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Mechanisms and evolution of hypoxia tolerance in family Cottidae Mandic, Milica 2008

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MECHANISMS AND EVOLUTION OF HYPDXIA TOLERANCE IN FAMILY COTTIDAE  by  Milica Mandic  B.Sc., The University of British Columbia, Vancouver, 2005  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES  (Zoology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April, 2008  © Milica Mandic, 2008  ABSTRACT A comparative phylogenetically independent contrast (PIC) analysis was employed to investigate the adaptive role of traits involved in hypoxia tolerance in sculpins, a group of closely related fish species that live in the nearshore marine environment. I demonstrated that there was a tight correlation between critical oxygen (02) tension (P eti t) and the distribution of species across an environmental gradient. Species of sculpins with the lowest P„, t inhabit the 02 variable intertidal zone, while species with higher P ent inhabit the 02 stable subtidal zone. Low Pcr t  values in sculpins were associated with enhanced 0 2 extraction capacity, with three principal  traits accounting for 83% of the variation in P ent : low routine 0 2 consumption rate (M0  ), high  mass specific gill surface area and high whole cell hemoglobin-oxygen (Hb-02) binding affinity. Variation in whole cell Hb-0 2 binding affinity was strongly correlated with the intrinsic affinity of Hb for 02 and not to differences in the concentration of the allosteric Hb modulators ATP and GTP. When environmental 02 dropped below a species' Pent, some species of sculpins behaviorally responded to the severe hypoxia by performing aquatic surface respiration (ASR) and aerial emergence. Although intertidal sculpins consistently performed these behaviors, the clustering of these species into a single phylogenetic Glade did not allow us to draw conclusions regarding the relationship between ASR, aerial emergence and P ent using PIC analysis. Three species of sculpins, which were chosen because of their low, medium and high P ent values, exhibited dramatically varied mortality rates when exposed to severe hypoxia equivalent to 40% of their respective P ent . Although ATP turnover rates were similar between the three species in the initial two hours of hypoxia exposure, the differences in the ability of the three species to survive severe hypoxia appeared to be associated with the concentration of on-board liver glycogen and the degree of liver glycogen depletion. However, when liver glycogen was  ii  assessed in twelve species of sculpins at normoxia and compared with P ent , there was no significant PIC correlation between  Pcnt  and liver glycogen.  Overall, I have shown that there is a clear relationship between Pent and the distribution of sculpins along the nearshore environment and that this is primarily related to differences in 02 extraction capacity. When 02 tensions are well below their  Pent,  there are dramatic differences in  behavioral, physiological and biochemical responses among these species of sculpins.  iii  TABLE OF CONTENTS Abstract ^  ii  Table of Contents ^  iv  List of Tables ^  vi  List of Figures ^  vi  List of Abbreviations ^ Acknowledgements ^  viii ix  Co-Authorship Statement ^ Chapter One: Overall Introduction ^ Environmental Hypoxia ^ Why Is Hypoxia Bad? ^ Determining Hypoxia Tolerance ^ Defenses Against Hypoxia ^ Behavioral and Morphological ^ Physiological ^ Biochemical ^ The Comparative Method and Assigning Adaptive Value ^ Sculpins: The Model System ^ Thesis Objectives ^ References ^  1 1 1 2 3 3 4 5 6 8 8 10  Chapter Two: Respiratory Adaptations to Hypoxia in Family Cottidae ^ 14 Introduction^ 14 Material and Methods ^ 16 Experimental Animals ^ 16 Experimental Protocols ^ 18 Whole Animal Respirometry (Pcrit and routine M O2 ) ^ 18 Blood and Tissue Sampling ^ 19 Preparation of RBC Hemolysates ^ 20 Analytical Procedures ^ 21 Gill Morphometrics ^ 21 Blood Hb-02 Affinity (P50) ^ 21 Blood [ATP], [GTP] and Hb Isoforms ^ 22 Magnesium, Met-Hb and RBC pHi ^ 23 Phylogenetic Analyses ^ 23 Phylogenetically Independent Contrasts ^ 24 Statistical Analysis ^ 25 Results ^ 25 Phylogeny and Species Distribution ^ 25 Critical Oxygen Tension ^ 26 iv  Stripped Hb-02 Binding Affinity and RBC Modulators ^ Discussion ^ References ^  27 29 46  Chapter Three: Behavioral, Physiological and Biochemical Strategies in Response to Hypoxia Exposure in Family Cottidae ^  50  Introduction ^ Material and Methods ^ Experimental Animals ^ Experimental Protocols ^ Series 1. Behavior ^ Series 2. Metabolic Fuel ^ Series 3. Relative Hypoxia Exposure ^ Analytical Procedures ^ Liver Metabolites ^ Red Blood Cell Hemoglobin Modulators ^ Statistical Analysis^ Results ^ Series 1. Behavior ^ Series 2. Metabolic Fuel ^ Series 3. Relative Hypoxia Exposure ^ Discussion ^ References ^  Chapter Four: General Discussion^  References ^  50 52 52 53 53 54 55 56 56 56 57 57 57 58 58 59 78 81  85  v  LIST OF TABLES Table 2-1. Fish weight and gill morphometrics from 12 species of sculpins. ^ 40 Table 2-2. Blood hematocrit, hemoglobin, mean cellular hemoglobin content, hemoglobin modulators, RBC intracellular pH from 11 species of sculpins. ^  41  Table 2-3. Hemoglobin isoforms from 11 species of sculpins. ^  42  Table 2-4. Hb-02 P50 and Hill coefficient in whole red blood cell, stripped blood, and reconstituted blood in 11 species of sculpins. ^  43  Table 2-5. Relationship between P„, t and hematological parameters, hemoglobin modulators, and hemoglobin isoforms of sculpins using conventional and phylogenetically independent contrast correlations. ^ 44 Table 2-6. Relationship between whole RBC Hb-02 P50 and hemoglobin modulators, hemoglobin isoforms, RBC intracellular pH and Hill coefficients of sculpins using conventional and phylogenetically independent contrast correlations. ^ 45 Table 3-1. Fish weight and behavioral responses from 11 species of sculpins. ^ 72 Table 3-2. Relationship between P cnt and percent of individuals performing aquatic surface respiration and aerial emergence using conventional and phylogenetically independent contrast correlations. 73 Table 3-3. Relationship between maximum habitat depth and percent of individuals of species performing aquatic surface respiration and aerial emergence using conventional and phylogenetically independent contrast correlations ^ 74 Table 3-4. Liver metabolites in 12 species of sculpins. ^  75  Table 3-5. Relationship between P cr it and liver metabolites using conventional and phylogenetically independent contrast correlations ^  76  Table 3-6. Hematocrit and red blood cell Mg 2+ in 0. maculosus, A. lateralis and B. cirrhosus exposed to normoxia and hypoxia. ^ 77  vi  LIST OF FIGURES  Figure 2-1. Phylogenetic relationship of 13 species of sculpins based on a maximum likelihood tree using cyt b sequences ^ 36 Figure 2-2. Relationship between P cnt and routine 1\:4 0, , Pent and mass specific gill surface area, and P cnt and whole blood Hb-02 P50 ^ 37 Figure 2-3. Relationship between whole blood Hb-02 P50 and stripped Hb-02 P50, and whole blood Hb-02 P50 and reconstituted Hb-02 P50.. ^ 38 Figure 2-4. Relationship between whole blood Hb-02 P50 and Hill's coefficient (n) measured in whole and stripped blood ^ 39 Figure 3-1. Red blood cell ATP and GTP in 0. maculosus, A. lateralis and B. cirrhosus exposed to normoxia and hypoxia equivalent to 40% of respective Pent^ 67 Figure 3-2. Liver ATP and CrP in 0. maculosus, A. lateralis and B. cirrhosus exposed to normoxia and hypoxia equivalent to 40% of respective Pcrit ^  68  Figure 3-3. Liver glycogen in 0. maculosus, A. lateralis and B. cirrhosus exposed to normoxia and hypoxia equivalent to 40% of respective Pent. ^ 69 Figure 3-4. Liver glucose in 0. maculosus, A. lateralis and B. cirrhosus exposed to normoxia and hypoxia equivalent to 40% of respective Pent. ^ 70 Figure 3-5. Liver lactate in 0. maculosus, A. lateralis and B. cirrhosus exposed to normoxia and hypoxia equivalent to 40% of respective Pent. ^ 71  vii  LIST OF ABBREVIATIONS ANOVA^analysis of variance ASR^aquatic surface respiration ATP^adenosine triphosphate BMSC^Bamfield Marine Sciences Centre °C^degrees Celsius CO2^carbon dioxide CrP^creatine phosphate cyt b^Cytochrome B  GTP^guanosine triphosphate GTR^general time reversal fl +^proton Hb^hemoglobin Hb P50^partial pressure at 50% saturation of hemoglobin by oxygen HPLC^high performance liquid chromatography MCHC^mean cellular hemoglobin content mg2+^magnesium Mo,  ^oxygen consumption rate  N^Hill's coefficient N2^nitrogen  02^oxygen PCR^polymerase chain reaction Pent^critical oxygen tension pHi^intracellular pH PIC^phylogenetically independent contrast P02^partial pressure of oxygen Ppt^parts per thousand RBC^red blood cell SE^Standard error  viii  ACKNOWLEDGEMENTS There is a good chance that I will not be able to adequately express my gratitude and admiration to the people that have been indispensable to me personally and academically for the past two years. Nonetheless, I would like to first thank my supervisor, Jeff Richards, for his guidance, knowledge, and wisdom. His open door policy and the ability to calm a flustered, stressed student have been lifesaving on more than one occasion. I am truly grateful. I would also like to thank Lindsay Jibb and Matthew Regan for their wonderful friendships. We have created many amazing memories together and although there are too many to name here, in my mind there are a few worth mentioning: night skiing at Cypress, sailing Erebus to Bowen Island, Montreal and the Square. Of course, I thank Ben Speers-Roesch for many intellectual and not-so-intellectual conversations, especially during the long hours in the `Nerdery'. And to Nann, Brian and Anne D., I could not have asked for better senior students to look up to nor better friends. Zelila bih takodje da se zahvalim svojoj porodici, posebno mami i tati, koji su mi uvijek pomogli kad mi je najvise trebalo. Hvala yam za podrsku, hvala yam sa strpljenje, i hvala mami za kuvanje i tati za kasne nocne voznje. Tata, mama, Aco i Bojane, puno vas volim, Svac.  ix  CO-AUTHORSHIP STATEMENT Chapter 2 and 3 of this thesis are co-authored. The research questions and the design of the experiments were conducted by Milica Mandic under the supervision of Jeffrey G. Richards. The vast majority of research and data analysis was accomplished by Milica Mandic. Anne E. Todgham provided expert advice regarding Family Cottidae in Chapter 2. Katherine A. Sloman provided expert advice and assisted with the behavioral experiment in Chapter 3. Following the conclusion of experiments, the manuscripts were written solely by Milica Mandic in consultation with Jeffrey G. Richards.  x  CHAPTER ONE: OVERALL INTRODUCTION ENVIRONMENTAL  HYPDXIA  Periods of low environmental oxygen (02; hypoxia) are common in the aquatic ecosystem, occurring in both freshwater and marine habitats. The aquatic environment is more susceptible to periodic hypoxia than terrestrial habitats due to relatively low capacitance of water for 02. In ice covered lakes and ponds, severe hypoxia can ensue for a period of 4 to 6 months due to a lack of 02 exchange with the atmosphere in combination with rotting dead plant material (van den Thillart et al., 1989). Seasonal fluctuations in environmental  02  also occur in  the highly vegetated small bodies of water that become isolated from the Amazon river when the water levels are low (Val, 1999). In addition to the long-term changes in 0 2 levels in the waters of the Amazon basin, there are dramatic diurnal fluctuations due to photosynthesis and respiration of organisms (Val, 1999). This has also been recorded in marine environments such as estuaries and tidepools isolated from the tide (Burggren and Roberts, 1991; Landry et al., 2007). In tidepools emerged at night, 0 2 levels can drop to nearly zero due to respiring biomass, while photosynthesis can cause 02 to reach supersaturating levels of 400 to 600 ton of  02  in  tidepools emerged during the day (Truchot and Duhamel-Jouve, 1980). As hypoxia is common in the aquatic environment and as the frequency of hypoxia increases in water systems worldwide as a product of wide spread eutrophication (Diaz, 2001), there is a pressing need to understand how animals respond and adapt to low environmental  02.  WHY IS HYPDXIA BAD? The primary threat an animal exposed to environmental hypoxia faces is the reduced capacity for ATP production to maintain normal cellular functioning because of the reduction in mitochondrial oxidative phosphorylation. As ATP becomes limiting, the general cellular response is a failure in ion motive ATPases such as the Na +/K+ ATPase which leads to an influx 1  of Na+ and efflux of K. ± (Krnjevic, 1993). This causes membrane depolarization and a large influx of Ca 2+ through voltage gated Ca 2+ channels (Boutilier and St-Pierre, 2000). Activation of Ca2+ dependent phospholipases and proteases due to increased Ca 2+ concentrations in the cell leads to further membrane depolarization and cellular swelling (Choi, 1995). As membranes rupture, necrotic cell death occurs causing the animal to die (Boutilier and St-Pierre, 2000). This pathway to necrotic cell death, elicited by a lack of 02, occurs within minutes of hypoxia exposure in hypoxia sensitive animals. Animals deemed hypoxia tolerant can employ a number of defense mechanisms to prolong survival in hypoxia. However, if the severity and duration of hypoxia exposure exceeds an animal's ability to defend itself, the hypoxia-initiated cascade of events leading to cellular death will also occur in hypoxia tolerant animals (Boutilier and St-Pierre, 2000). Since the pathway to necrotic cell death caused by limited cellular 0 2 is the same between a hypoxia tolerant and a hypoxia sensitive animal, this leads to the question of how an animal's tolerance to hypoxia is quantified and what mechanisms are utilized to prolong survival during hypoxia? DETERMINING HYPDXIA TOLERANCE An animal's tolerance to hypoxia can be predicted using several methods. One common technique is to determine an animal's critical 02 tension (Chapman et al., 2002; Saint-Paul, 1984; Ultsch et al., 1978). The critical 02 tension (P ent) is the threshold environmental 0 2 tension where an animal can no longer maintain 02 consumption rate ( M O2 ) independent of the environmental 02 tension. At Pcnt, M O2 begins to decrease in correspondence with a reduction in ambient 02 levels (POrtner and Grieshaber, 1993). The second common method of quantifying hypoxia tolerance is to determine the half maximal survival time (LT50; Bickler and Buck, 2007) when animals are exposed to a steady level of severe hypoxia. Although both techniques are valid predictors of hypoxia tolerance, one method over the other may preferentially be utilized 2  depending on which hypoxia defense mechanisms are under investigation. When the defensive responses are geared towards increasing the efficiency of 0 2 extraction from the environment to maintain routine metabolic rate, then P„, t , which quantifies the shift from routine M o, to depressed M o , should be used as a measure of an animal's ability to defend against changes in metabolic rate. When environmental 02 levels drop too low for an adequate supply of 02 to the tissues, however, other defensive mechanisms must be employed to maintain a balance between energy supply and demand. When investigating defensive responses that occur once routine K1 0, can no longer be maintained, LT 5 0 measure may be a more appropriate indicator of an animal's ability to survive periods of cellular 02 deficit. DEFENSES AGAINST HYPDXIA BEHAVIORAL AND MORPHOLOGICAL  Behavioral responses to low environmental 02, such as aquatic surface respiration and aerial emergence, have been observed in a number of fish species. Aquatic surface respiration (ASR), whereby a fish moves up the water column to ventilate at the water-air interface is an effective strategy of maximizing 02 extraction when the bulk water is hypoxic (Martin, 1995; Watters and Cech, 2003, Yoshiyama et al., 1995). Aquatic surface respiration is enhanced in some Amazon fish species, such as the Colossoma macropomum, through a morphological change of protruding the lower lip to more efficiently direct the surface layer of the water across the gills (Val, 1999). Aerial respiration is also an effective behavioral mechanism in maintaining 02 extraction since fish that actively emerge out of water are able to maintain an aerial metabolic rate that is close to their aquatic metabolic rate during normoxia (Martin, 1996; Sloman et al., 2008; Wright and Raymond, 1978; Yoshiyama and Cech, 1994). However, if pressure from aerial predation becomes too great (Kramer et al., 1983; Sloman et al., 2006; Yoshiyama et al., 1995), or if fish do not have the capacity to employ these behavioral strategies, a suite of 3  physiological and biochemical defenses are available to help cope with low environmental 02. PHYSIOLOGICAL  Modifications to the respiratory cascade have long been thought to be important in species that frequently encounter low environmental 02. A few studies have proposed that fish frequently exposed to bouts of hypoxia possess a large gill surface area for a greater capacity to extract 02 at lower tensions compared to fish living in well-oxygenated habitats that typically possess smaller gill surface areas (Chapman et al., 2002; Saint-Paul, 1984; Timmerman and Chapman, 2004). For example, the hypoxia tolerant Hoplias malabaricus possesses a greater respiratory surface area than the hypoxia sensitive Hoplias lacerdae due to greater filament length and larger total number of secondary lamellae (Fernandes et al., 1993). In addition, although Carassius carassius and Carassius auratus, two species well known for their ability to withstand prolonged, severe hypoxia, have a small respiratory surface area in well-aerated water, during an exposure to hypoxia an increase in the respiratory surface by a —7.5 fold occurs due to a remodeling of the gills. This is achieved through a decrease in intralamellar cell mass due to apoptosis and reduced cell proliferation, allowing protrusion of the secondary lamellae (Nilsson, 2007; Sollid et al., 2003). Fish species possessing hemoglobins (Hbs) with a high affinity for binding 02 can maintain 02 uptake during hypoxia to a greater degree than fish species with a lower Hb-02 binding affinity. Therefore, it has been suggested that hypoxia tolerant species have a higher Hb02 binding affinity than fish with a lower tolerance to environmental hypoxia (Hochachka and Somero, 2002; Jensen et al., 1998). The quintessential example of this is the hypoxia tolerant carp, Cyprinus carpio, which possesses a high Hb-02 binding affinity (P50 — 7 ton; Weber and Lykkeboe, 1978). Most likely the high Hb-0 2 binding affinity in Cyprinus carpio is due to specific amino acid sequence of the Hb protein, which has been shown to be the reason for the very high Hb-0 2 binding affinity in the bar-headed goose (Perutz, 1983). Since in many fish 4  species there are multiple Hb isoforms that exhibit functional heterogeneity, it has been proposed that isoform patterns play a large role in establishing the different Hb-0 2 binding affinities among fish species. Brix et al. (1999) demonstrated that triplefin fishes inhabiting the 02 variable intertidal possess a greater number of Hb isoforms that consist of a greater proportion of the higher Hb-02 binding affinity isoforms than the triplefin fish species located in the deeper dwelling 0 2 stable environments. Different Hb isoform patterns are also seen intraspecifically between individuals of a species reared under different 02 tensions. Higher Hb-02 binding affinity isoforms are expressed in individuals of Haplochromis ishmaeli raised in a hypoxic environment than those reared under normoxic conditions (Rutjes et al., 2007). Many fish species also have the ability to enhance Hb-02 binding affinity during hypoxia exposure through adjustments in concentrations of Hb modulators. The primary Hb modulators in fish are the organic phosphates ATP and GTP (Val, 2000), which bind to the cavity between two 13 chains of the Hb increasing the likelihood of the Hb remaining in the tense, deoxygenated state (Jensen et al., 1998). As environmental 02 decreases, there is a decline in red blood cell (RBC) organic phosphate concentration leading to a decrease in the interaction of the modulators with Hb (Tetens and Lykkeboe, 1981; Weber and Lykkeboe, 1978; Wood and Johansen, 1972). This causes an increase in Hb-02 binding affinity and therefore an increase in 02 extraction at the respiratory surface during hypoxia. BIOCHEMICAL  When environmental hypoxia becomes too severe for a fish to maintain adequate 02 extraction to support aerobic metabolism, a metabolic reorganization occurs in hypoxia tolerant species in order to maintain a balance between energy supply and demand (Hochachka et al., 1996). With a decrease in aerobic metabolism due to a lack of 02, there is an up-regulation in the 0 2 independent pathways of energy production, such as glycolysis and creatine phosphate (CrP) hydrolysis. However these pathways produce significantly less energy than oxidative 5  phosphorylation per unit glucose and a decrease in energy demand is necessary to match the decrease in energy supply. The reduction in energy utilization is primarily achieved through a regulated depression of metabolic rate by decreasing major energy consuming processes such as protein synthesis and ion pumping (Buck et al., 1993a; Buck et al., 1993b). This ability to depress metabolic rate has been considered one of the key hallmark defense mechanisms of a hypoxia tolerant animal (Boutilier and St-Pierre, 2000). THE COMPARATIVE METHOD AND ASSIGNING ADAPTIVE VALUE Because any of the previously mentioned behavioral, morphological, physiological and biochemical modifications can be clearly beneficial in allowing animals to exploit more 02 variable environments, many studies have referred to these traits as adaptations to hypoxia tolerance. However, in these studies the term adaptation was loosely applied to any traits that aided in the survival of an animal without careful consideration of phylogeny. Therefore, it is difficult to discern traits that are true adaptations to hypoxia compared to traits that are valuable defenses against hypoxia but are highly conserved across tolerant and intolerant species. If similar responses occur between species which consistently experience periods of low environmental 02 and those which are never exposed to hypoxic conditions, then most likely the responses are not adaptations to environmental hypoxia per se. The broad categorization of any trait which aids in the survival of an animal during an environmental perturbation as adaptive is not exclusive to hypoxia literature and has been prevalent in diving physiology prior to the study conducted by Mottishaw et al. (1999). Diving physiology literature provides a great example of the necessity of thoroughly testing the potential adaptive value of traits. Historically, the ability of pinnipeds to dive for a prolonged period of time has been attributed to two key 'adaptations', bradycardia and peripheral vasoconstriction (Scholander, 1963). Both responses have been observed in pinnipeds during forced laboratory dives and voluntary sea dives, leading to the general conclusion that bradycardia and peripheral 6  vasoconstriction are integral adaptations to diving. However, despite the importance of bradycardia and peripheral vasoconstriction to the dive response, Mottishaw et al. (1999) demonstrated that the two characteristics did not vary significantly among pinnipeds despite a large variation in species-specific dive duration. With the application of an analytical evolutionary analysis, phylogenetically independent contrasts (PIC), Mottishaw et al. (1999) were able to conclude that bradycardia and peripheral vasoconstriction were not adaptations to the dive response as has long been the belief of diving physiologists. However, spleen mass, blood volume and Hb pool size were shown to correlate with dive duration in a phylogenetically independent manner, suggesting an involvement of these traits in the adaptation of pinnipeds to diving. Clearly a similar approach of utilizing more stringent statistical methods such as PIC is vital in determining if the characteristics involved in prolonging survival during hypoxia could be the result of natural selection. The additional strength of using methods involving an understanding of the phylogenetic history is the ability to elucidate if characteristics under study have evolved independently numerous times only in the species that experience the relevant environmental perturbation. Part of the process involved in inferring evolutionary relationships among traits is employing a comparative method, ideally between closely related species that exhibit variation in the traits of interest (Garland et al., 2005). More closely related species are preferentially chosen to avoid the possibility that very distantly related species have undergone additional evolutionary change which may have confounding effects on the traits under investigation (Felsenstein, 1985; Garland et al., 2005). However, unless it is determined that all the species under study are equally distantly related from each other and form a 'star phylogeny', a serious statistical drawback of the comparative method is that the differential relatedness of the species creates phylogenetic non-independence (Felsenstein, 1985). Species cannot be treated as independent of each other since closely related species share more similar phenotypic and 7  genotypic characteristics than the more distantly related species. If the non-independence caused by a phylogenetic hierarchy is not corrected for, an increase in Type I error occurs in the data (Felsenstein, 1985; Garland et al., 1992; Garland et al., 2005). Application of the PIC method (Felsenstein, 1985) which uses phylogenetic information to transform species data into standardized independent contrasts effectively eliminates any non-independence from the data set. SCULPINS: THE MODEL SYSTEM A PIC corrected comparison of a group of closely related fish species, sculpins from the family Cottidae, was utilized in this thesis in an attempt to determine the adaptive value of traits long thought of as adaptations to hypoxia. Sculpins are an ideal comparative model for studying the evolutionary relationships among traits associated with hypoxia because of their differential distribution along the nearshore environment (Eschmeyer and Herald, 1983; Froese and Pauly, 2007). The nearshore displays a steep environmental gradient over a narrow geographical range. The intertidal zone shows severe diurnal fluctuations in 02, and is in close proximity with the subtidal zone that shows little fluctuation in 02 (Burggren and Roberts, 1991; Truchot and Duhamel-Jouve, 1980). Since species of sculpins exhibit a pattern of vertical zonation along the nearshore environment, they most likely experience drastically different selection pressures on the ability to tolerate fluctuating levels of environmental 0 2 . Thus they are an ideal group of species for studying adaptations to hypoxia. THESIS OBJECTIVES Despite an extensive mechanistic understanding of how hypoxia tolerant animals defend against hypoxia, there has been very little work conducted on the evolution of hypoxia tolerance in teleost fish. Focusing predominantly on characteristics involved in 0 2 extraction efficiency, the purpose of my thesis was to begin to elucidate the adaptation of sculpins to hypoxia using PIC. To accomplish this, the three main objectives of my thesis research were to: 1) determine if 8  species of sculpins exhibited variation in hypoxia tolerance and if this variation is correlated to their distribution along the nearshore environment, 2) correlate traits believed to be involved in hypoxia tolerance with the observed hypoxia tolerance using PIC, and 3) quantify the responses of sculpins exposed to severe hypoxia.  9  REFERENCES Bickler, P.E. and Buck, L.T. 2007. Hypoxia tolerance in reptiles, amphibians, and fishes: life with variable oxygen availability. Annu. Rev. Physiol. 69: 145-170. Boutilier, R.G. and St-Pierre, J. 2000. Surviving hypoxia without really dying. Comp. Biochem. Physiol. 126: 481-490. Brix, 0., Clements, K.D. and Wells, R.M.G. 1999. Haemoglobin components and oxygen transport in relation to habitat selection in triplefin fishes (Tripterygiidae). J. Comp. Physiol. B. 169: 329-334. Buck, L.T., Hochachka, P.W., Schon, A. and Gnaiger, E. 1993a. 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Time and tide wait for no fish: intertidal fishes out of water. Environ. Biol. Fishes. 44: 165-181. Martin, K.L.M. 1996. An ecological gradient in air-breathing ability among marine cottid fishes. Physiol. Zool. 69: 1096-1113. Mottishaw, P.D., Thornton, S.J. and Hochachka, P.W. 1999. The diving response mechanism and its surprising evolutionary path in seals and sea lions. Amer. Zool. 39: 434-450. Nilsson, G. E. 2007. Gill remodelling in fish - a new fashion or an ancient secret? J. Exp. Biol. 210: 2403-2409. Perutz, M.F. 1983. Species adaptation in a protein molecule. Mol. Biol. Evol. 1: 1-28. POrtner, H.O. and Grieshaber, M.K. 1993. Critical P02(s) in oxyconforming and oxyregulating animals: gas exchange, metabolic rate and the mode of energy production. Boca Raton, CRC Press. Rutjes, H.A., Nieveen, M.C., Weber, R.E., Witte, F. and Van den Thillart, G.E.E.J.M. 2007. Multiple strategies of Lake Victoria cichlids to cope with life long hypoxia include hemoglobin switching. Am. J. Physiol. 293: R1376-1383. Saint-Paul, U. 1984. Physiological adaptation to hypoxia of a neotropical characoid fish Colossoma macropomum, Serrasalmidae. Environ. Biol. Fishes. 11: 53-62. Scholander, P.F. 1963. The master switch of life. Sci. Amer. 209: 92-106. 11  Sloman, K.A., Mandic, M., Todgham, A.E., Fangue, N.A., Subrt, P. and Richards, J.G. 2008. The response of the tidepool sculpin, Oligocottus maculosus to hypoxia in laboratory, mesocosm and field environments. Comp. Biochem. Physiol. 149: 284-292. Sloman, K.A., Wood, C.M., Scott, G.R., Wood, S., Kajimura, M., Johannsson, 0.E., AlmeidaVal, V.M.F and Val, A.L. 2006. Tribute to R.G. Boutilier: the effect of size on the physiological and behavioural responses of oscar, Astronotus ocellatus, to hypoxia. J. Exp. Biol. 209: 11971205. Sollid, J., De Angelis, P., Gundersen, K. and Nilsson, G.E. 2003. Hypoxia induces adaptive and reversible gross morphological changes in crucian carp gills. J. Exp. Biol. 206: 3667-3673. Saint-Paul, U. 1984. Physiological adaptation to hypoxia of a neotropical characoid fish Colossoma macropomum, Serrasalmidae. Environ. Biol. Fishes. 11: 53-62. Tetens, V. and Lykkeboe, G. 1981. Blood respiratory properties of rainbow trout, Salmo gairdneri: responses to hypoxia acclimation and anoxic incubation of blood in vitro. J. Comp. Physiol. B. 145: 117-125. Timmerman, C.M. and Chapman, L.J. 2004. Hypoxia and interdemic variation in Poecilia latipinna. J. Fish. Biol. 65: 635-650. Truchot, J.P. and Duhamel-Jouve, A. 1980. Oxygen and carbon dioxide in the marine intertidal environment: diurnal and tidal changes in rockpools. Respir. Physiol. 39: 241-254. Ultsch, G.R., Boschung, H. and Ross, M.J. 1978. Metabolism, critical oxygen tension, and habitat selection in darters (Etheostoma). Ecology. 59: 99-107. Val, A.L. 2000. Organic phosphates in the red blood cells of fish. Comp. Biochem. Physiol. 125: 417-435. Val, A.L. 1999. Hypoxia adaptation in fish of the Amazon: a never-ending task. S. Afr. J. Zool. 33: 107-114. van den Thillart, G., van Waarde, A., Muller, H.J., Erkelens, C., Addink, A. and Lugtenburg, J. 1989. Fish muscle energy metabolism measured by in vivo 31 P-NMR during anoxia and recovery. Am. J. Physiol. 256: R922-R929. Watters, J.V. and Cech, Jr., J.J. 2003. Behavioural responses of mosshead and wolly sculpins to increasing environmental hypoxia. Copeia. 2003: 397-401. Weber. R.E. and Lykkeboe, G. 1978. Respiratory adaptations in carp blood: influences of hypoxia, red cell organic phosphates, divalent cations and CO2 on hemoglobin-oxygen affinity. J. Comp. Physiol. 128: 127-137. Wood, S.C. and Johansen, K. 1972. Adaptation to hypoxia by increased Hb-O 2 affinity and decreased red cell ATP concentration. Nature. 237: 278-279.  12  Wright, W.G. and Raymond, J.A. 1978. Air-breathing in a California sculpin. J. Zool. Biol. 203: 171-176. Yoshiyama, R.M. and Cech, Jr., J.J. 1994. Aerial respiration by rocky intertidal fishes of California and Oregon. Copeia. 1994: 153-158. Yoshiyama, R.M., Valpey, C.J., Schalk, L.L., Oswald, N.M., Vaness, K.K., Lauritzen, D. and Lirnm, M. 1995. Differential propensities for aerial emergence in intertidal sculpins (Teleostei; Cottidae). J. Exp. Mar. Biol. Ecol. 191: 195-207.  13  CHAPTER TWO: RESPIRATORY ADAPTATIONS TO HYPDXIA IN FAMILY COTTIDAE INTRODUCTION  The ability of an animal to acquire 02 from its environment has long been considered a major determinant of hypoxia tolerance (Hughes, 1973). Animals that possess a greater 02 extraction capacity are able to maintain a routine metabolic rate at lower 02 tensions and exploit more 02 variable environments (Hochachka and Somero, 2002; Hughes, 1973). Potential modification to any of the multiple sites along the respiratory cascade, from 02 uptake at the gills to 02 use by the final electron acceptor in the mitochondrial electron transport chain, can, in theory, lead to enhanced 02 extraction capacity from the environment and thereby increase hypoxia tolerance. Any mechanism that increases hypoxia tolerance may be a potential target of natural selection in organisms living in 0 2 variable environments. The Hb-02 binding system has long been considered a critical adaptation to low environmental 02. Generally, Hbs of active fish living in stable, well-oxygenated water possess low Hb-02 binding affinity, while fish that inhabit variable 02 environments, which routinely become hypoxic have high Hb-0 2 binding affinities (Hochachka and Somero, 2002; Powers, 1980; Wells, 1999). Interspecific variation in Hb-02 binding affinity can be attributed to differences in Hb multiplicity and heterogeneous Hb isoform expression (Brix et al., 1999; Wells et al., 1989; Wells, 1999) as well as mutations in amino acid sequences (Perutz, 1983). Apart from these intrinsic properties of the Hb, allosteric modulators, such as ATP and GTP, can also impact Hb-02 binding affinity. As effective modifiers of Hb-02 binding affinity, allosteric modulators may play an important role in the ability of Hb to efficiently function in 02 transport over a range of environmental 0 2 tensions (Wells et al., 1997).  A version of this chapter will be submitted for publication. Mandic, M., Todgham, A.E. and Richards, J.G. Respiratory Adaptations to Hypoxia in Family Cottidae.  14  Although there is a long standing contention that a high Hb-02 binding affinity is adaptive to surviving hypoxia, to date, most studies have compared distantly related species and therefore these studies have only been able to suggest that Hb plays a role in the evolution of tolerance to low 0 2 levels. Assigning adaptive value to a trait requires two important parameters: 1) the careful selection of closely related species that differ in the frequency and magnitude of their exposure to the environmental perturbation under study and 2) the ability to demonstrate that the trait of interest is exhibited only by the species that typically experience the environmental perturbation. However, to eliminate inflated Type 1 errors that occur due to the phylogenetic non-independence that exists between closely related species, phylogenetically independent contrasts (PIC) are applied to the analysis (Felsenstein, 1985; Garland et al., 1992). This maintains the advantages of comparing closely related species, which is a critical component in the determination of adaptation of a trait within a PIC study (Garland et al., 2005). For studying the evolution of hypoxia tolerance, fish inhabiting the nearshore marine environment present an ideal model system. The nearshore marine environment is a narrow geographical range encompassing two zones, the intertidal and the subtidal, that differ dramatically in the degree of variation in physical parameters such as 02, temperature, salinity and pH (Burggren and Roberts, 1991; Truchot and Duhamel-Jouve, 1980). The intertidal zone is under heavy influence of the daily ebb and flow of the tides, and rocky pools (tidepools) within this zone experience severe diurnal fluctuations in abiotic factors. Of particular importance to the present study, tidepools emerged at night experience drops in 02 to nearly zero within a few hours of emergence, while 0 2 in tidepools emerged during the day can reach supersaturating levels of 400 to 600 torr (Truchot and Duhamel-Jouve, 1980; personal observation). The degree of variation in environmental 02 is primarily dictated by the duration of emergence, therefore tidepools located high in the intertidal often experience more severe fluctuations in their environment than pools located lower in the 15  intertidal zone. The adjacent subtidal zone, however, shows little fluctuation in these physical parameters. The steep environmental gradient along the nearshore environment has a strong effect on the vertical distribution patterns of species (Brix et al., 1999; Doty, 1946; Stillman and Somero, 1996). A group of closely related fish species, sculpins from the family Cottidae, exhibit a pattern of vertical zonation with different species inhabiting different portions of the nearshore environment (Eschmeyer and Herald, 1983; Froese and Pauly, 2007). Given the distribution pattern along the nearshore environment, different species of sculpins experience dramatically different magnitudes and frequencies of variation in their physical environment. It is likely, therefore, that species found in the intertidal zone, which most commonly experience hypoxia in the environment, have evolved mechanisms to tolerate hypoxia, while species inhabiting the relatively 02 stable subtidal zone most likely lack these adaptations. The objectives of the study were to determine the characteristics that are related to hypoxia tolerance in 12 species of sculpins that are found in different parts of the nearshore marine environment. Components of the respiratory cascade, such as gill surface area and whole blood Hb-02 binding affinity were quantified in normoxia-acclimated sculpins and the application of PIC aided in assigning adaptive value to these characters. Intrinsic Hb-02 binding affinity and concentrations of RBC allosteric modulators (eg. ATP and GTP) were also investigated to determine the underlying cause of variation in whole blood Hb-02 binding affinity. MATERIAL AND METHODS EXPERIMENTAL ANIMALS  Marine sculpins were caught using handheld nets or seines during the lowest tidal cycles of June and August 2005, 2006 and 2007 at Ross Islets (48°52.4'N; 125°9.7'W), and Wizard's Rock (48°51.5'N; 125°9.4'W), near the Bamfield Marine Sciences Centre (BMSC), Bamfield, 16  British Columbia, Canada. Marine sculpins including tidepool (Oligocottus maculosus), fluffy (Oligocottus snyderi), and mosshead (Clinocottus globiceps) were caught in tidepools. The remaining marine sculpins were caught in the subtidal zone and include buffalo (Enophrys bison), padded (Artedius fenestralis), smoothhead (Artedius lateralis), cabezon (Scorpaenichthys marmoratus), Pacific staghorn (Leptocottus armatus), silverspotted (Blepsias cirrhosus) and scalyhead (Artedius harringtoni). The freshwater prickly sculpin (Cottus asper) was caught using baited minnow traps in Pachena Lake (48°50'11" N; 125°01'44" W) near BMSC during the same time period. Sculpins were transported to the University of British Columbia and the marine sculpins were held in re-circulating 12°C seawater (30 ppt salinity), which was obtained every two months from the Vancouver Aquarium. In 2006, shorthorn sculpins (Myoxocephalus scorpius) were brought in from the Atlantic Coast (Memorial University, Newfoundland) and held in the re-circulating saltwater system. Freshwater prickly sculpins were housed in an identical re-circulating system in 12°C freshwater. All sculpins were allowed to recover from transportation for at least 3 weeks before experimentation. Throughout the study period, fish were fed daily with bloodworms and frozen fish fillets, except 24 hours prior to experimental trials. Whole animal respirometry (Pcnt and routine M o ) was performed on sculpins captured in 2005 and sculpins captured in 2006 were terminally sampled for analysis of gill surface area, concentrations of Hb modulators, affinity of Hb for 0 2 in the presence and absence of HI) modulators, and Hb isoform profiles. To complete the Hb characterization, additional specimens were caught and terminally sampled in 2007 for measurements of RBC intracellular pH (pHi). The mean weights of fish used in 2006 are given in Table 2.1 and the weights of fish captured in 2005 and 2007 were generally not statistically different than those from 2006. Although there are statistical differences in some species, the variation in weight has no impact on  17  measurements of Pair (unpublished data). We also had some initial concern that fish might differ from year to year in their physiological responses to hypoxia; however, no appreciable difference in measurements of Pent from animals caught in separate years was observed (c.f. present study with Henriksson et al., In Press). All experimental procedures involving animals were done according to UBC protocol A05-0142. EXPERIMENTAL PROTOCOLS  Whole Animal Respirometry  (Pcra and  routine M O2 )  Most fish maintain a stable M 0, over a range of water 02 levels, termed the 02 independent pattern of M O2 , but as 02 drops below a threshold, M O2 decreases as water 02 levels decline. This is often referred to as the 02 dependent pattern of M O2 (POrtner and Grieshaber, 1993). The point at which M O2 transitions from being independent of environmental 02 to being dependent on environmental 02 is referred to as the critical 02 tension (Pcrit) and is considered one potential indicator of an animal's tolerance to hypoxia (Chapman et al. 2002; POrtner and Grieshaber, 1993). Pair was determined for each sculpin species by measuring  1VI in a sealed respirometer using a fiber optic 02 probe (FOXY-R, Ocean Optics Ltd., Florida, O2  USA). Briefly, fish were placed into a size-matched respirometer (20 mL/g) and held overnight under flow-through conditions (seawater for the marine sculpins and freshwater for the freshwater sculpins). Throughout the recovery and respirometry periods the respirometers were held in a temperature regulated water bath at 12°C. After the recovery period, the 02 probe was secured into the respirometer and the respirometer sealed to prevent 0 2 exchange between the inside of the respirometer and the outside water chamber. The 02 probe was connected to a data acquisition system that recorded the steady decline of 02 in the water as the fish consumed 0 2 in the respirometer. The experiment was terminated either when the fish lost equilibrium or when 18  there was no further 02 decline recorded by the probe. Mass specific M O2 was calculated over 10 minute sequential periods and Pcnt was determined as the first derivative of M 0 , vs. P0 2 using the visual basic program described by Yeager and Ultsch (1989). In our hands, we saw no differences in Pent determined using closed or open respirometry, therefore the potential accumulation of CO2 and ammonia that could occur during a typical trial does not affect M O2 . Routine M O2 was calculated over the 0 2 independent zone, which is well above the critical 0 2 threshold. Blood and Tissue Sampling To obtain resting, normoxic tissue samples, individual fish were housed overnight in sampling baskets, which were submerged in well-aerated 580 liter tanks containing appropriate water (seawater for marine sculpins and freshwater for freshwater sculpins). The sample baskets were 5 liter plastic chambers with mesh sides and a 1 liter basin at the bottom. To sample a fish, the chamber was carefully removed, confining the fish to the l liter basin and an overdose of benzocaine (250 mg/L, Sigma-Aldrich) was introduced into the chamber and the fish lost equilibrium within approximately one minute. The fish was removed, patted dry, weighed, and a blood sample was taken following caudal severance using a heparinized hematocrit (Hct) tube. Hematocrit tubes were placed on ice until processing could occur (<5 minutes). Liver, muscle, brain, heart and the left gill basket were dissected from each fish and immediately frozen in liquid N2 and stored at -80°C until analysis. The right gill basket was placed into Karnovsky's fixative (Karnovsky, 1965) for later determination of gill surface area (see below). From the sampled blood, 5 !IL was added to 1 mL of Drabkin's solution (Sigma-Aldrich, USA) for determination of whole blood [Hb]. Approximately 2 1AL of whole blood was set aside on ice for the analysis of whole blood Hb-0 2 affinity (see below), while the remainder of the blood was centrifuged (AUTOCRIT Ultra 3, Becton Dickinson and Company, New Jersey) at 19  13,700 g for 3 minutes. Hematocrit was obtained and RBCs were separated from plasma, and both were frozen in liquid  N2  and stored at -80°C.  On fish captured in 2007, blood samples were taken as described above, except whole blood was washed in heparinized Cortland saline (Wolf, 1963) and centrifuged at 5,000 g for 2 minutes at 5°C. The supernatant was drawn off and the RBC pellet was immediately frozen in liquid N2 for later determination of pHi. Preparation of RBC Hemolysates  Frozen RBC pellets were thawed on ice and 20 mM Tris buffer (pH 7.4) was added at 12 times the estimated RBC volume. Samples were vortexed and left on ice for 5 minutes before centrifugation at 15,000 g for 10 minutes at 4°C. From the resulting supernatant, aliquots (10-20 pL) were taken and frozen for determination of [ATP], [GTP], [Mg 2+ ] and [Hb]. Hemolysate [Hb] was used to standardize [ATP], [GTP] and [Mg 2+ ]. The remaining cell hemolysate was immediately stripped of Hb modulators (ATP and GTP) by loading the hemolysate onto Micro Bio-spin P30 Tris chromatography columns (Bio-Rad Laboratories) followed by centrifugation at 1000 g for 4 minutes at 4°C. Aliquots of the stripped Hb were set aside for the determination of stripped blood Hb-02 binding affinity (see below), met-Hb analysis, reconstituted Hb-02 binding affinity (see below) and Hb isoform analysis. All four aliquots were frozen and stored at -80°C. In order to determine if ATP and GTP were the primary RBC Hb modulators in sculpins, measured RBC [ATP] and [GTP] were added back to the stripped Hb hemolysates in an attempt to reconstitute whole RBC Hb-02 binding affinity. Red blood cell hemolysates were thawed on ice and [Hb] quantified immediately. Samples of stripped Hb were reconstituted to the same [ATP]/[Hb] and [GTP]/[Hb] ratio measure as whole cell lysates (see below) and immediately analyzed for Hb-02 binding affinity as described below. To verify [ATP] and [GTP] in the reconstituted samples, concentrations of total NTP were determined spectrophotometrically 20  using the enzyme-coupled assays (glyceraldehydes-3-phosphate dehydrogenase and phosphoglycerate phosphokinase) as described by Bergmeyer (1983). There was no significant difference between the nominal sum of [ATP] and [GTP] and measured total [NTP] (data not shown). ANALYTICAL PROCEDURES  Gill Morphometrics Total gill surface area was determined according to the protocol described by Hughes (1984). The total number of filaments along one side of the 2 nd gill was counted under a stereomicroscope and the length of every 5 th filament was measured. Under a compound microscope the average lamellar spacing was determined by measuring the distance occupied by 10 lamellae. Between 7 and 8 lamellae were dissected free of the filament and the total area of each lamella was determined using AutoMontage software (Syncroscopy, Maryland, USA). Total gill surface area was calculated from A = Lnbl, where L is the total filament length (mm) on all gill arches from both gill baskets, n is the number of lamellae/mm on both sides of the filament, and bl is the average bilateral surface area of the lamellae (mm 2 ; Hughes, 1984). Blood Hb-02 Affinity (P5o) The principles of measuring blood Hb-02 affinity (P50) in small volumes of blood (<2 pL) are outlined by Reeves (1980). Briefly, approximately liAL of blood was sandwiched between two gas permeable membranes and loaded into a prototype PWee50 generously loaned by Dr. P. Frappell (LaTrobe U.; Australia). The analysis chamber was regulated at 12°C, and CO2, 02, and N2 gases were mixed and delivered to the chamber by a Corning 192 Precision Gas Mixer. CO2 was kept constant at 0.5%, but 0 2 levels were varied throughout the experiment in order to determine the percent saturation of Hb at different 02 levels. Percent saturation of Hb was measured using absorption at wavelengths of 393 nm and 435 nm. Blood samples were exposed to approximately 7 to 9 different 02 tensions and measurements were recorded to 21  construct a linear section of the Hill plot for the determination of Hb  P50  (Frappell et al., 2002).  Hill's coefficient (n) was determined as the slope around half saturation in a plot of log(Y/(1-Y)) versus log P02. Blood 'ATP], [GTP] and Hb Isoforms High performance liquid chromatography (HPLC) using Gilson 322 was used to determine blood [ATP] and [GTP] and Hb isoform profiles, according to the protocols outlined by Feuerlein and Weber (1994). Separate HPLC runs were performed for nucleotide triphosphates and Hb isoform determinations. Briefly, for the analysis of ATP and GTP, aliquots of frozen RBC hemolysates were thawed and immediately deproteinized with 3% perchloric acid and then neutralized with 3M Tris-Base. The samples were centrifuged at 20,000 g for 5 minutes at 4°C to remove any precipitates and 20 IAL of the neutralized extract was injected onto an anion exchange Mono-Q 5/50 GL column (GE Health Care, USA). Following injection, buffer A (A=20 mM Tris, pH 8.0) was kept at 100% for 5 minutes. Between 5 and 22 minutes there was a linear decrease in buffer A to 70% buffer B (B=15 mM Tris, 0.5 mM NaCI, pH 8.0). Buffer B was then increased to 100% by 23.5 minutes and kept constant until 28.5 minutes when buffer A was increased to 100% by 30 minutes and maintained at that concentration for a subsequent 5 minutes (Feuerlein and Weber, 1994). Nucleotide triphosphates were detected at 254 nm and ATP and GTP peaks were identified by comparison to retention times of known standards and quantified by comparisons to a standard curve prepared daily. [ATP] and [GTP] were standardized to [Hb] determined on the RBC hemolysate. For Hb isoform analysis, prepared stripped hemolysates were thawed on ice and buffer A was added to dilute Hb sample 7.2 times. The solution was filtered through a 0.2 pm low protein binding syringe filter (Acrodisc, Pall Corporation, USA) and 20 kiL of the filtrate was injected onto the same anion exchange column used above. The HPLC separation protocol was identical to that used above and Hb isoform samples were monitored at 415nm. Hb isoforms were 22  identified according to the protocol described by Feuerlein and Weber (1994), who determined that the most cathodic isoforms will elute first and that the most anodic isoforms will elute last in an anion exchange column. To confirm that there was consistency between the current study and that of Feuerlein and Weber (1994), trout blood was run through the column, resulting in similar pattern of elution of isoforms between the two studies. Magnesium, Met-Hb and RBC pHi Red blood cell [Mg 2+ ] was determined in the frozen hemolysates using flame atomic absorption spectrometry (SpectrAA 240FS, Varian, Australia). Met-Hb was determined spectorphotometrically using the protocol established by Benesch et al. (1973) and samples always contained <15% met-Hb. To measure RBC pHi, approximately 100 [tL of metabolic inhibitor cocktail (POrtner et al., 1991) was added to frozen RBC pellets and vigorously mixed to facilitate cell lyses and left on ice for at least 10 minutes. Red blood cell pHi was measured using BMS 3 Mk2 (Radiometer, Copenhagen) capillary microelectrode at a regulated temperature of 12°C. Phylogenetic Analyses Genomic DNA was extracted from liver of 3 individuals of each species of sculpins using DNeasy Tissue Kit (Qiagen, Canada). Cytochrome b (cyt b) gene sequence was amplified from each genomic DNA sample by the polymerase chain reaction (PCR) using primers L 14724 and H15915 from Schmidt and Gold (1993). The PCR product was gel purified and extracted using GenElute Gel Extraction Kit (Sigma-Aldrich, USA) and sequenced directly using BigDye Terminator v3.1 chemistry and high throughput sequence analysis (Applied Biosystems 3730S 48-capillary sequencer, NAPS, UBC). For each sample, the resulting PCR product was sequenced in both directions and a consensus sequence generated. Sequences were aligned using ClustalW and formatted as a nexus file in Mega 3.1 (Kumar, Tamura and Nei, 2004).  23  Sequences were imported into PAUP* (version 4, Sinauer Associates, Inc. Publishers, USA) to construct both maximum likelihood and maximum parsimony gene trees. Although both analyses gave similar results, maximum likelihood gene tree was chosen and Modeltest (Posada and Crandall, 1998) was used to determine the likelihood ratio test that best fit the sequence data. The tree was constructed using the general time reversal (GTR) DNA substitution model with a gamma distribution of 1.075 and proportion of invariant sites set to 0.545. Nucleotide frequency was relatively evenly distributed (A=0.237, C=0.388, G=0.126, and T=0.249). A heuristics search was used to create the tree with bootstrap analysis of 100 pseudoreplicates and the starting tree option of stepwise addition. Bayesian trees were also created using MrBayes 3.0 (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003). Phylogenetically Independent Contrasts The maximum likelihood tree and branch lengths were imported into Mesquite (Maddison and Maddison, 2004) and the PDAP module (Midford et al., 2003) was used to analyze PIC. Additionally we imported 10,000 trees created through Bayesian analysis into Mesquite and performed the standardized contrasts on the simulated trees in order to determine the robustness of the PIC analysis in terms of the uncertainty in topology and branch lengths (Martins and Housworth, 2002). The PIC analysis using either maximum likelihood tree or Bayesian trees yielded similar results (Bayesian analysis not shown). The results of PIC analysis also did not change appreciably when branch lengths of the maximum likelihood tree were set to one (data not shown). Since analysis can only be performed on complete data sets, we pruned the phylogenetic tree in Mesquite to include only the species for which there were available character data. Therefore, we excluded the outgroup, Satyrichthys amiscus whose cyt b sequence (Accession No. AP004441) was used to root the tree. The cyt b sequence from Cottus bairdii (Accession No. AY833333) was included in order to resolve a polytomy between L. armatus, C. aspen and the 24  remainder of the sculpin family (cf. sculpin phylogeny in Kinziger and Wood, 2003). Cottus bairdii was subsequently pruned from the tree for PIC analysis. An additional tree was constructed (results not shown) containing only the species of sculpins with available character data and there was no significant effect on the results generated by PIC analysis. It is critical to verify if independent contrasts have been adequately standardized by plotting the absolute value of the standardized independent contrast versus its standard deviation (Garland et al., 1992). There was no significant linear or nonlinear trend in the independent contrasts of each character, therefore the contrasts were adequately standardized according to methods described by Garland et al. (1992). The standardized independent contrasts of different characters were subsequently plotted and analyzed by regression analysis that were forced through the origin. STATISTICAL ANALYSES  Data are presented as means ± standard error. Phylogenetically independent contrast correlations were analyzed for significance in Mesquite, while conventional (non-PIC) correlations were analyzed for significance using SigmaStat 3.0. Multiple linear regression models were developed on phylogenetically standardized contrasts and analyzed for significance using SPSS 11.0. Statistical significance was assumed at P<0.05. RESULTS PHYLOGENY AND SPECIES DISTRIBUTION  The present study utilized 12 species of sculpins for which a well-resolved phylogeny was developed using the cyt b gene (Fig. 2.1). Among the marine sculpins available, representatives were captured from various areas of the intertidal and subtidal environment with a good distribution of maximum depth along the nearshore environment (Fig. 2.1; maximum depth from Froese and Pauly, 2007).  25  CRITICAL OXYGEN TENSION  Critical 0 2 tension (Pent) varied between the 12 species of sculpins (Fig. 2.1) and was significantly related to their individual maximum depth distribution. There was a significant PIC correlation between published species maximum depth location (Froese and Pauly, 2007) in the nearshore environment and Pent (r2 =0.57, P=0.01, correlation not shown). Sculpins with high  Pent  values are only found in the subtidal and deeper water environments while sculpins with the low Pent values inhabit the intertidal zone (Fig. 2.1). Routine M oe ranged from 2.2 to 4.5 iimol/g/hr among the 12 species of sculpins (Fig. 2.1). Using conventional correlation analysis there was no significant relationship between  Pcnt  and routine M O2 (Fig. 2.2A, P=0.15); however, when corrected for the phylogenetic relationship between species using PIC, the positive relationship was significant (Fig. 2.2B, P=0.04, PIC). As routine M O2 increased there was a corresponding increase in There was an inverse relationship between  Pent  Pcnt•  and mass specific gill surface area (Fig.  2.2C, Table 2.1), such that as mass specific gill surface area decreased there was an increase in P ent .  The relationship was significant when tested with conventional correlation analysis  (P=0.04, Fig. 2.2C) and improved when corrected for phylogeny (P=0.03, Fig. 2.2D). The large differences in total surface area between species was primarily due to large differences in filament number, filament length, lamellar area and body weight (Table 2.1). No significant relationship existed between species weight and mass specific gill surface area (r 2 <0.01, P=0.94; data not shown), indicating that the variation in fish weight among species (see Table 2.1) had only a minor impact on mass specific gill surface area. Whole blood Hb-02 P50 ranged from 23 torr in the intertidal 0. maculosus to 58 torr in the subtidal B. cirrhosus (Table 2.4) and there was a significant positive correlation between whole blood Hb-0 2 P50 and Pent with both conventional correlation analysis (P = 0 .0 1 , Fig. 2.2E) 26  and with the application of PIC (P=0.03, Fig. 2.2F, PIC). The freshwater C. asper was removed from the correlation between P ent and whole blood Hb-02 P50 because it was found in a previous study that freshwater adaptation and exposure caused a thickening of the gill membrane leading to an increase in respiratory diffusion distance (Henriksson et al., In press). The thickening of the gills caused a mismatch between P c and whole blood Hb-0 2 P50 that decreased with acclimation to seawater. Variation in blood Hct (11 to 35%), [Hb] (0.1 to 1.3 mM), and a mean cellular Hb content (MCHC; 1.3 to 3.7) was observed among the species examined (Table 2.2); however, there was no relationship between these variables and  Pent  using either conventional or PIC  correlations (Table 2.5). Phylogenetically corrected multiple linear regression analysis revealed that whole blood Hb-02 P50,  routine M 0 and mass specific gill surface area combined explain 83% of the  variation in Pent among the species of sculpins (P=0.01). To maintain the number of species consistent, r 2 used for the multiple regression analysis was calculated after the removal of two species. Artedius harringtoni was removed due to lack of available data on whole blood Hb-02 P50, and  C. asper was removed due to confounding effects of freshwater adaptation on whole  blood Hb-0 2 P50 (see above). STRIPPED HB-02 BINDING AFFINITY AND RBC MODULATORS  Stripped Hb showed a similar pattern of Hb-02 P50 as whole blood Hb-02 P50, but at much reduced 02 tensions (Table 2.4). Both conventional (P=0.03, Fig. 2.3A) and phylogenetically independent contrasts (P=0.03, Fig. 2.3B) showed a significant positive correlation between whole blood Hb-02 P50 and stripped Hb-0 2 P50. Red blood cell [ATP]/[Hb] ranged between 0.69 and 2.43 in all species of sculpins except E. bison and S. marmoratus where [ATP]/[Hb] ratios were at nearly undetectable levels. There  27  was no significant relationship (conventional or PIC correlations) between [ATP]/[Hb] and P- cnt (Table 2.5), or between [ATP]/[Hb] and whole blood Hb-0 2 P50 (Table 2.6). Red blood cell [GTP]/[Hb] ranged between 0.10 and 0.51 among sculpins except in A. fenestralis which exhibited very high levels (2.33) of GTP and S. marmoratus that had barely detectable levels of GTP (0.02; Table 2.2). Red blood cell [Mg 24 ]/[Hb] did not vary appreciably between sculpins (Table 2.2). There was no correlation between [GTP]/[Hb] or [Mg 2± ]/[Hb] and Pent (Table 2.5, conventional and PIC) or between [GTP]/[Hb] or [Mg 2+ ]/[Hb] and whole blood Hb-0 2 P50 (Table 2.6, conventional and PIC). Red blood cell pHi did not vary appreciably among the sculpin species (ranging between 7.18 and 7.42; Table 2.2) and there was no significant correlation (conventional or PIC corrected) between pHi and whole blood Hb-0 2 P50 (Table 2.6). The estimated number of total Hb isoforms determined using anion-exchange chromatography varied between species but was generally between 3 and 10 isoforms (Table 2.3). The number of anodic Hb isoforms ranged between 2 and 9 among the sculpins examined, while cathodic Hb isoforms ranged between 0 in some species and up to 3 isoforms in other sculpin species (Table 2.3). There was no relationship (conventional or PIC) between the number of total, anodic, or cathodic isoforms and -Pcnt (Table 2.5) or whole blood Hb-0 2 P50 (Table 2.6). The addition of measured RBC [ATP] and [GTP] to stripped Hb lysate almost fully reconstituted the whole blood Hb-02 P50, and there was a significant correlation between reconstituted and whole blood Hb-0 2 P50 (conventional P=0.03, Fig 2.3C; PIC P=0.02, Fig. 2.3D). The correlations were significant despite two notable exceptions, E. bison and S. marmoratus, whose stripped Hb lysate we were unable to reconstitute back to whole blood Hb02 P50.  Although Hill's coefficient (n) did not vary to any great degree between different species of sculpins (Table 2.4), n did change depending on the amount of organic phosphates present in 28  the blood (Fig. 2.4). In whole blood, n was approximately 1.6 in all species examined (Table 2.4), and the removal of ATP and GTP increased n to about 2.6 (Fig. 2.4, Table 2.4) and this increase was seen across the species. When ATP and GTP were added back to stripped Hb lysates, n decreased back to approximately 1.7 (Table 2.4). There was no significant correlation between n in whole, stripped or reconstituted blood and whole blood Hb-0 2 P50 (Table 2.6, conventional and PIC). DISCUSSION Variation between sculpins in their ability to effectively extract 0 2 from the environment may play a crucial role in dictating species distribution along the marine nearshore environment. I demonstrated a phylogenetically independent relationship between Pent, habitat range, and the physiological parameters involved in environmental 0 2 extraction. Intertidal sculpins experiencing diurnal fluctuations in 02 possess a low Pcnt, indicating an ability to maintain a routine I■/1 02 at lower environmental 0 2 than subtidal or deeper water species that experience minimal fluctuations in 0 2 and possess higher P cnt values. Strong effects of hypoxia on species distribution have also been demonstrated in other intertidal organisms such as the triplefin fishes (Brix et al., 1999), coral reef fishes (Nilsson et al., 2007) and on the distribution of populations of Mytilus edulis (Altieri, 2006), with the more hypoxia tolerant species located in the 02 variable environments. Critical 02 tension is a composite measure of an animal's ability to extract 02 from the environment. Whole animal 0 2 extraction involves many components of the respiratory chain that can be differentially modified to maintain 02 uptake during environmental hypoxia (Hughes, 1973). In our hands, 83% of the variability in Pcnt among sculpins can be attributed to variation in routine 1\4 02 , mass specific gill surface area, and whole blood Hb-0 2 binding affinity with species possessing a low Pent having a low routine 1\4 02 , large gill surface areas, and high whole 29  blood Hb-02 binding affinities. Although it has long been assumed that adjustments to the respiratory chain which enhance 02 uptake would be adaptive to hypoxia survival (eg. Jensen, 1991 and Saint-Paul, 1984), this is the first study to demonstrate through phylogenetically independent contrasts convergent evolution of traits that aid in enhanced 0 2 extraction capacity. This higher 02 extraction capacity occurs in species of sculpins that are frequently exposed to 02 variable environments. Routine I■4 0, is an important factor shaping the respiratory cascade because it is an index of the ultimate demand for 02. In sculpins, although a simple correlation does not show a significant relationship, a phylogenetically independent correlation does indicate that there is a significant positive relationship between P ent and routine 1\/1 02 (Fig. 2.2B). A lower routine .  I\/1 0, could be beneficial to species that are frequently exposed to 0 2 variable environments, such .  as the intertidal sculpins, as a lower  Moe  can be maintained over a greater range of P0 2 values  than a higher M O2 . Gill surface area is an important factor influencing an animal's ability to extract 0 2 from the water. Within the sculpin family, there is a phylogenetically corrected significant correlation between Pent and mass specific gill surface area (Fig. 2.2D). This indicates that a larger gill surface area in sculpins with a low P ent is an adaptive modification to the respiratory chain as it provides an increased extraction efficiency of 02 from the environment. A large gill surface area has previously been demonstrated in fish species frequently exposed to bouts of hypoxia such as the Amazonian Colossoma macropomum (Saint-Paul, 1984), salt marsh dwelling Poecilia latipinna (Timmerman and Chapman, 2004b), Hoplias malabaricus (Fernandes et al., 1993), and populations of Pseudocrenilabrus multicolor, Gnathonemus victoriae and Petrocephalus catostoma inhabiting dense swamp regions (Chapman et al., 2002).  30  Conflicting views exist on whether or not evolutionary adaptation occurs in whole blood Hb-0 2 binding affinity. Jensen (1991) has proposed that since a high whole blood Hb-0 2 binding affinity is often associated with hypoxia tolerant animals there must be a strong positive selection on Hb-0 2 binding affinities. However, a recent study by Milo et al. (2007) found that whole blood Hb-0 2 binding affinity does not vary among different mammals, while cooperativity between Hb subunits (Hill's coefficient) varies drastically. Under changing physiological conditions, however, the opposite trend is noted and Hill's coefficient remains constant, while the greatest change occurs with whole blood Hb-0 2 binding affinity. Milo et al. (2007), therefore, conclude that evolutionary adaptations act on Hill's coefficient, while `physiological adaptations' act primarily on whole blood Hb-02 binding affinity. Within sculpins, there is little variation in Hill's coefficient (Fig. 2.4), while a clear phylogenetically independent correlation exists between Pcnt and whole blood Hb-0 2 binding affinity (Fig. 2.2F), suggesting that evolutionary adaptation within sculpins predominantly acts upon whole blood Hb-0 2 binding affinity. Sculpins that possess a higher P cnt have a lower Hb-02 binding affinity and are found lower in the nearshore environment. Meanwhile sculpins possessing higher Hb-02 binding affinity have an increased capacity to extract 0 2 from the environment and thereby can tolerate more hypoxic conditions such as the ones that are routinely encountered in isolated tidepools at night. Previous studies have suggested that an increase in 0 2 carrying capacity can be accomplished through an increase in blood [Hb] and Hct (Chapman et al. 2002; Hochachka and Somero, 2002; Timmerman and Chapman, 2004a). Although sculpins inhabiting the tidepools do show higher Hct and [Hb] than the subtidal and freshwater species there is no significant correlation between hypoxia tolerance and these blood parameters under resting normoxic conditions. This is unlike the study conducted by Chapman et al. (2002), where they found that fish species dwelling in hypoxic swamps showed greater 0 2 carrying capacity through higher 31  Hct and [Hb] than normoxic lake-dwelling fish species. In the sculpin family, it appears that the modifications to the 0 2 carrying capacity of blood is primarily achieved through changes in whole cell Hb-0 2 binding affinities. Whole blood Hb-0 2 binding affinity can be set by both the intrinsic properties of the Hb and the allosteric interactions between Hb and its modulators (Hochachka and Somero, 2002; Weber and Lykkeboe, 1978). Variation in whole blood Hb-0 2 binding affinity among different species of sculpins is primarily dictated by the intrinsic properties of the Hb protein. When all the major modulators of Hb are removed from the blood, the intrinsic (stripped) Hb-02 binding affinity significantly correlates with whole blood Hb-0 2 binding affinity in a phylogenetically independent manner (Fig. 2.3B). These differences in intrinsic Hb-02 binding affinity can be achieved through amino acid substitutions as seen in the bar-headed goose (Jessen et al., 1991; Perutz, 1983) or through variation in number and functional heterogeneity of Hb isoforms (Brix et al., 1999; Rutjes et al., 2007). Brix et al. (1999) found that the triplefin fishes located in 02 variable tidepools and shallow water possess a greater number of Hb isoforms that are predominantly the higher Hb-02 binding affinity cathodic isoforms. Meanwhile triplefin fishes inhabiting the mid-depth and deeper waters express the lower 0 2 affinity anodal isoforms and a decreasing number of Hb isoforms. In the Lake Victoria cichlid, Haplochromis ishmaeli, there is a functional switching of Hb isoforms during hypoxia acclimation to higher 0 2 affinity isoforms (Rutjes et al., 2007). In sculpins, although a variation is seen in the number of 1-1b isoforms and proportion of anodic to cathodic Hb isoforms, there is no relationship between the number of total, anodic, or cathodic Hb isoforms expressed by a species and hypoxia tolerance. This suggests that the variation in intrinsic Hb-02 binding affinity may be due to differences in amino acid substitutions, which will be the focus of future work. Whole blood Hb-02 binding affinity can also be dictated by the concentration of allosteric modulators such as ATP and GTP, whose binding to the Hb causes a reduction in 32  whole blood Hb-02 binding affinity. During hypoxia exposure, Hb-02 binding affinity increases through reductions in the allosteric modulators to bring about short-term improvements in 0 2 uptake from the environment (Weber and Lykkeboe, 1978). ATP and GTP are the two major Hb modulators in most sculpins examined in this study as indicated by our ability to fully reconstitute whole blood Hb-0 2 binding affinity by adding measured [ATP] and [GTP] back to stripped Hb (Fig. 2.3D). However we were unable to reconstitute whole blood Hb-0 2 binding affinity in two species, E. bison and S. marmoratus suggesting that these two species may possess different Hb modulators. Neither ATP or GTP showed a significant phylogenetically independent correlation to whole blood Hb-02 binding affinity, indicating that even though the modulators may contribute to the overall Hb-02 binding affinity, they probably do not determine the variation seen between the different species. Rutjes et al. (2007) has shown that hypoxia tolerant Lake Victoria cichlids have higher [ATP] and [GTP] under normoxic conditions compared with the relatively hypoxia intolerant salmonids. In sculpins a similar, although non-significant, trend is observed in RBC [ATP], with hypoxia tolerant species inhabiting the tidepools having higher [ATP] than the more hypoxia intolerant subtidal and freshwater species (Table 2.2). Higher [ATP] coupled with high whole blood Hb-02 binding affinity in hypoxia tolerant sculpins instill a significant capacity to endure not only severe hypoxia, but also large fluctuations in environmental 02. A decrease in Hb modulators during hypoxia exposure has been demonstrated in other fish species (Jensen and Weber, 1982; Lykkeboe and Johansen, 1975; Weber and Lykkeboe, 1978) and presumably a similar regulation of Hb modulators occurs in hypoxia tolerant sculpins providing a plasticity in Hb-0 2 binding affinities necessary for coping with fluctuating environmental 02. The removal of organic phosphates, ATP and GTP, also causes an increase in Hill's coefficient that is consistent in all species examined (Fig. 2.4). An increase in Hill's coefficient indicates an increase in the degree of cooperativity between Hb subunits, adding an increased 33  benefit to species frequently exposed to hypoxia. In other species of fish, such as Cyprinus carpio, an increase in Hill's coefficient due to a decrease in Hb modulators is only maintained at pH below 6.5, and reverses at pHs above 6.5 (Tan and Noble, 1973). However, when temperature decreases from 20°C to 10°C, the shift between higher Hill's coefficient in stripped Hb hemolysates to a higher Hill's coefficient in whole blood occurs at a pH of 7.0. Since cooperativity of Hb subunits clearly involves a complex interaction of many physiological parameters such as temperature and pH, it is not surprising that there are difference between the current study and that of Tan and Noble (1973). In sculpins, it is an advantage to have the ability to increase Hb subunit cooperativity due to a decrease in Hb modulators at a relevant physiological pH since presumably a decline in RBC ATP and GTP will occur during an exposure to hypoxia to increase whole blood Hb-02 binding affinity. Coupling an increase in Hb subunit cooperativity with an increase in whole blood Hb-0 2 binding affinity will enhance 02 extraction, aiding in survival during a bout of hypoxia. Low routine I\4 02 , high gill surface area, and high whole blood Hb-0 2 binding affinity .  are characteristics that hypoxia tolerant species possess prior to hypoxia exposure. These traits allow an animal to maintain a `normoxic' level of 0 2 consumption even at significantly reduced 0 2 tensions. This prolongs the period of time an animal can remain in low 0 2 environment prior to eliciting a hypoxia response, such as a down-regulation of Hb allosteric modulators or a decrease in metabolic rate. For animals living in the nearshore marine environment this is ideal, as it allows for the ability to cope with diurnal fluctuations in 0 2 levels without impacting cellular functioning. The focus of future work will be to examine a number of biochemical properties, such as the glycolytic enzyme capacity, during normoxia that may also help play a role in dictating hypoxia tolerance in sculpins. Additionally, future research will focus on  34  characterizing behavioral, physiological and biochemical responses of sculpins exposed to severe hypoxia.  35  Satynchthys amiscus  Depth^Pcrit^RMo2 Clinocottus globiceps^T^26.8- 3.3^3.4 10.3 -  99 ^  Oligocottus maculosus^T^25.9 +4.6^2.8 ± 0.2  ^ 100 ^ Oligocottus snyderi^27.1+3.8^3.0+0.2  loo  ^ Artedius lateralis^ 13^35.7_ 6.9^2.2+0.1 90  ^ - 84  Artedius fenestralis^55^35.4 r_ 3.3^2.3 ± 0.2  - 91 ^  Artedius harringtoni^21^48.5 1.6^2.3 + 0.2  Enophrys bison^  20^31.6=5.0^2.2±0.2  90  Myoxocephalus scorpius^  450^52.3:. 1.2^4.5 + 0.4  ^Scorpaenichfhys marmoratus^200^40.1 .,_ 3.3^4.4 ± 0.4 100  Blepsias cirrhoses^  ^ Leptocottus armatus^  150^44.4 +2.8^3.7 i 0.8  156^37.4± 1.2^2.5 ± 0.3  53  Cottus bairdii 96  Cottus asper^  RN^54.3+1.0^3.7 +0.5  0. 1  Figure 2-1. Phylogenetic relationship of 13 species of sculpins based on a maximum likelihood tree using cyt b sequences. Node bootstrap values are shown for groups with >50% support. S. amiscus is included as an outgroup species. Character data are presented for maximum depth of a species (meters), with T representing tidepool and FW representing freshwater. Pent (ton) and routine M O2 (tmol/g/hr) are also included with n = 6 to 9 except for A. lateralis and A. harringtoni where n = 4 and n = 3 respectively. Data are means ± SE.  36  60 -  100 -  A  B  80 50 -  o  60 -  •  40 -  40 -  •  20 -  a" 30-  ••  0-  •  -20 -  y = 5.15x+22.44 r2 = 0.20  20 -  10  •  •  •  •  •  y = 6.52x r2 = 0.35 p = 0.04  -40 -  p = 0.15  60 1 5^2.0^2.5^3.0^3.5^4.0^4.5^5.0^5.5^0^1^2^3^4^5^6 I  Routine metabolic rate (pmol/g/hr) ^Standardized contrast in routine metabolic rate (pmol/g/hr)  60 -  60 0 0  50 -  40 -  •  40  •  20 -  •  0-  C 0: 30 -  ms  -20 - 40-  20 -  y = -1.81x+176.03  e= 0.35  rn^  p = 0.04  10 40  ^  60^80  ^  co -a^-60-  •  ••  •  •  •  •  •  -ft—.^• y = -0.17x r2 = 0.38 p = 0.03  80 100^120^140^160 180 200^0^20^40^60^80^100^120 140 160  Mass specific gill surface area (mm 2 /g)^Standardized contrast in mass specific gill surface area (mm 2 lg)  60 -  E  r.•_•  60  0  50  •  3: •_.  40  40  •  20  0  •  0  0: 30 -  LN t.!  20 -  y = 0.63x + 9.94 r 2 =0.57 p = 0.01  10 20^30^40^50 Whole Cell P„(torr)  co  0  •  •  -20  y= 6.52x r 2 =0.45 = 0.03  -40 60  ^  0^5  10  15  20  25  30  35^40  Standardized contrast in whole cell P50 (torr)  Figure 2-2. Relationship between P„, t and routine M O2 (A,B), P cnt and mass specific gill surface area (C,D) and P cn t and whole blood Hb-02 P50 (E,F). (A,C,E) are conventional correlations and (B,D,F) are standardized independent contrasts. Error bars in (A,C,E) represent SE for x and y values. Dashed lines in (B,D,F) represent 95% prediction intervals.  37  ^ ^ ^  •^  70 -  -C_o .....^ 100^ 8 o.^ 80 -^ =^  A  B  60 50 40 U a) (7)^30 20 -  10  0o^  U -20 -o y = 2.58x + 24.56^a,^  • ^  y = 2.76x  p = 0.03  P50  Standardized contrast N st"-rippld 8 hem oglobin Pin (torr)^ 50 (torr)  8  70 ^C  .....^  120^  D  8 0_^100 -^  =^  60 -  is  ^7 50 -  1314^  80...---^ ...---U a,^ ....--..---,^ 60 -^ ----  c40 -  U  a) o 30  y = 0.83x - 6.86^-0 a)^-20 -^  20 -  N  •^  ...--r 2 = 0.44^ ti^-40^ .^ ---- ....-p = 0.03^ a ^13  60  .----  .....-•  ---- """ •  • til^20 ta ,...^ • ...., c^0 o^• u  = 40 a)^  0  •  r2 = 0.44 a^ p = 0.03 -o c^60 #^1^ i 4^6^8^10^12^II c n^0^2^4^6^10^12^14  Stripped hemoglobin  10  ...-•  .....--  •  • --r2 = 0.45^fi^-40^  1-§-1 ^  2  ..---  a,^ ...--U I a^60 -^---75^---- --- ^• 40 • c^ 20 in^ •  •  -----^ y = 0.49x ..■  r2 = 0.46 p = 0.02  1 10^20^30^40^50^60^0^20^ .i^ 40^60^80^100  cn  Reconstituted hemoglobin  P50  (torr)^ Standardized contrast in reconstituted hemoglobin  P50  (torr)  Figure 2-3. Relationship between whole blood Hb-0 2 P50 and stripped Hb-0 2 P50 (A,B) and whole blood Hb-02 P50 and reconstituted Hb-0 2 P50. (A,C) are conventional correlations and (B,D) are standardized independent contrasts. Error bars in (A,C) represent SE for x and y values. Dashed lines in (B,D) represent 95% prediction intervals.  38  70  -  60 50 40 30 20 10 1 2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2  n  Figure 2-4. Relationship between whole blood Hb-0 2 P50 and Hill's coefficient (n) measured in whole blood (open circle) and stripped (filled circle). Error bars around the symbols represent SE for x and y values.  39  Table 2-1. Fish weight and gill morphometrics from 12 species of sculpins. Weight  Filament number  Filament length  Lamellar area  0. maculosus  4.5+0.5  22±1  1.7+0.1  C. globiceps  1.6+0.2  25±1  O. snyderi  2.5+0.7  E. bison  Mass specific surface area  0.03+0.01  Total surface area 365+71  1.3+0.1  0.03+0.01  285+45  177+10  21+1  1.3+0.1  0.03+0.01  263+50  116+11  10.9+5.3  38+5  2.0+0.3  0.03+0.01  935+350  132+30  A. fenestralis  16.9+2.1  35+1  2.8+0.1  0.05+0.01  1613+135  101+10  A. lateralis  15.2+1.4  38+1  2.5+0.1  0.07+0.03  1703+701  105+34  L. armatus  48.2+2.1  67±2  4.1+0.1  0.09+0.01  6320+588  131+11  S. marmoratus  20.8+2.9  59+3  2.9+0.2  0.04+0.01  2211+429  106+10  B. cirrhosus  2.6+0.4  28±1  1.3+0.1  0.02+0.01  264+51  95+11  A. harringtoni  4.1+1.1  27±1  1.5+0.2  0.02+0.01  286+32  93+27  M scorpius  9.7+2.7  46±3  2.0+0.2  0.03+0.01  861+213  92+27  C. asper  19.3+1.8  33+1  2.3+0.1  0.06+0.01  1003+75  57±31  78+11  Data are means ± SE. Fish weight is presented in g, filament length in mm, lamellar area and total surface area in mm 2 and mass specific gill surface area is presented in mm 2/g. Sample size ranged from n = 8 to 9, except for A. lateralis where n = 4 and A. harringtoni where n = 3.  40  Table 2-2. Blood hematocrit, hemoglobin, mean cellular hemoglobin content, hemoglobin modulators, RBC intracellular pH from 11 species of sculpins. Hct^Hb^MCHC ATP/Hb GTP/Hb Mg 2 /Hb^pHi  0. maculosus^35±2^1.3±0.1^3.7+0.2^1.82+0.18 0.25+0.06^29±2^7.35±0.03 (8)^(5)^(3)^(8)^(8)^(5)^(12)  C. globiceps^35±1^1.3±0.1^3.4±0.3^2.43±0.09 0.51+0.03^37±2^7.26±0.05 (8)^(6)^(2)^(8)^(8)^(4)^(2)  0. snyderi^37±3^1.3+0.1^3.2±0.7^2.18±0.06 0.10±0.01^N/A^7.26±0.02 (8)^(3)^(2)^(3)^(3)^  (7)  E. bison^22+2^0.6+0.1^2.5±0.2^0.04±0.01 0.28±0.08^25±2^7.27 (8)^(7)^(7)^(6)^(6)^(3)  A. fenestralis^27±2^0.9±0.1^3.5±0.2^0.91±0.06 2.33±0.16^28±2^7.32±0.04 ( 8 )^(8)^(8)^(8)^(8)^(8)^(6)  A. lateralis^24±2^0.9±0.1^3.5±0.4^1.44±0.09 0.40±0.07^24±1^7.30±0.06 (4)^(4)^(4)^(3)^(3)^(4)^(4)  L. armatus^11±1^0.1±0.1^1.3±0.2^1.47±0.13 0.33±0.05^35±2^7.29±0.02 (8)^(8)^(7)^(8)^(8)^(8)^ (8) S. marmoratus^23±1^0.8±0.1^2.8+0.1^0.01±0.01 0.02±0.01^22±1^7.18 (8)^(16)^(15)^(7)^(7)^(7)  B. cirrhosus^29±1^0.7±0.1^2.7±0.1^1.38±0.04 0.18+0.02^N/A^7.42±0.02 (8)^(6)^(14)^(3)^(3)^  (12)  M scorpius^25±2^0.7±0.1^2.7+0.2^0.69±0.13 0.22±0.03^28+1^N/A (8)^(6)^(6)^(6)^(6)^(6)  C. asper^27±2^0.8±0.1^3.0±0.1^1.04±0.17 0.19±0.04^24+1^7.28±0.01 (8)^(15)^(15)^(8)^(8)^(7)^(8)  Data are means ± SE. Hematocrit (Hct) is presented in %, hemoglobin (Hb) in mM, mean cellular hemoglobin content (MCHC) in [Hb]/Hct, [ATP], [GTP] and [Mg 2+ ] are presented relative to [Hb]. pHi represents RBC intracellular pH. Numbers in brackets indicate sample size.  41  Table 2-3. Hemoglobin isoforms from 11 species of sculpins. Total Isoforms  Anodic Isoforms  Cathodic Isoforms  0. maculosus  4  4  0  C. globiceps  9  6  3  0. snyderi  4  3  1  E. bison  9  8  1  A. fenestralis  9  6  3  A. lateralis  10  7  3  L. armatus  5  5  0  S. marmoratus  4  4  0  B. cirrhosus  8  8  0  M scorpius  3  3  0  C. asper  10  9  1  A representative from each species was used to determine the total number of hemoglobin isoforms, as well as the number of cathodic and anodic isoforms.  42  Table 2-4. Hb-02 P50 and Hill coefficient (n) in whole red blood cell, stripped blood, and reconstituted blood in 11 species of sculpins. Whole  Stripped  Reconstituted  P50  P50  0. maculosus  23.3+0.8  C. globiceps  P50  n (whole)  n (stripped)  n (reconstituted)  3.5+0.3  18.9+0.5  1.56+0.03  2.39+0.18  1.66+0.06  31.4+0.4  3.1+0.1  19.2±2.5  1.67+0.05  2.48+0.05  1.64+0.02  0. snyderi  38.7+1.0  3.4+0.1  21.2+1.4  1.60+0.06  2.31+0.06  1.71+0.06  E. bison  38.7+1.5  5.6+0.2  8.9+0.7  1.47+0.06  2.27+0.08  1.62+0.12  A. fenestralis  49.7+1.2  3.7+0.1  27.9+1.0  1.42+0.02  2.40+0.16  1.52+0.01  A. lateralis  45.1+5.1  5.7+0.3  35.5+5.0  1.62+0.03  2.27+0.08  1.48+0.02  L. armatus  35.2+2.2  4.3+0.3  24.4+2.5  1.64+0.03  2.77+0.27  1.67+0.07  S. marmoratus  39.0+0.4  5.3+0.2  7.3+0.4  1.41+0.01  2.13+0.08  2.10+0.15  B. cirrhosus  57.5+2.4  11.7+0.2  56.0+1.3  1.65+0.06  2.33+0.15  1.71+0.04  M scorpius  50.7+1.8  10.7+0.4  40.7+3.8  1.43+0.04  1.98+0.08  1.59+0.04  C. asper  21.4+1.6  5.2+0.3  22.3+2.5  1.90+0.07  2.29+0.08  1.63+0.02  Data are means ± SE. All Hb-02 P50 values are represented in torr. For Hb-02 P50 and Hill coefficient (n) in whole red blood cell n = 6 to 9, and in stripped and reconstituted blood n = 3 to 4.  43  Table 2-5. Relationship between Pcnt and hematological parameters, hemoglobin modulators, and hemoglobin isoforms of sculpins using conventional and phylogenetically independent contrast (PIC) correlations. Conventional  PIC  Parameter  slope  r2  P  slope  r2  P  Hct  -0.53  0.16  0.23  0.31  0.04  0.57  Hb  -13.81  0.24  0.12  3.70  0.01  0.77  MCHC  -4.42  0.09  0.37  4.41  0.07  0.44  ATP/Hb  -5.79  0.21  0.15  -0.47  <0.01  0.92  GTP/Hb  -1.97  0.02  0.70  -0.50  <0.01  0.91  Mg 2±/Hb  -0.76  0.14  0.31  -0.46  0.05  0.55  Total Hb isoforms  0.17  <0.01  0.89  -0.45  0.02  0.66  Anodic Hb isoforms  1.24  0.07  0.43  -0.47  0.02  0.72  Cathodic Hb isoforms  -2.46  0.11  0.33  -1.32  0.02  0.64  44  Table 2-6. Relationship between whole RBC Hb-02 P50 and hemoglobin modulators, hemoglobin isoforms, RBC intracellular pH and Hill coefficients (n) of sculpins using conventional and phylogenetically independent contrast (PIC) correlations. Conventional  PIC  Parameter  slope  r2  P  slope  r2  P  ATP/Hb  -3.58  0.06  0.46  1.43  <0.01  0.83  GTP/Hb  0.02  0.08  0.41  3.80  0.04  0.54  Mg 2+/Hb  -0.34  0.03  0.68  0.21  0.10  0.80  Total Hb isoforms  -0.01  <0.01  0.99  -0.83  0.04  0.54  Anodic Hb isoforms  -0.18  <0.01  0.92  -1.35  0.07  0.43  Cathodic Hb isoforms  0.43  <0.01  0.88  -0.02  <0.01  0.99  RBC pHi  62.14  0.12  0.32  48.05  0.09  0.40  n (whole)  -38.79  0.25  0.11  -0.01  0.17  0.21  n  (stripped)  -16.89  0.09  0.37  -0.01  0.03  0.61  n  (reconstituted)  -7.24  0.01  0.76  -0.01  0.02  0.67  45  REFERENCES Altieri, A.H. 2006. Inducible variation in hypoxia tolerance across the intertidal-subtidal distribution of the blue mussel Mytilus edulis. Mar. Ecol. Prog. 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Jensen, F.B. 1991. Multiple strategies in oxygen and carbon dioxide transport by hemoglobin. In Physiological strategies for gas exchange and metabolism. (eds. A.J. Woakes, M.K. Grieshaber and C.R. Bridges) pp. 55-78. Cambridge: Cambridge University Press. Jensen, F.B. and Weber, R.E. 1982. Respiratory properties of tench blood and hemoglobin: adaptationto hypoxic-hypercapnic water. Mol. Physiol. 2: 235-250. Jessen, T.H., Weber, R.E., Fermi, G., Tame, J. and Braunitzer, G. 1991. Adaptation of bird hemoglobins to high altitudes: demonstration of molecular mechanism by protein engineering. Proc. Natl. Acad. Sci. USA. 88: 6519-6522. Karnovsky, M.J. 1965. A formaldehyde-glutaraldehyde fixative for high osmolarity for use in electron microscopy. J. Cell. Biol. 27: 137A. Kinziger, A.P. and Wood, R.M. 2003. Molecular systematics of the polytypic species Cottus hypselurus (Teleostei: Cottidae). Copeia. 3: 624-627. Kumar S., Tamura K., and Nei M. 2004. 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Coral Reefs. 26: 241-248. Perutz, M.F. 1983. Species adaptation in a protein molecule. Mol. Biol. Evol. 1: 1-28. POrtner, H.O, Boutlier, R.G, Tang Y. and Toews D. P. 1991. The use of fluoride and nitriloacetic acid in tissue acid-base physiology. II. Intracellular pH. Respir. Physiol. 81: 255-275. POrtner, H.O. and Grieshaber, M.K. 1993. Critical P0 2 (s) in oxyconforming and oxyregulating animals: gas exchange, metabolic rate and the mode of energy production. Boca Raton, CRC Press. Posada, D. and Crandall, K.A. 1998. Modeltest: testing the model of DNA substitution. Bioinform. 14: 817-818. Powers, D.A. 1980. Molecular ecology of teleost fish hemoglobins: strategies for adapting to changing environments. Amer. Zool. 20: 139-162. Reeves, R.B. 1980. A rapid micro method for obtaining oxygen equilibrium curves on whole blood. Respir. Physiol. 42: 299-315. Ronquist, F. and J. P. Huelsenbeck. 2003. MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinform. 19: 1572-1574. Rutjes, H.A., Nieveen, M.C., Weber, R.E., Witte, F. and Van den Thillart, G.E.E.J.M. 2007. Multiple strategies of Lake Victoria cichlids to cope with life long hypoxia include hemoglobin switching. Am. J. Physiol. 293: R1376-1383. Saint-Paul, U. 1984. Physiological adaptation to hypoxia of a neotropical characoid fish Colossoma macropomum, Serrasalmidae. Environ. Biol. Fishes. 11: 53-62. Schmidt, T.R. and Gold, J.R. 1993. Complete sequence of the mitochondrial Cytochrome b gene in the Cherryfin Shiner, Lythrurus roseipinnis (Teleostei: Cyprinidae). Copeia. 3: 880-883. Stillman, J. H. and Somero, G. N. 1996. Adaptation to temperature stress and aerial exposure in congeneric species of intertidal porcelain crabs (genus Petrolisthes): correlation of physiology, biochemistry and morphology with vertical distribution. J. Exp. Biol. 199: 1845-1855. Tan, A.L. and Noble, R.W. 1973. Conditions restricting allosteric transitions in carp hemoglobin. J. Biol. Chem. 218: 2880-2888. Timmerman, C.M. and Chapman, L.J. 2004a. Behavioural and physiological compensation for chronic hypoxia in the Sailfin Molly (Peocilia latipinna). Physiol. Biochem. Zool. 77: 601-610. Timmerman, C.M. and Chapman, L.J. 2004b. Hypoxia and interdemic variation in Poecilia latipinna. J. Fish. Biol. 65: 635-650.  48  Truchot, J.P. and Duhamel-Jouve, A. 1980. Oxygen and carbon dioxide in the marine intertidal environment: diurnal and tidal changes in rockpools. Respir. Physiol. 39: 241-254. Weber. R.E. and Lykkeboe, G. 1978. Respiratory adaptations in carp blood: influences of hypoxia, red cell organic phosphates, divalent cations and CO2 on hemoglobin-oxygen affinity. J. Comp. Physiol. 128: 127-137. Wells, R.M.G. 1999. Haemoglobin function in aquatic animals: molecular adaptations to environmental challenge. Mar. Freshwater. Res. 50: 933-939. Wells, R.M.G., Baldwin, J., Seymour, R.S. and Weber, R.E. 1997. Blood oxygen transport and hemoglobin function in three tropical fish species from northern Australian freshwater billabongs. Fish. Physiol. Biochem. 16: 247-258. Wells, R.M.G., Grigg, G.C., Beard, L.A. and Summers, G. 1989. Hypoxic responses in a fish from a stable environment: blood oxygen transport in the Antarctic fish Pagothenia Borchgrevinki. J. Exp. Biol. 141: 97-111. Wolf, K. 1963. Physiological salines for freshwater teleosts. Prog. Fish. Cultur. 25: 135-144. Yeager, D.P. and Ultsch, G.R. 1989. Physiological regulation and conformation: a BASIC program for the determination of critical points. Physiol. Zool. 62: 888-907.  49  CHAPTER THREE: BEHAVIORAL, PHYSIOLOGICAL AND BIOCHEMICAL STRATEGIES IN RESPONSE TO HYPDXIA EXPOSURE IN FAMILY COTTIDAE INTRODUCTION  Fish that experience hypoxia require a well-coordinated suite of responses to ensure survival. Upon exposure to hypoxia, fish may initially employ behavioral strategies of avoidance, followed by physiological and biochemical strategies to either enhance 02 uptake from the environment or initiate responses to reduce tissue and cellular reliance on 02. The common behavioral avoidance strategies utilized by fish include aquatic surface respiration (ASR) and aerial emergence (Martin, 1995; Watters and Cech, 2003; Yoshiyama et al., 1995), both of which allow an animal to access more 0 2 rich environments. Aquatic surface respiration involves a fish selectively accessing the better oxygenated water-air interface, while voluntarily emergence involves moving out of the water to respire in air. Although there is some discrepancy between studies, fish that actively emerge are generally able to maintain 0 2 consumption rates in air that are similar, or reduced only by 25%, compared to those measured in water (Martin, 1996; Sloman et al., 2008; Wright and Raymond, 1978; Yoshiyama and Cech, 1994). In fact, tidepool sculpins forcibly emerged on moist substrate for 72 hours showed no measurable increase in whole body lactate levels suggesting that the fish are able to extract sufficient 0 2 to maintain a completely aerobic metabolism under the relatively inactive conditions of aerial emergence (Sloman et al., 2008). Although there appears to be a limited physiological cost to aerial emergence, emergence behavior does involve an increased risk of aerial predation (Sloman et al., 2006; Yoshiyama et al., 1995). In response to perceived predation, fish will delay the performance of these behaviors (Sloman et al., 2008), and thus  A version of this chapter will be submitted for publication. Mandic, M. Sloman, K.A. and Richards, J.G. Behavioral, Physiological, and Biochemical Adjustments in Response to Hypoxia Exposure in Family Cottidae.  50  must invoke physiological or biochemical adjustments to survive periods of environmental hypoxia. The ability to enhance 02 uptake during confinement in hypoxia is primarily mediated by modifications to Hb-02 binding affinity. Reductions in RBC Hb modulators, such as ATP and GTP, occur in many fish species during hypoxia exposure, causing an increase in Hb-02 binding affinity leading to increased 02 loading from the hypoxic environment (Jensen and Weber, 1982; Lykkeboe and Johansen, 1975; Weber and Lykkeboe, 1978). Tetens and Lykkeboe (1981) demonstrated a tight correlation between a stepwise increase in Hb-02 binding affinity and a decrease in RBC [ATP] in trout (Oncorhynchus mykiss). Decreases in RBC [ATP] and [GTP] directly affect Hb-02 binding affinity through a reduction in the allosteric binding of the modulators to the cavity between the 13 chains of the Hb molecule, and indirectly through an alteration of Donnan distribution of H + across the cell membrane resulting in an elevated RBC pH (Hochachka and Somero, 2002; Jensen et al., 1998). If modulation of Hb-02 binding affinity is not sufficient to maintain adequate 02 supply to tissues to meet metabolic requirements, an up-regulation of 0 2 independent pathways for ATP production is one important component in prolonging survival during hypoxia. As 0 2 becomes limiting, there is a switch in energy provision from one based on oxidative phosphorylation to one based primarily on substrate level phosphorylation (i.e. glycolysis and CrP hydrolysis; Hochachka et al., 1996). Another important defense mechanism against hypoxia is a downregulation in cellular energy demand, primarily achieved through a significant decrease in major energy consuming processes such as protein synthesis and ion pumping (Buck et al., 1993a; Buck et al., 1993b, Lewis et al., 2007). Decreasing energy demand while attempting to maximize 0 2 independent energy production are critical biochemical adjustments essential to hypoxia survival when processes involved in maximizing 02 uptake fail.  51  Comparison among species of sculpins from the family Cottidae is an ideal way to understand the adaptive traits involved in hypoxia tolerance. The distribution of sculpins along the marine nearshore environment is associated with critical 02 tension  (Pcnt),  such that the  species with lowest Pent are found in the highly 0 2 variable tidepools and the species with higher Pcnt inhabit  the 02 stable subtidal zone (see Chapter 2). Previous work with sculpins has focused  on elucidating the physiological characteristics of fish under normoxic conditions that might be of adaptive value for hypoxia survival, such as gill surface area and Hb-02 binding affinity (see Chapter 2). The present study focused on the behavioral, physiological and biochemical defenses of different species of sculpins to hypoxia exposure. Specifically, I examined the relationship between Pcnt and behavioral responses, such as ASR and aerial emergence, in 12 species of sculpins. Hepatic glycogen and CrP, were also assessed in different sculpins to determine if there is a relationship between capacity to sustain substrate-level phosphorylation and  Pcnt• Three  species of sculpins, which were chosen because of their low, medium and high P en t values, were exposed to severe hypoxia and the concentrations of Hb modulators and glycolytic metabolites were analyzed. MATERIAL AND METHODS EXPERIMENTAL ANIMALS  Marine and freshwater sculpins were obtained near Bamfield Marine Sciences Centre (BMSC), Bamfield, British Columbia, Canada and housed at University of British Columbia as described in Chapter 2. Briefly, marine sculpins were caught using handheld nets or seines during the lowest tidal cycles of June and August 2006 at Wizard Islet (48°51.5'N; 125°9.4'W) and Ross Islets (48°52.4'N; 125°9.7'W), while freshwater sculpins, Cottus asper, were caught using baited minnow traps in Pachena Lake (48°50'11" N; 125°01'44" W). Marine sculpins obtained are representatives inhabiting various areas of the marine nearshore environment and 52  the specific maximum depth for each species is listed in Chapter 2 Figure 2.1. Briefly, Oligocottus maculosus, Oligocottus snyderi, and Clinocottus globiceps are found in the high to low tidepool areas, while the remainder of the species are found in the subtidal or deeper water, including Enophrys bison, Artedius fenestralis, Artedius lateralis, Artedius harringtoni, Scorpaenichthys marmoratus, Leptocottus armatus, and Blepsias cirrhosus (Eschmeyer and Herald, 1983; Froese and Pauly, 2007). An additional deep-water marine sculpin species, Myoxocephalus scorpius, was brought in from the Atlantic coast (Memorial University; Newfoundland). Three separate experiments were conducted. In experimental series 1, ASR and aerial emergence behaviors of 12 species of sculpins were assessed in response to declining 02. In experimental series 2, the concentration of available on-board fuels in the liver were determined for all 12 species of sculpins. In experimental series 3, Hb modulators and liver metabolites were measured in three species of sculpins that differ in their P ent , 0. maculosus, A. lateralis and B. cirrhosus, when subjected to severe hypoxia at 02 tensions equivalent to 40% of respective  Pcnt  levels for up to 6 hours. This relative level of hypoxia was expected to elicit a measurable hypoxic response that could potentially be sustained by each species for number of hours. EXPERIMENTAL PROTOCOLS  Series 1. Behavior The threshold 02 tension at which individual sculpin displayed ASR and aerial emergence behavior was determined using published protocols (Sloman et al., 2006; Watters and Cech, 2003; Yoshiyama et al., 1995). Briefly, individual fish were weighed and placed into a 60 liter aquarium which was maintained at 12°C through partial submergence in a temperature regulated wet table. The aquarium was subdivided into 4 zones and a variation in water depth across the zones was achieved with a 30° angled ramp. Zone 1 included a strip of the entire water column with decreasing water depth in subsequent zones until zone 4 represented an area where 53  the ramp emerged from the water. Small stones covered the ramp to provide a more natural substrate for the fish and three sides of the aquarium were covered with black plastic to minimize disturbance during the behavior trials. Fish were allowed to acclimate to the aquarium for one hour before experimentation. During the acclimation period, air was bubbled into the water through an air stone situated along the bottom of zone 1. At the start of the behavioral trial, air was switched to nitrogen (N2) and water 0 2 concentrations were decreased to near zero over a 1 hour period. Water 0 2 concentrations were monitored using an Oxyguard Handy MKIII 0 2 probe to ensure a consistent rate of 02 decrease between trials. The 02 probe was placed in different sections of the aquarium during a control trial and there was no noticeable difference in 02 levels between the zones. Every two minutes during a trial, behavior, 02 concentration, and the zone in which the fish was located were recorded. The behavioral responses under observation were ASR and aerial emergence. The 02 tension at which fish performed ASR was defined as when fish were seen ventilating at the surface of the water, and the 02 tension at which aerial emergence occurred was defined as when the head and gill operculae were completely emerged and exposed to the air. The experiment was terminated and 0 2 concentration was noted when fish either emerged or lost equilibrium. Series 2. Metabolic Fuel  To determine the quantity of on-board metabolic fuel in 12 species of sculpins, I examined liver from the same fish that were terminally sampled for blood and various tissues in Chapter 2. Details regarding experimental set-up and results on various measurements on blood and gills have previously been reported in Chapter 2. In the present study, liver samples were analyzed for [ATP], [CrP], [glycogen] and [glucose].  54  Series 3. Relative Hypoxia Exposure  For this study, we chose three species of sculpins that varied in their Pent and were found on separate clades of the sculpin phylogenetic tree (Chapter 2; Figure 2.1). 0. maculosus had the lowest Pent at 25.9 ton, A. lateralis possessed a P ent at 35.7 torr and B. cirrhosus had the highest P ent at 44.4 torn (Chapter 2). The three species of sculpins were selected to determine if the biochemical response caused by hypoxia at a level equal to 40% of a species' respective Pent varied between the species. Individual fish were placed in well-aerated 5 liter plastic chambers with mesh sides and a 1 liter basin in the bottom and were submerged in 69 liter glass aquaria. For each species, fish were equally divided in two separate aquaria and temperature was maintained at 12°C. Fish were allowed a 24 hour recovery period from handling during which time air was bubbled into all aquaria to maintain 02 levels at approximately air-saturated water. At the onset of the experimental trial, air was switched to  N2 and  declined to levels corresponding to 40% of  within half an hour 0 2 levels in all aquaria  Pent  values for each species (0. maculosus = 10.4  ton; A. lateralis = 14.9 ton; B. cirrhosus = 17.6 ton). 02 levels in the aquaria were monitored with an Oxyguard Handy MKIII 02 probe and maintained at this level for up to 6 hours. For all three species of sculpins, fish were meant to be terminally sampled at normoxia, 2, and 6 hours of hypoxia. However, B. cirrhosus did not survive past 2 hours of hypoxia and a separate trial was conducted with this species to obtain samples from normoxia and 1 and 2 hours of hypoxia. There was no significant difference in samples from normoxia and 2 hours of hypoxia between the two trials for B. cirrhosus (t-test), therefore all samples have been combined. To sample a fish, individual chambers were removed from the aquaria and an overdose of benzocaine (250 mg/L, Sigma-Aldrich) was added to the water remaining in the basin. Fish lost equilibrium within a minute, at which point it was removed from the water, patted dry and weighed (0. maculosus = 5.3±0.3 g; A. lateralis = 8.4±0.5 g; B. cirrhosus = 55  8.0+0.6 g). Blood was sampled via caudal severance using a heparinized Hct tube and placed on ice. Samples of liver, muscle, heart and brain were dissected and immediately frozen in liquid N2 and stored at -80°C until further analysis. Hematocrit tubes containing blood were centrifuged at 13,700 g for 3 minutes at which point Hct was calculated and packed RBC were separated from the plasma and both were frozen in liquid N2 and stored at -80°C. ANALYTICAL PROCEDURES  Liver Metabolites Frozen liver samples from both Series 2 and Series 3 experiments were weighed and immediately sonicated on ice in 500 piL of 8% perchloric acid for 5 seconds using a Kontes Micro Ultrasonic Cell Disrupter (Kontes, Vineland, New Jersey). An aliquot of the homogenate was taken for later glycogen digestion and frozen at -80°C. The remaining homogenate was centrifuged at 20,000 g for 5 minutes at 4°C, and the supernatant neutralized with 3M K2CO3. Neutralized extracts were centrifuged to remove the precipitates and frozen at -80°C until later analysis. Liver samples from Series 2 were assayed for ATP, CrP, glycogen and glucose and samples from Series 3 were assayed for ATP, CrP, glycogen, glucose and lactate. Neutralized extracts were assayed spectrophotometrically for ATP, CrP, glucose and lactate according to protocols outlined in Bergmeyer (1983). The homogenate set aside for glycogen analysis was thawed, digested to glucose using amyloglucosidase and then assayed for glucose. Glycogen was expressed as limo' glycosyl units/g wet weight. Red Blood Cell Hemoglobin Modulators Red blood cell [ATP] and [GTP] were determined using high performance liquid chromatography (HPLC; Gilson 322) according to protocols outlined in Feuerlein and Weber (1994) with modifications presented in Chapter 2. Magnesium concentrations were determined on RBC hemolysates using flame atomic absorption spectrometry (SpectrAA 240FS, Varian, Australia). 56  STATISTICAL ANALYSIS  Phylogenetically independent contrast correlations were analyzed in the PDAP module (Midford et al., 2003) in Mesquite (Maddison and Maddison, 2004) using the maximum likelihood tree created in Chapter 2 (Figure 2.1). Conventional correlations (non-PIC) were analyzed in SigmaStat 3.0. Results from Series 3 were analyzed with a one-way ANOVA (SigmaStat 3.0) with time as the independent variable followed by the Holm-Sidak pos-hoc test. A two-way ANOVA could not be performed due to differing time points during hypoxia between the three species of sculpins. Alpha was set at 0.05 for all statistical tests. RESULTS SERIES 1. BEHAVIOR  Aquatic surface respiration was performed by 100% of the individuals of the three tidepool species, 0. maculosus, 0. snyderi and C. globiceps (Table 3.1). The remaining eight species of sculpins displayed ASR behavior at lower frequency, ranging from 0% of the individuals of S. marmoratus and B. cirrhosus to 88% of the individuals of C. asper. The 02 tension at which ASR first occurred varied between species of sculpins, ranging from 23.3 torr in C. globiceps to 7.3 ton in A. fenestralis. Aerial emergence was consistently performed by the three tidepool species, 0. maculosus, 0. snyderi and C. globiceps, as well as L. armatus and C. asper and only a small percent of the individuals of E. bison and M scorpius (Table 3.1). The remaining species of sculpins lost equilibrium without displaying aerial emergence (data not shown). Of the species which performed aerial emergence, the 02 tension threshold for aerial emergence did not vary between the species and was around 10 torn. No correlation existed between percent of individuals of a species performing either ASR or aerial emergence and  Pent  (Table 3.2; conventional and PIC correlations). In addition, there was no correlation (conventional or PIC) between the percent of individuals of a species performing these behaviors and the species' maximum depth (a proxy measure for habitat distribution; Table 3.3). 57  SERIES 2. METABOLIC FUEL  There was a high degree of variation in liver [glycogen], [CrP], [glucose] and [ATP] among the normoxia acclimated sculpins (Table 3.4), but there was no correlation between  Pcrit  and liver metabolites (Table 3.5; conventional and PIC). SERIES 3. RELATIVE HYPDXIA EXPOSURE  There was differential survival among the three species of sculpins upon exposure to 40% of their respective  P cn t.  0. maculosus survived the full 6 hour hypoxia exposure with no  visible signs of distress, while 2 out of the 6 individuals of A. lateralis died between the 2 and 6 hour exposure. One hundred percent mortality was observed in B. cirrhosus shortly after the 2 hour exposure to hypoxia (data not shown). Red blood cell [ATP] did not decrease significantly in 0. maculosus during 6 hours of hypoxia (Fig. 3.1A). However, within 2 hour exposure to hypoxia, RBC [ATP] significantly decreased by 41% in A. lateralis and 66% in B. cirrhosus (Fig. 3.1A). Red blood cell GTP significantly declined within 2 hour exposure to hypoxia in 0. maculosus and A. lateralis and within 1 hour hypoxia exposure in B. cirrhosus (Fig. 3.1 B). The relative magnitude of decrease in RBC [GTP] was similar in all three species and ranged between 69% to 74%. There was no significant change in RBC Mg2+ during hypoxia exposure in 0. maculosus, A. lateralis and B. cirrhosus (Table 3.6). Hematocrit levels did not significantly change during hypoxia exposure in 0. maculosus and B. cirrhosus (Table 3.6), but in A. lateralis, Hct levels significantly increased within 6 hours of hypoxia exposure. Upon hypoxia exposure, liver ATP decreased significantly within 2 hours in 0. maculosus and A. lateralis (Fig. 3.2A). From 2 to 6 hours, ATP remained constant in A. lateralis, but significantly increased in 0. maculosus. In B. cirrhosus, ATP significantly declined during the first hour of hypoxia but remained steady for the duration of the exposure (Fig. 3.2A). In all three species, there was a consistent 80 to 87% decrease in liver CrP during the first 2 hours of 58  hypoxia, but in 0. maculosus and A. lateralis (Fig. 3.2B) the levels remained constant between 2 and 6 hr hypoxia exposure. Liver CrP decreased significantly within the first hour of hypoxia exposure in B. cirrhosus with no further decline in CrP (Fig. 3.2B). In normoxic acclimated sculpins there was large variation in liver [glycogen] among the three species of sculpins, with 0. maculosus possessing the highest and B. cirrhosus possessing the lowest amount of hepatic glycogen (Fig. 3.3). There was a significant decrease in liver [glycogen] in 0. maculosus by 6 hours of hypoxia exposure. In B. cirrhosus, however, a significant drop in [glycogen] was observed within the first hour of hypoxia and by 2 hours there was a 75% decline in glycogen from normoxic levels. There was no significant change in liver [glycogen] in A. lateralis. Liver [glucose] remained constant throughout hypoxia in 0. maculosus and B. cirrhosus (Fig. 3.4). In A. lateralis, there was no significant change in [glucose] between normoxic and hypoxic fish. Liver [lactate] significantly increased during hypoxia exposure in 0. maculosus and B. cirrhosus (Fig. 3.5). Within 2 hours of hypoxia exposure, there was a 32% increase in lactate in 0. maculosus and a 126% increase in B. cirrhosus. There was an apparent increase in [lactate] in A. lateralis during hypoxia, although this trend was not significant. DISCUSSION Surviving periods of hypoxia involves employing a complex suite of responses to either extend an animal's ability to remain active or to defend against consequences of limited cellular 02. Sustaining routine M O2 in face of hypoxia can be achieved through behavioral responses such as ASR and aerial emergence or physiological adjustments such as enhancement in 02 extraction. However, if 02 levels drop below an animal's Pcnt, a depression in metabolic rate and an increase in substrate level phosphorylation are utilized to prevent a catastrophic energy loss leading to necrotic cell death. Many authors (Jensen, 1991; Martin, 1995; Saint-Paul, 1984;  59  Wood, 1980) have argued adaptive value of behavioral, physiological, and biochemical traits for hypoxia survival, but have done so only in a qualitative fashion that lacks a thorough statistical analysis. The current study, as well as Chapter 2, are the first to employ a phylogenetically independent analysis of carefully selected species to ascertain the adaptive value of characteristics that have been long thought of as critical to an animal's ability to survive hypoxia. A small number of studies have suggested that differences in ASR and aerial emergence behaviors in sculpins are adaptive and result from different selection pressures created by the differential distribution of the species of sculpins along the intertidal zone (Martin, 1996; Watters and Cech, 2003). The results of the current study support these assumptions to the extent that the tidepool species consistently exhibit these behaviors while the subtidal and deeper water species do not. However, PIC analysis showed no correlation between the presence of these behaviors and Pcnt, nor between the presence of the behaviors and the distribution of the species of sculpins along the vertical tidal zone (Table 3.2 and 3.3). Therefore, there is no conclusive support for ASR or aerial emergence behaviors being adaptive to hypoxia survival. This may, in part, be due to the limited number of species that consistently display these behaviors thus affecting our ability to detect significance. Despite the lack of a relationship between P„i t and the behavioral responses of sculpins to hypoxia, within the tidepool species examined, 0. maculosus, C. globiceps and 0. snyderi, there is interesting variation in the pattern of behavioral avoidance that is worthy of discussion. Although all the tidepool species possess similar Pcnt values (0. maculosus = 25.9 torr; C. globiceps = 26.8 ton; 0. snyderi = 27.1 torr; Chapter 2) there are differences in the 02 threshold at which ASR and emergence behaviors are displayed. Despite low intraspecific variation, there is variation among the tidepool species in the 02 tensions at which they display avoidance behaviors, suggesting that there are negative consequences associated with employing these avoidance responses. For example, 0. maculosus are typically found high in the intertidal zone, 60  inhabiting barren tidepools that lack vegetation as protective covering. As a result, 0. maculosus may be more susceptible to aerial predation, and therefore may elect to delay the onset of ASR and aerial emergence (Yoshiyama et al., 1995). Aerial predation has been thought to be a factor in delaying ASR and aerial emergence in other fish, such as Mugil cephalus and juvenile Astronotus ocellatus (Shingles et al., 2005; Sloman et al., 2006). In addition to aerial predation, the variation in 02 thresholds for the avoidance behaviors may also be due to the differences in the efficiency of 02 extraction in the three species of sculpins. In 0. snyderi there is very little difference between ASR and emergence 02 thresholds compared with the other two species, suggesting that in 0. snyderi ASR may not be as efficient for maximizing 02 uptake. This could be due to differences in head morphology and will be investigated as a potential explanation for the variation in the behavioral responses of the three tidepool species. Sculpins inhabiting the 02 stable subtidal and deeper water environments exhibited high variation in the number of individuals performing ASR and aerial emergence during exposure to hypoxia. Although a few subtidal species, S. marmoratus and B. cirrhosus, were never seen to ASR or aerial emerge, other species exhibited variation in this behavior, such that only a percentage of the population performed these behaviors in response to hypoxia (Table 3.1). Martin (1996) demonstrated high mortality rates in three species of subtidal and deeper water sculpins, Icelinus borealis, Jordania zonope and Chitonotus pugentensis when emerged for less than 2 hours, suggesting that subtidal and deeper water sculpins cannot aerially respire as efficiently as the tidepool species, which can survive out of water for at least 72 hours without any measurable deleterious effects (Martin, 1993; Martin, 1996; Sloman et al., 2008). It will be the focus of a future investigation to understand this differential capacity in aerial respiration by comparing species to determine what features, such as gill structure, may differ and therefore be essential for effective aerial emergence.  61  There were differences in hypoxia survival in three species of sculpins, 0. maculosus, A. lateralis, and B. cirrhosus, exposed to the same relative levels of severe hypoxia. This suggests  that despite experiencing hypoxia at levels equivalent to 40% of their respective P er i l s, the three species of sculpins differ in their physiological and biochemical defense mechanisms for coping with severe 02 deficit. Among the three species examined there was a relationship between a species' Pcnt and their ability to survive hypoxia for a prolonged period of time, such that no mortality occurred in the species with the lowest P cri t , 0. maculosus, while 100% mortality occurred in the species with the highest P ent , B. cirrhosus, shortly after the initial two hours of hypoxia exposure. Since the sculpins were subjected to hypoxia based on the same percent of Pcnt, the  differences in mortality rates reveal dramatically varied defensive capabilities to  hypoxia when 02 levels drop below Pcnt levels. One mechanism for combating hypoxia and maximizing 02 uptake is the modulation of blood 02 carrying capacity, through an increase in Hct (Tetens and Lykkeboe, 1981; Brauner and Wang, 1997) or an increase in Hb-0 2 binding affinity via decreases in Hb allosteric modulators (Jensen and Weber, 1982; Lykkeboe and Johansen, 1975; Weber and Lykkeboe, 1978). Despite previous work showing that an increase in Hct is beneficial in increasing blood 02 carrying capacity (Brauner and Wang, 1997), an increase in Hct does not appear to be the primary means of modulation of blood 0 2 carrying capacity in sculpins, since levels remained constant in 0. maculosus and B. cirrhosus, and were only seen to significantly increase in A. lateralis by 6  hours of hypoxia exposure (Table 3.6). However, all three species of sculpins exhibited a decline in RBC [ATP] and [GTP] (Fig. 3.1), which should bring about an increase in Hb-02 binding affinity and enhance 0 2 uptake at low environmental 0 2 . This is not surprising since a decrease in Hb modulators has been noted in many fish species, including Antarctic fish, such as Pagothenia borchgrevinki, which inhabit an 0 2 stable environment, indicating that a hypoxia  induced decrease in Hb modulators is a highly conserved trait amongst fishes (Wells et al., 62  1989). However, there was interspecific variation in RBC [ATP] decline at 2 hours of hypoxia exposure, with a significant 41% and 66% decrease in A. lateralis and B. cirrhosus respectively and a non-significant decrease in 0. maculosus. There appears to be a relationship between the degree of RBC [ATP] decrease and P ent among the three species, such that the species with highest P ent , B. cirrhosus, exhibited the highest decrease in RBC [ATP] and the species with lowest P ent , 0. maculosus, exhibited the lowest decrease in RBC [ATP]. An animal's ability to survive hypoxia is not only dependent upon maximizing 02 uptake but also on the metabolic organization of the tissues (Hochachka and Somero, 2002). An important aspect of this metabolic organization is the ability to maintain metabolic ATP production during hypoxia exposure through an up-regulation of substrate level phosphorylation while limiting tissue energy demands via metabolic rate suppression (Boutilier, 2001; Hochachka et al., 1996; Hochachka and Somero, 2002). As a result, it has been postulated that there should be a relationship between substrate availability, in particular liver glycogen, and an animal's hypoxia tolerance. A survey of different fish species supports this contention with more hypoxia tolerant fish such as Carassius carassius and Carassius auratus possessing higher liver glycogen levels than more hypoxia intolerant fish such as Oncorhynchus mykiss and Gadus morhua (van den Thillart and van Raaij, 1995). Although there are large variations in liver glycogen, glucose and CrP and some variation in liver ATP between the 12 species of sculpins acclimated to normoxia in the present study (Table 3.4), there is no phylogenetically independent correlation between these liver substrates and P ent . This suggests that the amount of on-board fuels may not play a dominant role in dictating P ent in sculpins. However, P ent may not be the relevant measure in this case as it is a surrogate measure of whole animal 02 extraction and not necessarily reflective of the biochemical ability to alter energy use and provision at the tissue. Therefore, the focus of future work will be to employ another measure of hypoxia tolerance,  63  such as the LT50, in order to determine the relationship between substrate availability and hypoxia tolerance in sculpins. When liver ATP turnover, calculated from ATP utilization, CrP breakdown and lactate accumulation, is compared between 0. maculosus, A. lateralis and B. cirrhosus exposed to two hours at 40% of their respective Pent, there was little difference among species, with ATP turnover rates of 1.0 iimol/g/hr, 0.9 jimol/g/hr and 1.5 gmol/g/hr for 0. maculosus, A. lateralis and B. cirrhosus respectively. Overall, independent of the differences in P„, t , the rate of ATP turnover was roughly constant between the three species exposed to hypoxia based on the same percent of Pent. However, despite the lack of variation in liver ATP turnover, there was a considerable difference in mortality rates between the three species of sculpins. This suggests that differential defensive mechanisms do exist between the species, with the increased survivorship in sculpins that possess a low  Pcrit  being a result of higher liver [glycogen] during  hypoxia. Although there appeared to be a relationship between mortality rates and on-board levels of liver glycogen among these three species (Fig. 3.3), the relationship did not exist between liver [glycogen] and P e ri t when examined broadly across 12 species of sculpins. This suggests that the link between liver [glycogen] and P ent is not a generality but a phenomenon specific to the three species chosen for this experiment. Liver glycogen mobilization appears to be an important defensive strategy against severe hypoxia in 0. maculosus and B. cirrhosus. In both species, liver [glycogen] decreased dramatically over the duration of hypoxia exposure, although in B. cirrhosus the precipitous drop in glycogen occurred much sooner, at two hours of exposure to hypoxia, than in 0. maculosus where glycogen did not significantly decrease until six hours of hypoxia exposure. In A. lateralis, a sculpin species with a mid-range  Pent, however,  there was no significant change in  liver [glycogen]. This may signify that A. lateralis relies primarily on the endogenous activation of glycogen breakdown in other tissues as the fuel source for ATP production during severe 64  hypoxia. On the other hand, it is difficult to draw concrete conclusions regarding liver glycogen in A. lateralis since the large variation may mask any potential mobilization of liver glycogen. This variability has been shown in other species, such as Oncorhynchus mykiss (Dunn and Hochachka, 1986), Fundulus heteroclitus (Fangue et al., In Press), Rana temporaria (Smith, 1950), and in both A. lateralis and B. cirrhosus sampled under normoxia on two separate occasions (cf. Fig. 3.3 and Table 3.4). Since glycogen content appears to be variable between individuals of the same species, it is not surprising that there is no definitive association between liver glycogen and P cnt in sculpins. Liver lactate significantly accumulated in 0. maculosus and B. cirrhosus, with an indication of a similar trend in A. lateralis in response to hypoxia (Fig. 3.5). However, the absolute increase of lactate was negligible in all three species over the entire hypoxia exposure. Despite the large liver glycogen depletions in 0. maculosus and B. cirrhosus, [glucose] remained constant in the liver (Fig. 3.4) and [lactate] did not appreciably change, suggesting that the breakdown of liver glycogen into glucose was primarily destined for utilization by other tissues during hypoxia. We quantified the behavioral response of 12 species of sculpins that range in their  Pcrit  values and have determined that, contrary to the literature, no relationship exists between the location of a species along the marine nearshore environment or  P cn t to  the performance of ASR  and aerial emergence. However, since only a limited number of species exhibited these behaviors, this had an impact on our ability to detect significance and is worthy of further investigation. Exposing 0. maculosus, A. lateralis, and B. cirrhosus, to the same relative level of hypoxia revealed dramatically different mortality rates despite a similar ATP turnover rate in the initial two hours of hypoxia. Comparison of on-board liver [glycogen] among the three species would suggest that a species' ability to survive prolonged period of time in severe hypoxia is due to a higher liver [glycogen]. However, there is no correlation between liver [glycogen] at 65  normoxia and Pcnt when examining broadly across 12 species of sculpins.  66  1.6 -  A  1.4 1.2 1.0 -  .0  a: 0.8 1itt 0.6 -  a ....................................  a  0.4 0.2  N  7  ^0.0  ^  0.25 -  B  0.20 -  .0 0.15 I  0  C.9 0.10 -  as  0.05 • •. b C  00.  b  0.00  N^1^2^  6  Time in hypoxia (h)  Figure 3-1. Red blood cell ATP (A) and GTP (B) in 0. maculosus (circle, dotted line), A. lateralis (triangle, solid line) and B. cirrhosus (square, dashed line) exposed to normoxia and hypoxia equivalent to 40% of respective P ent (0. maculosus = 10.4 ton; A. lateralis = 14.9 ton; B. cirrhosus = 17.6 ton). For ATP and GTP n = 4 to 10, except for 0. maculosus at 6h hypoxia where n = 2. Data was lost for A. lateralis at 6h hypoxia. Overlapping points are offset. Different letters indicate a significant effect of time within a species. Data are means ± SE.  67  ▪ ▪ 1.2 —  A  1.0 z u  )  0.8 -  6 .  a) 0.6 o  E  • 0  0.4 0.2 b 0.0 3.0 —  B  a 2.5 -  71i Co • 2.0 7.  a1  4  0) 1.5 0  E  -  9:  0.5 -  b  0.0 N  ....................  1^2  ^  6  Time in hypoxia (h)  Figure 3-2. Liver ATP (A) and CrP (B) in 0. maculosus (circle, dotted line), A. lateralis (triangle, solid line) and B. cirrhosus (square, dashed line) exposed to normoxia and hypoxia equivalent to 40% of respective P cr jt (0. maculosus = 10.4 ton; A. lateralis = 14.9 ton; B. cirrhosus = 17.6 ton). For ATP and CrP n = 6 to 10, except for A. lateralis at 6h hypoxia where n = 4. Overlapping points are offset. Different letters indicate a significant effect of time within a species. Data are means ± SE.  68  •  300 =1) 250 to 15 200 o  E  150 -  w wo -  a) 0 U >.  50 0 N  1  2  6  Time in hypoxia (h)  Figure 3-3. Liver glycogen in 0. maculosus (circle, dotted line), A. lateralis (triangle, solid line) and B. cirrhosus (square, dashed line) exposed to normoxia and hypoxia equivalent to 40% of respective P cr jt (O. maculosus = 10.4 torr; A. lateralis = 14.9 torr; B. cirrhosus = 17.6 ton). For glycogen n = 6 to 10, except for A. lateralis at 6h hypoxia where n = 4. Overlapping points are offset. Different letters indicate a significant effect of time within a species. Data are means ± SE.  69  6— Tu 5 —  =  -  747.  t; 4-  3  cs)  o  E  3-  cl.) 2— ci)  0  c.)  a 0 N  1^2  ^  6  Time in hypoxia (h)  Figure 3-4. Liver glucose in 0. maculosus (circle, dotted line), A. lateralis (triangle, solid line) and B. cirrhosus (square, dashed line) exposed to normoxia and hypoxia equivalent to 40% of respective P„, t (0. maculosus = 10.4 ton; A. lateralis = 14.9 torn; B. cirrhosus = 17.6 ton). For glucose n = 6 to 10, except for A. lateralis at 6h hypoxia where n = 4. Overlapping points are offset. Different letters indicate a significant effect of time within a species. Data are means ± SE.  70  •  • •  2.0 —  en  u)  1.5 —  tc;  •o to E a) e  as  0.5 —  0.0 N  1^2  ^  6  Time in hypoxia (h)  Figure 3-5. Liver lactate in 0. maculosus (circle, dotted line), A. lateralis (triangle, solid line) and B. cirrhosus (square, dashed line) exposed to normoxia (-167 ton) and hypoxia equivalent to 40% of respective Pcnt (0. maculosus = 10.4 ton; A. lateralis = 14.9 torr; B. cirrhosus = 17.6 ton). For lactate n = 6 to 10, except for A. lateralis at 6h hypoxia where n = 4. Overlapping points are offset. Different letters indicate a significant effect of time within a species. Data are means ± SE.  71  Table 3-1. Fish weight and behavioral responses from 11 species of sculpins. Weight  ASR  OUT  % ASR  % OUT  0. maculosus  2.9+0.6  15.1+1.4  8.6+0.9  100  100  C. globiceps  1.5+0.2  23.3+1.0  10.0+0.4  100  100  O. snyderi  2.4+0.3  15.1+1.1  13.4+1.1  100  100  E. bison  2.4+0.2  11.7  12.3  17  17  A. fenestralis  16.2+0.6  7.3  17  0  A. lateralis  13.9+0.3  13.1+1.8  67  0  L. armatus  14.4+3.7  9.2+1.0  50  100  S. marmoratus  21.8+3.9  0  0  B. cirrhosus  2.1+0.2  0  0  M. scorpius  7.8+1.5  8.3+1.0  10.0+1.2  38  38  C. aspen  10.4+1.5  11.3+1.3  9.3+0.8  88  100  10.0+1.0  Data are means ± SE. Weight is presented in grams, aquatic surface respiration (ASR) and aerial emergence (OUT) are presented in torn, and % ASR and % OUT represent the percent of individuals performing ASR or aerial emergence. 6 to 8 individuals were tested for each behavioral analysis.  72  Table 3-2. Relationship between P ent and percent of individuals performing aquatic surface respiration (ASR) and aerial emergence (OUT) using conventional and phylogenetically independent contrast (PIC) correlations. Conventional Parameter  slope  r2  ASR  -0.09  0.13  % OUT  -0.05  0.05  PIC Slope  r2  0.28  0.04  0.02  0.68  0.51  0.01  <0.01  0.94  73  Table 3-3. Relationship between maximum habitat depth and percent of individuals of species performing aquatic surface respiration (ASR) and aerial emergence (OUT) using conventional and phylogenetically independent contrast (PIC) correlations. Conventional  PIC  Parameter  slope  r2  P  Slope  r2  P  ASR  -0.07  0.06  0.49  0.02  0.01  0.81  % OUT  -0.04  0.01  0.74  0.03  0.01  0.75  74  Table 3-4. Liver metabolites in 12 species of sculpins. ATP  CrP  Glycogen  Glucose  0. maculosus  1.5±0.2  3.5±0.6  250.4±38.2  2.0±0.3  C. globiceps  0.6±0.2  0.7±0.1  80.0±23.5  1.7±0.3  0. snyderi  0.9±0.2  0.9±0.3  229.8±52.5  2.0±0.4  E. bison  0.7±0.1  1.6±0.4  159.3±27.1  1.6±0.4  A. fenestralis  1.9±0.3  3.1±1.2  352.4±57.4  1.0±0.1  A. lateralis  1.4±0.3  2.2±0.6  328.7±64.4  1.0±0.2  L. armatus  1.0±0.1  0.6±0.1  98.2±17.0  1.4±0.2  S. marmoratus  1.3±0.1  0.4±0.1  394.2±34.6  1.4±0.2  B. cirrhosus  0.8±0.1  0.7±0.1  126.3±29.0  4.2±0.4  A. harringtoni  1.1±0.1  0.5±0.1  87.6±15.3  5.4±0.3  M scorpius  1.1±0.1  0.5±0.1  296.2±37.3  0.3±0.1  C. aspen  1.7±0.2  0.5±0.1  278.7±74.9  1.6±0.2  Data are means ± SE. Liver metabolites are presented in limolig of wet tissue. Sample size for liver metabolites ranged from n = 6 to 9, except for A. harringtoni where n = 2.  75  Table 3-5. Relationship between -Pcnt and liver metabolites using conventional and phylogenetically independent contrast (PIC) correlations. Conventional  PIC Slope  r2  0.44  4.34  0.04  0.50  0.28  0.08  -3.36  0.21  0.14  0.01  0.01  0.72  0.01  0.01  0.73  1.18  0.03  0.59  1.12  0.04  0.53  Parameter  slope  r2  ATP  5.95  0.06  CrP  -4.76  Glycogen Glucose  76  Table 3-6. Hematocrit and red blood cell Mg 2+ in 0. maculosus, A. lateralis and B. cirrhosus  exposed to normoxia and hypoxia. 0. maculosus Parameter  N  2h  A. lateralis  B. cirrhosus  6h  N  2h  6h  N  lh  2h  Hct  48±2a 42±3 a  45±3 a  33+2a  33±2 a  42±1 b  35±3 a  38±2 a  39±2 a  Mg 2+/Hb  26±3a 29±1 a  29±4 a  25±2a  23± 1 a  N/A  16±2a  19±3 a  18± 1 a  Data are means ± SE. Hematocrit (Hct) is presented in % and [Mg 2 1 is presented relative to [Hb]. For Hct n = 6 to 10 except for A. lateralis at 6h hypoxia where n = 4 and for [Mg 2+ ]/Hb n = 4 to 10 except for 0. maculosus at 6h hypoxia where n = 2. 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Dunn, J.F. and Hochachka, P.W. 1986. Metabolic responses of trout (Salmo gairdneri) to acute environmental hypoxia. J. Exp. Biol. 123: 229-242. Eschmeyer, W.N. and Herald, E.S. 1983. A field guide to Pacific Coast fishes: North America. New York: Houghton Mifflin Company. Feuerlein, R.J., and Weber, R.E. 1994. Rapid and simultaneous measurement of anodic and cathodic haemoglobins and ATP and GTP concentrations in minute quantities of fish blood. J. Exp. Biol. 189: 273-277. Froese, R. and D. Pauly. Eds. 2007. FishBase. www.fishbase.org , version (10/2007). Fangue, N.A., Mandic, M., Richards, J.G., and Schulte, P.M. In Press. Intraspecific variation in swimming performance and energetics as a function of temperature in killifish, Fundulus heteroclitus. Physiol. Biochem. Zool. Hochachka, P.W., Buck, L.T., Doll, C.J. and Land, S.C. 1996. Unifying theory of hypoxia tolerance: molecular/metabolic defense and rescue mechanisms for surviving oxygen lack. Proc. Natl. Acad. Sci. USA. 93: 9493-9498. Hochachka, P.W and Somero, G.N. 2002. Biochemical adaptation: mechanism and process in physiological evolution. New York: Oxford University Press. Jensen, F.B. 1991. Multiple strategies in oxygen and carbon dioxide transport by hemoglobin. In Physiological strategies for gas exchange and metabolism. (eds. A.J. Woakes, M.K. Grieshaber and C.R. Bridges) pp. 55-78. Cambridge: Cambridge University Press. Jensen, F.B., Fago, A. and Weber, R.E. 1998. Hemoglobin structure and function. In Fish Respiration. (eds. S.F. Perry and B. Tufts) pp. 1-40. San Diego: Academic Press. Jensen, F.B. and Weber, R.E. 1982. Respiratory properties of tench blood and hemoglobin: . adaptationto hypoxic-hypercapnic water. Mol. Physiol. 2: 235-250. 78  Lewis, J.M., Costa, I., Val, A.L., Almeida-Val, V.M.F., Gamperl, A.K., and Driedzic, W.R. 2007. Responses to hypoxia and recovery: repayment of oxygen debt is not associated with compensatory protein synthesis in the Amazonian cichlid, Astronotus ocellatus. J. Exp. Biol. 210: 1935-1943. Lykkeboe, G. and Johansen, K. 1975. Functional properties of hemoglobins in the teleost Tilapia grahami. J. Comp. Physiol. 104: 1-11. Maddison, W. P. and Maddison, D. R. 2004. Mesquite: A modular system for evolutionary analysis. Version 1.02. http://mesquiteproject.org. Martin, K.L.M. 1993. Aerial release of CO 2 and respiratory exchange ration in intertidal fishes out of water. Env. Biol. Fish. 37: 189-196. Martin, K.L.M. 1995. Time and tide wait for no fish: intertidal fishes out of water. Environ. Biol. Fishes. 44: 165-181. Martin, K.L.M. 1996. An ecological gradient in air-breathing ability among marine cottid fishes. Physiol. Zool. 69: 1096-1113. Midford, P.E, Garland, T., Jr. and Maddison, W. P. 2003. PDAP Package. Saint-Paul, U. 1984. Physiological adaptation to hypoxia of a neotropical characoid fish Colossoma macropomum, Serrasalmidae. Environ. Biol. Fishes. 11: 53-62. Shingles, A., McKenzie, D.J., Claireaux, G. and Domenici, P. 2005. Reflex cardioventilatory responses to hypoxia in the flathead gray mullet (Mugil cephalus) and their behavioral modulation by perceived threat of predation and water turbidity. Physiol. Biochem. Zool. 78, 744-755. Sloman, K.A., Mandic, M., Todgham, A.E., Fangue, N.A., Subrt, P. and Richards, J.G. 2008. The response of the tidepool sculpin, Oligocottus maculosus to hypoxia in laboratory, mesocosm and field environments. Comp. Biochem. Physiol. 149: 284-292. Sloman, K.A., Wood, C.M., Scott, G.R., Wood, S., Kajimura, M., Johannsson, 0.E., AlmeidaVal, V.M.F and Val, A.L. 2006. Tribute to R.G. Boutilier: the effect of size on the physiological and behavioural responses of oscar, Astronotus ocellatus, to hypoxia. J. Exp. Biol. 209: 11971205. Smith, C.L. 1950. Seasonal changes in blood sugar, fat body, liver glycogen, and gonads in the common frog, Rana Temporaria. J. Exp. Biol. 26: 412-429. Tetens, V. and Lykkeboe, G. 1981. Blood respiratory properties of rainbow trout, Salmo gairdneri: responses to hypoxia acclimation and anoxic incubation of blood in vitro. J. Comp. Physiol. 145: 117-125. van den Thillart, G. and van Raaij, M. 1995. Endogenous fuels: non-invasive versus invasive approaches. (eds. P.W. Hochachka and T.P. Mommsen). Pp. 33-57. Amsterdam: Elsevier. 79  Watters, J.V. and Cech, Jr., J.J. 2003. Behavioural responses of mosshead and wolly sculpins to increasing environmental hypoxia. Copeia. 2003: 397-401. Weber, R.E. and Lykkeboe, G. 1978. Respiratory adaptations in carp blood: influences of hypoxia, red cell organic phosphates, divalent cations and CO2 on hemoglobin-oxygen affinity. J. Comp. Physiol. 128: 127-137. Wells, R.M.G, Grigg, G.C., Beard, L.A. and Summers, G. 1989. Hypoxic responses in fish from a stable environment: blood oxygen transport in the Antarctic fish Pagothenia borchgrevinki. J. Exp. Biol. 141: 97-111. Wood, S.C. 1980. Adaptation of red blooc cell function to hypoxia and temperature in ectothermic vertebrates. Amer. Zool. 20: 163-172. Wright, W.G. and Raymond, J.A. 1978. Air-breathing in a California sculpin. J. Zool. Biol. 203: 171-176. Yoshiyama, R.M. and Cech, Jr., J.J. 1994. Aerial respiration by rocky intertidal fishes of California and Oregon. Copeia. 1994: 153-158. Yoshiyama, R.M., Valpey, C.J., Schalk, L.L., Oswald, N.M., Vaness, K.K., Lauritzen, D. and Limm, M. 1995. Differential propensities for aerial emergence in intertidal sculpins (Teleostei; Cottidae). J. Exp. Mar. Biol. Ecol. 191: 195-207.  80  CHAPTER FOUR: GENERAL DISCUSSION The goal of the research presented in this thesis was to begin to understand the adaptations responsible for hypoxia tolerance in sculpins. To accomplish this, a number of traits were examined using the comparative method in combination with PIC analysis in closely related species of sculpins. The application of this approach is essential in order to determine which traits are potentially adaptive for hypoxia tolerance, because it is provides insight into two important pieces of information: 1) if the traits have independently evolved numerous times, and 2) if the traits conferring tolerance have evolved only in the species that experience environmental hypoxia. In sculpins, a strong correlation exists between  Pcrit and  the species  distribution along the nearshore environment. Species of sculpins inhabiting the 02 variable intertidal possess the lowest Pcrit, while species of sculpins inhabiting the subtidal and deeper water possess higher P„, t . This ecological framework has allowed me to clearly demonstrate that variation in the ability of various sculpins to extract 02 from their environmental is key to surviving low environmental 02. There are three principle components involved in the enhanced 02 extraction capacity in sculpins possessing a low P crit . According to phylogenetically independent multiple regression analysis, routine  M O2 , mass specific gill surface area and whole blood Hb-02 binding affinity  contribute to 83% of the variability in P ent among species of sculpins. It appears that a low routine 1C4 0, , high gill surface area and high Hb-0 2 binding affinity are critical traits involved in the ability of sculpins to survive variable 02 environments. To date, this is the first study to demonstrate the adaptive value of these traits to hypoxia tolerance. This is in agreement with previous authors (Chapman et al., 2002; Hochachka and Somero, 2002; Saint-Paul, 1984; Wells, 1999) who have suggested that 1■:4 0, , gill surface area and especially Hb, play an important role  81  in the evolution of hypoxia tolerance in fish, although these authors were not able to directly demonstrate the adaptive value of the traits. Hemoglobin has been considered for a long time one of the key proteins involved in the evolution of animals to environmental hypoxia. This is most likely true for sculpins, which show that the species with a lower P„, t possess a higher whole blood Hb-0 2 binding affinity while the species with a lower Pcnt possess the lower whole blood Hb-02 binding affinity. The variation in whole blood Hb-0 2 binding affinity in sculpins is primarily determined by the intrinsic properties of the hemoglobin protein and not through influences of allosteric modulators. Recent studies have demonstrated the importance of the multiplicity and heterogeneity of hemoglobin isoforms in determining whole blood Hb-02 binding affinity (Brix et al., 1999; Wells et al., 1989; Wells, 1999). However, in sculpins there is no evidence that the number of total isoforms or the proportion of anodic to cathodic isoforms of hemoglobin play a significant role in whole blood Hb-02 binding affinity. This suggests that the variation in the Hb-02 binding affinity among species may be due to differences in the amino acid sequences. Although allosteric modulators do not contribute to setting the variation in whole blood Hb-02 binding affinity in sculpins, they are vital components in decreasing the high intrinsic 02 binding affinity, allowing for unloading of 02 at the tissues. Since 02 levels can drop to near anoxia in tidepools emerged at night (Truchot and Duhamel-Jouve, 1980) sculpins must be equipped to tolerate hypoxic conditions well below the point where 02 can be efficiently extracted from the environment. Sculpins, specifically those species found in tidepools, employ behavioral responses to hypoxia such as ASR and aerial emergence. These avoidance behaviors are beneficial because they allow fish to maintain routine M O2 despite 02 tensions of the bulk water being at or below  Pcrit  (Martin, 1996; Sloman et al.,  2008; Wright and Raymond, 1978; Yoshiyama and Cech, 1994). Although Martin (1996) has 82  suggested that aerial emergence is an adaptation of sculpins to the fluctuating 02 environment, a lack of a phylogenetically independent correlation between the avoidance behaviors and Pent, suggests that ASR and aerial emergence may not play a large role in the evolution of hypoxia tolerance in sculpins. However, only a small number of species exhibited these behaviors, limiting our ability to detect significance, therefore allowing for the possibility that ASR and aerial emergence may still be important in the evolution of hypoxia tolerance in sculpins. However, for those sculpins that do not possess these behavioral responses to hypoxia, or for those that do but are restricted to hypoxic waters due to a risk of aerial predation, there must be physiological and biochemical defenses that allow sculpins to survive periods of environmental hypoxia. The key biochemical defenses for surviving low environmental 0 2 tensions is a large upregulation of 0 2 independent pathways for ATP production, such as substrate level phosphorylation, and a decrease in metabolic rate through a suppression of energy consuming pathways (Boutilier and St-Pierre, 2000; Hochachka and Somero, 2002). In order to understand how species of sculpins respond to hypoxia, 0. maculosus, A. lateralis and B. cirrhosus, were chosen based on the difference in their Pcnt and exposed to several hours of hypoxia. Despite experiencing 0 2 tensions that were equivalent to 40% of respective P ent , mortality rates varied dramatically between the three species of sculpins. There was no mortality in the species with the lowest P cnt , 0. maculosus, while the species with highest P ent , B. cirrhosus did not survive beyond a two hour hypoxia exposure. The large variation in mortality rates could be attributed to the dramatic differences in on-board liver [glycogen] between the three species of sculpins. However, liver [glycogen] was examined in twelve species of sculpins and PIC analysis showed that there is no relationship between normoxic levels of liver glycogen and Pent in sculpins. The focus of future work will be to examine in greater detail biochemical defense strategies across a number of sculpin species using phylogenetically independent correlations. 83  Clearly, the Pcrit of a sculpin is important as it is linked to the distribution of the species along the nearshore environment and 02 extraction capacity. However, P„, t may be just one proxy measurement for hypoxia tolerance, since it is clear that when species of sculpins are exposed to 02 tensions well below their P crit threshold mortality rates differ dramatically despite the level of hypoxia being equivalent among the species. Therefore, a secondary measure for hypoxia tolerance such as the LT50 will be determined among the species of sculpins. It will be interesting to examine if the variation in P ent is mirrored by a similar variation in LT 5 0 and how these two measurements of hypoxia are linked to various aspects of metabolic change during hypoxia. With the application of PIC analysis, it will be possible to determine the role of these metabolic defenses in the evolution of hypoxia tolerance in sculpins.  84  REFERENCES  Boutilier, R.G. and St-Pierre, J. 2000. Surviving hypoxia without really dying. Comp. Biochem. Physiol. 126: 481-490. Brix, 0., Clements, K.D. and Wells, R.M.G. 1999. Haemoglobin components and oxygen transport in relation to habitat selection in triplefin fishes (Tripterygiidae). J. Comp. Physiol. B. 169: 329-334. Chapman, L.J., Chapman, C.A., Nordlie, F.G. and Rosenberger, A.E. 2002. Physiological refugia: swamps, hypoxia tolerance and maintenance of fish diversity in the Lake Victoria region. Comp. Biochem. Physiol. A. 133: 421-437. Hochachka, P.W and Somero, G.N. 2002. Biochemical adaptation: mechanism and process in physiological evolution. New York: Oxford University Press. Martin, K.L.M. 1996. An ecological gradient in air-breathing ability among marine cottid fishes. Physiol. Zool. 69: 1096-1113. Saint-Paul, U. 1984. Physiological adaptation to hypoxia of a neotropical characoid fish Colossoma macropomum, Serrasalmidae. Environ. Biol. Fishes. 11: 53-62. Sloman, K.A., Mandic, M., Todgham, A.E., Fangue, N.A., Subrt, P. and Richards, J.G. 2008. The response of the tidepool sculpin, Oligocottus maculosus to hypoxia in laboratory, mesocosm and field environments. Comp. Biochem. Physiol. 149: 284-292. Truchot, J.P. and Duhamel-Jouve, A. 1980. Oxygen and carbon dioxide in the marine intertidal environment: diurnal and tidal changes in rockpools. Respir. Physiol. 39: 241-254. Wells, R.M.G. 1999. Haemoglobin function in aquatic animals: molecular adaptations to environmental challenge. Mar. Freshwater. Res. 50: 933-939. Wells, R.M.G., Grigg, G.C., Beard, L.A. and Summers, G. 1989. Hypoxic responses in a fish from a stable environment: blood oxygen transport in the Antarctic fish Pagothenia Borchgrevinki. J. Exp. Biol. 141: 97-111. Wright, W.G. and Raymond, J.A. 1978. Air-breathing in a California sculpin. J. Zool. Biol. 203: 171-176. Yoshiyama, R.M. and Cech, Jr., J.J. 1994. Aerial respiration by rocky intertidal fishes of California and Oregon. Copeia. 1994: 153-158.  85  

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