Figure 3.2. Critical thermal maximum (CTMax) in normoxia (100% air saturation) and hyperoxia (>200% air saturation) ................................................................................... 73  Figure 3.3. Hypoxia tolerance (% air saturation at LOE) of 8 species following acute exposure or acclimation (10 days) to 28, 31, 33, and 35˚C at INPA .............................. 74  Figure 3.4. Critical thermal maximum (CTMax) and hypoxia tolerance (% air saturation at LOE) of 15 species following 10 days of acclimation to 28, 31, 33, and 35˚C ........... 75  Figure 3.5. Severe hypoxia (0.4 ~ 0.6% air saturation) tolerance (time to LOE) of 5 species at 28˚C .............................................................................................................. 76  Figure 4.1. Critical thermal maximum (CTMax, ˚C) and hypoxia tolerance (% air saturation at LOE) measured at UBC and INPA ............................................................ 85  Figure 4.2. Schematic graph representing species’ potential risk level from climate change ............................................................................................................................ 80    xi List of Abbreviations  CLMax Chronic lethal maximum CTMax Critical thermal maximum DO  Dissolved oxygen HIF  Hypoxia-inducible factor HSP  Heat shock protein ILOS  Incipient lethal oxygen saturation IPCC  Intergovernmental panel on climate change LDH  Lactate dehydrogenase LOE  Loss of equilibrium NTP  Nucleoside triphosphates OCLTT Oxygen- and Capacity- Limited Thermal Tolerance  P50  Partial pressure of oxygen at which hemoglobin is 50% saturated with O2 Pcrit Critical oxygen tension Q10 Fold-factor by which a biological rate function increases over a 10˚C rise in temperature UILT  Upper incipient lethal temperature VEGF  Vascular endothelial growth factor  xii Acknowledgements First and foremost, I want to thank my supervisor Dr. Colin Brauner for his enormous support and patience during one of the toughest chapters of my life. Colin, you have been an inspiration both as a researcher and a mentor. I cannot express enough gratitude for believing in me throughout the project. Special thank you to Dr. Kevin Brix for igniting my passion for research. I also wish to thank my committee Dr. Chris Wood and Dr. Jeff Richards for guiding me into and throughout the wonders of the Amazon.  Additional thank you to Dr. Adalberto Val, Dr. Vera Almeida-Val, and everyone in their lab for hosting me at the Instituto Nacional de Pesquisas da Amazônia. My wild Amazon adventure wouldn’t have been possible if not for Drs. Chris Wood, Ora Johannsson, Richard Gonzalez, Bernd Pelster, and Márcio Ferreira. To the Braunerites (past and present), thank you all for sitting through my presentations and making the whole journey amazing. I would also like to thank all the members of the Comphy department, and my incredible friends and UBC K Grad group for emotional support, encouragement and unforgettable memories.   xiii         For my parents and my brother, My love My reason My world  1 Chapter 1: General Introduction 1.1 General Overview  The Amazon is one of the largest river systems in the world, contributing 20% of the global freshwater discharge to the ocean (Salati and Vose, 1984). It is also a hot spot of freshwater fish biodiversity, home to about 20% of the world’s freshwater species. There are an estimated 6000 ~ 8000 freshwater fish species inhabiting the river despite its challenging abiotic conditions (Reis, 2013). The water temperature in the Amazon basin is constant annually, ranging from 28 to 31˚C (Val and Almeida-Val, 1995). The floodplain lakes, where a large number of fishes rely on to spawn and feed, have greater diurnal variations in both temperature and oxygen. Temperatures in these areas are typically about 29 to 33˚C and dissolved oxygen levels can fluctuate from anoxia to hyperoxia and can vary dramatically with depth and season (Röpke et al., 2016; Val and Almeida-Val, 1995). Not surprisingly, the Amazon hosts some of the most thermal and hypoxia tolerant fish species on the planet. However, climate change is predicted to exacerbate already extreme environmental conditions, pushing species to their physiological limits. The global temperature is rising as a result of anthropogenic pollution, and the Amazon especially is predicted to experience a dramatic 2.2 to 7˚C temperature rise by the next century in the worst-case scenario (IPCC, 2014). In addition to temperature, climate change will likely be associated with a range of additional disturbances including increased eutrophication and nutrient input, further reductions in pH, flooding associated with sea-level rise, and a decrease in precipitation (Ficke et al., 2007), all of which are a concern for such a biodiverse region.   2 Perhaps the most critical disturbance that will be experienced by fish in these regions is the increase in water temperature which will be accompanied by a decrease in dissolved oxygen content due to a temperature-induced reduction in oxygen solubility (Carpenter, 1966; Garcia and Gordon, 1992), and increased eutrophication. Eutrophication results in increased biological oxygen demand, creating larger hypoxic zones, greater oxygen stratification, and increased diurnal fluctuations in oxygen levels (Ficke et al., 2007). Therefore this thesis focused on high temperature and low oxygen (hypoxia), two factors with directs impact on the physiology of my animals of interest - water-breathing ectotherms, as will be elaborated upon in the following discussion.  Recently, a number of studies have found that the physiological ability to cope with high temperatures and hypoxia may be mechanistically linked in fish. According to the Oxygen- and Capacity- Limited Thermal Tolerance hypothesis (OCLTT), oxygen delivery to tissues is thought to limit upper temperature tolerance in fish (Farrell, 2008; Pörtner, 2010; Pörtner and Knust, 2007). However, the direct relationship between thermal tolerance and hypoxia tolerance has not yet been investigated in a wide range of fish species. Climate change may induce both an increase in temperature as well as greater and more frequent bouts of hypoxia in tropical regions (IPCC, 2014). If so, these interacting abiotic factors might be lethal to Amazonian fishes that are already living close to their physiological limits both in terms of temperature and oxygen (Somero, 2010). Thus, the main focus of my thesis was to investigate the correlation between thermal tolerance and hypoxia tolerance in a range of ornamental Amazonian fishes to provide insight into how proposed changes in temperature and hypoxia predicted by climate change may affect fish from this incredibly biodiverse region.   3 Previously, most studies that have investigated tolerance to high temperature and hypoxia have done so by altering each parameter in isolation. However, in tropical regions, climate change may result in both of these parameters changing simultaneously and much less is known about how interacting stressors affect fish. Therefore, I examined the effect of increased temperature, both acute and following acclimation, on the ability of a range of Amazonian fishes to tolerate additional acute thermal and hypoxic stress. The ultimate goal of this thesis was to develop a relatively straightforward assay for identifying species that may be at greatest risk due to changes in ambient temperature and oxygen induced by climate change. The remainder of this chapter will review thermal tolerance, hypoxia tolerance, the physiological interaction between temperature and oxygen, and application to climate change, leading to the overall specific objectives of my thesis.  1.2 Thermal Tolerance Temperature affects biochemical, physiological and life history activities of all organisms, and large changes in temperature can often be lethal especially in poikilothermic fishes (Beitinger and Magnuson, 1975; Fry, 1947). An increase in water temperature consequently increases a fish’s biological reaction rates; Q10 is often used to quantify this effect and is typically around 2 in fish, meaning a 10˚C increase doubles reaction rates in the body (Clarke and Johnston, 1999; Schmidt-Nielsen, 1997). So as ambient water temperature increases, the oxygen consumption rate increases to meet the energy demand in fish. Temperature also affects protein structure, enzyme kinetics, membrane fluidity, mitochondrial respiration, and more (Schulte, 2011). Extreme  4 temperature can denature proteins, impair enzyme function (Fields, 2011), and increase membrane leakiness thereby affecting the mitochondrial electron transport system, and thus energy (ATP) production (Guderley, 2011). Due to these large influences on overall physiology, thermal tolerance strongly governs biogeographic distributions of freshwater fishes (Carpenter et al., 1992) and those that are not capable of moving to a more suitable environment must either acclimate or adapt to the higher temperatures. As temperature sets a fishes’ physiological limits and their ability to function within those limits, there is consequently an interest in quantifying thermal tolerance. Similar to hypoxia tolerance, several studies have investigated thermal tolerance of individual Amazonian species, but a single comparative study on a phylogenetically diverse group of species is lacking and is the focus of this thesis. Given the current high temperature of the Amazon and further predicted elevation due to climate change, this study focused on a current maximum river temperature of 31˚C (Val and Almeida-Val, 1995), and increases above that. There are two laboratory approaches that are primarily used to quantify upper temperature tolerance in fishes: The upper incipient lethal temperature (UILT) and critical thermal maximum (CTMax) (Becker and Genoway, 1979). The main disadvantage of the UILT technique is that it involves a direct transfer of fish to higher temperatures and measures time to mortality (Fry, 1947; Fry, 1967). This rapid change in temperature does not represent natural conditions and thus the UILT has been criticized for having little application in the field (Bennett and Judd, 1992). The CTMax method involves heating the water at a constant rate fast enough to allow body temperature to match environmental temperature and measures the temperature at  5 which the exposed fish loses equilibrium (Cox, 1974). The CTMax method is widely used as an indicator of thermal tolerance and can be used to compare among species and strains. As it is repeatable and easy to measure, I chose to use this metric as a proxy for acute thermal tolerance of a range of Amazonian species. The mechanistic cause of loss of equilibrium (LOE) is still unclear. Some studies propose that it is due to loss of aerobic scope at critical temperatures (Pörtner, 2010) (more to be discussed in section 1.3.2) or a failure of the central nervous system (Friedlander et al., 1976).  Regardless, such disability is environmentally relevant, as it will limit fish from escaping lethal conditions and likely result in death.   1.2.1 Effect of acclimation to higher temperatures on thermal tolerance It has been repeatedly shown that acclimation to higher temperature improves CTMax in fish (Beitinger and Bennett, 1999; Beitinger et al., 2000). Although the specific mechanisms are still unclear, the restoration of homeostasis during thermal acclimation appears to be associated with physiological and/or biochemical adjustments that offset the negative effects of elevated temperature. A relatively well-studied example would be heat stress-induced expression of heat-shock proteins (HSP). Fish are found to rapidly turn on HSP in their cells to protect proteins and membranes from temperature damage such as denaturation (Currie, 2011). However, the responses seem to be species- specific (Anttila et al., 2013; Fangue, 2006; LeBlanc et al., 2012), and thermal acclimation induces large interspecific variation in the HSP response (Beitinger et al., 2000).   6 Overall, the mechanisms and magnitude of response to thermal acclimation vary between species. If Amazonian fishes have the capacity to acclimate to higher temperature and to increase thermal tolerance, it will serve as a great advantage in dealing with the future temperature changes predicted due to climate change. To quantify the degree to which Amazonian fishes can thermally acclimate, I acclimated different species to temperatures above the current river maximum and assessed their CTMax.  1.2.2 Chronic lethal maximum Acute measurements of thermal tolerance, such as CTMax, do not directly address the potential for long-term survival in elevated temperatures, which are difficult to measure in the lab. A few methods have been developed that involve slower temperature change over a longer duration and employing mortality as an endpoint of thermal tolerance. Elliott’s hybrid temperature tolerance methodology exposed Atlantic salmon (Salmo salar) to a temperature change of 1˚C/h until a series of final constant temperatures, which were then held until mortality (Elliott, 1981; Elliott, 1991). In other studies, an even more gradual temperature increase of 1˚C/day was used until mortality (Fields et al., 1987; Grande and Andersen, 1991; Guest, 1985). These methods are different from UILT or CTMax as they provide considerable time for fish to acclimate (at least to some degree) to the increases in temperature during the trial. The maximum lethal temperature (noted as CLM: chronic lethal methodology) of Indonesian tropical marine fishes, determined by increasing temperature by 1.6˚C/day until 50% mortality, was 36 ~ 39˚C (Eme and Bennett, 2009). These values were lower but not too far off  7 their CTMax values of 40.1 ~ 43.4˚C in fish that had previously been acclimated to 31˚C for 14 days. Thus, the two methods yield similar values implying that they may be dependent upon similar mechanisms. In my study, I acclimated fish to higher temperatures by increasing temperature by 1˚C/day (Chapter 2) or 1˚C/12 hours (Chapter 3) up to a target temperature and then held the temperature constant for 10 days (Chapter 3) or 4 weeks (Chapter 2) to allow fish to acclimate. When more than 50% morbidity was observed during the acclimation period, the temperature was noted as the chronic lethal maximum (CLMax) and this was used as a more long-term upper thermal tolerance indicator.  1.3 Hypoxia Tolerance Low dissolved oxygen (DO) can occur in aquatic ecosystems due to natural phenomena and anthropogenic activities (Diaz and Rosenberg, 2008). With a few exceptions, fish depend on dissolved oxygen in the water for aerobic respiration. Hypoxia refers to low oxygen levels in either the external or internal environment. When water DO becomes too low (environmental hypoxia for fish), internal oxygen levels are reduced, called hypoxemia (Farrell and Richards, 2009). Since basic oxygen requirements differ between species and even individuals, it is difficult to define environmental hypoxia with a specific oxygen concentration. When there is a mismatch between oxygen supply and metabolic energy demand, fish respond with an array of behavioral, cardiovascular, respiratory, and metabolic adjustments (Perry, 2011; Richards, 2011; Wang and Richards, 2011).  8 As discussed previously, the Amazon floodplains that many fishes rely on often become hypoxic (Kramer et al., 1978; Röpke et al., 2016; Val and Almeida-Val, 1995). Primary producers are active during the day, creating hyperoxia, and respiration by microbes, plants and other organisms depletes the oxygen during the night. Amazonian fishes have evolved diverse mechanisms to respond to diurnal hypoxia by modifying one or more steps of the oxygen transport cascade, which are the biological steps involved in oxygen movement from the atmosphere to the mitochondrion. Well-studied mechanisms include hyperventilation, initiation of aquatic surface respiration (e.g. Colossoma macropomum, Sundin et al., 2000), air breathing by facultative air-breathers (Graham, 1997), the use of modified swim-bladders for aerial respiration (e.g. Arapaima gigas, Brauner, 2004), suppression of metabolic rate (Chippari-Gomes et al., 2005; Sloman et al., 2006), and activation of anaerobic metabolism (Almeida-Val et al., 1995; Chippari-Gomes et al., 2005). Several species and their responses in hypoxia have been investigated previously, but a single comparative study on a diverse number of Amazonian species is lacking and will be the focus of this thesis. Generally, two methods are widely used to measure hypoxia tolerance of fish: 1.measurement of time to loss of equilibrium (Time to LOE) and 2.incipient lethal oxygen saturation (ILOS). Both methods involve a rapid decrease in water oxygen levels, usually by using nitrogen gas to displace oxygen. Then, oxygen tension is either held at a constant target level and time to LOE is measured, or a slow constant rate of oxygen decrease is induced and the oxygen tension at which the fish exhibit LOE is noted. Although these methods might be informative when comparing similar species or subspecies, they may overlook the role of anaerobic metabolism and the potential for  9 acclimation during the trial. Some species recruit anaerobic metabolism when the oxygen level is low, and the magnitude of this recruitment and oxygen tension at which it is initiated are species-specific (Pörtner and Farrell, 2008; Verberk et al., 2013). The recorded onset of anaerobic metabolism of Amazonian fishes is within 1 ~ 8 hours of exposure to hypoxia (Almeida-Val and Val, 1993; Chippari-Gomes et al., 2005; Muusze et al., 1998; Val et al., 2015). Although it is ecologically relevant and important, in this study I tried to minimize the potential for use of anaerobic metabolism and focus mainly on oxygen limitations by restricting the duration of hypoxic trial to less than 20 minutes in total. Longer exposure to hypoxia also imposes greater potential for acclimation. Responses during hypoxia acclimation are important to consider, but I focused on acute hypoxia for consistent comparisons across species. Thus, I measured hypoxia tolerance by decreasing water oxygenation levels at a constant and relatively rapid rate from normoxia (approximately 6%/min, <20 minutes in total from normoxia to anoxia), and then measured the air saturation at which fish lost equilibrium as an indication of hypoxia tolerance (% air saturation at LOE).   1.3.1 Effect of an acute increase in temperature on hypoxia tolerance A further consideration when investigating hypoxia tolerance in fish is temperature as it affects different physiological processes, some of which are related to the oxygen transport cascade. First, temperature affects the dissolved oxygen concentrations in the water (Benson and Krause, 1984; Carpenter, 1966; Garcia and Gordon, 1992). The oxygen solubility declines as temperature increases, lowering the amount of dissolved oxygen in the water. Also, hemoglobin-oxygen binding affinity  10 decreases with an increase in temperature due to the exothermic nature of heme groups binding with oxygen. The hemoglobin-oxygen binding affinity is measured and described using P50, the partial pressure of oxygen at which the blood is 50% saturated with oxygen. An increase in P50 represents a decrease in oxygen affinity as the oxygen dissociation curve of the blood shifts to the right with increasing temperature. While the increase in P50 (i.e. loss of affinity) can potentially increase oxygen delivery to the tissues, it can also limit oxygen uptake at the gills (Soldatov, 2003).  An increase in environmental temperature has a direct effect of an ectotherm animal’s metabolic rate. A meta-analysis of marine benthic organisms showed a reduction in survival time in hypoxia when exposed to warmer temperatures due to increased oxygen requirements (Vaquer-Sunyer and Duarte, 2011). Thus as a result of increased temperature, the limited environmental oxygen combined with compromised oxygen-supplying mechanisms make it more challenging for fish to meet increased metabolic demand.  Although there have been selective pressures on Amazonian species to deal with environmental challenges, the associated adaptations may not be sufficient to deal with additional pressures associated with anthropogenic activities. Tropical regions are predicted to experience both an increase in temperature and an increase in the frequency and severity of aquatic hypoxia (IPCC, 2014). However, the effect of an increase in temperature on Amazonian fish’s ability to tolerate hypoxia has not been well studied. Therefore, I investigated hypoxia tolerance across a diverse range of Amazonian fish species after exposure to an acute increase in temperature. Given the known negative effect of an increase in temperature on oxygen uptake and delivery,  11 along with the effect of temperature on metabolic rate, I hypothesized that an acute increase in temperature would result in a reduction in hypoxia tolerance. This study measured percent air saturation (i.e. a unit comparable to partial pressure) at LOE as an indicator of hypoxia tolerance. It is important to note that oxygen solubility, and therefore water oxygen content at any given percent saturation or partial pressure, declines with temperature. For example water oxygen content at 100% air saturation is 7.82 mg/L at 28˚C; 7.43 mg/L at 31˚C; 7.18 mg/L at 33˚C; 6.95 mg/L at 35˚C in freshwater (Benson and Krause, 1984). Thus, for every 2˚C increase in temperature, there is approximately 0.2 mg/L less dissolved oxygen in the water. I acknowledge that this could have a potential effect in comparing data between temperature treatments, but the difference is minor and I chose to standardize for partial pressure rather than oxygen content, as the former is the driving force for diffusion.   1.3.2 Effect of acclimation to higher temperatures on hypoxia tolerance To further explore the effect of temperature on hypoxia tolerance, I also acclimated species to higher temperatures and assessed acute hypoxia tolerance. During acclimation to increased temperature, fish are known to undergo morphological and/or physiological changes associated with enhancing oxygen flux through the oxygen transport cascade, resulting in improved hypoxia tolerance. For example, Atlantic killifish (Fundulus heteroclitus) showed a decrease in hypoxia tolerance after acute exposure to higher temperatures, but warm acclimation (6 weeks) partially restored hypoxia tolerance in association with an increase in gill surface area (McBryan et al., 2016). An increase in compact myocardium capillary density with warm  12 acclimation (4 weeks) was also found in Atlantic salmon (Salmo salar), improving hypoxia tolerance by enhancing cardiac performance and thus oxygen supply to tissues (Anttila et al., 2015).  Thermal acclimation could also offset the negative effects of increased temperature on hemoglobin oxygen affinity by decreasing red blood cell NTP:hemoglobin ratio and increasing hematocrit (Chapman et al., 2002; Fyhn et al., 1979; Powers, 1980; Powers et al., 1979). Organic phosphates (ATP, GTP, collectively referred to as NTP) are hemoglobin allosteric modifiers that decrease the affinity for oxygen. During the wet season of the Amazon when oxygen level is low, silver mylossoma (Mylossoma duriventre) have lower concentrations of NTP in the blood for greater oxygen extraction from the water (Cavalcante-Monteiro et al., 1987). Many Amazonian species are also found to decrease organic phosphate levels following acclimation to increased temperatures (Val, 2000). Overall, adjustments are made to help extract oxygen from the environment, facilitate oxygen delivery and lower metabolic demand. Therefore, I hypothesized that thermal acclimation in Amazonian fishes would be associated with recovered or improved hypoxia tolerance.  1.4 Oxygen defining thermal limit: relationship between thermal tolerance and hypoxia tolerance Despite the significance of thermal tolerance in ectotherms, what determines the limit is currently unclear. The “Oxygen- and Capacity- Limited Thermal Tolerance” (OCLTT) hypothesis proposes that an animal’s aerobic scope (difference between maximal and standard metabolic rate) is limited by its ability to supply and deliver  13 sufficient oxygen to the tissues at both sides of the thermal window (Pörtner, 2010; Pörtner and Farrell, 2008; Pörtner and Knust, 2007). Many studies have shown that aerobic scope declines at high temperatures in aquatic ectotherms, which is associated with cardiorespiratory limitations and limited oxygen supply (Eliason et al., 2011; Farrell, 2009; Steinhausen et al., 2008). Thus, the upper thermal limit of a fish may result from hypoxaemia or a mismatch in oxygen demand versus supply.  The OCLTT hypothesis implies that an animal with a higher thermal tolerance should have a better mechanism to cope with decreasing oxygen supply at warmer temperatures. The inverse might also be true: an animal that is tolerant to low oxygen may have a higher thermal limit relative to an animal that is more sensitive to hypoxia. These ideas lead to a functional association between thermal tolerance and hypoxia tolerance, but little work has been done to investigate this relationship. To date, only one study has directly compared two traits at the whole animal level. Anttila et al. (2013) have shown a significant correlation between thermal tolerance and hypoxia tolerance as indicated by CTMax and time to LOE at a constant level of hypoxia in families of Atlantic salmon (Salmo salar). Currently, no one has investigated this relationship interspecifically in a phylogenetically diverse group of species. Thus, I investigated whether there is a correlation between thermal tolerance and hypoxia tolerance across a diverse group of Amazonian fish species.  1.4.1 Thermal tolerance in hyperoxia According to the OCLTT hypothesis, an animal’s thermal tolerance could be extended with a greater supply of oxygen at the higher temperatures. Some studies  14 have shown improved resistance to warming in the presence of elevated environmental oxygen, hyperoxia. Antarctic marine bivalve (Laternula elliptica) displayed a decrease in haemolymph oxygenation with temperature, with a significantly large reduction at 4.3˚C in normoxia (Pörtner et al., 2006). A similar decrease in haemolymph oxygenation was observed in hyperoxia (200% air saturation), but the large reduction did not occur until a higher temperature of 7˚C was reached. Additionally, the survival time of Goldfish (Carassius auratus) at 35˚C was significantly longer in the presence of elevated oxygen saturation compared to normoxic water (Weatherley, 1970). In the stenothermic Antarctic fish Pachycara brachycephalum, metabolic rate increased exponentially with warming in normoxia as expected (Mark et al., 2002). Although fish in hyperoxic water died at the same temperature as the normoxic group, they showed a suppressed response, resulting in a more linear increase in metabolic rate with warming. In the Amazon, floodplain oxygen levels can reach up to 250% air saturation due to photosynthesis during the day (Kramer et al., 1978; Val and Almeida-Val, 1995) and thus, fishes of that region may be routinely exposed to hyperoxia. Whether hyperoxia influences thermal tolerance in Amazonian fishes is unknown but is directly relevant to their life history especially considering that peak levels of hyperoxia occur mid-day when it is hottest. If hyperoxia alleviates thermal stress and allow fish to survive higher temperature, it is not only of ecological relevance, but it would further support the OCLTT hypothesis. As a way of indirectly measuring if thermal tolerance is associated with oxygen limitation, in the present study, some species were exposed to hyperoxia - higher ambient oxygen levels (>200% air saturation) – and CTMax was measured.    15 1.5 Application To Climate Change The main purpose of this study was to build a basic physiological foundation and investigate whether fishes have the capacity to acclimate to future predicted changes. If thermal tolerance and hypoxia tolerances are mediated through similar pathways, thermal acclimation may increase an animal’s ability to deal with lower oxygen. To be most beneficial for Amazonian fishes facing the predicted increases in temperature and more severe bouts of hypoxia associated with climate change, warm acclimation must not only enhance thermal tolerance, but also further enhance tolerance to hypoxia (Figure 1.1B). If thermal tolerance is improved, but hypoxia tolerance is reduced following acclimation to a higher temperature (Figure 1.1A), the latter may negate the benefits of the former in an environment like the Amazon where both temperature and hypoxia are likely to co-vary.     Figure 1.1 Acclimation to higher temperature (e.g. 33˚C) may (A) increase thermal tolerance but decrease hypoxia tolerance relative to that observed at 31˚C or (B) increase both hypoxia and thermal hypoxia tolerance.  Thermal acclimation has been shown to trigger physiological adjustments that benefit oxygen transfer to tissues and lowers oxygen demand in some species. Some    A B  16 studies have shown that acclimation to low oxygen improves temperature tolerance and vice versa. For example, Burleson & Silva (2011) demonstrated acclimation to moderate hypoxia (50% air saturation) for 7 days in the channel catfish (Ictalurus punctatus) resulted in a slightly elevated temperature (<0.5˚C) at which cardiovascular collapse was observed. Anoxia preconditioning of neurons in the locust increased the upper thermal limit of neuron function (Wu et al., 2002) and plant seedlings following heat acclimation survive anoxia longer (Banti et al., 2008).  Studies on fish as well as other animals provide evidence that some of the molecular and biochemical responses to either thermal or hypoxic stress are also inter-related and can be induced by one another. A few overlapping molecular pathways are those involving heat shock proteins (HSP) and hypoxia inducible factors (HIF). HSP can be induced by both heat and hypoxic shocks (Currie, 2011). For example, hypoxia exposure increased liver HSP levels in the mudminnow (Umbra limi) (Currie et al., 2010). Also, rats preconditioned to hypoxia induced HSP, which was associated with a subsequent increased survival time in heat stress (Wen et al., 2002). HIF regulates genes that promote oxygen transfer, anaerobiosis, and oxygen consumption rates upon hypoxia (Nikinmaa and Rees, 2005). HIF up-regulation is found to be necessary and beneficial in heat acclimation in nematodes (Treinin et al., 2003), suggesting that it is induced upon thermal stress as well. If acclimation to elevated temperature improves hypoxia tolerance and vice versa as suggested by these studies, this may be beneficial in fish coping with interactive effects of increased temperature and reduced environmental oxygen associated with  17 climate change. Thus, I compared both hypoxia and thermal tolerance following acclimation to higher temperatures.  1.6 Overall objectives I investigated the following 5 objectives at 2 different locations: The University of British Columbia (UBC) and Instituto Nacional de Pesquisas da Amazônia (INPA). Chapter 2 discusses objectives 1 ~ 4 and experiments conducted at UBC with 6 wild-caught Amazonian species. These fishes probably experienced more selection through the shipping process than those tested near the field in Chapter 3. Chapter 3 discusses objectives 1 ~ 5 and experiments conducted at INPA with 15 wild-caught Amazonian species. Although these fishes did not experience shipping stress, the experiments in this chapter were more constrained by field conditions. Combined, I selected 20 species that represent a broad phylogeny (Table 1.1): In each chapter, species in the taxonomic orders of Characiformes, Siluriformes and Perciformes were studied. Of the total 20 species, 17 are ornamental species that are commonly exported out from the Amazon and 3 (Chapter 3: A. ocellatus, B. amazonicus, and C. macropomum) are common aquaculture species in the Amazon.   Objective 1: To survey thermal tolerance and hypoxia tolerance of all species investigated at current river temperature.  After acclimation to 31˚C (Chapter 2: UBC) or natural diurnal temperature fluctuation 26 to 30˚C (Chapter 3: INPA), 20 Amazonian species were measured for critical thermal maximum (CTMax) as a proxy for thermal tolerance and % air saturation  18 at LOE as a proxy for hypoxia tolerance. As tropical species that experience warm temperatures annually, I hypothesized that all species would have a relatively higher CTMax than temperate species such as rainbow trout and Atlantic salmon (Currie et al., 1998; Elliott and Elliott, 1995). Also, given that the oxygen level fluctuates from anoxia to hyperoxia in nature (Kramer et al., 1978; Val and Almeida-Val, 1995), I hypothesized all species to be hypoxia tolerant, enduring oxygen levels close to anoxia.   Objective 2: To assess the effect of acclimation to higher temperatures on thermal tolerance. As shown in many other studies (Beitinger and Bennett, 1999; Beitinger et al., 2000), I hypothesized that thermal acclimation to 31, 33 or 35˚C (Chapter 2: 4 weeks & Chapter 3: 10 days) would increase CTMax relative to that in objective 1.  Objective 3: To assess the effect of acute exposure and acclimation to higher temperatures on hypoxia tolerance.  An increase in ambient water temperature is found to limit oxygen supply and/or increase metabolic demand (Schulte, 2011). Thus, I hypothesized that acute exposure to higher temperature would decrease a species’ ability to tolerate hypoxia at that temperature. Furthermore, similar to findings of temperate species (Anttila et al., 2015; McBryan et al., 2016), I hypothesized that acclimation to higher temperatures would increase hypoxia tolerance at that temperature.    19 Objective 4: To assess the correlation between thermal tolerance and hypoxia tolerance at different acclimation temperatures.  Given the findings that support the correlation between thermal and hypoxia tolerance (Anttila et al., 2013; Burleson and Silva, 2011), I hypothesized to find the same correlation in Amazonian fishes following acclimation to all temperatures (Chapter 2: 31, 33 or 35˚C and Chapter 3: 28, 31, 33, or 35˚C).   Objective 5: To assess the effect of hyperoxia on thermal tolerance.  As Amazonian fishes experience supersaturation during the day when it is the hottest in the field, and thermal tolerance may be limited by oxygen delivery, I hypothesize that CTMax to be greater in hyperoxia than normoxia.  Order Chapter 2: UBC Chapter 3: INPA Characiformes   Carnegiella strigata,  Nannostomus trifasciatus, Hemigrammus rhodostomus Brycon amazonicus, Carnegiella strigata,  Colossoma macropomum, Hemiodus gracilis,  Hyphessobrycon erythrostigma, Nannostomus eques,  Paracheirodon axelrodi  Siluriformes Corydoras julii Corydoras pulcher,  Corydoras schwartzi,  Corydoras splendens,  Otocinclus spp.  Perciformes Apistogramma viejita, Mikrogeophagus ramirezi Apistogramma borelli,  Apistogramma geophyra,  Astronotus ocellatus, Laetacara fulvipinnis  Table 1.1. List of 20 species investigated in this thesis categorized into taxonomic orders. One species (C. strigata) was measured at both UBC and INPA.   20 Chapter 2: The Relationship Between Thermal Tolerance and Hypoxia Tolerance in Amazonian Fishes at The University of British Columbia (UBC)  2.1  Introduction The Amazon river is one of the most biodiverse habitats in the world, home to about 20% of the world’s freshwater fish species (Reis, 2013). It is also characterized by extreme environmental conditions, such as high stable temperature and seasonal and daily hypoxia (Kramer et al., 1978; Val and Almeida-Val, 1995). Amazonian fishes have evolved diverse mechanisms to cope with these challenging conditions (Fyhn et al., 1979; MacCormack et al., 2003; Wood et al., 2009). However, climate change is predicted to exacerbate these already extreme conditions. The temperature of the Amazon is predicted to increase by up to 2.2 ~ 7˚C in the next century (IPCC, 2014). This increase in temperature is associated with a decrease in oxygen solubility (Benson and Krause, 1984; Carpenter, 1966; Garcia and Gordon, 1992) and an increase in eutrophication (Ficke et al., 2007), which are predicted to lead to more frequent and severe bouts of hypoxia. Climate change, therefore, may push native species to their physiological limits if they do not have the capacity to acclimate and adapt to these new conditions.  The capacity of Amazonian fish species to acclimate and adapt to a warmer and more hypoxic environment is unclear. Studies of other tropical ectotherms have shown that these animals currently live close to their upper thermal limits, and some above thermal optima (Somero, 2010). Their ability to acclimate to higher temperatures is also limited relative to temperate species (Stillman, 2003). Amazonian fishes, as tropical  21 stenothermic ectotherms, may be near their upper thermal limits with little room for acclimation. This chapter aims to address this issue by surveying the acute thermal and hypoxia tolerances of various Amazonian fishes and investigating the effect of increased ambient temperature, both upon acute exposure and after acclimation, on those parameters. The mechanistic basis of thermal tolerance is currently uncertain. Recent studies suggest a limitation of aerobic performance at extreme temperatures sets the thermal optima and limits, a concept embodied in the “Oxygen- and Capacity- Limited Thermal Tolerance hypothesis (OCLTT)” (Pörtner, 2010; Pörtner and Knust, 2007). According to the hypothesis, a mismatch in oxygen demand versus supply resulting from cardiorespiratory failure defines the upper thermal limit in some ectotherms (Eliason et al., 2011; Farrell, 2009; Steinhausen et al., 2008). Therefore, the hypothesis implies a functional association between the ability to tolerate low oxygen levels and elevated temperature. If hypoxia and thermal tolerances are mediated through similar pathways, acclimation to one stressor may enhance tolerance to another, which will serve as a benefit in dealing with climate change induced changes. Studies have found that acclimation to higher temperatures benefits oxygen transfer to tissues and lowers oxygen demand in an array of taxa (Banti et al., 2008; Burleson and Silva, 2011; Wu et al., 2002). To date, only one study has directly compared thermal and hypoxia tolerance in fish; Anttila et al. (2013) found a relationship between these two traits in families of Atlantic salmon. This chapter aims to provide insight into this relationship interspecifically in a phylogenetically diverse group of Amazonian teleost species in a controlled setting.  22 Specifically, the objectives of this chapter were: 1) to survey thermal tolerance (CTMax) and hypoxia (% air saturation at LOE) of Amazonian species at current river maximum temperature of 31˚C and determine the relationship between those two parameters; 2) to determine the effect of acute exposure (<1.5 hours) and acclimation (4 weeks) to higher temperatures of 33 and 35˚C on hypoxia tolerance; 3) to determine the effect of acclimation (4 weeks) to higher temperatures of 33 and 35˚C on thermal tolerance (please refer to section 1.6). To investigate these objectives, I chose to work on 6 ornamental Amazonian fishes at UBC in this chapter (Table 1.1). In the middle Rio Negro Basin (Amazonas, Brazil), the ornamental fish trade contributes over 60% of the total income (Chao and Prang, 1997). The ornamental fishes are typically found and caught in floodplains (várzeas), small streams (igarapés), and flooded forests (igapós) (Chao, 2001). These shallow swamp habitats experience greater fluctuations in oxygen and temperature diurnally than does the main river (Almeida-Val and Hochachka, 1995; Soares and Junk, 2000; Val and Almeida-Val, 1995). Climate change will cause more prominent environmental changes in these areas where many ornamental fishes are found. If fishes cannot adapt to the changes, the ornamental fish trade will be negatively impacted. Therefore, I used ornamental Amazonian fishes that are commonly exported from this region. To eliminate any biases that may be associated with genetic selection by ornamental fish culturists, I used wild individuals caught in the Amazon and air-shipped to the lab. Experiments were carried out in the aquatics laboratory at UBC, which allowed careful control of selected factors (temperature and oxygen) and other variables. While  23 it may not be sufficiently representative of the natural environment, it provided a controlled environment as a first step to investigate the relationship between thermal and hypoxia tolerance. Furthermore, the influence of acclimation to temperatures above the current average floodplain temperature of 31˚C on subsequent hypoxia and thermal tolerance was investigated to gain insight into how fishes of the Amazon may be affected by climate change.  2.2  Material & Methods Experiments were conducted at UBC. Approximately 100 fish of Carnegiella strigata (Hatchetfish; Mean weight = 0.215±0.010g), Nannostomus trifasciatus (Three-lined pencilfish; 0.152±0.003g), Hemigrammus rhodostomus (Rummy-nose tetra; 0.590±0.028g), Corydoras julii (Julii corys; 1.858±0.067g), Apistogramma viejita (Viejita; 0.931±0.053g), and Mikrogeophagus ramirezi (Ram cichlid; 0.722±0.030g) were air-shipped from Manaus, Brazil (Canadian Aquatics Importing, BC, Canada) to the Department of Zoology at UBC. Approximately 20 ~ 30% of the fish shipped were already dead upon arrival or in poor condition and died within few days. All fish were held in individual 60-L glass aquaria outfitted with charcoal filters and filled with dechlorinated City of Vancouver tapwater: [Na+]=170uM, [Cl-]=210uM, hardness, 30mg/L CaCO3 (Ou et al., 2015). All aquaria were maintained in a common environmental chamber on a 12L:12D photoperiod at 25°C. Fish were fed daily to satiation (Tetramin® Tropical Flakes, VG, USA) and fasted for 36-48 hours prior to all experiments. Water quality (O2, pH and ammonia) was measured weekly to ensure an adequate environment (85-100% air saturation, 6.9-7.1, 0~0.5mg/L respectively).  24 Treatment of all experimental animals was in accordance with the UBC animal care protocol #A15-0266.  2.2.1  Experimental design Fish were acclimated to target temperatures of 31, 33 or 35°C in 60-L aquaria (20 fish/aquaria) for 4 weeks prior to assessment of thermal tolerance and hypoxia tolerance. Since the current maximum river temperature is 31˚C, it was chosen as the “control” temperature for this study. To achieve these acclimation temperatures, electric heaters plugged into temperature controllers (FisherbrandTM TraceableTM Digital Temperature Controller, ON, Canada) were used to increase the temperature by 1˚C/day within the aquaria until the target temperature was reached at which point that temperature was maintained. Fish that were acclimated to 31, 33 or 35˚C were transferred to the experiment setups (within 1 min) and subjected to either thermal or hypoxia tolerance trials as described below. Fish that were acclimated to 31˚C were also acutely exposed to higher temperatures of 33 and 35˚C (0.2˚C/min) and then assessed for hypoxia tolerance.   2.2.2 Thermal tolerance To measure and compare species' thermal tolerance, a critical thermal maximum (CTMax) test was conducted. After 4 weeks acclimation to 31°C, 33˚C or 35˚C, 8 fish from each acclimation temperature were transferred to a 20-L glass aquaria and allowed an hour to acclimatize to the new environment. Tanks were initially held at the temperature to which fish were acclimated and water was well aerated. Then water  25 temperature was increased by approximately 0.2°C/min until loss of equilibrium (LOE; as described above) of each fish was observed. At LOE, the temperature was noted (indicative of CTMax), and the fish was removed (with care to not disturb the remaining fish) and then weighed. Throughout the CTMax trial, water was constantly bubbled with air to ensure adequate oxygenation and mixing of the water, and also to avoid supersaturation, which can occur with warming. This procedure continued until all fish in the tank experienced LOE.  The ideal rate of temperature increase during a CTMax trial varies with size and body type (Cox, 1974). The rate must be slow enough for internal body temperature to match the outer water temperature, but fast enough to prevent progressive thermal acclimation. The rate used in this study is consistent with that used with other similar sized tropical teleost (Becker and Genoway, 1979). During the process of acclimation to 33 and 35˚C, I observed high (>50%) morbidity of some species. In this case, the highest acclimation temperature that >50% fish were able to tolerate for 4 weeks was noted as a chronic lethal maximum (CLMax).   2.2.3 Hypoxia tolerance  To assess hypoxia tolerance, percent air saturation at which fish lose equilibrium was measured (% air saturation at LOE). Individual fish were transferred to 300-ml mesh-sided chambers submerged in a 40-L Plexiglas tank filled with fresh water and fish were allowed an hour to acclimatize. Water within the 40-L tank was continuously aerated and circulated with two submersible water pumps to ensure sufficient water flow through the chambers. Percent air saturation was monitored continuously using a fiber  26 optic oxygen probe (PreSens Precision Sensing, Regensburg, Germany) that was calibrated before every trial. The hypoxia tolerance trial was initiated by bubbling nitrogen gas into the water at a constant rate, resulting in a constant rate of reduction in water PO2 (expressed as % air saturation of the water) of 7%/min. At LOE (as indicated by the inability of the fish to maintain its dorso-ventral orientation even with a gentle movement of the container), the % air saturation was noted as the end point of hypoxia tolerance, and the fish was removed, euthanized and weighed. Under these conditions, complete anoxia was reached within 20 minutes. This relatively rapid rate of O2 depletion was chosen to prevent fish from acclimating to hypoxia during the protocol and confounding results.  Species acclimated to the control temperature of 31˚C were acutely exposed to higher temperatures of 33˚C or 35˚C and hypoxia tolerance was assessed. Fish were transferred to the 300-ml chambers as described above and water temperature in the 40-L tank was increased at 0.2˚C/min until desired temperatures (33˚C or 35˚C) were reached. After an hour, hypoxia tolerance (as described above) was conducted at the respective temperature.   2.2.4 Statistical analysis For both chapters, statistical analyses were performed using R software (version 0.98.1091), and graphs were created with Prism software (version 5.0a). Data have been expressed as the means ± SEM (standard error, n=6-8). Data were first analyzed using a two-way ANOVA with temperature or hyperoxic treatment and species as factors. If the interaction terms were significant, the data were separated and analyzed  27 independently using one-way ANOVA with temperature or hyperoxic treatment as a factor for each species. Then post hoc tests were carried out using Tukey’s HSD test.  Correlation between temperature and hypoxia tolerance was determined using the Pearson’s correlation test.  To test if thermal tolerance or hypoxia tolerance co-varies with body mass, I tested for a correlation between the phylogenetic independent contrast of thermal or hypoxia tolerance and absolute body mass (Felsenstein, 1985) and found no effect in all temperatures (p>0.05). To test for phylogenetic non-independence in thermal tolerance and hypoxia tolerance, I used Pagel’s lambda (Pagel, 1999) and found no significant phylogenetic signal for both variables (Thermal tolerance: 28˚C λ=0.303, p=0.415; 31˚C λ<0.000, p=1; 33˚C λ=0.110, p=0.717; Hypoxia tolerance: 28˚C λ<0.000, p=1; 31˚C λ=0.395, p=0.334; 33˚C λ=0.297, p=0.514). As a result, the body mass and the phylogenetic non-independence were not corrected for in the subsequent analysis. Tests for phylogenetic independent contrast and phylogenetic signal were performed in R using the ape and phytools package, respectively, using branch lengths proportional to time and mean values for each species. Here, I used composite phylogeny based on published data (Abe et al., 2014; Near et al., 2012; Near et al., 2013). However, for the genera where time-calibrated phylogenies have not been resolved (Apistogramma, Corydoras and Nannostomus), I used the tree topology of Open Tree of Life (Hinchliff et al., 2015) and branch lengths that added up to the age of the respective genera (Appendix A). In all cases, α was set at 0.05.  28 2.3 Result 2.3.1 Thermal tolerance and the effect of acclimation to higher temperatures Critical thermal maximum (CTMax) in 31˚C acclimated fish ranged from 38.2 to 41.1 ˚C for 6 species measured (Figure 2.1). C. strigata lost equilibrium at the lowest temperature of 38.2˚C, and N. trifasciatus slightly higher at 39.5˚C. C. julii and H. rhodostomus were very similar, 40.5˚C. Two cichlid species (A. viejita & M. ramirezi) exhibited a CTMax of 41.0 and 41.1˚C respectively.  In general, there was an increase in CTMax in species that could acclimate to higher temperatures (Figure 2.1). Five species (all except C. strigata) survived the 4-week acclimation to 33˚C. Of those, 3 species (N. trifasciatus, A. viejita, and M. ramirezi) increased CTMax relative to 31˚C acclimated fish. N. trifasciatus displayed the most prominent change with a 3˚C increase in CTMax, followed by A. viejita with a 1.1˚C increase and M. ramirezi with a 1.5˚C increase relative to 31˚C acclimated fish. Only one species (M. ramirezi) was capable of surviving the 4-week acclimation to 35˚C, and CTMax increased to 44˚C, a 1.3˚C increase from the 33˚C acclimation fish and a 2.8˚C increase over the 31 ˚C acclimated fish. On average, if fish survived acclimation to higher temperature, CTMax increased by 0.6˚C for every 1˚C increase in acclimation temperature. Moreover, the species’ chronic lethal maximum (CLMax) was positively correlated with CTMax measured in 31˚C acclimated fish (r=0.84, N=6, p=0.022). C. strigata, which had the lowest CTMax of the 6 species investigated (38.2˚C), also had the lowest CLMax of 31˚C. The 4 species with mid thermal tolerance (N. trifasciatus 39.5˚C; C. julii 40.2˚C; H. rhodostomus 40.4˚C; A. viejita 41˚C) had CLMax of 33˚C. M.  29 ramirezi which had the highest CTMax (41.1˚C) also had the highest CLMax of 35˚C and was the only species capable of the 4-week acclimation to 35˚C.  2.3.2 Hypoxia tolerance and the effect of an increase in temperature (either acute or following acclimation) All 6 species acclimated to 31˚C were hypoxia tolerant having an LOE <10% air saturation (Figure 2.2). Generally, cichlids (A. viejita & M. ramirezi) were the most hypoxia tolerant species, withstanding close to anoxia (LOE of 0.7 & 1.6% air saturation respectively). H. rhodostomus, C. julii, C. strigata, and N. trifasciatus had an LOE that ranged from 3.1 to 9.6% of air saturation respectively. Hypoxia tolerances were reduced in 3 of the 6 species following an acute increase to higher temperatures (Figure 2.2A). H. rhodostomus lost equilibrium at about 3% air saturation higher for every 2˚C warmer water. N. trifasciatus displayed the greatest change, losing equilibrium at about 5% air saturation higher following an acute exposure to 33 or 35˚C relative to values measured at 31˚C. A. viejita also lost equilibrium at a higher air saturation when exposed to either 33 or 35˚C acutely. The other 3 species (C. strigata, C. julii, and M. ramirezi) showed no effect of acute exposure to higher temperatures. Acclimation to either 33 or 35˚C did not improve hypoxia tolerance at those temperatures relative to that measured at 31˚C in any of the 6 species (Figure 2.2B). C. strigata could not be acclimated to temperatures above 31˚C, and thus cannot be compared. Acclimation to higher temperature of 33˚C had no effect on the 2 species (H. rhodostomus and C. julii). In the other 3 species, acclimation to higher temperatures  30 (33˚C for N. trifasciatus and A. viejita; 35˚C for M. ramirezi) resulted in reduced hypoxia tolerance at those temperatures relative to that measured at 31˚C. In fact, for 2 species (A. viejita and M. ramirezi) 4 weeks of acclimation to 33 and 35˚C respectively resulted in a further reduction in hypoxia tolerance relative to acute exposure to the same temperatures (Figure 2.2; denoted by †). For A. viejita, LOE was 1.13% air saturation following acute exposure to 33˚C and 1.42% air saturation following acclimation to 33˚C. In M. ramirezi, LOE was 1.63% air saturation following acute exposure to 35˚C and 4.15% air saturation following 35˚C acclimation.   2.3.3 Relationship between thermal tolerance and hypoxia tolerance I found a significant correlation between thermal and hypoxia tolerance of these 6 ornamental Amazonian species that had been acclimated to 31˚C (Figure 2.3; r=-0.448285, p=0.03425). The species with greater hypoxia tolerance (and thus lower LOE as % air saturation) also had the greatest thermal tolerance (as indicated by CTmax; e.g. A. viejita) and vice versa (e.g. C. strigata). Following a 4-week acclimation to 33˚C, however, there was no significant correlation thermal and hypoxia tolerance (Figure 2.3; r=0.0420425, p=0.9465). I could not test for correlation at higher temperatures at 35˚C, as only one species was able to acclimate to that temperature.   2.4 Discussion  This chapter surveyed thermal tolerance (CTMax) and hypoxia tolerance (% air saturation at LOE) of 6 ornamental Amazonian fishes acclimated to 31˚C. I found that all are of these fishes highly tolerant to high temperatures (section 2.4.1) and hypoxia  31 (section 2.4.2), and there was a correlation between thermal and hypoxia tolerance in these 6 species acclimated to 31˚C (section 2.4.3). The CTMax increased after acclimation to 33 and 35˚C as expected, but in most species only modestly (section 2.4.1.1). However, many species failed to survive in those temperatures over a long term (section 2.4.1.1: CLMax). An acute exposure (<1.5hours) to 33 and 35˚C decreased hypoxia tolerance at those temperatures in these 6 species relative hypoxia tolerance measured at 31˚C (section 2.4.2.1). In species that could acclimate to 33 and 35˚C (4 weeks), hypoxia tolerance at those temperatures also decreased relative to the hypoxia tolerance measured at 31˚C (section 2.4.2.2). In fact, 2 species yielded lower hypoxia tolerance after acclimation to higher temperature relative to the acute exposure to higher temperature values. I also found a correlation between thermal and hypoxia tolerance in the 6 species at 31˚C, but this was lost after acclimation to 33 and 35˚C.  2.4.1 Thermal tolerance The CTMax of 6 Amazonian species measured in this study range from 38.2 ~ 41.1˚C in fish that were acclimated to 31˚C (Figure 2.1). These values are higher than temperate species such as trout (Oncorhynchus mykiss) with a CTMax of 29.8˚C in fish that had been acclimated to 20˚C (Currie et al., 1998). Other tropical species such as Nile perch (Lates niloticus) have a similar CTMax of 38.6˚C from a habitat temperature of 27.5˚C (Chrétien and Chapman, 2016) and platyfish (Xiphophorus maculatus) of 41.5˚C in fish previously acclimated to 30˚C (Prodocimo and Freire, 2001). Eurythermal fishes also have similarly high CTMax values: 30˚C acclimated largemouth bass (Micropterus salmoides) and channel catfish (Ictalurus punctatus) exhibited CTMax  32 values of 38.5˚C and 40.3˚C respectively (Currie et al., 1998). Therefore, the CTMax of these investigated fishes are within the typical range of tropical species that correspond to their warm habitats.  2.4.1.1 Effect of acclimation to higher temperatures on thermal tolerance In species that could acclimate to higher temperatures (33 or 35˚C) for 4 weeks, there was a significant increase in CTMax (Figure 2.1), which has been observed in many other fishes (Beitinger and Bennett, 1999; Beitinger et al., 2000). Some species (N. trifasciatus, A. viejita, and M. ramirezi) had a greater capacity to increase their CTMax with acclimation than others (H. rhodostomus and C. julii), indicating species dependent plasticity. M. ramirezi acclimated to 35˚C had a CTMax of 44˚C, slightly below the most thermally tolerant species measured to date, the South West Hot Springs tilapia (Alcolapia grahami) with a CTMax of 45.6˚C in fish that had been acclimated to 40 ~ 41˚C in their natural environment (Wood et al., 2016).  Overall, CTMax increased by about 0.6˚C for every 1˚C increase in acclimation temperature that could be achieved. This value is relatively large compared to other species: 0.12 ~ 0.2 for temperate species (Brett, 1952; Currie et al., 1998); 0.26 ~ 0.36 for tropical species  (Eme and Bennett, 2009); 0.28 ~ 0.4 for eurythermal species (Bennett and Beitinger, 1997; Chrétien and Chapman, 2016; Currie et al., 2004). However, I found that the magnitude of change is species-specific. The greatest increase in CTMax of 1.6˚C was observed in N. trifasciatus from 31 to 33˚C acclimation. On the other hand, C. julii and H. rhodostomus were only able to increase CTMax by about 0.1˚C from 31 to 33˚C acclimation. Thus, I found evidence that a few species  33 have some capacity to increase thermal tolerance with acclimation to higher temperature, which could benefit them in dealing with climate change. The most significant finding of this study, however, is that 5 out of 6 species failed to survive 4 weeks in the highest temperature of 35˚C (CLMax). C. strigata could not survive 4 weeks of acclimation above 31˚C, and 4 species (N. trifasciatus, H. rhodostomus, C. julii, and A. viejita) not above 33˚C. The findings clearly indicate that these species have a limited capacity to acclimate and will likely struggle if temperatures rise much above the current river temperature (31˚C) for a long duration. Even M. ramirezi, which could withstand the highest temperature measured (35˚C), seemed to face physiological constraints in its ability to tolerate hypoxia at higher temperatures (Figure 2.2B).  There is a growing body of evidence that fish species living in a high and narrow thermal range, as in equatorial regions, may have a more narrow window of thermal tolerance and thus be more prone to the increases in temperature predicted by climate change (Somero, 2010). This is likely because they inhabit a thermal niche that is near their upper thermal limit (Deutsch et al., 2008; Neuheimer et al., 2011; Tewksbury et al., 2008; Wright et al., 2009). Results of this study indicate that some Amazonian fishes also have limited capacity to increase thermal tolerance following acclimation to higher temperatures. Although I only investigated 6 species here, the results indicate that chronic temperature elevations induced by climate change may impose a detrimental effect on a large number of the Amazonian species. The sample size may be small to predict population effects and the short exposure time neglects the possibility of adaptation or early life history acclimation, but the observed high morbidity is surprising  34 and may imply a lack of plasticity required to withstand temperature changes associated with climate change in many Amazonian species.    2.4.2 Hypoxia tolerance Amazonian fish species are known to be hypoxia tolerant to survive diurnally fluctuating oxygen levels in flooded forests and swamps (Kramer et al., 1978; Val and Almeida-Val, 1995). At 31˚C, all 6 species were able to tolerate below 10% air saturation during exposure to a rapid induction of hypoxia (<20 minutes). A few studies have used similar methods to measure hypoxia tolerance on other fish species. One study allowed sunfish species (family Centrarchidae) to consume oxygen until LOE in sealed respirometer following a metabolic rate measurement and achieved hypoxia tolerance of ~1 to 1.6kPa (~1 to1.6% air saturation) (Crans et al., 2015). Another study on Arctic Char (Salvelinus alpinus) achieved hypoxia tolerance of 6.8% air saturation by reducing water oxygen level by 0.2%/min until LOE after 2 hours at 13% air saturation (Anttila et al., 2015). These methods vary in exposure time to hypoxia, which could allow fish to acclimate and adjust to the change, thus a precaution should be taken for comparison between studies. Studies have reported rapid adjustments of blood parameters in hypoxia to enhance oxygen delivery to tissues. Prochilodus nigricans, an Amazonian species, increased hematocrit and hemoglobin concentration within an hour of exposure to hypoxia (30mmHg; 19% air saturation) (Val et al., 2015). Fish can also regulate red blood cell organic phosphates (NTP) levels quite rapidly. Rainbow trout (Oncorhynchus mykiss) significantly reduced blood NTP concentrations within an hour of exposure to  35 hypoxia (30mmHg; 19% air saturation) (Tetens and Lykkeboe, 1985). Amazonian fishes are observed to respond faster and to a greater degree than trout. For example, in a similar exposure P. nigricans was shown to significantly reduce red blood cell GTP levels within an hour of exposure to hypoxia (30mmHg; 19% air saturation), with further reductions over the next 3 hours to reach ¼ of the initial value (Val et al., 2015). Piranha (Pygocentrus nattereri) exposed to a similar level of hypoxia reduced red blood cell GTP levels significantly within 10 minutes, and ATP levels within 30 minutes (Val, 2000). Thus, Amazonian fishes seem to be adapted to make rapid regulations to suit diurnal hypoxic bouts. Some, if not all, fishes investigated in this study may also have the ability to make the same rapid changes within the hypoxic trial that lasted about 20 minutes. Currently, data on the hypoxia tolerance of Amazonian species using a common procedure are limited, precluding a comprehensive comparison. A number of studies have measured the critical oxygen tension (Pcrit), the PO2 at which fish are thought to change from an oxygen-regulator to an oxygen-conformer, as a proxy for hypoxia tolerance. Lower Pcrit values are indicative of greater hypoxia tolerance because it indicates that the fish is able to maintain oxygen consumption rates down to a lower environmental oxygen level. The Pcrit of some Amazonian species have been measured: oscar (A. ocellatus) 46mmHg (29% air saturation) at 28˚C (Scott et al., 2008); tambaqui (C. macropomum) 42mmHg (26% air saturation) at 30˚C (Saint-Paul, 1984); jeju (Hoplerythrinus unitaeniatus) 40mmHg (25% air saturation) at 25˚C (Oliveira et al., 2004). These values are lower than the hypoxia-sensitive species such as brook trout (Salvelinus fontinalis) with 75mmHg (47% air saturation) at 15˚C (Beamish, 1964) and dragonet (Callionymus lyra) with 125mmHg (78% air saturation) at 12˚C (Hughes and  36 Umezawa, 1968). However, there seems to be a large variation in Pcrit values between methods and physiological conditions of fishes (Rogers et al., 2016; Speers-Roesch et al., 2013). For example, the Pcrit of oscar is found to depend significantly on size and feeding (De Boeck et al., 2013; Scott et al., 2008). The Pcrit of hypoxia-sensitive rainbow trout (Oncorhyncus mykiss) ranges from 27mmHg (17% air saturation) at 20˚C to 118mmHg (74% air saturation) at 12˚C between studies (Marvin and Heath, 1968; Ott et al., 1980). Thus, a comparison of hypoxia tolerance between studies using Pcrit values should be done with caution.  2.4.2.1 Effect of an acute increase in temperature on hypoxia tolerance The effect of an acute increase in temperature resulted in the predicted reduction in hypoxia tolerance in 3 of 6 species (Figure 2.2A). A reduction in hypoxia tolerance with an increase in temperature could be due to hindered oxygen-supplying mechanisms. An increase in temperature to 33 and 35˚C caused a general decrease of blood oxygen affinity (or an increase in P50) in Amazonian species in vitro (Val et al., 2016). For example, the P50 of trahira (Hoplias malabaricus) was 7.4mmHg (5% air saturation) at 31˚C and increased to 11.6mmHg (7% air saturation) at 35˚C. An increase in P50 is favorable in delivering oxygen to the tissues, which could be beneficial in meeting increased oxygen demand in warmer water. Concurrently, it makes oxygen loading difficult at the gill, causing a greater problem in coping with an oxygen-depleted environment.  Temperature also increases metabolic demand, which will consequently hinder the ability to withstand hypoxia (Clarke and Johnston, 1999; Schmidt-Nielsen, 1997).  37 Previous studies found that the acute exposure to higher temperatures (15 minutes ~ 24 hours) increases resting metabolic rate in fishes (Clark et al., 2011; Healy and Schulte, 2012a; Lowe and Davison, 2006; Steinhausen et al., 2008). Overall, the Amazonian species investigated in this study likely experienced similar difficulties in extracting and transporting sufficient amounts of oxygen to meet an increased energy demand in warmer water, resulting in LOE at higher % air saturation.   2.4.2.2 Effect of acclimation to higher temperatures on hypoxia tolerance Thermal acclimation for 4 weeks to 33 and 35˚C appeared to have relatively little effect, or in some species, negative effects on hypoxia tolerance (Figure 2.2B). In the 3 of the 6 species (N. trifasciatus, A. viejita, and M. ramirezi), hypoxia tolerance was reduced after 33˚C acclimation relative to values measured after 31˚C acclimation. Acclimation to 33 or 35˚C further reduced hypoxia tolerance relative to acute exposures at those temperatures in 2 species (A. viejita and M. ramirezi respectively), indicating that fish may not have been acclimating to these temperatures but tolerating them for this duration. Thus, species responded differently to acclimation to higher temperatures but in all 6 species, it did not improve hypoxia tolerance as I had predicted. The effect of thermal acclimation on hypoxia tolerance observed in this study was in contrast with what has been found in temperate species, where acclimation to higher temperature not only improves CTMax but also increases hypoxia tolerance relative to that measured at a lower temperature (Anttila et al., 2015; McBryan et al., 2016). However, my findings are consistent with a previous study on tropical fishes (Nilsson et al., 2010) in which an increase in water temperature caused an increase in metabolic rate and a decrease in hypoxia tolerance in 2 coral reef species (Ostorhinchus  38 doederleini and Pomacentrus moluccensis), and acclimation to the same temperatures for 7 ~ 22 days did not change the results. Temperate species likely have greater scope for acclimation to higher temperatures than tropical species as they experience a greater range of temperatures in their niches (discussed in more detail in Chapter 3 section 3.4.1.2). Taken together, these data indicate that thermal acclimation in Amazonian species appears to have no effect, or a very minor effect, on hypoxia tolerance and support the idea that tropical species have limited ability to acclimate to changing environmental conditions (Neuheimer et al., 2011; Rummer et al., 2014; Somero, 2010; Stillman, 2003).   2.4.3 Relationship between thermal tolerance and hypoxia tolerance I found a strong correlation between thermal tolerance and hypoxia tolerance among 6 species as predicted (Figure 2.3; 31˚C: r=-0.8448285, N=6, p=0.03425). This is the first study to reveal this correlation across multiple species, especially in stenothermic tropical fishes. Result demonstrates that there is a link in tolerance to both environmental stressors at the level of the whole organism. It provides evidence that a species with greater ability to tolerate low oxygen, and thus supply tissues with oxygen, has a greater thermal limit, consistent with OCLTT hypothesis.  Interestingly, the correlation between thermal and hypoxia tolerance was lost with acclimation to higher temperatures (33˚C among 5 species that survived: r=0.0420425, p=0.9465). Only one species (M. ramirezi) was able to acclimate to 35˚C, so I was unable to compare thermal and hypoxia tolerance at this temperature. Thermal acclimation improved CTMax as demonstrated in many other studies (Beitinger and  39 Bennett, 1999). In contrast, thermal acclimation did not restore hypoxia tolerance in all species. These results contradict previous findings that suggest acclimation to one stressor improves the other (Anttila et al., 2015; Burleson and Silva, 2011; McBryan et al., 2016). In fact, acclimation to an elevated temperature further decreased hypoxia tolerance relative to acute thermal exposure in 2 species (A. viejita & M. ramirezi). Overall, I found 3 possible reasons for this loss of correlation at higher temperatures: 1) A decrease in power due to a reduced number of species because several had reached their CLMax, 2) The magnitude of change in CTMax and % air saturation at LOE associated with acclimation is species-specific, and 3) Although CTMax increases with thermal acclimation, hypoxia tolerance remains the same or even decreases (Figure 2.1; 2.2). These divergent responses to thermal acclimation contribute to a weaker correlation. As previously mentioned, the predicted increase in temperature of the Amazon by up to 2.2 ~ 7˚C by 2100 will likely be associated with more frequent and severe hypoxic bouts. Thus, acclimation to higher temperatures in these Amazonian fishes may be helpful in dealing with the elevated temperature predicted by climate change but not in dealing with the concurrent induced hypoxia; clearly, more research is required to address this point. It is important to note that these experiments were carried out under ideal constant conditions in the lab in fish that may have already been selected for hardiness through the transport process from the Amazon to UBC. Thus, it is possible that the findings here under-represent the potential impact of the combination of elevation in temperature and hypoxia. To address this, the next chapter focused upon more species of fish caught directly from the wild and investigated in the Amazon.  40        Figure 2.1. Critical thermal maximum (CTMax, ˚C) following 4 weeks of acclimation to 31, 33 and 35˚C (p<0.05) in 6 species of Amazonian fishes. The absence of a bar indicates that fish were not able to acclimate to the respective temperature for the 4 week duration and thus the bar preceding it represents the chronic lethal maximum (CLMax). Values are means ± SEM (n=5-8). * represents a significant difference between acclimation temperatures within a species (p<0.05).    31°C33°C35°C31°C33°C35°C31°C33°C35°C31°C33°C35°C31°C33°C35°C31°C33°C35°C05353739414345* * **CTMax (oC) 41 31°C33°C35°C31°C33°C35°C31°C33°C35°C31°C33°C35°C31°C33°C35°C31°C33°C35°C0246810121416***BAir Saturation at LOE (%)                                 Figure 2.2. Hypoxia tolerance (% Air saturation at loss of equilibrium (LOE)) of A) fish acclimated to 31˚C and measured at 31˚C or following acute exposure and measurement at 33 and 35˚C B) fish following 4 weeks of acclimation and measurement at 31, 33 and 35˚C. Control 31˚C values in panel B are the same data as presented in A but included for comparative purposes. The absence of a bar in B) indicates fish were not able to acclimate to the respective temperature for the 4-week duration. Values are means ± SEM (n=5-8). * represents a significant difference between temperatures within a species (p<0.05). † in bars of panel A indicate a significant difference in LOE between acute (panel A) and 4 week acclimated (panel B) fish at the same temperature within a species (p<0.05). 31°C33°C35°C31°C33°C35°C31°C33°C35°C31°C33°C35°C31°C33°C35°C31°C33°C35°C0246810121416****††AAir Saturation at LOE (%) 42    Figure 2.3. The relationship between critical thermal maximum (CTMax) and hypoxia tolerance (% Air saturation at loss of equilibrium (LOE)) in fish following 4 weeks of acclimation to 31 ˚C (black symbols, r=-0.8448285, N=6, p=0.03425), 33 ˚C (orange symbols, r=0.0420425, N=5, p=0.9465) and 35˚C (red symbols). 33 and 35˚C values are reproduced from figure 2.1 and 2.2. Values are means ± SEM (n=5-8). The number above a symbol represents the species: (1) Carnegiella strigata; (2) Hemigrammus rhodostomus; (3) Nannostomus trifasciatus; (4) Corydoras julii; (5) Apistogramma ramirezi; (6) Mikrogeophagus ramirezi.    Ch1 Correlation combined38 39 40 41 42 43 44 4501234567891011121331˚C33˚C35˚CCTMax (˚C)Air Saturation at LOE (%)2 6 5 3 4 1 6 6 5 4 3 2  43 Chapter 3: The Relationship Between Thermal Tolerance and Hypoxia Tolerance in Amazonian Fishes at the Instituto Nacional de Pesquisas da Amazônia (INPA)  3.1 Introduction The Amazon River is home to 20% of the world’s freshwater fishes and climate change is predicted to increase the temperature of the region from 2.2 to 7˚C in the next century which may also be associated with more frequent and severe bouts of hypoxia (IPCC, 2014). How these future changes will impact Amazonian fishes and whether these fishes have the capacity to cope with these changes are largely unknown. In Chapter 2 (UBC), I found that thermal tolerance (CTMax) and hypoxia tolerance (% air saturation at LOE) were correlated in 6 ornamental Amazonian species, implying a physiological linkage between mechanisms associated with tolerance of these two challenges. Acclimation to temperatures predicted to occur within the next century (33 and 35˚C) increased CTMax to some degree, but resulted in either no change or a reduction in hypoxia tolerance at those temperatures. The most significant finding of the study was that most species failed to survive in those higher temperatures for 4 weeks. Previous studies, as well as data from Chapter 2, suggest many tropical fishes, including these Amazonian fishes, have a limited ability to acclimate to higher temperatures and may be at risk (Nilsson et al., 2010; Rummer et al., 2014; Somero, 2010; Stillman, 2003).  The limitations of the data presented in Chapter 2 are: 1.these fish probably underwent tremendous stress – thereby possibly selection – during transport to UBC as 20 ~ 30% of the total fish died within days of arrival due to stress; 2.these fish were held  44 in the constant conditions of a lab for several months prior to experimentation, which does not represent a natural environment. To address this, I went to the Instituto Nacional de Pesquisas da Amazônia (INPA) in Manaus, Brazil where I could collect fish from their natural environment and hold them under conditions that were more representative of their natural environment. A total of 15 species were investigated, 3 of which are important aquaculture fishes in the region (Table 1.1). These diverse species were chosen to ensure phylogenetic variance, however 1 species was the same (C. strigata) and 3 species were from the same genus (Nannostomus, Corydoras, Apistogramma) as those investigated in Chapter 2.  The goals of this chapter were similar to those of Chapter 2, and were: 1) to perform a general survey of thermal tolerance and hypoxia tolerance of various Amazonian fishes at 28˚C (average of the natural diurnal temperature fluctuation), and to determine the relationship between those two parameters; 2) to determine the effect of acute exposure (<1.5 hours) and acclimation (10 days) to higher temperatures of 31, 33 and 35˚C on hypoxia tolerance; 3) to determine the effect of acclimation (10 days) to higher temperatures of 31, 33 and 35˚C on thermal tolerance. In addition, I investigated 4) the effect of hyperoxia on thermal tolerance (please refer to section 1.6).   3.2 Material & Methods Experiments were conducted in Manaus, Brazil in collaboration with the Instituto Nacional de Pesquisas da Amazônia (INPA). Twelve local species were caught by INPA fishermen: Carnegiella strigata (Hatchetfish; Mean weight: 0.544±0.021g, n=60); Nannostomus eques (Diptail pencilfish; 0.327±0.008g, n=60); Paracheirodon axelrodi  45 (Cardinal tetra; 0.104±0.004g, n=40); Hyphessobrycon erythrostigma (Bleeding heart tetra; 0.300±0.023g, n=35); Corydoras schwartzi (Schwartz’s cory; 1.667±0.043g, n=70); Corydoras splendens (Emerald cory; 6.436±0.542g, n=16); Coryodras pulcher (7.707±0.504g, n=23); Otocinclus spp. (Suckermouth cory; 0.458±0.024g, n=40); Hemiodus gracilis (Red tail hemiodus tetra; 2.903±0.208g, n=8); Apistogramma borelli (Umbrella cichlid; 0.303±0.010g, n=70); Apistogramma gephyra (1.580±0.144g, n=14); Laetacara fulvipinnis (1.042±0.061g, n=15). Three species were obtained from a commercial aquaculture farm (Sítio dos Rodrigues, Km 35, Rod. AM-010, Brazil): juvenile Brycon amazonicus (Brycon; 1.475±0.102g, n=40); juvenile Astronotus ocellatus (Oscar; 2.975±0.077g, n=50); juvenile Colossoma macropomum (Tambaqui; 0.579±0.022g, n=80). Fish were brought to INPA and held in INPA well water: [Na+]=35uM, [Cl-]=36uM, [Ca2+]=18uM, [Mg2+]=4uM, [K+]=16uM; pH 6.5 (Robertson et al., 2015). All fish were held under a natural 12L:12D photoperiod and natural diurnal temperature fluctuations of the Amazon, 26 to 30˚C (control). Fish were fed daily to satiation with commercial flakes (Pirà mirim, Guabi, Brazil) and fasted at least 24h prior to all experimentation. All procedures were in compliance with UBC animal protocol #A15-0266 and Brazilian national and Instituto Nacional de Pesquisas da Amazônia animal care regulations.  3.2.1 Experimental Design Each species was held for 7 to 10 days in 2-L glass aquaria (6 ~ 16 fish/aquaria) in an outdoor laboratory that experienced natural diurnal temperature fluctuations of 26 to 30˚C (control). Three 500-L flow-through tanks were used to acclimate fish to one of  46 three higher temperatures: 31, 33, or 35˚C. 8 of 15 species (C.strigata, N. eques, C. schwartzi, Otocinclus spp., P. axelrodi, H. erythrostigma, A. borelli, and C. macropomum; n=20 per spp.) were transferred to these acclimation tanks and held together in a common garden experimental design for 10 days. Initially, the water temperature was 28˚C and was heated at a rate of 1˚C/12hr using electric heaters and held at the respective target temperature using temperature controllers (FisherbrandTM TraceableTM Digital Temperature Controller). Fish acclimated to the natural diurnal temperature were assessed for hypoxia tolerance at 28˚C and thermal tolerance as described below. The 8 species acclimated to 31, 33, or 35˚C for 10 days were assessed for hypoxia tolerance at their respective acclimation temperatures and thermal tolerance as described below. The same species, except H. erythrostigma, were also acutely exposed (from 28 ˚C) to higher temperatures of 33 and 35˚C and then assessed for hypoxia tolerance at those temperatures. Due to the limited number of fish, hypoxia tolerance of H. erythrostigma could not be measured after acute exposure to higher temperatures.   3.2.2 Thermal tolerance  Following the 10-day acclimation period, fish were assessed for critical thermal tolerance (CTMax). After 10 days, 4 ~ 8 fish per species were netted out from the acclimation tank, transferred to 2-L glass aquaria and allowed an hour to acclimatize to the new environment. The same general protocol, temperature incremental rate, and endpoint assessment were used as in Chapter 2 (please refer to section 2.2.2 for protocols). Once the fish lost equilibrium, the temperature was noted and the fish was  47 removed and weighed. Some species exhibited high (>50%) morbidity during acclimation to 31, 33 or 35˚C. If this was the case, the temperature that >50% of the fish were not able to tolerate and survive for 10 days was noted as the chronic lethal maximum (CLMax).   3.2.2.1 Thermal tolerance in hyperoxia Fish held at the natural diurnal temperature fluctuation of 26 to 30˚C were transferred to fully aerated 28˚C water in 2-L glass aquaria and acclimatized for 1 hour. Oxygen was bubbled in to reach >200% air saturation, monitored by an optical oxygen meter (WTW Multi3410 meter, Weilheim, Germany). Immediately thereafter, water was maintained at >200% air saturation and CTMax was determined as described in section 2.2.2.  3.2.3 Hypoxia tolerance Following the 10-day acclimation period, hypoxia tolerance was assessed by determining the % air saturation at LOE. Fish were transferred to individual 200-ml chambers and 8 chambers were submerged in a 50-L Plexiglas tank. Water within the whole system was aerated and circulated using two aquarium water pumps, and the 200-ml chambers each had mesh netted sides to ensure water oxygen levels were consistent with that of the system water. Percent air saturation of the 50-L system was monitored continuously using a fiber optic oxygen probe (PreSens Precision Sensing, Regensburg, Germany) that was calibrated before every trial. After the fish were acclimatized to the chamber for one hour, nitrogen gas was bubbled into the 50-L tank  48 to decrease air saturation in the system. Please refer to the section 2.2.3 protocol for further information on the method used for % air saturation reduction rate and endpoint assessment.  The lowest water oxygen tension that could be reached in this system was 0.4 - 0.6% air saturation. In those fish that could tolerate this lowest level, the time to LOE at this tension range was used as the measurement of severe hypoxia tolerance. In all trials, once LOE was achieved, the % air saturation or time after 0.4 - 0.6% air saturation was recorded and fish were removed and weighed.  Species acclimated to a natural diurnal temperature of 26 to 30˚C were acutely exposed to higher temperatures of 33 or 35˚C at a rate of 0.2˚C/min. Once fish reached the target temperature, fish were given one hour to acclimate and then assessed for hypoxia tolerance (please refer to the section 2.2.3 protocol).  3.2.4 Statistical analysis Similar statistical analyses were carried out as Chapter 2 (please refer to section 2.2.4). Comparisons between the same species (C. strigata) or genus (Characiformes, Siluriformes, Perciformes) measured in the different chapters were done using Student’s t-test. Data have been expressed as the means ± SEM (standard error, n=4-8). In all cases, α was set at 0.05.   49 3.3 Result 3.3.1 Thermal tolerance and the effect of acclimation to higher temperatures The CTMax of 15 species held in the natural diurnal temperature was measured at 28˚C and ranged from 36.5 ~ 42.1˚C (Figure 3.1). The lowest CTMax values were observed for Otocicnlus spp., 36.5˚C, followed by C. pulcher and H. gracilis at 37.5˚C and C. strigata at 37.9˚C. Another 6 species (P. axelrodi, C. schwartzi, C. splendens, H. erythrostigma, N. eques, and B. amazonicus) had similar CTMax within 38.7 ~ 39.4˚C. Of the remaining 5 species, 4 were cichlids (Perciformes: A. borelli, A. ocellatus, A. geohpyra, and L. fulvipinnis) that had an even higher CTMax within 39.7 ~ 41˚C. The final species, juvenile tambaqui (C. macropomum), had the highest CTMax of 42.1˚C.  Of the 15 species, 8 were acclimated to higher temperatures of 31, 33 and 35˚C and were assessed for CTMax (Figure 3.1). All species displayed a significant increase in CTMax with an increase in acclimation temperature (p<0.000). In fish acclimated to 31˚C, CTMax increased by about 1.3˚C relative those measured at 28˚C. Of the 6 species that survived the 10 days of acclimation to 33˚C (all except C. strigata and Otocinclus spp.), CTMax was about 0.6˚C higher than for the 31˚C acclimated group. The only species that survived 35˚C acclimation was juvenile tambaqui (C. macropomum), and it showed the highest CTMax of 44.1˚C, which was a 2˚C increase from 28˚C treatment and about 1.2˚C above the values in the 31 and 33˚C treatments. On average, in those that could acclimate to higher temperatures, the CTMax increased by 0.4˚C for every 1˚C increase in acclimation temperature. Several of the species investigated here are the same or very closely related to those in Chapter 2, allowing a better comparison of thermal tolerance among locations.  50 The species acclimated to the same temperature of 31˚C with different acclimation duration (Chapter 2: 4 weeks & Chapter 3: 10 days) were compared (Table 3.2). The CTMax of the same species (C. strigata) or those from the same genus (Nannostomus, Corydoras, Apistogramma) or family (Tetra: P. axelrodi and H. erythrostigma) were significantly different among locations. In C. strigata and N. eques at INPA, the CTMax was approximately 1.5˚C higher than C. strigata and N. trifasciatus measured at UBC. In contrast, C. schwartzi, P. axelrodi, and A. borelli at INPA had a CTMax 0.5˚C lower than C. julii, H. rhodostomus, and A. viejita measured at UBC. H. erythrostigma at INPA also had a CTMax about 1˚C lower than H. rhodostomus at UBC.  The chronic lethal maximum (CLMax) was positively correlated to species’ acute thermal tolerance (CTMax) measured at 28˚C  (r=0.9331, N=8, p=0.00022), and values reported here are consistent with those reported in Chapter 2.  Of the 8 species that underwent acclimation to higher temperatures, 2 species that displayed the lowest CTMax at 28˚C (Otocinclus spp. 36.5˚C; C. strigata 37.9˚C) could not survive in temperatures above 31˚C for 10 days. The 5 species with mid thermal tolerance (P. axelrodi and C. schwartzi 38.7˚C; N. eques and H. erythrostigma 39.1˚C; A. borelli 39.7˚C) could survive only up to 33˚C. C. macropomum with the highest CTMax of 42.1˚C measured at 28˚C could survive all four acclimation temperatures (28, 31, 33, and 35˚C) investigated in this chapter.  3.3.1.1 Thermal tolerance in hyperoxia The CTMax values of 7 species were measured in normoxia and hyperoxia (>200% air saturation) after 10 days of acclimation to the natural diurnal fluctuation of  51 26 to 30˚C. Overall, hyperoxia had a significant effect on CTMax (p<0.0001). Specifically, 4 of the 7 species increased CTMax in hyperoxic water relative to normoxic water (Figure 3.2). CTMax increased by up to 2˚C in B. amazonicus and significantly increased by about 0.5˚C in C. strigata, P. axelrodi, and C. schwartzi. No significant change was observed in C.pulcher, A. borelli and C. macropomum.  3.3.2 Hypoxia tolerance and the effect of an increase in temperature (either acute or following acclimation) Hypoxia tolerance (% air saturation at LOE) of 15 species was assessed at 28˚C (Figure 3.4). H. gracilis displayed the lowest tolerance, with an LOE of 10.8% air saturation. In 9 species, LOE was between 5.1 and 1% air saturation. The remaining 5 species (A. ocellatus, L. fulvipinnis, A. gephyra, A. borelli, and C. macropomum) tolerated severe hypoxia  (0.4 ~ 0.6% air saturation) below which oxygen levels could not be reduced. For comparison, these 5 species have been included on the same graph with the others even though they did not lose equilibrium initially at that tension. However, for those 5 species that could withstand this lowest oxygen tensions, time to LOE was measured (Figure 3.5) and ranged from 5 (A. borelli and C. macropomum) to 24 minutes (L. fulvipinnis).  Hypoxia tolerance of 7 species was measured following acute transfer to the higher temperatures of 33 or 35˚C (Figure 3.3A). Acute exposure to 33˚C significantly reduced hypoxia tolerance (increase in % air saturation at LOE) in all species except Otocinclus spp. and C. schwartzi relative to that measured at the control temperature of 28˚C. All 7 species exhibited a decrease in hypoxia tolerance when acutely exposed to  52 35˚C. The greatest change was nearly a 5-fold decrease by C. schwartzi relative to the control temperature (1% at 28˚C to 4.5% air saturation at 35˚C). C. strigata exhibited the lowest hypoxia tolerance by losing equilibrium at 12.2% air saturation after acute exposure to 35˚C in comparison to 5.1% at 28˚C.  Acclimation for 10 days to higher temperatures of 31, 33 and 35˚C did not improve hypoxia tolerance relative to that measured at 28˚C in 8 species (Figure 3.3B). In 4 species (C. strigata, Otocinclus spp., N. eques, and C. schwartzi), there was no significant difference in hypoxia tolerance between 28 and 31˚C acclimated fish. In the other 4 species (P. axelrodi, H. erythrostigma, A. borelli, and C. macropomum), acclimation to 31˚C resulted in a reduction in hypoxia tolerance relative to that measured at 28˚C. Of the 5 species that survived 33˚C acclimation, 4 species (N. eques, C. schwartzi, H. erythrostigma, and A. borelli) showed a further reduction of hypoxia tolerance and 1 species (C. macropomum) showed no change at that temperature relative to 31˚C. Only C. macropomum could be acclimated to 35˚C, and I observed no change in hypoxia tolerance relative to the 31 and 33˚C acclimation groups. Furthermore, thermal acclimation to 33 or 35˚C did not result in improved hypoxia tolerance in any of the 8 species relative to acute exposure to the same temperatures (Figure 3.3; denoted by †). The hypoxia tolerance of C. strigata could not be determined at higher temperatures, as it did not survive either 33 or 35˚C acclimation. The hypoxia tolerance of H. erythrostigma could not be compared between acute exposures and acclimation to higher temperatures because of the limited number of fish. Otocinclus spp., N. eques, and P. axelrodi acclimated for 10 days had the same hypoxia tolerance  53 as those acutely exposed to the same temperatures (33 or 35˚C). For C. schwartzi, A. borelli, and C. macropomum, acclimation to 33˚C yielded lower hypoxia tolerance than when they were acutely exposed to 33˚C. C. macropomum acclimated to 35˚C also exhibited a significantly lower hypoxia tolerance relative to acute exposure. Results of hypoxia tolerance following 31˚C acclimation at UBC and INPA were compared (Table 3.1). Except for 2 species (P. axelrodi and A. borelli), hypoxia tolerance was significantly different from those measured at UBC. Species measured at INPA were generally more tolerant to hypoxia than those measured at UBC. Fish measured at INPA were exposed to different water conditions, photoperiod, acclimation duration, and had different size range.  3.3.3 Relationship between thermal tolerance and hypoxia tolerance I found a significant correlation between % air saturation at LOE and CTMax between 15 species at 28˚C including the 5 species that could tolerate severe hypoxia (A. borelli, A. ocellatus, A. gephyra, L. fulvipinnis, and C. macropomum) (Figure 3.4; r=-0.7146, p=0.0028). In fish acclimated to either 31 or 33˚C, there was no significant relationship between % air saturation at LOE and CTMax (Figure 3.4; 31˚C: r=-0.4423, p=0.2725 & 33˚C: r=-0.665, p=0.1491). Furthermore, no correlation was found between time to LOE in fish that could tolerate the severe hypoxia and CTMax (Figure 3.5; r=-0.085, p=0.892).   54 3.4 Discussion  This chapter surveyed thermal tolerance (CTMax) and hypoxia tolerance (% air saturation at LOE) of 12 ornamental and 3 aquaculture Amazonian fishes acclimated to natural diurnal temperature fluctuation of 26 to 30˚C. I found that all are highly tolerant to high temperatures (section 3.4.1) and extreme hypoxia (section 3.4.2), and there was a clear correlation between thermal and hypoxia tolerance (section 3.4.3). Eight species were further investigated for the capacity to respond to predicted increases in temperature associated with climate change. Acclimation to higher temperatures for 10 days and exposure to hyperoxia at 28˚C both increased the CTMax (section 3.4.1.1 and 3.4.1.2). However, many species failed to survive at higher temperatures over a long term: 2 species not above 31˚C, 5 species not above 33˚C, and only one species survived above 35˚C  (section 3.4.1.1: CLMax). An acute exposure (<1.5hours) to 33 and 35˚C decreased hypoxia tolerance in all species (section 3.4.2.1) at those temperatures relative to hypoxia tolerance at 28 C. Acclimation (10 days) to 31, 33, and 35˚C also decreased hypoxia tolerance in most species (section 3.4.2.2) at those temperatures relative to that at 28˚C. In fact, 3 species yielded lower hypoxia tolerance after acclimation to 33˚C relative to that determined after acute exposure to 33˚C, and the same was true for 1 species after acclimation to 35˚C. I also found a significant correlation between thermal and hypoxia tolerance in the 15 species at 28˚C, but this was lost after acclimation to 31, 33 and 35˚C.    55 3.4.1 Thermal tolerance In 15 species exposed to the natural diurnal temperature fluctuation, the CTMax was measured at 28˚C and ranged from 36.5 to 42.1˚C (Figure 3.4). This range was similar to that found in Chapter 2 (Figure 2.3) and within the range observed in other tropical fishes (Eme and Bennett, 2009; Prodocimo and Freire, 2001). Campos et al. (2016) acclimated P. axelrodi to 29˚C and found a CTMax value of about 39˚C, which is remarkably similar to the CTMax value of 38.7˚C that I determined for the same species at 28˚C (Figure 3.3). Despite the fact that Campos et al. (2016) used a faster heating rate and longer acclimation duration (1˚C/hour; 2 weeks) relative to that used in this study (0.2˚C/min; 10 days), results were similar. It implies the temperature incremental rate and acclimation duration used in this study is sufficient for fish to respond to thermal stress, at least in this particular species. Overall, the thermal tolerance data in this study are consistent with that found at UBC (Chapter 2) and by other investigators. In a previous study on adult oscar (A. ocellatus; 16.49g), maximum temperature (1˚C/hour increment; endpoint as an onset of opercular spasm) of about 41˚C was found after 4 days of acclimation to 30˚C (Gutierre et al., 2016). The same study also found that juvenile oscar (3.92g) were slightly less tolerant than adults with a maximum temperature of 40.5˚C. The CTMax of juvenile oscar (2.975g) measured in this study was 39.7˚C after 10 days of acclimation to 26 to 30˚C. These findings suggest that adults have a slightly higher CTMax than juveniles. As area/volume ratio can affect the time it takes for the temperature to equilibrate throughout the entire fish’s body, body size is considered to be an important factor when measuring thermal tolerance (Becker and Genoway, 1979), and could potentially explain the difference between these values.  56 The effect of body size on CTMax however is inconsistent in other species (Anttila et al., 2013; Ospína and Mora, 2004; Underwood et al., 2012). Another potential explanation for the difference is the effect of body size on hypoxia tolerance. Studies found that oscar have size-dependent hypoxia tolerance: larger individuals are more tolerant to hypoxia than smaller individuals (please refer to section 3.4.2). This may translate to their thermal tolerance if oxygen limitation does correlate to thermal tolerance as suggested by the OCLTT, and could explain lower CTMax observed in juveniles in this study.  3.4.1.1 Effect of acclimation to higher temperatures on thermal tolerance One of the goals of the present chapter was to broaden the investigation of increased temperature effect on the CTMax across a wide range of Amazonian species in a more natural environment. Of the species that survived higher acclimation temperatures for 10 days, the CTMax slightly increased (Figure 3.1). The CTMax increased by about 0.4˚C for every 1˚C increase in acclimation temperature across species, lower than the result found at UBC (Chapter 2; 0.6˚C) but similar to other tropical fishes (Eme and Bennett, 2009). The amount of time required to acclimate depends on species, and the direction and magnitude of temperature change. For example, it took 21 days for channel catfish (Ictalurus punctatus) to re-acclimate from 10 to 20˚C and increase CTMax, but only 3 days from 30 to 35˚C (Bennett et al., 1998). Barrionuevo and Fernades (1998) found that full acclimation takes less than a week for most species, and even as short as 1 ~ 3 days in tropical fishes. I also observed a large variation in the magnitude of change in CTMax with acclimation temperature at UBC  57 (please refer to 2.4.1.1). The lower value found in this chapter suggests that 10 days may not be sufficient for some species to fully acclimate, but it is also important to note that the two chapters studied different species that may have varying responses to acclimation. Results also indicate that in those species that could acclimate to higher temperatures, there was some capacity to increase thermal tolerance. However, only a handful of species measured were able to acclimate and survive at higher temperatures (CLMax) for 10 days. Similar to the findings at UBC, I observed population morbidity in the process of thermal acclimation for 8 of 15 species (Figure 3.4). All 8 species could be acclimated to 31˚C, which is the current river maximum temperature (Val and Almeida-Val, 1995). Two species (C. strigata and Otocinclus spp.) could not survive temperatures above 31˚C for 10 days, and 5 species (N. eques, P. axelrodi, C. schwartzi, H. erythrostigma, and A. borelli) could not survive above 33˚C. Oliveira et al., (2008) showed that lethal temperature of P. axelrodi is 33.7˚C, the same species in which I also observed a large morbidity over 10 days at 33˚C, even though this was about 6˚C lower than its CTMax.  Only one species tested (C. macropomum) could survive temperatures up to 35˚C. Thus, even a short acclimation period of 10 days to higher temperatures appears to be lethal for most Amazonian fishes. Study location seems to not affect their long-term thermal tolerance as same species (C. strigata) or species from the same genus measured at UBC (Chapter 2) had similar CLMax.  Several studies found that stenothermal ectotherms, such as these Amazonian fishes, could be more vulnerable to warming as they live in an environment with little temperature variation throughout the year (Somero, 2010; Tewksbury et al., 2008).  58 Ospína and Mora (2004) found that tropical fishes have little intraspecific variation in CTMax, suggesting limited capability to adapt to higher temperatures. Nilsson et al. (2010) observed an increase in mortality in one coral reef species (Ostorhinchus doederleini) after 8 days at 32˚C whereas the other species (Pomacentrus moluccensis) from the same environment could survive up to 22 days. Rummer et al. (2014) found 100% mortality of one coral reef species (Acanthochromis polyacanthus) within 7 days at 34˚C while other 5 species survived 14-day acclimation to the same temperature. These results imply that some tropical species may face lethal temperatures in the future, but the duration and magnitude of thermal exposure to be lethal is species-specific and needs to be further investigated. There is a possibility of adaptation, selection, or ontogenetic acclimation occurring in real life, but it is evident that many tropical fishes, including Amazonian, could be endangered by the direct effects of climate change. Species tested in this chapter were compared to those from the same genus or family tested in Chapter 2 at UBC at 31˚C (Table 3.1). C. strigata and N. eques measured at INPA had significantly higher CTMax than C. strigata and N. trifasciatus measured at UBC. The 4 other species (C. schwartzi, P. axelrodi, H. erythrosigma, and A. borelli) had lower CTMax than those measured at UBC (C. julii, H. rhodostomus, and A. viejita). Although closely related, all except for C. strigata are different species and as mentioned above, the capacity to acclimate can be species-dependent. The studies in the two chapters also differ in the acclimation duration, water condition, and animal holding history. It is currently unclear how all these parameters can affect CTMax, but a direct comparison should be taken with caution.   59  3.4.1.2 Thermal tolerance in hyperoxia In 4 of 7 species tested, the CTMax was significantly increased in supersaturated water (>200% air saturation), indicating that greater oxygen availability alleviates some of the acute thermal stress (Figure 3.2). Several studies have looked into the effect of available environmental oxygen on CTMax, but the results are contradictory. Hyperoxia had no effect on CTMax in Chaenocephalus aceratus (icefish) and Notothenia coriiceps (Devor et al., 2015). Other studies found that limited oxygen in the water (hypoxia) decreases CTMax, but excess oxygen (hyperoxia) has no effect. For example, the CTMax of several temperate freshwater fishes was 4.1 ~ 6.2˚C lower in hypoxia (1.2mg/L; 13% air saturation) but remained the same in hyperoxia (12mg/L; 132% air saturation) relative to that in normoxia (Rutledge and Beitinger, 1989). The CTMax of killifish (Fundulus heteroclitus) decreased in hypoxia (0.8mg/L; 9% air saturation) but was unchanged in hyperoxia (Healy and Schulte, 2012b). Brijs et al. (2015) found that European perch (Perca fluviatilis) in hyperoxia (200% air saturation) exhibited a twofold increase in aerobic scope, but no difference in CTMax. However, in another study, the CTMax of the same species increased slightly by 0.9˚C in hyperoxia (200% air saturation) (Ekström et al., 2016). Currently, the data imply that the CTMax is, to some degree, affected by impaired oxygen uptake and/or delivery in some fishes.  The studies to date have looked at only whole-animal thermal responses to hyperoxia, so more in-depth investigation is required. For example, a species’ capacity to increase arterial blood oxygen content in hyperoxia is not clear. If blood were already fully saturated in normoxia in the fishes that showed no response (C. pulcher, A. borelli,  60 and C. macropomum), then excess oxygen in the water would have had only a minor   additional benefit in supplying tissues with oxygen. A study found that exposure to hyperoxia (360mmHg; 225% air saturation) for 15 days had no effect on blood NTP levels in oscar, but there was a significant reduction in tambaqui (Marcon and Val, 1996). Hyperoxia can induce oxidative cell damage, so an increase in NTP level reduces blood affinity and thus potentially minimizes tissue damage. However, changes in NTP levels during hyperoxia among studies are also variable (reviewed by Val, 2000). The mixed results imply that rather than oxygen being a sole determining factor, multiple limiting mechanisms contribute to thermal tolerance. Friedlander et al. (1976) found local heating of the Goldfish brain induced the same response as those observed during heat stress, including loss of equilibrium. Therefore, in addition to oxygen limitation, temperature-dependent failure of the central nervous system could be setting thermal tolerance, and these may be species-specific.  3.4.2 Hypoxia tolerance The goal of this section was to further investigate hypoxia tolerance and the influence of increased temperature in Amazonian fishes in a more natural environment. This chapter looked at 15 Amazonian species that are known to use different mechanisms to cope with hypoxia and inhabit areas with different physical water characteristics. Fish were acclimated to a natural diurnal temperature fluctuation of 26 to 30˚C and were tested for hypoxia tolerance at 28˚C (control). Hypoxia tolerance  61 ranged from near anoxia to 11% air saturation (Figure 3.4), similar to results found at UBC (Chapter 2; Figure 2.3). Lower values of blood P50 are thought to be associated with greater hypoxia tolerance as they indicate higher affinity of the hemoglobin for oxygen. From unpublished data by A.L. Val, whole blood P50 has been reported to be 13mmHg (8% air saturation) for tambaqui (C. macropomum), 14mmHg (9% air saturation) for oscar (A. ocellatus), and 28mmHg (18% air saturation) for cardinal tetra (P. axelrodi) at 28˚C (reported in Robertson et al., 2015). Consistently, I also found that tambaqui and oscar are more hypoxia tolerant than cardinal tetra at 28˚C: Tambaqui and oscar lost equilibrium at 6 and 17 minutes respectively in severe hypoxia (Figure 3.5; 0.4 ~ 0.6% air saturation), while cardinal tetra lost equilibrium at 2.6% air saturation (Figure 3.4). Other Amazonian species are found to have similar or even lower blood P50 values (Val et al., 2016). The hypoxia-sensitive rainbow trout (O. mykiss) has a higher P50 or lower hemoglobin-oxygen binding affinity with 25 ~ 31mmHg (16 ~ 19% air saturation) at 20˚C (Cameron, 1971; Eddy, 1971). A study on different species of sculpins found that the hypoxia tolerance, measured by Pcrit, is correlated to whole blood Hb-O2 P50 and environmental oxygen levels (Mandic et al., 2009). Overall, these results imply that P50 may be indicative of whole-animal hypoxia tolerance to some degree. Another study reported that the P50 of Amazonian fishes relate to the rate of water flow in which the fish resided (Powers et al., 1979). Species that are found in slow-flowing littoral water have low P50 values such as Curimatus spp. with 6mmHg (4% air saturation), Perciformes with 4 ~ 10mmHg (3 ~ 6% air saturation), and armored catfish (Hoplosternum littorale) with 9.6mmHg (6% air saturation). Species that inhabit  62 moderately flowing water streams such as tambaqui (C. macropomum) have slightly higher P50 of 12mmHg (8% air saturation). Those that are found in rapid streams such as Hemiodus spp. have the highest of 22.5mmHg (14% air saturation). I also found a similar trend of hypoxia tolerance amongst species: Cichlids (Perciformes) and tambaqui had the highest tolerance (5 ~ 24 minutes in 0.4 ~ 0.6% air saturation; catfish species (Corydoras spp.) had mid-tolerance (1 ~ 4.5% air saturation); Hemiodus gracilis had the lowest (10.8% air saturation) (Figure 3.4; 3.5). In the Amazon, water flow is correlated with oxygen levels: slow-flowing waters (e.g. floodplains) tend to have fluctuating oxygen levels and are often hypoxic, while rapid flowing waters (e.g. main river) tend to be more oxygenated. These results indicate that species’ P50 and hypoxia tolerance may be related to their habitat oxygen levels. In addition to hemoglobin-oxygen affinity, some fishes in hypoxia can regulate other pathways to improve oxygen delivery such as HIF and vascular endothelial growth factor (VEGF). VEGF is a signal protein that stimulates the formation of blood vessels (vasculogenesis and angiogenesis), which could enhance oxygen delivery to tissues. A significant increase in expression of HIF and VEGF was observed in the liver of oscar following acute hypoxia (3 hours in 6.5% air saturation) (Baptista et al., 2016). Overall, the Amazonian species are equipped with an enhanced oxygen delivery system that can be modified rapidly to cope with frequent hypoxic bouts. When oxygen is too low to produce enough energy, many Amazonian species revert to anaerobic metabolism. In hypoxia, oscar reduce the liver activity of malate dehydrogenase (MDH) and citrate synthase (CS), indicative of down-regulation of oxidative metabolism (Baptista et al., 2016). Lactate dehydrogenase (LDH) catalyzes  63 the interconversion of pyruvate to lactate, with specific isoforms playing different roles in accordance with the oxygen availability. LDH-A, which converts pyruvate to lactate, is the key enzyme of the anaerobic end of glycolysis. On the other hand, LDH-B converts lactate to pyruvate and is more active in aerobic conditions. Amazonian fishes can adjust and regulate these isoforms according to environmental conditions and tissues (reviewed by Val et al., 1998). They have been documented to have two general models for LDH distribution during hypoxia: 1) Predominance of LDH-B in critical tissues such as the heart and brain to maintain aerobic metabolism; 2) High levels of LDH-A and low expression of LDH-B in all tissues, suggesting activation of anaerobic metabolism (Reviewed by Almeida-Val and Val, 1993). Cichlids (A. ocellatus, L. fulvipinnis, A. gephyra, and A. borelli) were generally the most hypoxia tolerant species in this study. In general, Amazonian cichlids stay in hypoxic lakes throughout all seasons and are considered to be good anaerobes (Chippari-Gomes et al., 2005). In hypoxia, some cichlids suppress all metabolic pathways, including anaerobic pathways, like the Cichlasoma amazonarum that showed an overall reduction of LDH activity after a long-term hypoxia exposure (50 days at 36mmHg; 23% air saturation) (Almeida-Val et al., 1995). Many cichlids, on the other hand, suppress aerobic metabolism and use large muscle and liver glycogen storage for energy production (Chippari-Gomes et al., 2005; Muusze et al., 1998). These metabolic changes are induced rapidly in Amazonian fishes. Prochilodus nigricans in hypoxia (30mmHg; 19% air saturation) showed a 3 fold increase in plasma lactate within an hour (Val et al., 2015). In this study, the cichlids were exposed to 20 minutes of gradual hypoxia and additional species-specific time (5 ~ 24 minutes) in severe hypoxia (Figure  64 3.5). During this duration, the cichlids may have either reverted to metabolic depression and/or began using their glycogen storage for anaerobic energy production, allowing them to be one of the more tolerant species.  Of those cichlid species, oscar (A. borelli) is congeneric to A. ocellatus that is unique in that it presents a positive relationship between hypoxia tolerance and size (Almeida-Val et al., 2000; Sloman et al., 2006). Larger individuals (300-500g) can tolerate up to 6 hours in complete anoxia at 28˚C, and have a higher capacity to reduce standard metabolic rate and revert to anaerobic metabolism in hypoxia (<6% air saturation at 28˚C) than juveniles (<200g) (Muusze et al., 1998). An up-regulation of LDH-A was found in white and heart muscles of juveniles (38g) exposed to hypoxia (20% air saturation), indicative of an onset of anaerobic metabolism (Almeida-Val et al., 2011). Contrastingly, adults (148g) did not regulate LDH levels, suggesting that they use other mechanisms to survive hypoxia. Another study found that the Pcrit of larger individuals (388g) is 34.3mmHg (21% air saturation), which is significantly lower than that of smaller individuals (129g) with 74.4mmHg (47% air saturation) (Scott et al., 2008). This reflects the natural environment they experience during development: adults are found in hypoxic waters whereas juveniles are more active and are found in superficial water body layers with greater oxygen availability. Here, I provide additional evidence that juveniles (mean weight: 2.975g) are less tolerant than adults tested in previous experiments, but they can nevertheless withstand severe hypoxia (0.4 ~ 0.6% air saturation) for about 17 minutes (Figure 3.5).  Tambaqui (C. macropomum) expands the lower lip to skim the well-oxygenated surface in hypoxia (23% air saturation) and can maintain the same blood oxygen  65 content as in normoxia at 28 ~ 30˚C (Val, 1995). However, those denied access to the surface in the same hypoxia treatment had a 35% reduction in blood oxygenation and a significant increase in mortality after 24 hours was observed. I found that juvenile tambaqui without access to the surface still have exceptional hypoxia tolerance, lasting about 6 minutes in 0.4 ~ 0.6% air saturation (Figure 3.5). They also have other strategies to deal with hypoxia such as modifying blood parameters to increase blood oxygen carrying capacity and use of anaerobic metabolism (Affonso et al., 2002; Almeida-Val et al., 1990), which are likely to still function regardless of access to the surface.  The Amazonian catfish species investigated in this study (Otocinclus spp., C. pulcher, C. splendens, and C. schwartzi) are observed to perform surface respiration regularly even in normoxia. Without access to the surface, Otocinclus spp. and C. pulcher lost equilibrium at 4.5%, C. splendens at 2.4%, and C. schwartzi at 1% air saturation. Other studies have found that these facultative air-breathers are still hypoxia tolerant without surface access by increasing ventilation, suppressing metabolism, and/or using anaerobic metabolism. Another Amazonian catfish, the armoured catfish (Glyptoperichthyes gibbceps) under hypoxia exhibited an increase in ventilation rate and increase in plasma lactate concentration (MacCormack et al., 2003). Hoplosternum thoracatum reduced oxygen consumption by about 40% in PO2 of 40mmHg (25% air saturation) (Gee and Graham, 1978) and Hoplosternum littorale by 83% in 30mmHg (19% air saturation) (Brauner et al., 1995). In a preliminary trial, I observed that C. splendens and C. pulcher with access to the surface increased the air-breathing frequency and did not lose equilibrium for more than 2 hours in severe hypoxia (0.4 ~  66 0.6% air saturation). Facultative air-breathers were relatively less tolerant to hypoxia without access to air, but regardless one of the more tolerant groups in the species measured in this study.  3.4.2.1 Effect of an acute increase in temperature on hypoxia tolerance Overall, acute exposure to higher temperature negatively affected hypoxia tolerance in 7 species assessed (Figure 3.3A). Hypoxia tolerance was maintained the same after an acute exposure to 33˚C relative to 28˚C in 2 species (Otocinclus spp. and C. Schwartzi) and was reduced in 5 species (C. strigata, N. eques, P. axelrodi, A. borelli, and C. macropomum). All 7 species significantly reduced hypoxia tolerance after acute exposure to 35˚C relative to 28˚C. The effect on hypoxia tolerance is more prominent when the magnitude of temperature change is greater. It is also consistent with this study’s previous findings carried out at UBC (Chapter 2; Figure 2.2A). There are currently insufficient data that directly address the effect of acute thermal exposure on the physiology of Amazonian fishes. However, as discussed in the previous chapter, increasing the ambient water temperature generally reduces the available amount of oxygen in the water (Benson and Krause, 1984; Carpenter, 1966; Garcia and Gordon, 1992), negatively affects the animal’s oxygen delivery mechanisms (Val et al., 2016) and increases metabolic demand (Clark et al., 2011; Clarke and Johnston, 1999; Healy and Schulte, 2012a; Lowe and Davison, 2006; Schmidt-Nielsen, 1997; Steinhausen et al., 2008). These Amazonian fishes likely experienced a threshold where they no longer can match oxygen supply with demand at higher temperatures, resulting in LOE at higher % air saturation.   67  3.4.2.2 Effect of acclimation to higher temperatures on hypoxia tolerance An animal will try to re-establish homeostasis disturbed by thermal stress during acclimation. Some species, mostly temperate, are found to undergo physiological adjustments during thermal acclimation and improve hypoxia tolerance (Anttila et al., 2015; McBryan et al., 2016). To assess Amazonian species’ capacity for thermal acclimation, this study measured hypoxia tolerance after 10 days of acclimation to higher temperatures of 31, 33 and 35˚C (Figure 3.3B). Hypoxia tolerance after acclimation to 31˚C remained the same as that in 28˚C in 4 species, and was reduced in the other 4 of the 8 species measured. Temperatures of 28 and 31˚C are within the present river thermal regime (Val and Almeida-Val, 1995), implying some species are able to sustain and preserve physiological mechanisms within this range.  The 5 species that could acclimate to 33˚C all decreased hypoxia tolerance relative to that measured at 28˚C (C. macropomum) or 31˚C (N. eques, H. erythrostigma, C. schwartzi, and A. borelli). Only C. macropomum could be acclimated to 35˚C, but showed no difference in hypoxia tolerance at 35˚C relative to that in fish acclimated to 31 or 33˚C. Moreover, after acclimation to 33 and 35˚C, 3 species (C. schwartzi, A.borelli and C. macropomum) were less tolerant to hypoxia than when acutely exposed to the same temperature (Figure 3.3; denoted by †), indicating that acclimation to those temperatures is not complete. Species had varying responses to thermal acclimation but overall, acclimation had similar or more severe consequences on hypoxia tolerance relative to acute exposure to the same temperature.  68 Results show that these Amazonian species have no or limited capacity to acclimate to higher temperatures and restore hypoxia tolerance as also seen in Chapter 2. A few previous studies along with results of the present study show that the response to thermal acclimation is not uniform across fish taxa, especially in stenothermic species (Neuheimer et al., 2011; Nilsson et al., 2010; Somero, 2010; Stillman, 2003). Metabolism of temperate and sub-arctic cunners (Tautogolabrus adspersus and Gadus ogac) increased after 6 weeks of acclimation to warmer water (49 and 44% respectively) (Corkum and Gamperl, 2009). In hypoxia (20% air saturation), one of those species (T. adspersus) was not able to reduce oxygen consumption at a higher temperature as efficiently as it did at lower temperature: 52% at 1˚C and 10% reduction at 8˚C. The coral reef elasmobranchs, coral reef teleosts, and Antarctic fishes are found to lack metabolic compensation with a change in temperature, which is disadvantageous in oxygen-depleted water (Nilsson et al., 2010; Tullis and Baillie, 2005; Wilson et al., 2002). Rummer et al. (2014) found a significant reduction of aerobic scope in 6 coral reef species after 14-day acclimation to temperatures above the current thermal range (31, 33 and 34˚C). Nile tilapia (Oreochrormis niloticus) increased oxygen uptake and Pcrit with temperature following 4 weeks of acclimation (Fernandes and Rantin, 1989). These results imply many species, mostly stenotherm, lack the ability to adjust metabolic demand in warmer water, which consequently can hinder hypoxia tolerance. However, the ability to suppress metabolic rate with temperature does not always indicate acclimation capacity. A previous study found that a tetra species (Paracheirodon simulans) increased metabolic rate with an increase in acclimation  69 temperatures, implying a similar low resilience to chronic exposure to warming waters (Campos et al., 2016). Surprisingly, the same study found that another tetra species (Paracheirodon axelrodi) could be acclimated to 35˚C for 2 weeks - a species which this study was only able to acclimate up to 33˚C – and found that the fish maintained metabolic rate from 25 ~ 35˚C. It is also surprising to note that these results are opposite of their habitat conditions: P. simulans inhabits warmer waters that can surpass 35˚C, while P. axelrodi inhabits cooler waters that do not exceed 30˚C. It suggests that thermal tolerance cannot be predicted solely by the fish’s ability to suppress metabolism with acclimation. Clearly, a further investigation on other physiological parameters is required to better understand Amazonian fishes’ capacity to thermally acclimate. I observed a difference in hypoxia tolerance between similar species measured at UBC and INPA (Table 3.2). C. strigata and N. trifasciatus/eques have significantly higher CTMax at INPA compared to UBC (1.4 and 1.5˚C difference respectively). This may be associated with their significantly greater hypoxia tolerance at INPA relative to UBC (3 and 5.2% air saturation difference respectively). I did not observe the same trend in the other 3 species where differences were generally minor. For example, A. viejita tested at UBC had a CTMax 0.5˚C higher and was 0.6% less hypoxia tolerant than that of A. borelli measured at INPA. There are many factors that can contribute to the difference between chapters such as acclimation duration, water chemistry, and food. Fish tested at UBC likely experienced handling and shipping stress, where selection for stronger individuals could have occurred. Also, different species were held in a common tank during the acclimation treatment due to limited space at INPA. The  70 outcomes however are generally very similar and the relative hypoxia tolerance between species is the same between chapters. However, the conditions at INPA are closer to real field conditions and thus likely more valuable in predicting effects related to climate change.   3.4.3 Relationship between thermal tolerance and hypoxia tolerance I found evidence of some, but not a complete, involvement of inter-related pathways between thermal and hypoxia tolerance. As seen at UBC, I found a strong correlation between thermal and hypoxia tolerance among 15 species at 28˚C (Figure 3.4: r=-0.7146, N=15, p=0.0028). It provides evidence that the linkage between two functions could be observed in the lab as well as in nature. However, the correlation was eliminated following 10 days of acclimation to higher temperature (31˚C: r=-0.4422868, N=8, p=0.2725; 33˚C: r=-0.6654924, N=6, p=0.1491). Also as seen at UBC, an improvement of acute temperature tolerance (CTMax) after acclimation to higher temperatures did not confer an improvement or even a recovery of hypoxia tolerance. This could explain the loss of correlation between temperature and hypoxia tolerance after acclimation to higher temperatures. It seems that there is a limited capacity for acclimation of one trait – thermal or hypoxia tolerance – to act positively on another, at least in these Amazonian fishes.  In addition, I found no correlation between time to LOE in severe hypoxia (0.4 ~ 0.6% air saturation) and CTMax (Figure 3.5: r=-0.085, N=5, p=0.892). The ability to take up and utilize oxygen is futile in an anoxic environment. The 5 species were probably exploiting anaerobic pathways to supply energy when very little oxygen was available.  71 In fact, 4 of the 5 species are cichlids, which are known to have great anaerobic capacity in hypoxia (Almeida-Val et al., 1995; Chippari-Gomes et al., 2005). The time to withstand severe hypoxia and anoxia then depends on their ability to suppress oxygen consumption and to mobilize anaerobic metabolism. Therefore, severe hypoxia tolerance does not define thermal limitation of these species, and the two parameters are no longer comparable. Results found in this study further support that thermal tolerance might be aerobically determined.                  72      Figure 3.1. Critical thermal maximum (CTMax, ˚C) at 28˚C and following 10 days of acclimation to 31, 33 and 35˚C (p<0.05) in 8 species of Amazonian fishes. The absence of a bar indicates that fish were not able to acclimate to the respective temperature for the 10-day duration and thus represents the chronic lethal maximum (CLMax). Values are means ± SEM (n=5-8). * represents a significant difference between acclimation temperatures within a species (p<0.05).        28°C31°C33°C35°C28°C31°C33°C35°C28°C31°C33°C35°C28°C31°C33°C35°C28°C31°C33°C35°C28°C31°C33°C35°C28°C31°C33°C35°C28°C31°C33°C35°C05353739414345******CTMax (oC)*** 73      Figure 3.2. Critical thermal maximum (CTMax, ˚C) in normoxia (100% air saturation) and hyperoxia (>200% air saturation). Normoxia CTMax values are reproduced from figure 3.4. * represents significant difference between two treatments (p<0.05). Mean ± SEM.     Ch2 Hyperoxia CTMaxCorydoras pulcherCarnegiella strigataParacheirodon axelrodiCorydoras schwartziBrycon amazonicusApistogramma borelliColossoma macropomum05353739414345 NormoxiaHyperoxia*CTMax (oC)* ** 74     Figure 3.3. Hypoxia tolerance (% Air saturation at loss of equilibrium (LOE)) of A) fish acclimated to 28˚C and measured at 28˚C or following acute exposure and measurement at 33 and 35˚C B) fish following 10 days of acclimation and measurement at 31, 33 and 35˚C. Control 28˚C values in panel B are the same data as presented in A but included for comparative purposes. The absence of a bar in B) indicates fish were not able to acclimate to the respective temperature for the 4-week duration. Values are means ± SEM (n=4-8). * represents a significant difference between temperatures within a species (p<0.05). † in bars of panel A indicate a significant difference in LOE between acute (panel A) and 10 days acclimated (panel B) fish at the same temperature within a species (p<0.05). A. borelli and C. macropomum at 28˚C maintained equilibrium for some time in 0.4 ~ 0.6% air sat. (please refer to Figure 3.5). 28°C31°C33°C35°C28°C31°C33°C35°C28°C31°C33°C35°C28°C31°C33°C35°C28°C31°C33°C35°C28°C31°C33°C35°C28°C31°C33°C35°C28°C31°C33°C35°C02468101214** ** ***† †† †AAir Saturation at LOE (%)28°C31°C33°C35°C28°C31°C33°C35°C28°C31°C33°C35°C28°C31°C33°C35°C28°C31°C33°C35°C28°C31°C33°C35°C28°C31°C33°C35°C28°C31°C33°C35°C02468101214*******BAir Saturation at LOE (%) 75      Figure 3.4. The relationship between critical thermal maximum (CTMax, ˚C) and hypoxia tolerance (% Air saturation at loss of equilibrium (LOE)) in fish following 10 days of acclimation to diurnal fluctuation of 26 ~ 30˚C and measured at 28˚C (black symbols, r=-0.7146, N=15, p=0.0028), 10 days of acclimation to 31˚C (blue symbols, r=-0.4422868, N=8, p=0.2725), 33 ˚C (orange symbols, r=-0.6654924, N=6, p=0.1491) and 35˚C (red symbols). 33 and 35˚C values are reproduced from figure 3.1 and 3.3. Values are means ± SEM (n=4-8). The number above a symbol represents the species: (1) Hemiodus gracilis; (2) Otocinclus spp.; (3) Corydoras pulcher; (4) Carnegiella strigata; (5) Nannostomus eques; (6) Paracheirodon axelrodi; (7) Corydroas splendens; (8) Brycon amazonicus; (9) Corydoras schwartzi; (10) Hyphessobrycon erythrostigma; (11) Apistogramma borelli; (12) Astronotus ocellatus; (13) Apistogramma geophyra; (14) Laetacara fulvipinnis; (15) Colossoma macropomum.     Ch2 Correlation combined36 37 38 39 40 41 42 43 44 45012345678910111213 28˚C31˚C33˚C35˚CCTMax (˚C)Air Saturation at LOE (%)2 6 5 3 4 1 15 6 7 8 9 10 11 12 13 14 15 15 15 11 9 5 10 11 9 4 6 5 10 2  76       Figure 3.5. Severe hypoxia (0.4 ~ 0.6% air saturation) tolerance (time to loss of equilibrium, min) at 28˚C of 5 species (r=-0.085, N=5, p=0.892).     39 40 41 42 43024681012141618202224r = -0.9612SEr=0.1593w/o Nannostomusr=-0.8684SEr=0.2479t=-3.5t0.05(2),4=2.78reject H0=no correlationApistogramma borelliAstronotus ocellatusApistogramma geophyraLaetacara fulvipinnisColossoma macropomumCTMax (oC)LOE (min) 77       Species  UBC  CTMax (˚C) Weight (g) INPA  CTMax (˚C) Weight (g)  p Carnegiella strigata  38.2±0.1  0.246±0.018  39.6±0.0  0.463±0.047   ***  Nannostomus trifasciatus / eques  39.5±0.2  0.150±0.007   41.0±0.0  0.347±0.016   ** Corydoras julii / schwartzi 40.4±0.1  1.675±0.102   39.9±0.1  1.742±0.055   * Hemigrammus rhodostomus / Paracheirodon axelrodi / Hyphessobrycon erythrostigma  41.4±0.1  0.693±0.095  39.9±0.1  0.093±0.005   39.3±0.2  0.361±0.052    *** / ** Apistogramma viejita / borelli  41.0±0.1  0.722±0.107  40.5±0.1  0.350±0.018   ** Table 3.1. Comparison of critical thermal maximum (CTMax, ˚C) measured at UBC following 4 weeks of acclimation to 31˚C and at INPA following 10 days of acclimation to 31˚C. Values are reproduced from figures 2.1 and 3.1. *p<0.05; **p<0.01; ***p<0.0001.        78    Species UBC  % Air Sat. Weight (g) INPA  % Air Sat. Weight (g)  p Carnegiella strigata  8.1±0.5  0.223±0.016  5.1±0.7  0.516±0.052   **  Nannostomus trifasciatus / eques  9.3±0.5  0.144±0.010  4.0±0.3  0.312±0.018   *** Corydoras julii / schwartzi  4.9±0.5  1.565±0.158  1.5±0.3  1.946±0.098   *** Hemigrammus rhodostomus /  Paracheirodon axelrodi /  Hyphessobrycon erthrystigma  3.1±0.2  0.526±0.054  4.8±0.3  0.108±0.005  2.6±0.4  0.361±0.054   NS / *** Apistogramma viejita / borelli  0.7±0.0  0.977±0.116  1.2±0.3  0.283±0.034   NS Table 3.2. Comparison of % air saturation at LOE measured at UBC following 4 weeks of acclimation to 31˚C and at INPA following 10 days of acclimation to 31˚C. Values are reproduced from figures 2.2 and 3.2. *p<0.05; **p<0.01; ***p<0.0001; NS=Not significant.        79 Chapter 4: General Conclusion The Amazon River is one of the most biodiverse aquatic ecosystems in the world despite its challenging abiotic conditions. The river temperature remains high within 27 to 31˚C, and oxygen level oscillates diurnally and between microhabitats (Röpke et al., 2016; Val and Almeida-Val, 1995). Climate change is predicted to cause not only a large temperature increase but also further exacerbate fluctuations in water oxygen saturation and solubility. These disturbances may ultimately affect the distribution and abundance of Amazonian fishes. Thus, it is important to understand the underlying mechanisms between thermal tolerance and hypoxia tolerance as well as species’ capacities to acclimate and adapt to the environmental changes.  This study investigated 17 ornamental and 3 aquaculture fish species of the Amazon. I found that species that are thermally sensitive are also hypoxia sensitive and vice versa, implying a functional association at the whole-organism level within the current thermal range of the region. When the data from Chapter 2 and Chapter 3 are combined together, I again found a correlation at 31˚C despite different acclimation duration and location (Figure 4.1 r=-0.6075961, p=0.02118), further supporting the hypothesis that the thermal limit may be a result of inability to supply and deliver sufficient amount of oxygen to tissues. The responses to increased temperature due to climate change however will likely vary among species (Figure 4.2). Chronic exposure to higher temperatures (10 days at 31, 33, and 35˚C or 4 weeks at 33 and 35˚C) increased acute thermal tolerance (CTMax) to varying degrees in all 13 species measured, but resulted in chronic morbidity in most species at some or all of the higher temperatures. Acute exposure to  80 the higher temperatures of 33 and 35˚C (<1.5 hours) reduced acute hypoxia tolerance (% air saturation at LOE) in 10 of 12 species, but acclimation to the same temperatures generally had the same effect or even a further reduction in hypoxia tolerance measured at those temperatures. Despite the correlation between two physiological parameters within the current river thermal regime (28 or 31˚C), acclimation to higher temperature enhanced acute thermal tolerance but decreased acute hypoxia tolerance (Figure 4.1 indicated by arrows), implying that two traits may be partially related.  One of the most important findings of my thesis was that many Amazonian species lack the capacity to acclimate and survive at higher temperatures. Of the 13 species that were acclimated to higher temperatures, 2 species had the chronic lethal maximum of 31˚C, 9 species of 33˚C, and only 2 species above 35˚C. I found that the species’ CLMax is correlated to its acute thermal tolerance, CTMax (please refer to section 2.3.1 and 3.3.1): The species with low acute thermal tolerance, and thereby low acute hypoxia tolerance, have low CLMax and vice versa. Thus, the species’ potential risk levels facing climate change could be relatively categorized by measuring their current acute thermal tolerance and acute hypoxia tolerance (Figure 4.2).  Figure 4.2. Schematic graph representing species’ potential risk level due to climate change according to their current acute thermal and hypoxia tolerance. % Air Saturation at LOECTMaxModerateHigh RiskLow Risk 81  There is growing evidence that fish species living in a high and narrow thermal range, like that of the Amazon, may be especially prone to increases in temperature associated with climate change (Somero, 2010). This is likely because they inhabit a thermal niche that is near their upper thermal limit and experience relatively small temperature fluctuations within their life span (Deutsch et al., 2008; Neuheimer et al., 2011; Tewksbury et al., 2008; Wright et al., 2009). Overall, results indicate that many of these Amazonian species are near their upper thermal limit, and a temperature increase of 2.2 ~ 7˚C above the current river maximum of 31˚C, as predicted in the worst-case scenario according to the IPCC (2014), may prove to be detrimental for many species. This will consequently affect the large ornamental fishery and aquaculture trades of the region specifically and negatively impact one of the world’s hotspots of freshwater fish biodiversity.   4.1 Future Research This study investigated the effect of predicted temperature changes by the end of next century on thermal tolerance and hypoxia tolerance of Amazonian fishes. In addition to increased temperature, climate change is associated with more frequent and severe hypoxic bouts. Lower environmental oxygen is another critical abiotic factor that could be detrimental to these fishes as all of them depend on aerobic respiration. Studies suggest that two stressors – temperature and hypoxia - combined may act synergistically, producing a greater effect than the sum of independent effects (reviewed by McBryan et al., 2013). The response may be species-specific as shown by Anttila et  82 al., 2015 on the effect of high temperature and/or overnight hypoxia in landlocked salmon (Salmo salar m. sebago) and Atlantic char (Salvelinus alpinus). Salmon significantly increased hypoxia tolerance with warm acclimation and also with warm acclimation combined with overnight hypoxia. On the other hand, the hypoxia tolerance of stenothermic Atlantic char did not change after warm acclimation, but increased after acclimation to the combination of the two stressors. Thermal tolerance (CTMax) increased in all cases for both species. A further study on the combined effect of hypoxia and thermal acclimation is required to more accurately represent and predict the potential impacts of climate change on these stenothermic tropical fish species.   The results of this thesis were derived from short time scale experiments. The acclimation period in this thesis was between 10 days and 4 weeks, and whereas it is generally assumed that the former results in complete thermal acclimation (Barrionuevo and Fernandes, 1998), it may be that longer duration acclimation alters this response. It is possible that some acclimation could occur through the process of developmental or transgenerational plasticity and this will be the next step to investigate. In fact, recent studies on damselfish, stenothermic coral reef species, suggest that there is a limitation in short-term and even developmental acclimation research (Munday, 2014). Transgenerational acclimation, a non-genetic inheritance, occurs when the environment experienced by parents influences the phenotype of their offspring (Salinas et al., 2013). While developmental acclimation had no effect on the aerobic scope of fish, transgenerational acclimation increased the aerobic scope and restored reproductive and offspring attributes (Donelson et al., 2014; Munday, 2014). Moreover, a gradual warming over generations resulted in greater plasticity of the same reproductive  83 attributes than transgenerational acclimation (Donelson et al., 2016). More studies are required to understand whether Amazonian fishes also have similar transgenerational acclimation capacity. Furthermore, climate change will occur slowly and species have more time to make intrinsic changes over generations. Selection for tolerant genotypes for both temperature and hypoxia over ecologically relevant time scale will be vital. More molecular and genomic work is required to understand and predict the adaptation capacity of fishes of the Amazon.   A high rate of latitudinal shift in distribution and abundance of marine aquatic species in order to find more thermally suitable habitats is predicted to occur due to climate change (Cheung et al., 2009; Fernandes et al., 2013). The possibility of latitudinal migration of the Amazonian fishes is more difficult and complex to predict. The floodplains are important for the fishes of the Amazon as they rely on those areas to spawn and feed (Lowe-McConnell, 1987; Winemiller and Jepsen, 1998). Previous climatic variability in the Amazon such as drought, had a large influence on fish communities and thus fisheries, mainly due to the impact on the floodplains (Freitas et al., 2013; Pinaya et al., 2016). One study estimates that the droughts in the Amazon caused by climate change could cause a loss of 7 to 12% of fish species by 2070 (Freitas et al., 2013). According to the IPCC (2014), a large proportion of South America will experience similar climatic changes, but there is still a lack of detailed information on the future climatic changes of the main river as well as the floodplains of the region. Thus, it is important to construct database of different habitats and regions to understand and model potential for migration of the Amazonian fishes.  84 This thesis has investigated the interaction of thermal tolerance and hypoxia tolerance, and the potential physiological consequences of climate change in a diverse range of Amazonian fish species. The groundwork of this thesis should be taken further to better understand the ecophysiological tolerances of these species and identify the areas inhabited by those that are most at risk. These habitats should be protected and additional pressures reduced that otherwise threaten the populations such as fishing and habitat destruction. Ultimately, this thesis should be used to propose a conservational strategy or potential solution in protecting the fish species that may be most vulnerable to climate change.              85     Figure 4.1. The relationship between critical thermal maximum (CTMax, ˚C) and hypoxia tolerance (% Air saturation at loss of equilibrium (LOE)) in fish following 10 days of acclimation to diurnal fluctuation of 26 ~ 30˚C and measured at 28˚C (black symbols, r=-0.7146, N=15, p=0.0028), and following either 10 days or 4 weeks of acclimation to 31˚C (blue symbols, r=-0.6075961, N=14, p=0.02725), 33 ˚C (orange symbols, r=-0.05455648, N=11, p=0.8734) and 35˚C (red symbols). Closed symbols represent 4-week acclimation data measured at UBC (Chapter 2) and open symbols represent 10-day acclimation data measured at INPA (Chapter 3). Arrows connect same species measured at different temperatures. All values are reproduced from figure 2.3 and 3.4. 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