Anoxia Survival Strategies in the Grey Carpet Shark (Chiloscyllium punctatum) and the Epaulette Shark (Hemiscyllium ocellatum)
Author Chapman, Clint Allan
Published 2009
Thesis Type Thesis (PhD Doctorate)
School School of Physiotherapy and Exercise Science
DOI https://doi.org/10.25904/1912/594
Copyright Statement The author owns the copyright in this thesis, unless stated otherwise.
Downloaded from http://hdl.handle.net/10072/366452
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ANOXIA SURVIVAL STRATEGIES IN THE GREY
CARPET SHARK (Chiloscyllium punctatum) AND THE
EPAULETTE SHARK (Hemiscyllium ocellatum)
By
Clint Allan Chapman
B. Sc. (Hons) Grad. Cert. (Marine Studies)
A thesis submitted for the degree of Doctor of Philosophy
School of Physiotherapy and Exercise Science
Griffith University, Gold Coast, Australia
May, 2009
Declaration
The work presented in this thesis is, to the best of my knowledge and belief, original and my work, except as acknowledged in the text. I thereby declare that this material has not been submitted, either in whole or in part, for a degree at this or any other University.
______
Clint Allan Chapman Date
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Acknowledgements
I would like to acknowledge the following people for their tireless effort and patience, whom without, this study would not have been possible.
To my family. Mum, Dad, Georgie, Siemon and Skyla. Thank you for always showing interest and understanding during the ups and downs. You all continually listened and offered constant advice and encouragement. You were a great distraction from the stress and offered an escape when I needed it most. Thank you so much for just being yourselves. I love you all.
To my Blake. How can I thank the greatest woman in the world? You have made this journey with me and helped me every step along the way. You have given me constant love and support and have had more patience than anyone could ask for. You have been there to brainstorm and to hold me up when I was down. I will always appreciate the effort and involvement you have had in my work. Most of all I want to thank you for coming into my life and sharing this journey with me. I will love you always.
To my friends. Thank you for having constant understanding and cheering me up when I needed it. It is a sign of the true friends that I have, after all the times you have all been there for me. Thank you.
To my supervisor Gillian. Thank you for providing all the lab equipment and support for the research. For spending tireless hours revising my work and providing direction and focus to my writing.
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To Seaworld. Trevor, Marnie and Aileen. Thank you for accommodating and supporting my research. I am incredibly grateful for the trust that was placed in me to allow me to conduct my research at all hours of the day and night. I felt very welcome and appreciate all the time that you spent with me. I highly value the relationships I have made with SeaWorld and its staff and hope it continues into the future. Thank you.
To UnderWater World. Andreas and Richard. Thank you for your assistance and support. You assisted in my research at crucial times and were always willing to provide resources. Thank you for the time spent discussing my research plans and assisting in housing my animals. I deeply appreciate the great lengths that were taken to house a number of animals so that were available at any time. Thank you.
To Adeeb Girges. Thank you for your efforts and tutelage in laboratory procedures. I learnt invaluable knowledge on laboratory standards and the meticulous art that is laboratory science from you. It was a pleasure to work in the lab with you.
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Abstract
Most vertebrates exhibit a negligible tolerance to anoxic conditions. The epaulette shark (Hemiscyllium ocellatum), however, is exposed to severe hypoxia in its natural environment and has developed adaptive strategies to cope with these conditions, making this species the only currently known anoxia tolerant elasmobranch. The grey carpet shark
(Chiloscyllium punctatum) is a closely related species inhabiting ecologically similar environments. Anecdotal evidence suggests some degree of tolerance to low oxygen conditions occurs in this species. This thesis examined the degree of anoxia tolerance in the grey carpet shark along with the haematological and physiological responses of the grey carpet shark and the epaulette shark to anoxia and re-oxygenation.
The effect of seasonal temperature on the duration of anoxia tolerance in both species was examined. Both species were exposed to a number of anoxic regimes and re-oxygenation at moderate (23C), intermediate (25C) and high (27C) temperatures. Total time to loss of righting reflex (LRR) and ventilation rates were measured in adult epaulette sharks, along with adult and juvenile grey carpet sharks. Anoxia tolerance times of both species were temperature dependent, with a significant reduction in the time to LRR occurring at higher temperatures. While both species had similar times to LRR at 23°C, epaulette sharks had a significantly greater time to LRR at higher temperatures. Juvenile grey carpet sharks appear to possess little tolerance to anoxia. Neither juvenile nor adult grey carpet sharks entered into ventilatory depression.
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The haematological responses of wild and captive populations of both species were examined in response to anoxia and re-oxygenation. The epaulette shark showed evidence of erythrocyte swelling, while the grey carpet shark had a significant increase in erythrocyte concentrations due to a release of erythorcytes into the circulation and/or haemoconcentration of the blood in response to anoxia. Plasma glucose concentrations were maintained in epaulette sharks and wild grey carpet sharks during anoxia but increased significantly during re-oxygenation. Captive grey carpet sharks had an immediate increase in plasma glucose concentrations after anoxia, which was sustained during the re-oxygenation period. Lactate concentrations significantly increased in all animals after anoxia, reaching a peak at 2 hours of re-oxygenation.
Since anoxia compromises the supply of ATP, the maintenance of ion homeostasis may be compromised during prolonged anoxia. A significant increase in plasma potassium concentrations were observed in the grey carpet shark immediately following anoxia and in the epaulette shark after 2 hours of re-oxygenation. No differences in plasma sodium concentrations were observed in either species, although a decrease in plasma chloride occurred after 2 hours of re-oxygenation in the grey carpet shark. Plasma magnesium concentrations significantly increased in both species immediately following anoxia and for 2 hours of re-oxygenation, while plasma calcium only increased in the epaulette shark during re-oxygenation. With the exception of chloride in the grey carpet shark, all plasma electrolyte concentrations were restored during re-oxygenation in both species.
One plausible hypothesis for the increase in erythrocytes observed in the grey carpet shark in response to anoxia is the release of erythrocytes from a storage site. Changes in spleen and liver weight and haemoglobin concentrations were measured to determine if they
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function as erythrocyte stores. While significant increases in haematological parameters were observed, no significant differences were observed in organ weights or haemoglobin concentrations in response to anoxia.
The up-regulation of protective proteins has been observed to protect vital organs during events of cellular stress in many vertebrates. The heat shock protein 70 kDa (Hsp70) response to anoxic stress was characterised in both species using two different protocols.
Hsp70 concentrations of both species were determined via western blotting on the plasma separated blood and additionally in the cerebellum and the ventricle of the heart of the grey carpet shark. No significant differences in Hsp70 concentrations were observed in the blood of either species. Furthermore, no significant differences in Hsp70 concentrations were observed in the cerebellum or the ventricle of the grey carpet shark in response to anoxia.
This study reported for the first time a significant tolerance to anoxia in the grey carpet shark and demonstrated the effect of temperature on the duration of anoxia tolerance in both species. A reduction in the duration of anoxia tolerance was identified in both species at higher temperatures, although the effect was more pronounced in the grey carpet shark. This study identified unique changes in haematological parameters and plasma constituents in both species in response to anoxia and normoxic re-oxygenation. While the epaulette shark possesses energy conserving strategies present in other anoxia tolerant vertebrates, such as metabolic and ventilatory depression, the grey carpet shark does not. This study concluded that the grey carpet shark possesses an intermediate tolerance to anoxia, which would prolong survival during events of naturally occurring hypoxia encountered from the ambient environment on coral reef flats, mangrove swamps and seagrass beds.
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Statement of contributions of jointly authored work contained in this thesis
G.M.C. Renshaw was responsible for was responsible for providing feedback and suggestions to drafts of the manuscript and providing equipment and laboratory facilities.
C. O’Leary was responsible for providing the laboratory facilities used to perform plasma electrolyte analysis in Chapter 4.
B. Bynon was responsible for processing the samples to determine the plasma electrolyte concentrations in Chapter 4.
C.A. Chapman was responsible for the remainder of the work.
Published works by the author incorporated into this thesis
Chapman CA, Renshaw GMC. In press. Haematological responses of the grey carpet shark (Chiloscyllium punctatum) and the epaulette shark (Hemiscyllium ocellatum) to anoxia and re- oxygenation exposure. J Exp Zoo.
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Table of contents Acknowledgements ______ii Abstract ______iv Statement of contributions of jointly authored work contained in this thesis ______vii Published works by the author incorporated into this thesis______vii List of figures ______xiii List of tables ______xvi GENERAL INTRODUCTION ______i
CHAPTER ONE ______1
Literature review: Anoxic stress and survival strategies in vertebrate animals______1
1.1 Introduction ______2
1.2 Normoxic energy metabolism ______3
1.3 Hypoxic and anoxic cell death______3
1.4 Apoptotic cell death ______5
1.5 Anoxia intolerant vertebrates ______6
1.6 Anoxia intermediate vertebrates ______8
1.7 Anoxia tolerant vertebrates______9
1.8 The effect of temperature on anoxia tolerance ______13
1.9 Tropical hypoxia on coral reef flats ______14
1.10 The epaulette shark (Hemiscyllium ocellatum) ______16
1.10.1 Anoxia tolerance of the epaulette shark ______16 1.11 The grey carpet shark (Chiloscyllium punctatum) ______19
1.11.1 Anoxia tolerance of the grey carpet shark ______20 1.12 Anoxia and haematology ______21
1.13 Hypoxia induced erythrocyte concentrations ______22
1.13.1 Erythropoiesis ______22 1.13.2 Haemoconcentration ______24 1.13.3 Erythrocyte reservoirs ______24 1.13.4 The elasmobranch spleen ______25 1.13.5 Epigonal and Leydig’s organ ______26
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1.14 Plasma constituents ______27
1.14.1 Glucose ______27 1.14.2 Lactate ______28 1.14.3 Plasma electrolytes ______29 1.15 Heat shock proteins ______32
1.15.1 Heat shock protein 70 kDa ______33 1.15.2 Hsp70 and hypoxia ______34 1.16 References cited ______36
CHAPTER TWO ______46
The physiological tolerance of the grey carpet shark (Chiloscyllium punctatum) and the epaulette shark (Hemiscyllium ocellatum) to anoxic exposure at three seasonal temperatures ______46
2.1 Summary ______47
2.2 Introduction ______49
2.3 Materials and methods ______52
2.3.1 Animal collection and age ______52 2.3.2 Temperature ______53 2.3.3 Anoxic exposure ______53 2.3.4 Statistics ______56 2.4 Results ______56
2.4.1 Experiment 1. Time to loss of righting reflex during an “open ended” anoxic exposure at three temperatures ______56 2.4.2 Experiment 2. Ventilation rates during an “open ended” anoxic exposure at 23C ______59 2.4.3 Experiment 3. Ventilation rates during 1.5 hours of anoxia followed by 2 hours of normoxic re- oxygenation at 23°C or 25°C ______64 2.4.4 Behavioural characteristics of the epaulette shark to anoxic exposure ______68 2.4.5 Behavioural characteristics of the grey carpet shark to anoxic exposure ______68 2.5 Discussion ______69
2.6 Literature cited______76
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CHAPTER THREE ______78
Haematological responses of the grey carpet shark (Chiloscyllium punctatum) and the epaulette shark (Hemiscyllium ocellatum) to anoxia and re-oxygenation exposure _____ 78
3.1 Summary ______79
3.2 Introduction ______80
3.3 Materials and methods ______84
3.3.1 Study animals and locations ______84 3.3.2 Anoxic challenge and re-oxygenation ______85 3.3.3 Blood sampling and haematology ______86 3.3.4 Statistics ______88 3.4 Results ______88
3.4.1 Haematocrit ______88 3.4.2 Red blood cell count ______91 3.4.3 Haemoglobin ______93 3.4.4 Calculated erythrocytic indices ______95 3.4.5 Glucose ______96 3.4.6 Lactate ______99 3.5 Discussion ______102
3.5.1 Haematological differences in control animals from captive and wild populations of two species of shark 102 3.5.2 Haematological responses to anoxic challenge and re-oxygenation ______103 3.5.3 Glucose ______107 3.5.4 Lactate ______109 3.5.5 Conclusions ______112 3.6 Literature cited______114
CHAPTER FOUR ______119
Plasma electrolyte balance of the epaulette shark (Hemiscyllium ocellatum) and the grey carpet shark (Chiloscyllium punctatum) in response to anoxia and normoxic re- oxygenation ______119
4.1 Summary ______120
4.2 Introduction ______122
4.3 Materials and methods ______124
4.3.1 Study animals and location ______124
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4.3.2 Anoxic exposure ______125 4.3.3 Blood analysis ______125 4.3.4 Statistics ______127 4.4 Results ______127
4.4.1 Plasma potassium levels ______127 4.4.2 Calcium ______129 4.4.3 Sodium ______131 4.4.4 Chloride ______131 4.4.5 Magnesium ______132 4.5 Discussion ______135
4.6 Literature cited______143
CHAPTER FIVE ______146
The absence of splenic and hepatic contraction to release erythrocytes into the circulation of the grey carpet shark (Chiloscyllium punctatum) in response to anoxia and re- oxygenation ______146
5.1 Summary ______147
5.2 Introduction ______148
5.3 Materials and methods ______151
5.3.1 Study animals and location ______151 5.3.2 Anoxic exposure ______151 5.3.3 Blood sampling and haematology ______152 5.3.4 Tissue analysis ______153 5.3.5 Statistics ______154 5.4 Results ______155
5.4.1 Haematology of blood samples ______155 5.4.2 Splenic and hepatic tissue analysis ______157 5.5 Discussion ______158
5.6 Literature cited______164
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CHAPTER SIX ______168
Characterisation of the heat shock protein (Hsp70) response in the brain, heart and blood following anoxia and re-oxygenation in the grey carpet shark (Chiloscyllium punctatum) and the epaulette shark (Hemiscyllium ocellatum) ______168
6.1 Summary ______169
6.2 Introduction ______170
6.3 Materials and methods ______172
6.3.1 Study animals and location ______172 6.3.2 Anoxic exposure ______173 6.3.3 Blood sampling and haematology ______173 6.3.4 Two anoxic episodes ______174 6.3.5 Standardised anoxic episode ______174 6.3.6 SDS Page electrophoresis ______175 6.3.7 Immuno-detection ______176 6.4 Results ______177
6.4.1 Two anoxic episodes ______177 6.4.2 Standardised anoxic episode ______179 6.5 Discussion ______181
6.5.1 Two anoxic episodes ______181 6.5.2 Standardised anoxic episode ______184 6.5.3 Conclusion ______185 6.6 Literature cited______186
GENERAL DISCUSSION ______189
Literature cited ______201
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List of figures
Chapter 1 Literature review: Anoxic stress and survival strategies in vertebrate animals
Figure 1.1 Tropical hypoxia on coral reef flats 15 Figure 1.2 The epaulette shark (Hemiscyllium ocellatum) 16 Figure 1.3 The grey carpet shark (Chiloscyllium punctatum) 19
Chapter 2 The physiological tolerance of the grey carpet shark (Chiloscyllium punctatum) and the epaulette shark (Hemiscyllium ocellatum) to anoxic exposure at three seasonal temperatures
Figure 2.1 Time to loss of righting reflex during anoxic exposure in two adult 58 shark species at three temperatures
Figure 2.2 Time to loss of righting reflex during anoxic exposure in juvenile 59 and adult grey carpet sharks at three temperatures
Figure 2.3 Ventilation rates of adult epaulette sharks in response to anoxia at 60 23C
Figure 2.4 Ventilation rates of adult grey carpet sharks in response to anoxia 62 at 23C
Figure 2.5 Ventilation rates of juvenile grey carpet sharks in response to 63 anoxia at 23C
Figure 2.6 (A)Ventilation rates of adult epaulette sharks in response to 1.5 65 hours anoxia and 2 hours re-oxygenation at 23C (B) Ventilation rates of adult epaulette sharks in response to 1.5 hours anoxia and 2 hours re-oxygenation at 25C
Figure 2.7 (A) Ventilation rates of adult grey carpet sharks in response to 1.5 67 hours anoxia and 2 hrs re-oxygenation at 23C (B) Ventilation rates of adult grey carpet sharks in response to 1.5 hours anoxia and 2 hrs re-oxygenation at 25C
Chapter 3 Haematological responses of the grey carpet shark (Chiloscyllium punctatum) and the epaulette shark (Hemiscyllium ocellatum) to anoxia and re-oxygenation exposure
Figure 3.1 (A) Percent haematocrit in control animals for two species of 90 sharks from two environments (B) Percent haematocrit in response to anoxic challenge and re- oxygenation in two species of sharks from two environments
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Figure 3.2 (A) Red blood cell concentrations in control animals for two 92 species of shark from to environments (B) Changes in red blood cell concentrations in response to anoxic challenge and re-oxygenation in two species of sharks from two environments
Figure 3.3 (A) Haemoglobin concentrations for control animals in two species 94 of shark from two environments (B) Changes in haemoglobin concentrations in response to anoxic challenge and re-oxygenation in two species of sharks from two environments
Figure 3.4 (A) Plasma glucose concentrations for control animals in two 98 species of sharks from two environments (B) Changes in plasma glucose concentrations in response to anoxic challenge and re-oxygenation in two species of sharks from two environments
Figure 3.5 (A) Changes in plasma lactate concentrations for control animals in 101 two species of sharks from two environments (B) Changes in plasma lactate concentrations in response to anoxic challenge and re-oxygenation in two species of sharks from two environments
Chapter 4 Plasma electrolyte balance of the epaulette shark and the grey carpet shark in response to anoxia and normoxic re-oxygenation
Figure 4.1 Changes in plasma potassium concentrations in response to anoxia 128 and re-oxygenation or control treatments in the epaulette shark
Figure 4.2 Changes in plasma potassium concentrations in response to anoxia 128 and re-oxygenation or control treatments in the grey carpet shark
Figure 4.3 Changes in plasma calcium concentrations in response to anoxia 130 and re-oxygenation or control treatments in the epaulette shark
Figure 4.4 Changes in plasma chloride concentrations in response to anoxia 132 and re-oxygenation or control treatments in the grey carpet shark
Figure 4.5 Changes in plasma magnesium concentrations in response to 133 anoxia and re-oxygenation or control treatments in the epaulette shark
Figure 4.6 Changes in plasma magnesium concentrations in response to 134 anoxia and re-oxygenation or control treatments in the grey carpet shark
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Chapter 5 The absence of splenic and hepatic contraction to release erythrocytes into the circulation of the grey carpet shark (Chiloscyllium punctatum) in response to anoxia and re-oxygenation
Figure 5.1 Changes in percent hematocrit in response to anoxia and re- 155 oxygenation or control treatments in the grey carpet shark
Figure 5.2 Changes in haemoglobin concentrations in response to anoxia and 156 re-oxygenation or control treatments in the grey carpet shark
Figure 5.3 Changes in red blood cell concentrations in response to anoxia and 157 re-oxygenation or control treatments in the grey carpet shark
Chapter 6 Characterisation of the Hsp70 response in the brain, heart and blood following anoxia and re-oxygenation in the grey carpet shark (Chiloscyllium punctatum) and the epaulette shark (Hemiscyllium ocellatum)
Figure 6.1 Time to loss of righting reflex in the grey carpet shark during two 178 episodes of anoxia
Figure 6.2 Hsp70 concentration in the cerebellum, ventricle and the blood 178 following two anoxic exposures in the grey carpet shark
Figure 6.3 Changes in Hsp70 concentrations in the blood of the grey carpet 180 shark in response to anoxia and re-oxygenation
Figure 6.4 Changes in Hsp70 concentrations in the blood of the grey carpet 181 shark in response to anoxia and re-oxygenation
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List of tables
Chapter 2 The physiological tolerance of the grey carpet shark (Chiloscyllium punctatum) and the epaulette shark (Hemiscyllium ocellatum) to anoxic exposure at three seasonal temperatures
Table 2.1 The time to loss of righting reflex during anoxia at three different 57 temperatures, 23C, 25C and 27C in the adult epaulette shark (H. ocellatum), adult grey carpet shark (C. punctatum) and juvenile grey carpet shark
Chapter 3 Haematological responses of the grey carpet shark (Chiloscyllium punctatum) and the epaulette shark (Hemiscyllium ocellatum) to anoxia and re-oxygenation exposure
Table 3.1 Erythrocytic indices of the epaulette shark (Hemiscyllium 95 ocellatum) in response to anoxia and normoxic re-oxygenation
Table 3.2 Erythrocytic indices of the grey carpet shark (Chiloscyllium 96 punctatum) in response to anoxia and normoxic re-oxygenation
Chapter 4 Plasma electrolyte balance of the epaulette shark and the grey carpet shark in response to anoxia and normoxic re-oxygenation
Table 4.1 Percent changes in plasma electrolyte concentrations of the 135 epaulette shark (Hemiscyllium ocellatum) and the grey carpet shark (Chiloscyllium punctatum) in response to anoxia and re- oxygenation
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GENERAL INTRODUCTION
Most animals require a constant supply of oxygen to sustain large quantities of cellular energy (ATP) for survival and function. During events of reduced oxygen availability
(hypoxia) and in the complete absence of oxygen (anoxia), ATP is not produced as rapidly as it is consumed. Subsequently, prolonged periods of hypoxia and anoxia can lead to the depletion of ATP, which results in a loss of cellular homeostasis. As a consequence, cell membrane integrity and cellular degradation occurs, signifying the necrotic cell state.
While mammals have a negligible tolerance to hypoxia and anoxia (Lutz et al., 2003), research into the comparative physiology of non-mammalian species encountering reduced oxygen in their natural environment has recognised that some vertebrates have developed a repertoire of specialised adaptive strategies to prolong their tolerance to hypoxic and anoxic conditions. The investigation of such animals provides an understanding of the underlying mechanisms involved in the diverse array of strategies that specialised vertebrates have evolved to deal with oxygen deprivation.
Comparison of hypoxia- and anoxia-tolerant species has led to the identification and characterization of numerous novel mechanisms, which aid in cellular protection and survival during events of hypoxic or anoxic stress. Within the subclass elasmobranchii, which includes the sharks, rays and skates, the epaulette shark (Hemiscyllium ocellatum, Bonnaterre, 1788) is the only reported anoxia tolerant species (Renshaw et al., 2002).
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The grey carpet shark (Chiloscyllium punctatum, Müller and Henle, 1838) is a close relative to this anoxia-tolerant species, however it’s tolerance to episodes of anoxic exposure had not been investigated. Therefore this thesis aimed to examine and characterise the extent of anoxia tolerance in the grey carpet shark in order to identify and comparare the physiological strategies that both species use in order to survive periods of anoxic challenge.
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CHAPTER ONE
Literature review: Anoxic stress and survival strategies in vertebrate animals
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Chapter 1: Literature review
1.1 Introduction
With the exception of high altitudes, oxygen is generally readily available within the terrestrial environment. Therefore most vertebrates rarely encounter situations that impose a severe hypoxic insult. However within aquatic environments, such as under lakes with frozen surfaces, in shallow semi-enclosed bodies of water, estuaries, abyssal depths and isolated reef lagoons, vertebrates are exposed to natural cyclic events of hypoxic and anoxic stress as a result of reduced oxygen saturation in their ambient environment. To survive in these environments, these animals have adapted physiological protective mechanisms.
The most well characterised anoxia tolerant vertebrates are naturally exposed to anoxia at extremely low temperatures. Both the crucian carp (Carcassius carassius) and freshwater turtles in the genus Chrysemys, can survive periods of anoxia at low temperatures for up to several months (Jackson, 2000; Lutz et al., 2003). At low temperatures, metabolic rates are dramatically reduced, which subsequently reduces ATP demand and consumption.
This reduction in metabolism helps to conserve ATP and prolong survival during periods of anoxia. Exposure to anoxia at warmer temperatures has been observed to dramatically reduce the anoxia tolerance of these animals (Herbert and Jackson, 1985; Bickler, 1992).
Interestingly, some vertebrates have evolved a set of unique strategies, which enable them to tolerate periods of severe hypoxia and anoxia in a highly metabolicly active environment, with temperatures reaching up to 30C (Nilsson and Renshaw, 2004). A comparative analysis of hypoxia tolerant vertebrates in tropical environments may reveal a greater repertoire of protective mechanisms to prolong survival and reduce cellular damage.
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Chapter 1: Literature review
1.2 Normoxic energy metabolism
All vertebrate animals rely on a constant and continual supply of cellular energy to survive. During normoxia (normal oxygen conditions), cellular energy is predominantly supplied via aerobic metabolism, which is the breakdown of substrates such as glucose, in the presence of oxygen to yield large quantities of adenosine triphosphate (ATP). Although background anaerobic ATP synthesis is present at low levels, aerobic metabolism is the most efficient pathway for ATP production. Under aerobic conditions, one molecule of glucose can yield 38 molecules of ATP, while anaerobic metabolism only produces two molecules of ATP for every glucose molecule (Lutz and Nilsson, 1997).
In order to maintain the energy budget, the production of ATP must equal the energy consumption of the body. Due to the reliance on aerobic metabolic pathways to produce large quantities of ATP, any changes in oxygen availability may have dramatic effects on the production of ATP and subsequently have deleterious effects to cell function and survival.
1.3 Hypoxic and anoxic cell death
During hypoxia and anoxia, there is insufficient oxygen available to maintain aerobic respiration (Schmidt-Nielsen, 1983). Thus, any continued ATP synthesis relies on the up- regulation of the anaerobic process of glycolysis. Compared to aerobic pathways, anaerobic metabolism yields significantly smaller quantities of ATP, and can therefore only make up the energy deficit for a short time.
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Chapter 1: Literature review
Once ATP levels fall and are insufficient to supply energy demand, ion homeostasis is jeopardised. The efficiency of ion pumps is compromised and there is a net movement of ions across the cell membrane down their concentration gradients (Lutz, 1992; Wasser and Heisler,
1997). Consequently, there is an increase in extracellular potassium, caused by potassium diffusing out of the cell, and intracellular sodium, caused by diffusion into the cell, resulting in cellular depolarisation (Wasser and Heisler, 1997). This depolarisation in conjunction with further decreases in intracellular ATP results in a failure of calcium regulating mechanisms, including energy dependant calcium exclusion and the release of bound calcium from the endoplasmic reticulum (Lutz, 1992). This rapid rise in calcium causes multiple dysfunctional effects, including the stimulation of phospholipid hydrolysis and a rise in harmful free fatty acids (particularly arachidonic acid), which leads to the formation of free radicals (Lutz, 1992;
Wasser and Heisler, 1997).
As cell ion homeostasis is disrupted due to reduced ATP supplies, the effect of cellular depolarisation is accompanied by the influx of other ions such as chloride following sodium ions into the cell along its electrical gradient. This influx is then accompanied by osmotically driven water, which causes cell swelling that can lead to structural cell damage and finally, cell death via necrosis (Lutz, 1992).
During anaerobic glycolysis, small quantities of ATP and pyruvate are produced.
When pyruvate cannot enter the aerobic respiration circle due to an absence of oxygen, it is converted predominantly to lactic acid by the enzyme lactic dehydrogenase (Schmidt-Nielsen,
1983). Lactic acid dissociates into a hydrogen ion and a negatively charged lactate ion.
Accumulation of H+ from prolonged anaerobic metabolism can cause a reduction in intracellular pH, known as metabolic acidosis (Jensen, 2004). Changes in intracellular pH can
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Chapter 1: Literature review have widespread metabolic effects. Intracellular acidosis has been observed to alter enzyme activity and protein structure, disrupt the stability of cell and organelle membranes, inhibit mitochondrial respiration, increase free radical production and play a role in the activation of mitochondrial apoptotic pathways (Kubasiak et al., 2002), which may ultimately result in cell death.
1.4 Apoptotic cell death
During hypoxic and anoxic stress, necrotic cell death occurs as a direct result of ATP depletion, cellular injury and loss of ion homeostasis. Following stress, cells are re-exposed to a stable environment and aerobic respiration resumes to replenish ATP levels, which drive either cellular recovery or energy requiring apoptotic pathways. Apoptotic cell death is characterised by slower, distinct morphological changes to the cell, which is typified by DNA fragmentation and cell shrinkage, without the loss of membrane integrity or activation of an inflammatory process (Regula et al., 2003). In teleost fishes, apoptosis has been reported following hypoxia in a broad range of tissues, including the central nervous system, skeletal and cardiac muscle and the gill epithelium (Sollid et al., 2003; Lu et al., 2005).
Following hypoxic and anoxic stress, the exact mechanisms/stimuli that trigger the activation of apoptotic cell death have yet to be fully understood. However, it appears that they receive stimuli from DNA damage, growth factor deprivation, increases in reactive oxygen species and intracellular calcium, engagement of cell surface death receptors and endoplasmic reticulum permeabilisation (Nicholson & Thornberry, 1997). Irrespective of the exact mechanism required to induce apoptotic cell death following reduced oxygen
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Chapter 1: Literature review conditions, the permeabilisation of the mitochondrial membrane appears to be the point of no return in the apoptotic cell death process.
Within the mitochondria, inactive apoptotic constituents, such as cytochrome c, pro- caspases, diablo/smac and apoptosis inducing factor, are segregated from the highly reactive cell cytoplasm, thus preventing their activation (Regula et al., 2003). Upon a pro-apoptotic signal the outer mitochondrial membrane undergoes a brief permeabilisation, which permits the release of these potentially hazardous factors into the cytoplasm (Regula et al., 2003). The release of these mitochondrial constituents activates a cascade of events involving the dismantling of critical homeostatic and repair processes of the cell (Nicholson & Thornberry,
1997) as well as cleaving key structural components, resulting in the systematic and orderly disassembly of the dying cell, which collectively manifest the apoptotic phenotype (Nicholson
& Thornberry, 1997).
1.5 Anoxia intolerant vertebrates
The brain is the most complex organ in the body and is involved in the control and regulation of all the other organs and tissues. Therefore, the brain is the most vital organ for survival and function. As a consequence, the brain is in a continually active state and therefore requires a continual and constant supply of energy (Lutz and Nilsson, 1997). In the mouse (Mus musculus) and the gold fish (Carassius auratus) the central nervous system is responsible for 8.5% and 7.4% total energy consumption, respectively (Mink et al., 1981).
With the exception of primates, the brain makes up 0.1-1% of the vertebrate body weight, however it is responsible for 1.5-8.5% of the total energy consumption (Mink et al., 1981).
Lutz and Nilsson (1997) therefore concluded that the metabolic rate of the brain for
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Chapter 1: Literature review vertebrates is commonly 10-30 times higher than that of the whole body. This high-energy consumption of the brain therefore makes it a very vulnerable organ during events of cellular stressors that inhibit energy supply. Interruptions in the energy supply to the brain can result in rapid and irreversible damage. Under normoxic conditions, more than 95% of the brain
ATP is produced aerobically. In order to maintain the brain’s high energy levels, a continual supply of oxygen and metabolic fuel is required for aerobic production of ATP. Factors such as hypoxia, asphyxia and ischemia, which can affect the delivery of these substances, can jeopardise the production and availability of energy to the brain.
The mammalian brain can only withstand a few minutes of anoxia before cellular damage begins to occur (Lutz and Nilsson, 1997). However, this intolerance to anoxia is not limited to mammals and is true for most vertebrate animals. In short, when oxygen is removed, brain ATP levels are consumed very quickly. This reduction in ATP causes a subsequent failure of ATP dependent ion exchangers. As a result, ionic homeostasis is compromised and depolarisation occurs. With the onset of a brain depolarisation, neurotransmitters (either inhibitory or excitatory) are released indiscriminately, which induces the depolarisation of neighbouring cells. The onset of depolarisation when ATP levels are depleted signifies a complete loss of ionic homeostasis and allows ions to move freely across their electrical and concentration gradients. Osmotically driven water enters the cells, which causes disruption of membrane integrity and ultimately, cell death.
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Chapter 1: Literature review
1.6 Anoxia intermediate vertebrates
Interestingly, some species appear to possess an intermediate tolerance to periods of anoxia. The most recent and well-known example is the leopard frog (Rana pipiens), which can survive anoxia for 3 hours at room temperature (Lutz and Reiners, 1997) and 30 hours at
5ºC (Hermes-Lima and Storey, 1996). The depletion of ATP levels in the leopard frog are similar to those observed in mammals (Siesjo, 1978) and anoxia-intolerant fish (Van Raaij et al., 1994). Unlike anoxia-tolerant species, the leopard frog does not appear to conserve brain
ATP levels. Instead, ATP levels slowly decline immediately upon exposure to anoxia.
Interestingly, ATP depletion takes place very rapidly in anoxia-intolerant vertebrates, whereas
ATP depletion in these intermediate animals takes place over hours instead of minutes.
Therefore upon anoxic exposure this the leopard frog (R. pipiens) is essentially slowly dying.
In the anoxia-intermediate frog, homeostatic maintenance of intracellular potassium significantly delays the onset of depolarisation (Knickerbocker and Lutz, 2001). The maintenance of potassium is due to a retarded leakage of potassium pumps (50% decrease in potassium leakage), which reduces the rate of extracellular potassium accumulation, prolonging depolarisation and conserving energy expenditure from the reduced activity of ion pumps. Similarly, anoxia-tolerant turtles show a 70% decrease in potassium leakage during events of anoxia (Pek and Lutz, 1997). The leopard frog has been observed to maintain potassium homeostasis for up to 3 hours as ATP levels slowly decline. When ATP levels fall to 35% of normoxic values, a threshold is reached and ion homeostasis is lost (Knickerbocker and Lutz, 2001) resulting in a massive release of inhibitory and excitatory neurotransmitters such as GABA and glutamate, respectively (Lutz and Reiners, 1997), similar to the anoxic catastrophe of the mammalian brain.
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Chapter 1: Literature review
One charcateristic that gives the leopard frog their intermediate anoxia-tolerance is their ability to undergo what appears to be metabolic depression. The leopard frog can undergo an overall metabolic depression primarily via hypofusion of skeletal muscles
(Donohoe and Boutilier, 1998; Donohoe et al., 1998). Wegner and Krause (1993) also reported evidence of a reduction in neuronal activity in the frog brain during the onset of early anoxia. Similarly, Okada and McDougal (1971) observed a suppression of action potentials.
Currently the mechanisms that control these reductions in metabolism and neuronal activity have not been identified. In the turtle brain, adenosine is released early and acts as a retaliatory metabolite to restore energy balance (Lutz and Nilsson, 1997; Sweeny, 1997).
However, in the leopard frog, adenosine is not released until after ATP has been almost completely depleted (Lutz and Reiners, 1997). In the turtle brain, adenosine plays an important role in channel arrest (Pek and Lutz, 1997), however adenosine does not appear to lower potassium leakage in the leopard frog (Pek and Lutz, 1998). Therefore, the role of adenosine in the leopard frog is currently unknown and this late release may be a pathological event in the leopard frog associated with energy failure.
1.7 Anoxia tolerant vertebrates
Some vertebrates have evolved strategies of surviving natural periods of hypoxic and anoxic exposure. Three of the most well studied examples of anoxia tolerance are the crucian carp (C. carassius) and the freshwater turtles, the painted turtle (Chrysemys picta) and the red-eared slider turtle (Trachemys scripta). From current literature, the two most significant defences against reduced oxygen availability are the down-regulation of energy consumption
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Chapter 1: Literature review and the up-regulation of energetic efficiency in ATP producing pathways (Lutz, 1992). In order to maintain energy balance during prolonged hypoxia and anoxia, the reduction in energy supply must be matched with a reduction in energy consumption. Anoxia-tolerant animals have been characterised to enter into an energy conserving state of metabolic depression or hypometabolism following the onset of anoxia (Lutz and Nilsson, 1997).
The brain of the crucian carp shows a 30-40% decrease in ATP turnover during prolonged periods of anoxia (Nilsson et al., 1993). Interestingly, the crucian carp maintains an active state during anoxia, which means some areas of the brain must remain metabolically active. The crucian carp uses neurotransmitters and neuromodulators to control and suppress the central nervous system and subsequent ATP consumption in specific areas of the brain such as the auditory and visual systems (Nilsson et al., 1993).
It has also been shown that adenosine levels in the crucian carp increase following anoxic exposure. Adenosine appears to play a role in metabolic depression by suppressing the release of the excitatory neurotransmitter, glutamate (Oshima, 1989; Nilsson et al., 1994). A neuronal inhibitor, -amino butyric acid (GABA), has been observed to increase in particular areas of the brain of the crucian carp during anoxia, while excitatory neurotransmitters such as glutamate remain low (Nilsson, 1992; Nilsson and Lutz, 1993). GABA also increases in the anoxia tolerant turtle brain to a much greater extent during anoxia and renders the turtle in a comatose state (Nilsson et al., 1990). Evidence also suggests ion channel pumping may be suppressed in the anoxia-tolerant turtle during prolonged anoxia. This involves the down regulation of the permeability of various neuronal ion channels including potassium, sodium and calcium (Lutz et al., 2003) and may yield a significant energy saving. Other additional
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Chapter 1: Literature review energy saving strategies include bradycardia, reduced cardiac output and ventilatory depression (Soderstrom et al., 1999; Routley et al., 2002).
The up-regulation of anaerobic metabolism in response to reduced oxygen availability has been well documented (Lutz and Nilsson, 1997). Anoxia-tolerant and -intolerant vertebrates alike both increase anaerobic metabolism in response to reductions in oxygen saturation. In anoxia-intolerant species, the up-regulation of anaerobic metabolism is limited, while anoxia-tolerant animals have been observed to possess a larger anaerobic capacity
(Lutz, 1992). The anoxia-tolerant turtle brain has much higher levels of lactate dehydrogenase and hexokinase than anoxia-intolerant vertebrates to increase its anaerobic capacity (Lutz,
1992).
Anoxia-tolerant vertebrates have been reported to possess significantly larger glycogen stores than anoxia-intolerant vertebrates. The crucian carp has the largest liver glycogen stores ever recorded for use during anoxic exposure (Hochachka and Somero,
1984). During anoxia, the crucian carp and turtle brain becomes hyperglycaemic, accompanied by an adenosine-mediated increase in cerebral blood flow (Nilsson et al., 1990;
Hylland et al., 1994). These glycogen stores and increases in glucose are thought to be involved in supplying fuel for up-regulated anaerobic metabolism during anoxia. Elevated adenosine-mediated cerebral blood flow thereby increases the delivery of these metabolic fuels during anoxia.
Accompanying these dramatic increases in anaerobic metabolism is the production of anaerobic by-products such as lactic acid. Elevated adenosine-mediated cerebral blood flow is also involved in removing these toxic anaerobic by-products from critical organs. However,
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Chapter 1: Literature review during periods of prolonged anoxia, accumulation of these by-products can quickly become toxic, which may disrupt the acid-base balance within the body. Johnston and Bernard (1983) observed that the crucian carp deals with these increases in anaerobic by-products by possessing high levels of alcohol dehydrogenase, which converts lactate to ethanol in the muscle tissues. The ethanol diffuses out of the body through the gills and eliminates the accumulating lactic acid, therefore protecting the acid-base balance (Johnston and Bernard,
1983), which allows these species to remain relatively active during prolonged anoxia. This conversion of lactate to ethanol has only been observed in the crucian carp and the less anoxia-tolerant goldfish (Carassius auratus).
The anoxia-tolerant turtle undergoes a period of up-regulation of anaerobic metabolism. However, as the turtle cannot eliminate lactic acid accumulation, anaerobic metabolism is highly regulated to reduce the incidence of toxic accumulation and disruption to the acid-base balance (Lutz and Nilsson, 1997). Anaerobic metabolism can therefore not sustain ATP levels during prolonged periods of anoxia and the anoxia-tolerant turtle undergoes a significant metabolic depression in order to reduce ATP consumption (Nilsson et al., 1990). Reduced ATP demand therefore minimises the rate of anaerobic metabolism required to supply sufficient ATP and subsequently reduce lactic acid accumulation. This hypometabolism renders the turtle in a comatose state, however over prolonged periods of anoxia, lactate levels within the body rise dramatically high (Ultsch and Jackson, 1982). The turtle therefore possesses a significant buffering capacity to maintain the acid-base balance during these times (Jackson and Heisler, 1983). Within the anoxia-tolerant turtles, intracellular and extracellular buffering is effectively due to high concentrations of bicarbonate and non-bicarbonate buffers, such as calcium and magnesium. Additionally, the turtle has also been observed to possess significant bicarbonate sources in the pericardial and
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Chapter 1: Literature review peritoneal fluids. Finally, a predominant source of additional buffering has recently been observed from the turtle skeleton and shell (Jackson, 2000).
Maintaining the energy balance within the body is the key requirement to surviving prolonged periods of anoxic exposure. Anoxia-tolerant species have developed a number of different strategies of dealing with these stressful conditions. The two most significant defence strategies are the reduction of ATP requirements and the up-regulation of anaerobic
ATP producing pathways and dealing with their toxic effects.
1.8 The effect of temperature on anoxia tolerance
Teleosts in the genus Carassius and freshwater turtles in the genera Chrysemys and
Trachemys (Lutz and Nilsson, 1997) reside in temperate environments and have evolved their anoxia tolerance in response to over-wintering in freshwater at temperatures close to 0C. The crucian carp and freshwater turtle can survive for several months in anoxia at temperatures of less than 10C. However, as temperature increases (20-25C), the anoxic survival time is significantly reduced to a couple of days due to a subsequent increase in metabolic rate (Lutz and Nilsson, 1997).
Some vertebrates have evolved a substantial tolerance to severe hypoxia at high temperatures. In freshwater environments, some African cichlids, such as tilapia
(Oreochromis niloticus) are renowned for their hypoxia tolerance at high temperatures as an adaptation to hypoxic exposure in tropical lakes (Verheyen et al., 1994; Chapman et al.,
1995). Similarly, many freshwater teleosts in the Amazon basin are also exposed to natural
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Chapter 1: Literature review periods of hypoxia (Muusze et al., 1998). Investigation into the strategies employed by these freshwater teleosts to survive prolonged periods of hypoxia include morphological and anatomical adaptations (e.g. air breathing), behavioural adapatations (e.g. avoidance and aquatic surface respiration) and physiological adaptations (e.g. metabolic depression and elevated anaerobic metabolism).
In tropical marine environments a number of habitats have been reported as being periodically hypoxic. These include abyssal depths, mangrove swamps, seagrass beds, and coral reef flats. Within these environments, a number of marine teleosts have been reported to possess some degree of hypoxia tolerance. Ultsch et al. (1981) reported the toadfish (Opsanus tau) can tolerate hypoxia, but not anoxia, at high temperatures, while Nilsson and Ostlund-
Nilsson (2004) and Nilsson et al. (2004) reported hypoxia tolerance in approximately 31 teleost species across seven families, inhabiting coral reef flats. Within elasmobranchs, the torpedo ray (Torpedo marmorata) has demonstrated a tolerance to severe hypoxia in temperate conditions (Hughes and Johnston, 1978), however the epaulette shark
(Hemiscyllium ocellatum) has previously been the only elasmobranch species reported to possess a significant tolerance to anoxia at tropical temperatures.
1.9 Tropical hypoxia on coral reef flats
Fringing reef crests protect islands and coral cays, such as Heron Island in the
Capricorn Bunker Group, from the surrounding ocean currents. During high tides, highly oxygenated oceanic water is able to consume the height of the reef crest allowing circulation throughout the reef lagoon. During periods of low tide, the reef crest obstructs this oxygenated water flow from entering the reef lagoon, resulting in an isolated pool of water.
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Chapter 1: Literature review
During diurnal low tides, photosynthetic algae, such as macro algae and symbiotic zooxanthellea within corals produce significant levels of oxygen to sustain a normoxic environment. However, during nocturnal low tides, respiration by coral polyps and trapped fauna quickly consumes the dissolved oxygen, creating a hypoxic environment (from 6.8
-1 -1 mgO2. L to 2.1 mgO2. L ) (Wise et al., 1998) (Figure 1.1). It is within this extreme environment that the epaulette shark maintains its normal nocturnal activity (Last and
Stevens, 1994; Wise et al., 1998).
High Tide
Water Level Oceanic Water
Reef Flat Reef Crest A) High tide, day
Low Tide Water
Level Oceanic Water
Reef Flat Reef Crest B) Low tide, day
Low Tide Water Hypoxic reef lagoon Level Oceanic Water
Reef Flat Reef Crest C) Low tide, Night
Figure 1.1. Tropical hypoxia on coral reef flats. A) The reef flat is oxygenated by oceanic water during diurnal high tide. B) Photosynthetic algae and zooxanthellae oxygenate the reef flat during diurnal low tide. C) Respiration of resident fauna on the reef flat creates a hypoxic environment during nocturnal low tides.
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Chapter 1: Literature review
1.10 The epaulette shark (Hemiscyllium ocellatum)
The epaulette shark (H. ocellatum) is a benthic elasmobranch within the family
Hemiscylliidae (Last and Stevens, 1994) (Figure 1.2). It is a long, slender shark characterised by a light tan colouration, widely spaced dark spots over the body and a large black ocellus above each pectoral fin. This species has a broad distribution predominantly in the tropical regions of New Guinea and northern Australia (Last and Stevens, 1994). The epaulette shark is a nocturnal feeder common in coral reefs and on shallow reef platforms. It reaches an average size of approximately 107 cm in total length, reaching sexual maturity at around 60 cm in total length.
A B
Figure 1.2. The epaulette shark (H. ocellatum). A) Juvenile. B) Adult.
1.10.1 Anoxia tolerance of the epaulette shark
Wise et al. (1998) demonstrated that the epaulette shark is tolerant to mild and severe cyclic exposures to hypoxia, which has been proposed to be an adaptation to the repeated occurrence of hypoxia in its natural habitat (Routley et al., 2002; Nilsson and Renshaw,
2004). Within elasmobranchs, the epaulette shark (H. ocellatum) has previously been the only species reported to possess a significant tolerance to severe and repeated hypoxia and even anoxia at temperatures of up to 28C for up to 46 minutes (Renshaw et al., 2002).
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Chapter 1: Literature review
Protective mechanisms elicited in the brain by hypoxic and anoxic insult have been extensively studied in the epaulette shark. During exposure to moderate hypoxic insult, increased gill perfusion and increased ventilation frequency provides short-term tolerance via increasing the oxygen uptake at the respiratory surfaces. Under more severe hypoxia, anaerobic metabolism is up-regulated to facilitate the lack of aerobic ATP production as oxygen levels decline further (Wise et al., 1998). During exposure to extreme hypoxia (2.2
-1 mg O2 l at 25ºC) ventilatory depression, increased anaerobic metabolism (Routley et al.,
2002), and bradycardia (Soderstrom et al., 1999) occur. The onset of these protective mechanisms is closely linked to the onset of neuronal hypometabolism (Mulvey and
Renshaw, 2000), in particular of motor nuclei, indicated by a loss of the animals righting reflex, and some sensory nuclei in the brain (Nilsson and Renshaw, 2004). Renshaw et al.
(2002) observed that animals treated with the adenosine antagonist, aminophylline, had an extended time to loss of righting reflex (LRR) during anoxia compared to control groups, providing evidence of adenosine as a metabolic depressant in the epaulette shark. These mechanisms may provide protection from ATP depletion during severe hypoxia and anoxia and a tolerance to oxidative stress upon re-oxygenation.
During deep metabolic depression, the epaulette shark reduces brain function, demonstrated by neuronal metabolism in motor but not sensory regions (Mulvey and
Renshaw, 2000) to conserve ATP and subsequently this could be expected to extend the duration of cellular homeostasis and prolong survival. Neuronal hypometabolism in selective brain areas aids in maintaining ATP levels and reducing neuronal depolarisation during hypoxia and anoxia. No evidence of neuronal apoptosis has been reported in the epaulette shark following both hypoxia and anoxia (Renshaw and Dyson, 1999; Kerrisk and Renshaw, unpublished).
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Chapter 1: Literature review
Anoxia intermediate vertebrates display a prolonged slow death, leading towards the complete loss of ATP, ion homeostasis and finally the necrotic phenotype. The protective strategies observed in the epaulette shark indicate that this species has specialised mechanisms that reduce ATP expenditure and protect cellular structure and function during events of anoxic stress. These mechanisms allow the epaulette shark to prolong survival in anoxia and is therefore characterised as a true anoxia-tolerant vertebrate.
Like most ectothermic vertebrates, the metabolic rate of the epaulette shark is predominantly determined by ambient temperatures. Therefore, with increased tempature a subsequent increase in energy consumption occurs due to elevated metabolic rates. Although seasonal variation exists, the distribution and habitat of the epaulette shark in tropical reef lagoons exposes these animals to cyclic periods of severe hypoxia at high temperatures. While the effects of temperature have been well documented in the anoxia-tolerant carp and turtles, the effects of temperature on the anoxia tolerance of the epaulette shark had not been examined. Therefore, this study examined the anoxia tolerance of the epaulette shark in response to three naturally encountered temperatures (23C, 25C and 27C).
AIM 1: Characterise the affect of temperature on the anoxia tolerance of the epaulette
shark.
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Chapter 1: Literature review
1.11 The grey carpet shark (Chiloscyllium punctatum)
The grey carpet shark (C. punctatum) is a close relative to the epaulette shark, both residing in the family Hemiscylliidae (Last and Stevens, 1994) (Figure 1.2). The grey carpet shark has a similar distribution to the epaulette shark. It is locally found in Australian waters from Moreton Bay (Queensland), throughout northern Australian waters to Shark Bay
(Western Australia) (Last and Stevens, 1994). They commonly frequent seagrass beds, sandy/muddy bottoms and reef flats. The grey carpet shark is an oviparous shark (Last and
Stevens, 1994), which displays age dimorphism. The neonatal and juvenile animals have a cream colouration with dark brown/black dorsal bands running perpendicular to the body midline. At approximately 30-40 cm in total length, a gradual colour metamorphosis occurs.
Adults appear distinctly different with a brownish/grey colouration on the dorsal surface and pale brown ventrally.
A b)B
Figure 1.2. The grey carpet shark (C. punctatum). A) Juvenile. B) Adult.
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Chapter 1: Literature review
1.11.1 Anoxia tolerance of the grey carpet shark
This study is the first to examine whether the grey carpet shark (C. punctatum) is also tolerant to anoxia. The grey carpet shark has been reported to inhabit a wide range of habitats however it is uncommon on hypoxic reef flats. Therefore, the grey carpet shark was assumed to possess limited tolerance to declines in oxygen saturation. However anecdotal evidence from fishermen prompted Last and Stevens (1994) to report a possible tolerance of this species to survive out of water for considerable periods. Interestingly, grey carpet sharks have been occasionally observed on coral reef flats during hypoxic low tides (personal observation). Therefore, in order to use the grey carpet shark as a comparative species to the epaulette shark, it is important to understand and quantify the anoxia tolerance of this animal.
AIM 2: Quantify the anoxia tolerance of the grey carpet shark by monitoring ventilation frequency and the time until the loss of righting reflex under anoxic
conditions.
The grey carpet shark has a broad geographical distribution range, from tropical to subtropical climates. The broad distribution of the grey carpet shark exposes these vertebrates to a broad range of ambient temperatures. However the effect of ambient temperature on the anoxia tolerance of the grey carpet shark had not been examined.
AIM 3: Characterise the affect of temperature on the anoxia tolerance of the grey carpet shark.
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Chapter 1: Literature review
1.12 Anoxia and haematology
The blood plays a significant role in the physiological response of the body to events of stress. In teleost fishes, changes in haematological parameters occur following exposure to stressors such as capture and handling (Bourne, 1986; Frisch and Anderson, 2005), asphyxiation (Soivio et al., 1974) exercise (Yamamoto, 1987; Milligan and Girard, 1993;
Wells and Baldwin, 2006), surgery (Cooper and Morris, 1998), changes in water quality
(Avilez et al., 2004) and hypoxia (Soivio et al., 1974; Yamamoto et al., 1983; Affonso et al.,
2002; Baker et al., 2005). These stressors have been shown to elicit changes in hematocrit, haemoglobin and erythrocyte concentrations, which increase the oxygen affinity, absorption and carrying capacity of the blood. These changes in haematological parameters are caused by the movement of fluid from the plasma (Swift and Lloyd, 1974; Nikinmaa and Tervonen,
2004; Tervonen et al., 2006), increased cell volume (Fievet et al., 1987; Fuchs and Albers,
1988; Salama and Nikinmaa, 1990) and also the release of red blood cells (RBCs) from a storage reservoir (Yamanmoto et al., 1980; Yamanmoto et al., 1983; Yamamoto, 1987; Kita and Itazawa, 1989; Yamamoto and Itazawa, 1989; Pearson and Stevens, 1991).
During periods of reduced oxygen availability the blood acts to increase oxygen absorption and delivery, increase the circulation of metabolic fuels and remove metabolic waste products. Previous studies on elasmobranchs have reported no changes in haematological parameters in response to declines in oxygen saturation (Hughes and
Johnston, 1978; Butler et al., 1979; Baldwin and Wells, 1990; Wise et al., 1998; Routley et al., 2002). These studies however, only reported changes in blood parameters during varying severities of hypoxia. In the anoxia-tolerant epaulette shark, no changes in haematocrit were observed following an episode of hypoxia (Routley et al., 2002), although the haematological
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Chapter 1: Literature review response to a period of anoxia had not been investigated in this species. Furthermore, no studies have investigated the re-oxygenation recovery response of elasmobranchs to anoxic exposure.
AIM 4: Characterise the haematological response to anoxia and re-oxygenation in the epaulette shark and the grey carpet shark
1.13 Hypoxia induced erythrocyte concentrations
In response to a number of stressful stimuli, many vertebrates show an increase in the number of erythrocytes (RBCs) per unit volume of blood. This can be caused by (i) an increase in the production of RBCs via erythropoietic pathways (Soldatov, 1996), (ii) a decrease in plasma volume (haemoconcentration) due to fluid shifts and/or increased diuresis
(Swift and Lloyd, 1974; Nikinmaa and Tervonen, 2004; Tervonen et al., 2006), and finally
(iii) liberation of RBC from storage organs into the circulation (Yamamoto et al., 1983;
Pearson and Stevens, 1991). In response to periods of reduced oxygen availability, many vertebrates have been observed to show an increase in the number of RBCs per unit volume of blood via one or a combination of the mechanisms mentioned above.
1.13.1 Erythropoiesis
The blood constitutes only 2% of the total body weight of fish as opposed to 7-8% in mammals (Harms et al., 2002). In higher vertebrates, the primary haematopoietic site is the bone marrow, while lymphatic tissues such as the thymus, spleen and lymph nodes are predominantly involved in white blood cell production (Fange and Pulsford, 1983). However, in elasmobranchs, the production site of blood is not fully understood, as cartilaginous fishes
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Chapter 1: Literature review do not possess bone marrow and the production of blood cells has been observed in a number of different tissues (Zapata, 1981). In elasmobranchs, lymphoidal tissue gives rise to all of the cellular constituents of the blood. Haematopoietic sites in elasmobranchs common with other vertebrates include the thymus and the spleen (Fange and Johansson-Sjobeck, 1975) with the addition of two unique organs called the Leydig and epigonal organs (Zapata, 1981; Mattisson and Fange, 1982). Walsh and Luer (2004) observed frequent mitotic activity in elasmobranch peripheral blood, supporting mitotic replication as well as maturation of erythrocytes in the peripheral blood of elasmobranchs (Saunders, 1966; Stokes and Firkin, 1971; Glomski et al.,
1992).
During events of hypoxia and anoxia, energy-consuming mechanisms are significantly reduced. The production of new RBCs is an extremely energetically expensive process.
Therefore, during periods of reduced oxygen availability, erythropoietic activity is often suppressed (Lai et al., 2006). Following re-oxygenation however, prolonged changes in RBC and haemoglobin concentrations have been attributed to increased erythropoietic activity.
Soldatov (1996) reported an increase in RBC division after 2 hours, and observed a 37% increase in RBC concentrations after 6 hours of hypoxia. Elevated erythropoietic activity following a hypoxic event increases the RBC and haemoglobin concentrations within the blood circulation, which subsequently increases the oxygen carrying capacity of the blood and acts as a protective mechanism against any additional hypoxic events.
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Chapter 1: Literature review
1.13.2 Haemoconcentration
Another mechanism accounting for an increase in RBC concentration is a reduction in plasma volume via haemoconcentration (Nikinmaa and Tervonen, 2004).
Haemoconcentration of the blood following hypoxia may be due to either increased diuresis or fluid shifts out of the blood plasma. Swift and Lloyd (1974) reported an increase in urine flow in response to hypoxia in rainbow trout, while Kirk (1974) observed water movement from the plasma into the tissues during hypoxic exposure in the channel catfish (Ictalurus punctatus). Tervonen et al. (2006) concluded that these increases in RBC numbers per unit volume of blood would increase the amount of oxygen carried by the blood in unit time. In elasmobranchs, Cross et al. (1969) reported that there was no change in renal function in response to hypercapnia in the anoxia-intolerant dogfish (Squalus acanthias). However, fluid shifts in response to severe hypoxia or anoxia, have not been reported in elasmobranchs.
1.13.3 Erythrocyte reservoirs
Within mammals, the spleen is involved in the filtering and destruction of abnormal cells, such as old and defective RBCs, the production of new erythrocytes, acting as a blood storage reservoir and the production of lymphocytes and antibodies in the immune response
(Barcroft and Barcroft, 1923). The spleen has been observed to contract and release RBC into the circulation in response to a variety of stimuli, including periods of severe exercise, trauma, physical constraint or confinement and oxygen deprivation (Barcroft and Stephens, 1927;
Froelich et al., 1988; Kuwahira et al., 1999). The adaptive significance of the splenic reservoir is to fine tune the blood respiratory system to short-term variations in metabolic rate via an increase in the oxygen carrying capacity of the blood.
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Chapter 1: Literature review
The spleens of teleosts and elasmobranchs were originally characterised as having insufficient muscularisation of the capsule and trabeculae to cause a splenic contraction, comparable to those of the mammalian spleen (Yoffey, 1929). However, the ability of the spleen to act as a RBC storage reservoir and the presence of a splenic contraction in response to metabolically demanding stimuli has now been well characterised in many teleost fishes
(Yamamoto et al., 1980; Yamamoto et al., 1983; Yamamoto, 1987; Kita and Itazawa, 1989;
Yamamoto and Itazawa, 1989; Pearson and Stevens, 1991).
In response to stimuli such as exercise and hypoxia, Yamamoto et al. (1980) and
Gallaugher et al. (1992) observed that the release of RBCs via splenic contraction was a graded response. Gallaugher et al. (1992) concluded that increased swimming performance in rainbow trout was attributed to the degree of splenic contraction and the subsequent increase in hematocrit. Therefore, it appears that the release of RBCs into the circulation during metabolically demanding events such as severe exercise and oxygen deprivation can provide a significant increase in the oxygen carry capacity of the blood.
1.13.4 The elasmobranch spleen
While it is known that the spleen of the dogfish (Scyliorhinus canicula) has a role in
RBC production (Fange and Johansson-Sjobeck, 1975), Butler et al. (1979) reported no changes in erythrocyte concentrations in response to hypoxia in the same species.
Consequently, Butler et al. (1979) suggested that elasmobranchs, in general, may not possess the ability to undergo a splenic contraction, a mechanism utilised in higher vertebrates.
Interestingly, Nilsson et al. (1975) observed a splenic contraction by catecholamines and
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Chapter 1: Literature review artificial nerve stimulation in both Squalus acanthias and Scyliorhinus canicula in perfused and isolated spleens. In contradiction to these findings, Opdyke and Opdyke (1971) observed no release of RBCs from the spleen in Squalus acanthias in response to electrical stimulation of the splenic pedicle or catecholamine infusion, supporting findings by Butler et al. (1979).
Therefore it appears that both these species possess the mechanisms to induce a splenic contraction by administering electrical and chemical stimulation, however, the ability of the elasmobranch spleen to act as a RBC reservoir or to contract in response to metabolically demanding stimuli, such as hypoxia or exercise has not been observed.
1.13.5 Epigonal and Leydig’s organ
Elasmobranchs have two accessory immunological structures not found in other vertebrates: the epigonal organ and Leydig's organ. The epigonal organ is an elongate, paired, pinkish-white structure located beneath the kidneys. Leydig's organ is located along the top and bottom of the oesophagus. The roles of the epigonal and Leydig’s organ are still largely controversial. Researchers generally agree on a predominantly granulopoietic role in both organs (Fange, 1968; Mattisson and Fange, 1982), however the erythropoietic function has only been studied in a very small number of animals. Many authors suggest that these organs are granulopoietic, but also produce lymphocytes (Bolton, 1926; Fange, 1968; Mattisson and
Fange, 1982) while others suggest an erythropoietic function (Matthews and Parker, 1950).
Fange and Johansson-Sjobeck (1975) observed erythropoiesis in the Leydig organ of splenectomized dogfish (Scylorhinus canicula). However, Zapata (1981) found no evidence of erythropoietic activity in either the epigonal or the Leydig organ in two species of elasmobranchs (Raja clavate and T. marmorata).
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Chapter 1: Literature review
Most shark species have both epigonal and Leydig's organs, but a few have one or the other. The presence of an epigonal and/or Leydig’s organ within either the epaulette shark or the grey carpet shark has not currently been examined. Neither organ have been observed to contain significant numbers of erythrocytes and even though the epigonal and Leydig’s organ may play a role in erythropoiesis, neither organ appears to acts as a RBC reservoir.
AIM 5: Investigate the presence erythrocyte reservoirs in the grey carpet shark in response to anoxic exposure
1.14 Plasma constituents
1.14.1 Glucose
Hypoxia tolerant turtles and teleost fishes elevate their blood glucose levels during periods of reduced oxygen availability (Lutz et al., 2003; Chippari-Gomes et al., 2005) in order to increase fuel supplies for up-regulated glycolytic production of ATP (Lutz et al.,
2003). In contrast, blood glucose concentrations in elasmobranchs such as the dogfish (S. canicula) and the epaulette shark remained constant during events of hypoxia (Butler et al.,
1979; Routley et al., 2002). It has therefore been suggested that elasmobranchs lack the ability to increase blood glucose levels in response to hypoxic exposure (Butler et al., 1979; Routley et al., 2002).
Interestingly, while Butler et al. (1979) and Routley et al. (2002) reported increases in plasma lactate concentrations, signifying the up-regulation of anaerobic metabolism, constant plasma glucose concentrations were observed throughout hypoxic exposure. Therefore this constant glucose state may simply reflect a highly sensitive release to balance glucose levels.
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Chapter 1: Literature review
Under complete anoxia, a heavier burden is placed on the metabolic breakdown of glucose by anaerobic glycolysis and therefore, may require an increased release of glucose into the blood.
This study will monitor blood glucose levels in response to anoxia and re-oxygenation in the epaulette shark and the grey carpet shark, which has not previously been examined.
1.14.2 Lactate
Lactic acid is a metabolic endpoint of anaerobic metabolism and can be used to provide a measure of the severity of hypoxic and anoxic insult. Lactic acid production significantly increases as anaerobic glycolysis is up-regulated during low oxygen saturation.
The blood acts to remove anaerobic by-products, such as lactic acid, from the tissues to reduce the risk of toxic accumulation. Within the blood, lactic acid dissociates into a lactate anion and H+, which can cause a reduction in pH of the extracellular fluid. Acidosis of the extracellular fluid quickly results in a decline in intracellular pH of RBCs. This may cause deleterious effects such as a loss of cellular ATP, contraction of the RBC membrane skeleton, cell swelling (Skelton et al., 1998) and decreased RBC deformability, subsequently increasing blood viscosity (Reinhart et al., 2002).
In elasmobranchs, lactate concentrations rise quickly when they are exposed to acute stressors such as capture and handling (Piiper and Baumgarten, 1968; Piiper et al., 1972; Cliff and Thurman, 1984; Hoffmayer and Parsons, 2001), fasting and confinement (Martini, 1974), transport (Cliff and Thurman, 1984) and hypoxia (Hughes and Johnston, 1978; Wise et al.,
1998). These increases in plasma lactates are attributed to increases in anaerobic metabolism during stress. The lactate concentrations of both the epaulette shark (Wise et al., 1998) and the grey carpet shark (Ulyate, unpublished) have been measured in response to graded
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Chapter 1: Literature review increases in hypoxia. In both these studies an increase in hypoxic insult resulted in a graded increase in plasma lactate concentration. These findings suggest that there is a correlation between hypoxic exposure and the level of anaerobic metabolism. However the level of lactate production in response to a more severe stress such as anoxia has not been observed in these two species of elasmobranchs.
AIM 6: Characterise the changes plasma glucose and lactate in response to anoxia and re-oxygenation in the epaulette shark and the grey carpet shark
1.14.3 Plasma electrolytes
Plasma electrolyte concentrations have been repeatedly measured in vertebrates following hypoxic and anoxic exposure. Changes in plasma electrolytes have been reported to have protective and degenerative implications for cellular survival during events of reduced oxygen availability. Changes in plasma electrolyte concentrations have been reported to be attributed to (i) a loss of transmembrane ion homeostasis, (ii) elevated blood buffering and
(iii) endocrine activation of ion channels to induce cell swelling. In response to periods of anoxia, vertebrates have been observed to show significant changes in plasma electrolyte concentrations as a consequence of one or a combination of the mechanisms mentioned above.
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Chapter 1: Literature review
A) Loss of ion homeostasis
In anoxia-tolerant vertebrates, such as the teleosts in the genus Carassius and freshwater turtles in the genera Chrysemys and Trachemys, plasma electrolytes have been observed to be involved in a number of physiological functions during anoxic exposure.
During prolonged anoxia, ATP levels fall and are insufficient to supply energy demand (as described above; page 3). In brief, as ATP levels are exhausted, ion homeostasis is jeopardised and the efficiency of ion pumps is compromised, resulting in a net movement of ions across the cell membrane down their concentration gradients (Lutz, 1992; Wasser and
Heisler, 1997). A loss in ion homeostasis is characterised by an increase in extracellular potassium, caused by potassium diffusing out of the cell, and intracellular sodium, caused by diffusion into the cell, resulting in a cellular depolarisation (Wasser and Heisler, 1997).
Depolarisation and further decreases in intracellular ATP results in a failure of calcium regulating mechanisms, including energy dependant calcium exclusion and the release of bound calcium from the endoplasmic reticulum (Lutz, 1992). Cellular depolarisation is accompanied by the influx of other ions, such as chloride, following sodium ions into the cell along its electrical gradient. This influx is then accompanied by osmotically driven water, which causes cell swelling that can lead to structural cell damage and necrotic cell death (Lutz, 1992). Jackson and Heisler (1982) and Jackson and Ultsch (1982) reported a decrease in plasma sodium and an increase in plasma potassium concentrations following prolonged anoxia in C. picta bellii. Jackson and Heisler (1983) also reported a reduction in chloride concentrations along with an increase in potassium concentrations within the plasma.
Taken together, these observations support a loss of transmembrane ion gradients, characterising the disruption of ionic homeostasis.
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Chapter 1: Literature review
B) Erythrocyte volume
It has been proposed that cell swelling in response to hypoxia and anoxia is predominantly attributed to either a fall in extracellular pH or the loss of Na+/K+ channel function, due to depleted ATP levels, leading to the accumulation of intracellular sodium (as mentioned above). This influx in sodium is followed by chloride, which causes an osmotic influx in water resulting in cell swelling (Wood and Perry, 1985). Within teleost fishes, RBC swelling is caused by the activation of β-adrenergic Na+/H+ exchangers in the RBC membrane
(Fievet et al., 1987). The accumulation of anaerobic metabolites such as H+, caused by the up- regulation of anaerobic metabolism, results in a decrease in the RBC intracellular pH (Jensen,
2004). The Na+/H+ exchanger is mediated by an increase in catecholamines (noradrenaline and adrenaline) in the blood, which activates the release of H+ from the RBC in exchange for extracellular sodium (Fievet et al., 1987; Chiocchia and Motais, 1989; Salama and Nikinmaa,
1990). The elimination of intracellular H+ results in an extracellular acidification and an intracellular alkalisation, thereby protecting and maintaining the intracellular pH.
An increase in plasma catecholamines resulting in cell swelling has been well characterised in response to reduced oxygen availability in teleost fishes (Fievet et al., 1987;
Chiocchia and Motais, 1989; Salama and Nikinmaa, 1990). However, Tufts and Randall
(1989) and Wood et al. (1994) reported the absence of a detectable β-adrenergic Na+/H+ exchanger in the anoxia-intolerant dogfish (S. canicula). Perry and Gilmour (1996) also reported no change in catecholamine level in the dogfish (Squalus acanthias) in response to hypoxia. However the catecholamine-stimulated activation of H+/Na+ exchangers in an anoxia-tolerant elasmobranch such as the epaulette shark had not been examined.
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C) Plasma buffers
While activation of β-adrenergic Na+/H+ exchangers may protect intracellular pH, some anoxia-tolerant vertebrates also have specialised mechanisms to maintain the acid-base balance of the blood. Within the anoxia-tolerant turtles, intracellular and extracellular buffering is effectively due to high concentrations of bicarbonate and non-bicarbonate buffers, such as calcium and magnesium. Jackson (2000) reported significant bicarbonate sources in the pericardial and peritoneal fluids and additional non-bicarbonate buffering (calcium and magnesium) from the turtle skeleton and shell. Jackson and Heisler (1983) also reported an increase in plasma calcium and magnesium concentrations, which act as non-bicarbonate buffers responsible for maintaining pH levels, reducing acidification during events of anoxia.
In elasmobranchs, the effects of low oxygen saturation on plasma electrolytes have been little studied.
AIM 7: Characterise changes in plasma electrolytes in response to anoxia and re-
oxygenation in the epaulette shark and the grey carpet shark.
1.15 Heat shock proteins
Heat shock proteins (HSPs) were first observed as a result of heat stress, however subsequent studies have observed HSPs to be synthesised in response to a wide range of physiological and environmental stressors within a wide range of animals and tissues. Not all
HSPs are stress inducible, however, those that are respond to stressors such as exposure to pollutants, toxins, heavy metals and changes in pH, temperature, salinity, hypoxia, cellular
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Chapter 1: Literature review energy depletion and also psychological stress such as exposure to predators, pathogens, and overcrowding (Feder and Hofman, 1999).
During cellular stress events, HSPs play a number of protective roles within the cell.
Stress inducible heat shock proteins predominantly act as molecular chaperones, binding to proteins and stabilising their structure (Feder and Hofman, 1999). Heat shock proteins are also involved in down-regulating protein synthesis and assisting in the repair of damaged proteins by participating in the refolding process and inhibiting abnormal protein interactions (Craig,
1985). Furthermore, Marx (1983) observed HSPs within the nucleus and suggested HSP involvement in DNA and RNA protection during cellular stress.
HSPs exist in constitutive form at all times and are involved in maintaining cellular homeostasis by assisting in protein folding, cellular repair and protein transport (Latchman,
1999). The common mode of action of stressors towards the induction of HSP synthesis appears to be that they are all proteotoxic, resulting in damage to proteins. Therefore, the HSP stress response is not the primary response, but a secondary consequence to toxicity damages following alteration of cellular function.
1.15.1 Heat shock protein 70 kDa
Heat Shock Protein 70 kDa (Hsp70) is the most widely understood family of the HSPs and its production is significantly up-regulated in response to a broad array of stressors.
Recent research has demonstrated that constitutive levels of Hsp70 are present within cells throughout all stages of normal cell function and maintenance (Iwama et al., 1999). Hsp70 is found in both the nucleus and the cytoplasm of the cell, often in association with cytoskeleton
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Chapter 1: Literature review filaments (Marx, 1983). However, controversy still exists as to whether Hsp70 is also present within all organelles.
Hsp70 has been identified in tissues such as skeletal and cardiac muscle, and organs such as the brain, liver, kidneys, gills, spleen, small intestine, bladder, adrenal gland and skin
(Craig, 1985; Airaksinen et al., 1998; Currie et al., 2000; Delaney and Klesius, 2004). Hsp70 has also been successfully measured in blood cells. Kohan et al. (1991) found that erythrocytes accumulated the greatest amount of HSP mRNA compared to gill and liver tissue. Furthermore, Currie et al. (2000) observed Hsp70 mRNA concentrations in erythrocytes reflected the concentrations found in other tissues such as the heart, brain and liver.
1.15.2 Hsp70 and hypoxia
Cells, and hence overall body function and maintenance, are dependent on protein function and integrity (Iwama et al., 1999). During events of cellular stress, these activities are disturbed or inhibited and it has been determined that Hsp70 has a major association with proteins and consequently cellular protection from such stress events. In mammals and teleosts, the Hsp70 response to oxygen deprivation has been well characterised. It has been repeatedly reported that stress proteins in the mammalian heart and brain increase in response to hypoxia and brief ischemic stress (Marber et al., 1995). Interestingly, the HSP response in teleosts is in many cases both species and tissue specific. Delaney and Klesius (2004) observed an increase in Hsp70 in response to hypoxia in the blood and brain of juvenile Nile tilapia (Oreochromis nilaticus), while rainbow trout and Chinook salmon (Oncorhynchus tshawytscha) cells or tissues did not show elevated Hsp70 levels in response to hypoxia
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Chapter 1: Literature review
(Currie and Tufts, 1997; Airaksinen et al., 1998; Gamperl et al., 1998; Currie et al., 1999).
The heat shock protein response has been little studied in elasmobranchs. Renshaw et al.
(2004) reported a significant increase in Hsp70 levels in the cerebellum of the epaulette shark in response to two sequential exposures of anoxia. Furthermore, upon the administration of the adenosine antagonist (aminophylline), the brain energy charge was significantly lower and the Hsp70 induced was signficantly higher. Therefore, the Hsp70 response in the cerebellum of the epaulette shark contributes as a neuronal protective response during anoxia.
Interestingly, no change in Hsp70 was observed in other areas of the brain, such as the brain stem, indicating a heterogenous HSP response within different regions of the brain following an anoxic challenge.
It is widely accepted that the main role of Hsp70 is to maintain homeostasis by assisting in protein folding, preventing inappropriate protein associations and playing a role in protein transport (Feder and Hofman, 1999; Latchman, 1999). It has therefore been suggested that species and tissue specific differences in Hsp70 levels are a consequence of the type and severity of stress imposed to an animal and its physiological protective response to these events. While the Hsp70 response has been characterised in the brain of the epaulette shark, the presence of an inducible Hsp70 response in other vital organs had not been examined.
Furthermore, the presence of a protective Hsp70 response in the grey carpet shark during anoxia had not previously been investigated. The present study therefore aims to investigate the Hsp70 response to anoxic stress and re-oxygenation in a number of different tissues in two closely related elasmobranch displaying differences in anoxia tolerance.
AIM 8: Characterise the Hsp70 response to anoxia in the epaulette shark and the grey carpet shark.
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Chapter 1: Literature review
1.16 References cited
Affonso EG, Polez VLP, Correa CF, Mazon AF, Araujo MRR, Moraes G, Rantin FT. 2002. Blood parameters and metabolites in the teleost fish Colossoma macropomum exposed to sulfide or hypoxia. Comp Biochem Physiol C 133:375-382. Airaksinen S, Råbergh CM, Sistonen L, Nikinmaa M. 1998. Effects of heat shock and hypoxia on protein synthesis in rainbow trout (Oncorhynchus mykiss) cells. J Exp Biol 201(17):2543-2551. Avilez IM, Altran AE, Aguiar LH, Moraes G. 2004. Hematological responses of the neotropical teleosts matrinxa (Brycon cephalus) to environmental nitrite. Comp Biochem Physiol 139:135-139. Baker DW, Wood AM, Kieffer JD. 2005. Juvenile Atlantic and shortnose sturgeons (Family Acipenseridae) have different hematological responses to acute environmental hypoxia. Physiol Biochem Zool 78:916-925. Baldwin J, Wells RMG. 1990. Oxygen transport potential in tropical elasmobranchs from the Great Barrier Reef: Relationship between haematology and blood viscosity. J Exp Mar Bio and Ecol 144:145-155. Barcroft J, Barcroft H. 1923. Observations on the taking up of carbon monoxide by the haemoglobin in the spleen. J Physiol 58:138-144. Barcroft J, Stephens JG. 1927. Observations upon size of the spleen. J Physiol 64:1-22. Bickler PE. 1992. Cerebral anoxia tolerance in turtle: regulation of intracellular calcium and pH. Am J Physiol 263:R1298-R1302. Bourne PK. 1986. Changes in haematological parameters associated with capture and captivity of the marine teleost, Pleuronectes platessa L. Comp Biochem Physiol A 85:435-43. Bolton LL. 1926. Observations on the anatomy of Hexanchus corinus: Note on the structure of the lymphoid organ (Organ of Leydig) and spleen of Hexanchus corinus. J Anat 61:40-63. Butler PJ, Taylor EW, Davison W. 1979. The effect of long term, moderate hypoxia on acid base balance, plasma catecholamines and possible anaerobic end products in the unrestrained dogfish Scyliorhinus canicula. J Comp Physiol B 132:297-303.
Clint Chapman 36
Chapter 1: Literature review
Chapman LJ, Kaufman LS, Chapman CA, Mckenzie FE. 1995. Hypoxia tolerance in twelve species of east African cichlids: Potential for low oxygen refugia in Lake Victoria. Conserv Biol 9(5):1274-1287. Chiocchia G, Motais R. 1989. Effect of catecholamine on deformability of red cells from trout: relative roles of cyclic AMP and cell volume. J Physiol (Paris) 412:321-332. Chippari-Gomes AR, Gomes LC, Lopes NP, Val AL, Almeida-Val VMF. 2005. Metabolic adjustments in two Amazonian cichlids exposed to hypoxia and anoxia. Comp Biochem Physiol B 141:347-355. Cliff G, Thurman GD. 1984. Pathological and physiological effects of stress during capture and transport in juvenile dusky shark, Carcharhinus obscurus. Comp Biochem. Physiol A 78:167-173. Cooper AR, Morris S. 1998. The blood respiratory, haematological, acid-base and ionic status of the Port Jackson shark, Heterodontus portusjacksoni, during recovery from anaesthesia and surgery: a comparison with sampling by direct caudal puncture. Comp Biochem Physiol 119:895-903. Craig EA. 1985. The heat shock response. CRC Crit Rev Biochem 18(3):239-280. Cross CE, Packer BS, Linta JM, MurdaughV, Robin ED. 1969. H+ buffering and excretion in response to acute hypercapnia in the dogfish Squalus acanthias. Am J Physiol 216:440-452. Currie S, Tufts BL, Moyes CD. 1999. Influence of bioenergetic stress on heat shock protein gene expression in nucleated red blood cells of fish. Am J Physiol 276:R990-996. Currie S, Moyes CD, Tufts BL. 2000. The effects of heat shock and acclimation temperature on hsp70 and hsp30 mRNA expression in rainbow trout: in vivo and in vitro comparisons. J Fish Biol 56:398-408. Currie S, Tufts BL. 1997. Synthesis of stress protein 70 (hsp70) in rainbow trout (Oncorhynchus mykiss) red blood cells. J Exp Biol 200:607-614. Delaney MA, Klesius PH. 2004. Hypoxic conditions induce Hsp70 production in blood, brain and head kidney of juvenile Nile tilapia Oreochromis niloticus (L.). Aqua 236:633- 644. Donohoe PH, Boutilier RG. 1998. The protective effects of metabolic rate depression in hypoxic cold submerged frogs. Resp Pysiol 111:325-336.
Clint Chapman 37
Chapter 1: Literature review
Donohoe PH, West TG, Boutilier RG. 1998. Respiratory, metabolic, acid-base correlates of aerobic metabolic rate reduction in overwintering frogs. Am J Physiol 274:R704- R710. Fange R. 1968. The formation of eosinophilic granuloctyes in the oesophageal lymphomyeloid tissue of the elasmobranchs. Acta Zool (Stockholm) 49:155-161. Fange R, Johansson-Sjobeck ML. 1975. The effect of splenectomy on the hematology and on the activity of delta-aminolevulinic acid dehydratase (ALA-D) in hemopoietic tissues of the dogfish, Scyliorhinus canicula (Elasmobranchii). Comp Biochem Physiol A 52:577-80. Fange R, Pulsford A. 1983. Structural studies on lymphoyeloid tissues of the dogfish, Scyliorhinus canicula L. Cell Tissue Res 230:337-351. Feder ME, Hofmann GE. 1999. Heat-shock proteins, molecular chaperones, and the stress response. Annu Rev Physiol 61:243-282. Fievet B, Motais R, Thomas S. 1987. Role of adrenergic-dependent H+ release from red cells in acidosis induced by hypoxia in trout. Am J Physiol 252:R269-275. Frisch A, Anderson T. 2005. Physiological stress response to two species of coral trout (Plectropomus leopardus and Plectropomus maculatus). Comp Biochem Physio A 140:317–327. Froelich JW, Strauss HW, Moore RH, McKusick KA. 1988. Redistribution of visceral blood volume in upright exercise in healthy volunteers. J Nucl Med 29:1714-1718. Fuchs DA, Albers C. 1988. Effect of adrenaline and blood gas conditions on red cell volume and intra-erythrocytic electrolytes in the carp, Cyprinus carpio. J Exp Biol 137:457- 477. Gallaugher P, Axelsson M, Farrell AP. 1992. Swimming performance and haematological variables in splenectomised rainbow trout, Oncorhynchus mykiss. J Exp Biol 171:301- 314. Gamperl AK, Vijayan MM, Pereira C, Farrell AP. 1998. Beta-receptors and stress protein 70 expression in hypoxic myocardium of rainbow trout and chinook salmon. Am J Physiol 274:R428– R436. Glomski CA, Tamburlin J, Chainani M. 1992. The phylogenetic odyssey of the erythrocyte. III. Fish, the lower vertebrate experience. Histol Histopathol 7(3):501-528. Harms C, Ross T, Segars A. 2002. Plasma biochemistry reference values of wild bonnethead sharks, Sphyrna tiburo. Vet Clin Path 31(3):111-115.
Clint Chapman 38
Chapter 1: Literature review
Herbert CV, Jackson DC. 1985. Temperature effects on the responses to prolonged submergence in the turtle Chrysemys picta bellii. I: Blood acid-base and ionic changes during and following anoxic submergence. Physiol Zool 58(6):655-669. Hermes-Lima M, Storey KB. 1996. Relationship between anoxia exposure and antioxidant status in the frog Rana pipiens. Am J Physiol 271:R918-R925. Hochachka PW, Somero GN. 1984. Biochemical Adaptation. Princeton University Press, Princeton, NJ. Hoffmayer ER, Parsons GR. 2001. The physiological response to capture and handling stress in Atlantic sharpnose shark, Rhizoprionodon terraenovae. Fish Physiol Biochem 25:277-285. Hughes GM, Johnston IA. 1978. Some responses of the electric ray (Torpedo marmorata) to low ambient oxygen tensions. J Exp Biol 73:107-117. Hylland P, Nilsson GE, Lutz PL. 1994. Time course of anoxia induced increase in cerebral blood flow rate in turtles: evidence for a role of adenosine. J Cerebral Blood Flow Metab 14:877-881. Iwama GK, Vijayan MM, Forsyth RB, Ackerman PA. 1999. Heat shock proteins and physiological stress in fish. Am Zool 39:901-909. Jackson DC. 2000. Living without oxygen: lessons from the freshwater turtle. Comp Biochem Physiol 125:299-315. Jackson DC, Heisler N. 1982. Plasma ion balance of submerged anoxic turtles at 3C: the role of calcium lactate formation. Respir Physiol 49(2):159-174. Jackson DC, Heisler N. 1983. Intracellular and extracellular acid-base and electrolyte status of submerged anoxic turtles at 3C. Respir Physiol 53(2):187-201. Jackson DC, Ultsch GR. 1982. Long-term submergence at 3ºC of the turtle, Chrysemys picta bellii, in normoxic and severely hypoxic water. II. Extracellular ionic responses to extreme lactic acidosis. J Exp Biol 96:29-43. Jensen FB. 2004. Red blood cell pH, the Bohr effect, and other oxygenation-linked
phenomena in blood O2 and CO2 transport. Act Physiol Scand 182:215-227. Johnston IA, Bernard LM. 1983. Utilization of the ethanol pathway in carp following exposure to anoxia. J Exp Biol 104:73-78. Kirk WL. 1974. The effects of hypoxia on certain blood and tissue electrolytes of channel catfish, Ictalurus punctatus (Rafinesque). Trans Am Fish Soc 103:593-600.
Clint Chapman 39
Chapter 1: Literature review
Kita J, Itazawa Y. 1989. Release of erythrocytes from the spleen during exercise and splenic constriction by adrenaline infusion in the rainbow trout. Jap J Ichthyol 36:48-52. Knickerbocker DL, Lutz PL. 2001. Slow ATP loss and the defense of ion homeostasis in the anoxic frog brain. J Exp Biol 204:3547-3551. Koban M, Yup AA, Agellon LB, Powers DA. 1991. Molecular adaptation to environmental temperature: heat shock response of the eurythermal teleosts Fundulus heteroclitus. Mol Mar Biol Biotech 1:1-17. Kubasiak LA, Hernandez OM, Bishopric NH, Webster KA. 2002. Hypoxia and acidosis activate cardiac myocyte death through the Bcl-2 family protein BNIP3. Proc Natl Acad Sci 99(12):825–830. Kuwahira I, Kamiya U, Iwamoto T, Moue Y, Urano T, Ohta Y, Gonzalez NC. 1999. Splenic contraction-induced reversible increase in haemoglobin concentration in intermittent hypoxia. J Appl Physiol 86(1):181-187. Lai JC, Kakuta I, Mok HO, Rummer JL, Randall D. 2006. Effects of moderate and substantial hypoxia on erythropoietin levels in rainbow trout kidney and spleen. J Exp Biol 209:2734-2738. Last PR, Stevens JD. 1994. Sharks and Rays of Australia. CSIRO Division of Fisheries. Melbourne, Australia. Latchman DS. 1999. Stress proteins an overview. In: Stress Proteins. Handbook of Experimental Pharmacology. Ed Latchman, DS. Springer-Verlag. Berlin. Lu G, Mak YT, Wai SM, Kwong WH, Fang F, James A, Randall D, Yew DT. 2005. Hypoxia- induced differential apoptosis in the central nervous system of the sturgeon (Acipenser shrenckii). Micro Res Tech 68(5):258-263. Lutz PL. 1992. Mechanisms for anoxic survival in the anoxic vertebrate brain. Annu Rev Physiol 54:619-637. Lutz PL, Nilsson GE, Prentice H. 2003. The Brain without Oxygen: Causes of Failure and Molecular Mechanisms for Survival. 3rd edn. Boston: Kluwer Academic Publishers. Lutz PL, Nilsson GE. 1997. Contrasting strategies for anoxic brain survival-glycolysis up or down. J Exp Biol 200:411-419. Lutz PL, Reiners R. 1997. Survival of energy failure in the anoxic frog brain: delayed release of glutamate. J Exp Biol 200:2913-2917.
Clint Chapman 40
Chapter 1: Literature review
Marber MS, Mestril, R, Chi S, Sayen MR, Yellon DM, Dillmann WH. 1995. Overexpression of the rat inducible 70kD heat stress protein in a transgenic mouse increases the resistance of the heart to ischemic injury. J Clin Invest 95:1446-1456. Martini FH. 1974. Effects of capture and fasting confinement on an elasmobranch, Squalus acanthias. Ph.D thesis, Cornell University, New York. Marx JL. 1983. Surviving Heat Shock and Other Stresses. Heat Shock: From Bacteria to Man, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 251-253. Matthews LH, Parker HW. 1950. Note on the anatomy and biology of the basking shark (Cetorhinus maximus). J Proc Zool Soc Lond 120:535-376. Mattisson A, Fange R. 1982. The cellular structure of the Leydig organ in the shark, Etmopterus spinax (L.). Biol Bull 162:182-194. Milligan CL, Girard SS. 1993. Lactate metabolism in rainbow trout. J Exp Biol 180:175–193.
Mink JW, Blumenschine RJ, Adams DB. 1981. Ratio of central nervous system to body metabolism in vertebrates: its constancy and functional basis. Am J Physiol 241:R203- R212. Mulvey JM, Renshaw GMC. 2000. Neuronal oxidative hypometabolism in the brainstem of the epaulette shark (Hemiscyllium ocellatum) in response to hypoxic pre-conditioning. Neuroscience Letters 290:1-4. Muusze B, Marcon J, Van den Thillart G, Almeida-Val V. 1998. Hypoxia tolerance of Amazon fish repirometry and energy metabolism of the cichlid Astronotus ocellatus. Comp Biochem Physiol 120A:151-156. Nicholson DW, Thornberry NA. 1997. Caspases: killer proteases. Trends Biochem Sci 22(8):299-306. Nikinmaa M, Tervonen V. 2004. Regulation of blood haemoglobin concentration in hypoxic fish: Proceedings of the 7th International Symposium on Fish Physiology, Toxicology, and Water Quality. U.S. Environmental Protection Agency, Ecosystems Research Division. 243-252. Nilsson GE. 1992. Evidence for a role of GABA in metabolic depression during anoxia in crucian carp (Carassius carassius). J Exp Biol 164:243-259. Nilsson GE, Alfaro AA, Lutz PL. 1990. Changes in turtle brain neurotransmitters and related substances during anoxia. Am J Physiol 259:R376-R384.
Clint Chapman 41
Chapter 1: Literature review
Nilsson GE, Hobbs JP, Munday PL, Ostlund-Nilsson S. 2004. Coward or braveheart: extreme habitat fidelity through hypoxia tolerance in a coral dwelling goby. J Exp Biol 207:33-39. Nilsson GE, Hylland P, Lofman CO. 1994. Anoxia and adenosine induce increased cerebral blood flow rate in crucian carp. Am J Physiol 267:R590-R595. Nilsson GE, Lutz PL. 1993. The role of GABA in hypoxia tolerance, metabolic depression and hibernation – possible links to neurotransmitter evolution. Comp Biochem Physiol C 105:329-336. Nilsson GE, Ostlund-Nilsson S. 2004. Hypoxia in paradise: widespread hypoxia tolerance in coral reef fishes. Proc R Soc Lond Biol 271:30-33. Nilsson GE, Renshaw GMC. 2004. Hypoxia survival strategies in two fishes: extreme anoxia tolerance in the north European crucian carp and the natural hypoxic preconditioning in a coral-reef shark. J Exp Biol 207:3131-3139. Nilsson GE, Rosen P, Johansson D. 1993. Anoxic depression of spontaneous locomotor activity in crucian carp quantified by a computerized imaging technique. J Exp Biol 180:153-162. Nilsson S, Holmgren S, Grove DJ. 1975. Effects of drugs and nerve stimulation on the spleen and arteries of two species of dogfish, Scyliorhinus canicula and Squalus acanthias. Act Physiol Scand 95:219-30. Opdyke DF, Opdyke NE. 1971. Splenic responses to stimulation in Squalus acanthias. Am J Physiol 221:623-625. Okada Y, McDougal DB. 1971. Physiological and biochemical changes in frog sciatic nerve during anoxia and recovery. J Neurochem 18:2335-2353. Oshima N. 1989. Adensoine inhibits the release of neurotransmitters from melanosome- aggregating nerves of fish. Comp Biochem Physiol 93:207-211. Pearson MP, Stevens ED. 1991. Size and hematological impact of the splenic erythrocyte reservoir in rainbow trout, Oncorhynchus mykiss. Fish Physiol Biochem 9:39-50. Pek M, Lutz PL. 1997. Role of adenosine in “channel arrest” in the anoxic turtle brain. J Exp Biol 200:1913-1917. Pek M, Lutz PL. 1998. K+-ATP channel activation provides transient protection in anoxic turtle brain. Am J Physiol 44:R2023-R2027.
Clint Chapman 42
Chapter 1: Literature review
Perry S, Gilmour K. 1996. Consequences of catecholamine release on ventilation and blood oxygen transport during hypoxia and hypercapnia in an elasmobranch Squalus acanthias and a teleost Oncorhynchus mykiss. J Exp Biol 199:2105-2118. Piiper J, Baumgarten D. 1968. Analysis of brachial gaseous exchange in an elasmobranch fish: Scyliorhinus stellaris. J Physiol (Paris) 60:518. Piiper J, Meyer M, Drees, F. 1972. Hydrogen ion balance in the elasmobranch Scyliorhinus stellaris after exhausting activity. Resp Physiol 16, 290-303. Regula KM, Ens K, Kirshenbaum LA. 2003. Mitochondria-assisted cell suicide: a license to kill. J Mol Cell Cardiol 35(6):559-567. Reinhart WH, Gaudenz R, Walter R. 2002. Acidosis induced by lactate, pyruvate or HCl increases blood viscosity. J Critical Care 17(1):68-73. Renshaw GMC, Dyson SE. 1999. Increased nitric oxide synthase in the vasculature of the epaulette shark brain following hypoxia. Neurorep 10(8):1707-1712. Renshaw GMC, Kerrisk CB, Nilsson GE. 2002. The role of adenosine in the anoxic survival of the epaulette shark, Hemiscyllium ocellatum. Comp Biochem Physiol B 131:133- 141. Renshaw GM, Warburton J, Girjes A. 2004. Oxygen sensors and energy sensors act synergistically to achieve a graded alteration in gene expression: consequences for assessing the level of neuroprotection in response to stressors. Front Biosci 9:110-116. Routley MH, Nilsson GE, Renshaw GMC. 2002. Exposure to hypoxia primes the respiratory and metabolic responses of the epaulette shark to progressive hypoxia. Comp Biochem Physiol A 131:313-321. Salama A, Nikinmaa, M. 1990. Effect of oxygen tension on catecholamine-induced formation of cAMP and on swelling of carp red blood cells. Am J Physiol 259:C723-726. Saunders DC. 1966. Elasmobranch blood cells. Copeia 66(2):348-351. Schmidt-Nielsen K. 1983. Animal Physiology: Adaptation and Environment. 5th ed. University Press, Cambridge. Siesjo BK. 1978. Brain energy metabolism. Chichester: Wiley. Skelton MS, Kremer DE, Smith EW, Gladden LB. 1998. Lactate influx into red blood cells from trained and untrained human subjects. Med Sci Sports 30(4):536-542. Soderstrom V, Renshaw GMC, Nilsson GE. 1999. Brain blood flow and blood pressure during hypoxia in the epaulette shark Hemiscyllium ocellatum, a hypoxia-tolerant elasmobranch. J Exp Biol 202:829-835.
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Chapter 1: Literature review
Soivio A, Westman K, Nyholm K. 1974. Changes in haematocrit values in blood samples treated with and without oxygen; a comparative study with four salmonid species. J Fish Biol 6:763. Soldatov AA. 1996. The effect of hypoxia on red blood cells of flounder: a morphologic and autoadiographic study. J Fish Biol 48:321-328. Sollid J, De Angelis P, Gundersen K, Nilsson GE. 2003. Hypoxia induces adaptive and reversible gross morphological changes in crucian carp gills. J Exp Biol 206:3667- 3673. Stokes EE, Firkin BG. 1971. Studies of the peripheral blood of the Port Jackson shark (Heterodontus portusjacksoni) with particular reference to the thrombocyte. Br J Haematol 20:427-435. Sweeny MI. 1997. Neuroprotective effects of adenosine in cerebral ischemia: window of opportunity. Neurosci Biobehav Rev 21:207-217. Swift DJ, Lloyd R. 1974. Changes in urine flow rate and haematocrit value of rainbow trout Salmo gairdneri (Richardson) exposed to hypoxia. J Fish Biol 6:379-387. Tervonen V, Vuolteenaho O, Nikinmaa M. 2006. Haemoconcentration via diuresis in short- term hypoxia: A possible role for cardiac natriuretic peptide in rainbow trout. Comp Biochem Physiol A 144:86-92. Tufts BL, Randall DJ. 1989. The functional significance of adrenergic pH regulation in fish erythrocytes. Can J Zool 67:235-238. Ultsch GR, Jackson DC, Moalli R. 1981. Metabolic oxygen conformity among lower vertebrates: the toadfish revisited. J Comp Physiol 142:439-443. Ultsch GR, Jackson DC. 1982. Long term submergence at 3°C of the turtle Chrysemys picta belli, in normoxic and severely hypoxic water. In: Survival, Gas Exchange and Acid- base Status. J Exp Biol 96:11-28. Van Raaij MTM, Bakker E, Nieveen MC, Zirkzee H, Van den Tillart GEEJM. 1994. Energy status and free fatty acid patterns in tissues of common carp (Cyprinus carpio, L.) and rainbow trout (Oncorhynchus mykiss, L.) during severe oxygen restriction. Comp Biochem Physiol 109A:755-767. Verheyen E, Blust R, Decleir W. 1994. Metabolic rate, hypoxia tolerance and aquatic surface respiration of some lacustrine and riverine African cichlid fishes (Pisces: Chichlidae). Comp Biochem Physiol 107A:403-411.
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Chapter 1: Literature review
Walsh CJ, Luer CA. 2004. Elasmobranch hematology: Indentification of cell types and practical applications. In: Elasmobranch Husbandry Manual. Ed. Smith M, Warmolts D, Thoney D, Hueter R. Ohio Biological Survey, Columbus Ohio. Wasser JS, Heisler N. 1997. Effects of anoxia on intracellular free Ca2+ in isolated cardiomyocytes from turtles. Comp Biochem Physiol 116(4):305-312. Wegner G, Krause U. 1993. Environmental and exercise anaerobsis in frogs. In: Hochachka PW, Lutz PL, Sick TJ, Rosenthal M, Thillart G. Surviving Hypoxia. Boca Raton, FL. CRC Press:217-236. Wells RMG, Baldwin J. 2006. Plasma lactate and glucose flushes following burst swimming in silver trevally (Pseudocaranx dentex: Carangidae) support the "releaser" hypothesis. Comp Biochem Physiol A 143:347-352. Wise G, Mulvey JM, Renshaw GMC. 1998. Hypoxia tolerance in the epaulette shark (Hemiscyllium ocellatum). J Exp Zool 281:1-5. Wood CM, Perry SF. 1985. Respiratory, circulatory and metabolic adjustments to exercise in fish. In: Gilles R. Circulation, Respiratory, and Metabolism. Springer-Verlag, Berlin: 1-22. Wood CM, Perry SF, Walsh PJ, Thomas S. 1994. HCO3- dehydration by the blood of an elasmobranch in the absence of a Haldane effect. Resp Physiol 98:319-337. Yamamoto K. 1987. Contraction of spleen in exercised cyprinid. Comp Biochem Physiol A 87:1087-1097. Yamamoto K, Itazawa Y, Kobayashi H. 1980. Supply of erythrocytes into the circulating blood from the spleen of exercised fish. Comp Biochem Physiol A 65:5-11. Yamamoto K, Itazawa Y, Kobayashi H. 1983. Erythrocyte supply from the spleen and hemoconcentration in hypoxic yellowtail. Mar Biol 73:221-226. Yamamoto K, Itazawa I. 1989. Erythrocyte supply from the spleen of exercised carp. Comp Biochem Physiol A 92:139-144. Yoffey JM. 1929. A contribution to the study of the comparative histology and physiology of the spleen, with reference chiefly to its cellular constituents. J Anatomy 88(3):314- 346. Zapata A. 1981. Ultrastructure of elasmorbranch lymphoid tissue. 2. Leydig’s and epigoneal organs. Devel Comp Immun 5:43-52.
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CHAPTER TWO
The physiological tolerance of the grey carpet shark (Chiloscyllium punctatum) and the epaulette shark (Hemiscyllium ocellatum) to anoxic exposure at three seasonal temperatures
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Chapter 2: Anoxia tolerance at three different seasonal temperatures
2.1 Summary
The effect of seasonal temperature on the anoxia tolerance of two closely related elasmobranchs, the epaulette shark (Hemiscyllium ocellatum) and the grey carpet shark
(Chiloscyllium punctatum) were examined. The tolerance time, indicated by the time to loss of righting reflex (LRR), was examined in the adult epaulette shark as well as adult and juvenile grey carpet sharks in response to an open ended anoxic challenge at three seasonal temperatures, moderate, intermediate and high corresponding to 23C, 25C and 27C respectively. In a second series of experiments, the ventilation rates of both species were measured at two seasonal temperatures (23C or 25C) during a standardised (1.5 hour) anoxic challenge and during 2 hours of re-oxygenation in order to characterise the relationship between ventilatory depression and temperature for both shark species.
During the open ended anoxic challenge at three seasonal temperatures, the time to
LRR in the epaulette shark was 178.33 18.6 minutes at 23C, 112.50 20.7 minutes at 25C and 56.25 18.9 minutes at 27C. This indicated a significant reduction in the anoxia tolerance time of the epaulette shark at intermediate and high seasonal temperatures. The time to LRR in the adult grey carpet shark during the open ended anoxic challenge was only similar to that of the epaulette at 23C (172.0 11.0 minutes) and was significantly less than that of the epaulette shark at 25C (53.33 18.6 minutes) and 27C (26.86 5.8 minutes).
Additionally, juvenile grey carpet sharks had a significantly lower tolerance compared to adult grey carpet sharks, indicated by a reduced time to LRR at 23C (36.42 6.4 minutes),
25C (22.83 1.6 minutes) and 27C (14.0 1.5 minutes).
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Chapter 2: Anoxia tolerance at three different seasonal temperatures
During the standardised anoxic challenge at two seasonal temperatures, the epaulette shark entered into ventilatory depression significantly earlier at a higher temperature than at a lower one (60 minutes at 23C; 30 minutes at 25C). Throughout the subsequent 2 hours of re-oxygenation, epaulette sharks previously exposed to anoxia at 23C had no significant increase in ventilation rates above their pre-experimental levels or the level of the control group. However, after anoxic challenge at 25C followed by normoxic re-oxygenation at
25C epaulette sharks showed a significant increase in ventilation rates during re- oxygenation. In contrast, the grey carpet shark displayed no evidence of ventilatory depression during anoxia at 23C or 25C. However, during re-oxygenation, the grey carpet shark had significantly elevated ventilation rates above their pre-experimental levels and that of control animals at 23C and 25C.
These data demonstrate that the anoxia tolerance times of both the epaulette shark and the grey carpet shark were temperature dependent, with a significant reduction in the time to
LRR occurring at higher temperatures. Adult epaulette sharks had a significantly greater time to LRR at higher temperatures compared to adult grey carpet sharks. While juvenile grey carpet sharks appear to possess little tolerance to anoxic exposure, adult grey carpet sharks do possess a tolerance to anoxia at lower temperatures (23°C) that is similar to that of epaulette sharks, although neither juvenile nor adult grey carpet sharks enter into a ventilatory depression.
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Chapter 2: Anoxia tolerance at three different seasonal temperatures
2.2 Introduction
Diminished oxygen availability to cells and tissues (hypoxia) and the complete absence of oxygen (anoxia) are physiological stressors that can cause a significant and rapid reduction in the production and supply of ATP (Lutz and Nilsson, 1997). There follows a consequent loss of cellular homeostasis and membrane integrity, which subsequently leads to cellular degradation and death. Few vertebrate animals possess the ability to tolerate severe reductions in oxygen saturation. However, some aquatic vertebrates are periodically exposed to reduced oxygen conditions in their natural environment and as a consequence, have developed specialised protective mechanisms to survive prolonged periods of anoxia.
Anoxia-tolerant vertebrates survive prolonged periods without oxygen by maintaining/conserving brain ATP levels. This is partly accomplished via varying degrees of metabolic depression, indicated by a reduction in physical (gill ventilation and movement)
(Routley et al., 2002) and neuronal activity (Lutz and Nilsson, 1997). The strategies employed by hypoxia- and anoxia-tolerant animals differ in the extent to which such metabolic depression occurs (Lutz and Nilsson, 1997; Nilsson and Renshaw, 2004).
The most well studied examples of anoxia-tolerant vertebrates are the teleosts in the genus Carassius and freshwater turtles in the genera Chrysemys and Trachemys (Lutz and
Nilsson, 1997). These vertebrates reside in temperate environments and are exposed to anoxia during over-wintering in freshwater ponds and streams at extremely low temperatures. The anoxia-tolerant crucian carp (Carcassius carassius) and the freshwater turtles (Chrysemys picta and Trachemys scripta) can survive in anoxia for months at temperatures close to 0C, however as temperatures increase, the crucian carp and freshwater turtles survival times are
Clint Chapman 49
Chapter 2: Anoxia tolerance at three different seasonal temperatures reduced to days at room temperature (20C - 25C). This reduction in survival time has been attributed to a temperature induced increase in metabolic rate, depleting ATP supplies (Lutz and Nilsson, 1997).
Some tropical vertebrates have evolved a substantial tolerance to severe hypoxia at temperatures between 20C and 30C. In aquatic environments, teleosts such as the Oscar cichlid (Astronotus ocellatus), elephant-nose fish (Gnathonemus petersii) and toadfish
(Opsanus tau) (Ultsch et al., 1981) can tolerate hypoxia, but not anoxia, at high temperatures
(Nilsson, 1996). Within elasmobranchs, the torpedo ray (Torpedo marmorata) has been observed to tolerate severe hypoxia in temperate conditions for several hours (Hughes and
Johnston 1978), however, the epaulette shark (Hemiscyllium ocellatum) has previously been the only elasmobranch species reported to possess a significant tolerance to anoxia at tropical temperatures (Renshaw et al., 2002).
The anoxia tolerance of the epaulette shark provides an adaptive advantage to periodic exposure to severe hypoxic stress within its natural environment on coral reef flats (Wise et al. 1998; Renshaw et al., 2002). Following prolonged periods of hypoxia and anoxia, the epaulette shark reduces its energy consumption by undergoing ventilatory depression
(Routley et al., 2002), accompanied by bradycardia (Soderstrom et al., 1999) and neuronal hypometabolism (Mulvey and Renshaw, 2000). In response to anoxia, Renshaw et al. (2002) observed that the epaulette shark enters a phase of ventilatory depression and withstood an average of 46.3 minutes of anoxia at 28C before loosing it’s righting reflex, which was closely linked to the onset of neuronal hypometabolism (Mulvey and Renshaw, 2000).
However it was noted that outside the summer season, the time to loss of righting reflex
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Chapter 2: Anoxia tolerance at three different seasonal temperatures
(LRR) was sometimes in excess of 5 hours (Renshaw, personal communication). Thus, further investigation was required.
Recently, anoxia tolerance in another tropical elasmobranch, the grey carpet shark
(Chiloscyllium punctatum) has been reported (Chapman and Renshaw, in press, Chapter 3).
This species is a close relative to the epaulette shark inhabiting intertidal areas, including sandy and rocky substrates, seagrass areas as well as coral reefs (Last and Stevens, 1994).
Chapman and Renshaw (in press, Chapter 3) observed that this species tolerated anoxia for
1.5 hours at 24C, however the tolerance of this species to a more prolonged period of anoxia over a range of seasonal temperatures had not been examined.
Like most ectothermic vertebrates, the metabolic rate of the epaulette shark and the grey carpet shark is predominantly determined by ambient temperatures. Both species have a broad geographical distribution throughout tropical regions. The epaulette shark has a broad distribution, predominantly in the tropical regions of New Guinea and northern Australia, while the grey carpet shark is locally found in Australian waters from Moreton Bay
(Queensland), throughout northern Australian waters to Shark Bay (Western Australia) (Last and Stevens, 1994). This distribution exposes both species to seasonal fluctuations in ambient water termperatures ranging from 19C to 30C (Last and Stevens, 1994). High temperatures have a significant impact on the anoxia tolerance of ectothermic vertebrates due to an increase in temperature induced metabolic rate, which can significantly affect energy consumption during natural events of hypoxic and anoxic esposure (Schmidt-Neilsen, 1983; Lutz and
Nilsson, 1997; Carlson and Parsons, 1999). Therefore, the present study investigated the anoxia tolerance of the epaulette shark and the grey carpet shark at three temperatures most commonly encountered during spring (23-25C), autumn (23-25C) and summer (25-27C).
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Chapter 2: Anoxia tolerance at three different seasonal temperatures
The present study examines the anoxia tolerance of the grey carpet shark by measuring gill ventilation rates and the total time to LRR compared to the anoxia-tolerant epaulette shark. While the effects of temperature on metabolic rate in elasmobranchs have been reported in a number of species (Butler and Taylor, 1975; Du Preez et al., 1988; Hopkins and Cech, 1994; Carlson and Parsons, 1999; Lowe and Goldman, 2001), this is the first study to examine to effect of seasonal temperature on the anoxia tolerance of the epaulette shark and the grey carpet shark.
2.3 Materials and methods
2.3.1 Animal collection and age
Grey carpet sharks and epaulette sharks were supplied by UnderWater World
Aquarium (Mooloolaba, Sunshine Coast) and Sea World Aquarium (Main Beach, Gold
Coast). Additional sharks were collected from Heron Island in the Capricorn bunker group
(latitude 23° 27’ S, longitude 151° 55’ E) (GBRMPA Permit # GO4/12675.1) and from
Moreton Bay (latitude 27º 28’ 43’’ S, longitude 153º 23’ 84’’ E) (DPI&F Permit
#PRM38182I). Experiments requiring age determination were conducted only on captive bred animals from commercial aquaria. Epaulette sharks were approximately 3-4 years of age and had an average length of 66.4 7.4 cm and weight of 1.0 0.5 kg. Juvenile grey carpet sharks ranged in age from 1-3 months old (length, 27.8 1.6 cm; weight, 65.0 12.3 g), while adult grey carpet sharks were approximately 3-4 years in age (length, 102.2 10.3 cm; weight, 4.8
1.3 kg). All animals were kept in constant sea water flow through aquaria with no additional
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Chapter 2: Anoxia tolerance at three different seasonal temperatures light sources in their outside habitats, relying on a normal photoperiod. Food was withheld for
24 hours before experiments were performed.
2.3.2 Temperature
Experiments on wild caught animals were conducted throughout the spring, summer and autumn months to allow the animals to be naturally acclimated to experimental temperatures used in this study. Captive sharks were kept at a controlled temperature which mimicked the natural environment, reflecting a seasonal temperature cycle for these animals.
Experiments on captive animals were also conducted throughout the spring, summer and autumn months.
2.3.3 Anoxic exposure
Juvenile grey carpet sharks were exposed to anoxia or control treatments in 40 litre glass tanks, while adult grey carpet sharks and epaulette sharks were exposed to anoxia in 200 litre containers, all of which were covered in plastic wrap and covered with semi-permeable lids. Each tank was filled with normoxic seawater, which was circulated around the tank using a submersible 228-power head pump (CCC Pty. Ltd. Australia) to ensure uniform conditions throughout. Nitrogen gas was bubbled through the water to displace saturated oxygen and create an anoxic environment (experimental). Alternatively, air was bubbled through the water to maintain a normoxic environment (control). The dissolved oxygen concentration
[dO2] levels and temperature were measured and recorded each minute using TPS WP-90 oxygen probes (TPS Pty. Ltd. Australia). In the anoxic tanks, the [dO2] was maintained below
-1 -1 0.02 mg L whereas the [dO2] of the control tanks was maintained above 7.0 mg L . Animals
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Chapter 2: Anoxia tolerance at three different seasonal temperatures were closely pair matched for length and then randomly selected from the pair for either experimental (anoxia) or control (normoxia) treatment.
A) Experiment 1. Time to loss of righting reflex during an “open ended” anoxic exposure at three temperatures
Anoxia tolerance was measured at three seasonal temperatures (23C, 25C and 27C) by measuring the total time to LRR in an open ended anoxic treatment on 17 epaulette sharks
(n= 10 captive; n=7 wild) and 19 adult grey carpet sharks (n=13 captive; n=6 wild). Total time to LRR were also measured on 36 juvenile grey carpet sharks that were all bred and raised in captivity. Experimental animals were exposed to anoxia until the animals lost their righting reflex as described by Renshaw et al. (2002). Breifly, animals were quickly transferred to treatment tanks and their righting reflex was tested every 15 minutes after an initial 30 minute period. Righting reflex was lost when an animal could be placed onto its dorsal surface and not right itself.
B) Experiment 2. Ventilation rates during an “open ended” anoxic exposure at 23C
Ventilation rates were determined by counting contractions of the buccal chamber, which were recorded at rest prior to capture, immediately upon entering the treatment tank
(control or anoxic exposure), at 5 minutes and subsequently at 15 minute intervals for the remainder of the experiment. Ventilation rates of 14 epaulette sharks (n=10 captive; n=4 wild), 12 adult grey carpet sharks (n=8 captive; n=4 wild) and 17 juvenile grey carpet sharks
(captive) were measured in an open ended anoxic treatment, until the animals lost their righting reflex, at 23C. Control animals were placed into normoxic tanks for an equal time to
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Chapter 2: Anoxia tolerance at three different seasonal temperatures their anoxia matched counterparts. The Q10 temperature coefficient was measured using the rate of change in ventilation rates at 23C and 25C using the following equation:
Where R is the rate and T is the temperature (C).
C) Experiment 3. Ventilation rates during 1.5 hours of anoxia followed by 2 hours of normoxic re-oxygenation at 23°C or 25°C
Ventilation rates were measured prior to capture and handling to determine resting ventilation frequencies in 19 epaulette sharks (n=10 captive; n=9 wild) and 14 grey carpet sharks (n=10 captive; n=4 wild). Ventilation rates were measured immediately upon entering the treatment tank (control or anoxic exposure), at 5 minutes and subsequently at 15 minute intervals for a standardised 1.5 hour treatment at 23C and 25C. Animals were then placed back into separate 200 L normoxic tanks for a re-oxygenation recovery period. Following re- oxygenation, ventilation rates were recorded every 15 minutes for 2 hours to calculate the estimated oxygen demand. Since gill ventilation is affected by a number of factors, including dissolved gases in the environment and metabolic demand (Burleson and Smatresk, 2000, review; Sundin and Nilsson, 2002, review; Vulesevic et al., 2006), changes in gill ventilation rates act to increase oxygen absorption and carbon dioxide release. It has been shown that gill ventilation rates are closely correlated with oxygen absorption (Dixon and Renshaw, unpublished; Perry and Wood, 1989; Robinson and Davison, 2007), therefore the present study examined changes in ventilation rates above pre-experimental ventilation frequencies to determine the ventilatory demand and subsequent oxygen replenishment during re-
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Chapter 2: Anoxia tolerance at three different seasonal temperatures oxygenation in response to control and experimental treatment, which is referred to in the present study as the estimated oxygen demand.
2.3.4 Statistics
Data was analysed using a one-way ANOVA with a 95% confidence interval and a
Mixed Model Repeated Measures ANOVA with a posthoc adjustment (Bonferroni). Data are represented as mean standard deviation.
2.4 Results
2.4.1 Experiment 1. Time to loss of righting reflex during an “open ended” anoxic exposure at three temperatures
There were no significant differences between wild and captive sharks of either species
(or age) in the time to LRR in reponse to anoxia. The mean time to LRR of the epaulette shark was inversely proportional to temperature (Table 2.1; Figure 2.1). During anoxia at 23C, the mean time to LRR of the epaulette shark was 178.33 ( 18.6) minutes, which was significantly longer than the mean time to LRR in epaulette sharks exposed to anoxia at 25C
(112.50 20.7 minutes) (p<0.001) and at 27C (56.25 18.9 minutes). Similarly the mean time to LRR was significantly longer (p<0.001) during anoxia at 25C than 27C in the epaulette shark. The adult grey carpet shark also had significantly reduced mean times to LRR as temperature increased. At 23C, the mean time to LRR (172 11.0 minutes) was significantly longer than at 25C (53.33 18.6 minutes) and 27C (26.86 5.8 minutes)
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Chapter 2: Anoxia tolerance at three different seasonal temperatures
(p<0.001). Similarly, the mean time to LRR at 27C was significantly reduced from the mean time to LRR at 25C (p<0.001).
The mean time to LRR in the adult grey carpet shark paralleled the pattern observed for the epaulette shark, however with increasing temperatures the mean time to LRR was significantly reduced in the adult grey carpet shark compared to the epaulette shark. No significant differences were observed between the mean time to LRR in the epaulette shark and the grey carpet shark at 23C. At 25C the epaulette shark had a significantly longer mean time to LRR than the grey carpet shark (p<0.01). Similarly, the epaulette shark had a significantly longer mean time to LRR than the grey carpet shark at 27C (p<0.001).
Table 2.1. Time to loss of righting reflex during anoxia at 3 different temperatures, 23C, 25C and 27C in the adult epaulette shark (H. ocellatum), adult grey carpet shark and juvenile grey carpet shark (C. punctatum) (± Standard deviation).
Temperature
23C (min-1) 25C (min-1) 27C (min-1)
Epaulette shark (Adult) 178.33 ± 18.6 112.5 ± 20.7 56.25 ± 18.9
Grey carpet shark (Adult) 172 ± 11.0 53.33 ± 18.6 26.86 ± 5.8
Grey carpet shark (Juvenile) 36.42 ± 6.4 22.83 ± 1.6 14 ± 1.5
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Chapter 2: Anoxia tolerance at three different seasonal temperatures
Time to loss of righting reflex during anoxic exposure in two adult shark species at three temperatures 250
200
150 * † H. ocellatum C. punctatum
Time(min) 100 † * † # *
50 * † #
0 23 25 27 Temperature (oC) Figure 2.1. Time to loss of righting reflex during anoxic exposure at 23C, 25C and 27C on the adult epaulette shark (H. ocellatum) and the adult grey carpet shark (C. punctatum). Symbols indicate significant differences from epaulette sharks at 23C (*), from grey carpet sharks at 23C (†) and from epaulette sharks at each respective temperature (#).
A significant difference was observed in the mean total time to LRR between juvenile and adult grey carpet sharks exposed to anoxia at 23C, 25C and 27C (p<0.001) (Figure
2.2). Similar to adult grey carpet sharks, the mean time to LRR of the juvenile grey carpet shark was inversely proportional to temperature. During anoxia at 23C, the mean time to
LRR of the juvenile grey carpet shark was 36.42 ( 6.4) minutes, which was significantly longer than the mean time to LRR in juvenile grey carpet sharks exposed to anoxia at 25C
(22.83 1.6 minutes) (p<0.001) and at 27C (14.0 1.5 minutes). In comparison to adult grey carpet sharks, juveniles lost their righting reflex significantly earlier at each respective temperature (Figure 2.2).
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Chapter 2: Anoxia tolerance at three different seasonal temperatures
Time to loss of righting reflex during anoxic exposure in juvenile and adult grey carpet sharks at three temperatures 200 180
160
140 C. punctatum 120 (Adult) C. punctatum 100 (Juvenile)
Time(min) 80 *
60 * 40 * † * * † 20 0 23 25 27 Temperature (oC) Figure 2.2. Time to loss of righting reflex during anoxic exposure at 23C, 25C and 27C on adult and juvenile grey carpet sharks (C. punctatum). Symbols indicate significant differences from adult grey carpet sharks at 23C (*) and from juvenile grey carpet sharks at 23C (†).
2.4.2 Experiment 2. Ventilation rates during an “open ended” anoxic exposure at 23C
Prior to treatment, the mean resting ventilation rate of the adult epaulette shark at
23°C (control and experimental) was 29.29 ( 1.4) beats min.-1 (Figure 2.3). Immediately after animals were placed into the aquaria, a transient increase in mean ventilation rates occurred in both the anoxia treated group (79.14 4.6 beats min.-1; 23C) and control group
(64.43 5.7 beats min.-1; 23C), however animals exposed to sudden anoxia had significantly higher ventilation rates throughout the post-transfer period than did control animals
(p<0.001). In the control group, the transient increase in ventilation rate remained elevated above the resting ventilation rate for 5-10 minutes before a gradual decline in which the ventilation rate returned to mean resting ventilation levels after approximately 135 minutes.
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Chapter 2: Anoxia tolerance at three different seasonal temperatures
The mean ventilation rates in the control group showed no significant differences from mean resting ventilation rates for the remainder of the experiment.
Ventilation rates of adult epaulette sharks in response to anoxia at 23C 90
80
70
)
1 - 60
50 Anoxia Control 40
30 Ventilation (beats min
20 10 *
0 Rest 0 5 10 30 45 60 75 90 105 120 135 150 165 Time (min) Figure 2.3. The ventilation rate (beats min.-1) of anoxia treated and control epaulette sharks (H. ocellatum) to the time of loss of righting reflex at 23C. Symbols indicate the onset of ventilatory depression (*). Note: The x-axis is not to scale.
In the epaulette shark experimental group, ventilation rates began to decline by 5 minutes of anoxic exposure (56.14 3.4 beats min.-1; 23C). From this time point onwards, mean ventilation rates of anoxia treated epaulette sharks were significantly lower than control groups (p<0.001). After approximately 60 minutes of anoxia, the mean ventilation rates of the epaulette sharks were significantly lower (20.43 4.5 beats min.-1; 23C) than their mean resting ventilation levels, signifying the onset of ventilatory depression, which continued to decrease gradually to 15.57 4.1 beats min.-1 at 23C until righting reflex was lost at the mean time of 165 minutes.
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Chapter 2: Anoxia tolerance at three different seasonal temperatures
At 25C, the epaulette shark had a mean resting ventilation rate (control and experimental) of 27.71 ( 2.3) beats min.-1. After rapid transfer to the treatment tank, the epaulette shark displayed similar transient increases in ventilatory frequency, before gradually declining back to resting levels by 15 minutes in anoxia. Comparison of ventilatory depression during anoxia at 23C and 25C revealed that mean ventilation rates were significantly reduced below mean resting values after 60 minutes and 30 minutes respectively
(p<0.01) (Figure 2.6b). These data suggest that ventilatory depression occurred earlier at a higher temperature. Furthermore, during ventilatory depression, the mean ventilation rates at
25C were significantly lower (14.83 1.3 beats min.-1) than that of animals at 23C, (17.6
2.5 beats min.-1) (p<0.01). Taken together these data indicate that at 25C epaulette sharks enter into a deeper ventilatory depression much earlier than animals exposed to anoxia at
23C. The Q10 calculated for the epaulette shark was 1.3.
In the grey carpet shark, there was no significant difference between mean resting ventilation rates of adult animals (24.4 6.1 beats min.-1) and juveniles (26.41 4.1 beats min.-1) in normoxia at 23C (Figure 2.4 and 2.5). The mean resting ventilation rates of adult grey carpet sharks were not significantly different for control and experimental animals. After transfer to aquaria, the mean ventilation rates of adult grey carpet sharks increased significantly (p<0.001) from mean resting values in both control (58.0 3.8 beats min.-1;
23C) and experimental animals (65.33 3.2 beats min.-1; 23C). Following this initial increase, ventilation rates gradually returned to resting levels and remained there, in both experimental and control animals. Ventilation rates of anoxia treated adult grey carpet sharks had returned to mean resting values by 105 minutes. Mean ventilation rates in anoxia treated adult grey carpet sharks showed no significant differences from mean resting ventilation rates
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Chapter 2: Anoxia tolerance at three different seasonal temperatures for the remainder of the experiment. Therefore, no ventilatory depression occurred in the grey carpet shark and ventilation rates slowly returned to resting levels.
Ventilation rates of adult grey carpet sharks in response to anoxia at 23C 80
70
60
)
1 -
50 Anoxia 40 Control
30 Ventilation (beats min 20
10
0 Rest 0 5 15 30 45 60 75 90 105 120 135 150 165 Time (min) Figure 2.4. The ventilation rate (beats min.-1) of anoxia treated and control grey carpet sharks (C. punctatum) to time of the loss of righting reflex at 23C. Note: The x-axis is not to scale.
Comparison of ventilation rates of adult grey carpet sharks at 23C and 25C revealed that there was no significant difference in mean ventilation rates of animals exposed to anoxia and their respective control groups at either temperature throughout the treatment. However, at 25C mean ventilation rates of adult grey carpet sharks exposed to both experimental and control treatment returned to mean resting levels significantly earlier than animals at 23C
(p<0.001). The Q10 calculated for the grey carpet shark was 1.5.
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Chapter 2: Anoxia tolerance at three different seasonal temperatures
In juvenile grey carpet sharks at 23C, there were no significant differences in mean resting ventilation values between control and experimental animals (Figure 2.5). Following transfer to the aquaria, both control (65.6 2.7 beats min.-1) and experimental (55.58 4.3 beats min.-1) groups had a transient increase in mean ventilation rates, although the increased ventilation rate of anoxia treated juvenile grey carpet sharks were significantly lower
(p<0.001) than that of the control animals. Mean ventilation rates gradually declined in both control and experimental animals throughout the time course. Experimental animals had significantly lower ventilation rates at all sample points throughout the experiment even though ventilation did not return to pre-treatment levels until 105 minutes. Mean ventilation rates of the anoxia treated group were not significantly different to mean resting values at the sample point immediately before the LRR (31 3.2 beats min.-1) while the control mean ventilation rate was still elevated (p<0.001).
Ventilation rates of juvenile grey carpet sharks in response to anoxia at 23C 80
70
60
)
1 -
50 Anoxia 40 Control
30 Ventilation (beats Ventilation (beats min 20
10
0 Rest 0 5 15 30 45 Time (min) Figure 2.5. The ventilation rate (beats min.-1) of anoxia treated and control juvenile grey carpet sharks (C. punctatum) to time of the loss of righting reflex at 23C. Note: The x-axis is not to scale.
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Chapter 2: Anoxia tolerance at three different seasonal temperatures
2.4.3 Experiment 3. Ventilation rates during 1.5 hours of anoxia followed by 2 hours of normoxic re-oxygenation at 23°C or 25°C
Ventilatory depression occurred after 60 minutes of anoxia in the epaulette shark at
23C, however at 25C the onset of ventilatory depression was significantly earlier (30 minutes). Following anoxia at 23C, the mean ventilation rates during normoxic re- oxygenation were not significantly different at any time point than that of epaulettes sharks exposed to either 1.5 hours of anoxia treatment or control animals (Figure 2.6A).
Subsequently, the ventilatory demand calculated from changes in ventilation frequency above resting values during re-oxygenation were not significantly different between anoxia treated
(9.71 9 beats min.-1 above resting ventilation) and control epaulette sharks (12.82 5.4 beats min.-1 above resting ventilation) at 23C.
In contrast, anoxia treated epaulette sharks at 25C had no significant differences in mean ventilation rates above resting values from control animals during 45 minutes of re- oxygenation (Figure 2.6B). At 1 hour of re-oxygenation however, anoxia treated epaulette sharks had significantly higher ventilation rates above their resting values than control animals throughout the remainder of the re-oxygenation period. Increased ventilation frequency above resting values following re-oxygenation revealed an increased respiratory demand significantly larger in experimental animals (32.94 6.1 beats min.-1 above resting ventilation) than control (21.41 3.1 beats min-1 above resting ventilation) epaulette sharks at
25C. Furthermore, the ventilatory demand of anoxia treated epaulette sharks at 23C was significantly lower than that of animals exposed to anoxia at 25C. After 2 hours of normoxic re-oxygenation, ventilation rates were still significantly elevated above resting values in epaulette sharks exposed to anoxia at 25C.
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Chapter 2: Anoxia tolerance at three different seasonal temperatures
A Ventilation rates of adult epaulette sharks in response to 1.5 hours anoxia and 2 hours re-oxygenation at 23C 60 Anoxia Re-oxygenation 50
)
1 -
40
30 Anoxia 20 Control
10
0 Ventilation above resting (beats min -10 Rest 0 5 15 30 45 60 75 90 5 15 30 45 60 75 90 105 120
-20 Time (min) B Ventilation rates of adult epaulette sharks in response to 1.5 hours anoxia and 2 hours re-oxygenation at 25C 60 Anoxia Re-oxygenation
50
)
1 - 40
30 Anoxia 20 Control
10
0
Ventilation above resting (beats min -10 Rest 0 5 15 30 45 60 75 90 5 15 30 45 60 75 90 105 120
-20 Time (min) Figure 2.6. Ventilation frequency above resting values of the epaulette shark (H. ocellatum) during 1.5 hours of anoxic exposure and 2 hours of re-oxygenation in normoxia. A) Anoxic exposure and re-oxygenation at 23C in the epaulette shark. B) Anoxic exposure and re-oxygenation at 25C in the epaulette shark.
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Chapter 2: Anoxia tolerance at three different seasonal temperatures
During 1.5 hours of anoxic exposure, ventilation rates were not significantly lower than resting ventilation rates at any time point in the adult grey carpet shark at either 23C or
25C, signifying the absence of ventilatory depression. During the re-oxygenation period, the mean ventilation rates of the adult grey carpet shark were significantly increased in experimental and control groups compared to their respective pre-experimental mean values at both 23C and 25C (Figure 2.7A, B). No significant differences were observed in mean ventilation rates between treatment and control animals for the first 15 minutes of re- oxygenation at 23C and the first 5 minutes at 25C. Following this time interval, mean ventilation rates were significantly higher in treatment animals than control grey carpet sharks for the remainder of the re-oxygenation period at both 23C and 25C (Figure 2.7A, B).
Therefore the ventilatory demand during re-oxygenation by anoxia treated grey carpet sharks at 23C (23.83 4.0 beats min.-1 above resting ventilation) and at 25C (19.9 3.3 beats min.-
1 above resting ventilation) were significantly greater than their respective control groups
(9.14 3.8 beats min-1 above resting ventilation, 23C; 4.24 4.6 beats min-1 above resting ventilation, 25C). Finally, no significant differences were observed in the ventilatory demand of anoxia treated grey carpet sharks at 23C and 25C. After 2 hours of normoxic re- oxygenation, ventilation rates were still significantly elevated above resting values in adult grey carpet sharks exposed to 1.5 hours of anoxia at both 23C and 25C (Figure 2.7A, B).
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Chapter 2: Anoxia tolerance at three different seasonal temperatures
A Ventilation rates of adult grey carpet sharks in response to 1.5 hours anoxia and 2 hours re-oxygenation at 23C 40 Anoxia Re-oxygenation
35
)
1 - 30
25
20 Anoxia Control 15
10
5
Ventilation above resting (beats min 0 Rest 0 5 15 30 45 60 75 90 5 15 30 45 60 75 90 105 120 -5 Time (min) B Ventilation rates of adult grey carpet sharks in response to 1.5 hours anoxia and 2 hrs re-oxygenation at 25C 40 Anoxia Re-oxygenation
35
)
1 -
30
25
20 Anoxia Control 15
10
5 Ventilation above resting (beats min 0
-5 Rest 0 5 15 30 45 60 75 90 5 15 30 45 60 75 90 105 120 Time (min) Figure 2.7. Ventilation frequency of the adult grey carpet shark (C. punctatum) above resting values during 1.5 hours of anoxic exposure and 2 hours of re-oxygenation in normoxia. A) Anoxic exposure and re-oxygenation at 23C in the grey carpet shark. B) Anoxic exposure and re-oxygenation at 25C in the grey carpet shark.
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Chapter 2: Anoxia tolerance at three different seasonal temperatures
2.4.4 Behavioural characteristics of the epaulette shark to anoxic exposure
Initially, grey carpet shark juveniles and adults responded to anoxic exposure with a burst of activity, irrespective of the treatment temperature. This activity lasted for a minimum of 15 seconds and a maxiumum of 3 minutes in the epaulette shark exposed to anoxia at 23°C.
The epaulette shark typically remained motionless throughout the remainder of the experiment until righting reflex was finally lost.
2.4.5 Behavioural characteristics of the grey carpet shark to anoxic exposure
Initially, grey carpet shark juveniles and adults responded to anoxic exposure with a burst of activity, irrespective of the treatment temperature. This active period lasted a minimum of 15 seconds in both adults and juveniles and a maximum of 3 minutes in juvenile animals and 7 minutes in adults. Additionally, animals had random small bursts of activity during the anoxic exposure. Both adults and juveniles then typically defecated and/or vomited; this occurred at approximately 20 minutes in juveniles and at approximately 57.5 minutes in adults after transfer to sudden anoxia at 23°C, possibly signalling the autoregulatory shutdown of the digestive system and clearing of the digestive tract. Finally the animals typically had one last burst of activity immediately before losing their righting reflex. Following the LRR, any further exposure to anoxia resulted in the shutdown of gill ventilation after approximately 15-30 seconds in both adult and juvenile grey carpet sharks.
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Chapter 2: Anoxia tolerance at three different seasonal temperatures
2.5 Discussion
During anoxic exposure the epaulette shark enters into a protective state of neuronal hypometabolism which would maintain brain energy charge and subsequently prolong survival (Renshaw et al., 2002). Renshaw et al. (2002) observed the epaulette shark to tolerate an average of 46.3 minutes of anoxia at 28°C, before losing their righting reflex, signifying the induction of neuronal hypometabolism. The present study observed similar tolerance times until righting reflex was lost at 27°C (56.25 20.7 minutes), however at lower temperatures the time to LRR was significantly increased. At 25°C, the mean time to LRR of the epaulette shark was 112.5 ( 20.7) minutes, while at 23°C the mean time to LRR was significantly longer still (178.33 18.6 minutes). Reductions in anoxia tolerance time have been observed in other anoxia tolerant animals such as Carassius carassius and turtles in the genera Chrysemys and Trachemys as a consequence of elevated temperature (Lutz and
Nilsson, 1997). The present study observed an increase in temperature to greatly reduce the anoxia tolerance of the epaulette shark, which may be attributed to a temperature induced increase in metabolic rate. Consequently, the epaulette shark withstood significantly longer periods of anoxia as temperature decreased from 27°C to 23°C. It is likely that this inverse relationship between time to LRR and temperature was due to a reduction in ATP consumption at lower temperatures, forestalling ATP depletion and the onset of the “anoxia catastroph” (Lutz and Nilsson, 1997).
The grey carpet shark was not known to survive exposure to hypoxic and anoxic conditions until recently. They have been observed to frequent shallow seagrass areas in estuaries (1-2m) and are periodically found over coral reef flats (Last and Stevens, 1994) where oxygen levels have been reported to reach 2.0 mg. L-1 (Parsons, 1987; Wise et al.,
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Chapter 2: Anoxia tolerance at three different seasonal temperatures
1998; Carlson and Parson, 2003). The mean time to LRR in the adult grey carpet shark was also significantly affected by changes in temperature between 23°C and 27°C. At 23°C the grey carpet shark had a mean time to LRR of approximately 172.0 ( 11.0) minutes.
However, like the epaulette shark, the mean time to LRR was significantly reduced in the grey carpet shark during anoxia at each increasing temperature value. At 23°C the grey carpet shark had a similar time to LRR as those observed in the epaulette shark. However at 25°C and 27°C the epaulette had a significantly longer tolerance time than the grey carpet shark until time to LRR occured. This suggests that the grey carpet shark is more sensitive to changes in temperature during anoxic exposure than the epaulette shark, which possesses a significantly greater tolerance to anoxia over a wider temperature range.
Interestingly, the LRR in the grey carpet shark was followed by an almost immediate cessation of gill ventilation and the animal required an increase in water over the gills and manual lateral swimming stimulation to be revived. This suggests that the LRR in response to anoxia in this species may not be a result of metabolic depression to conserve ATP, as observed in the epaulette shark (Renshaw et al., 2002), but may be due to severely low/depleted neuronal energy levels.
Juvenile grey carpet sharks lost their righting reflex significantly earlier than adult grey carpet sharks at 23°C, 25°C and 27°C. Therefore the adult grey carpet sharks can tolerate a significantly longer period of anoxic challenge before loosing their righting reflex at similar temperatures. During anoxia, larger grey carpet sharks may have a significant advantage over smaller individuals, due to the smaller supply of anaerobic fuel in juvenile grey carpet sharks accompanied by a much faster accumulation of anaerobic endproducts (lactate and H+) due to their higher mass-specific metabolic rate (Nilsson and Ostlund-Nilsson, 2008). In addition,
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Chapter 2: Anoxia tolerance at three different seasonal temperatures little is known about the biological requirements and habitat preferences of this species throughout maturation and the increase in anoxia tolerance of adult grey carpet sharks may be due to an ontogenetic shift in lifestyle and habitat.
During hypoxic exposure, ventilation rates in the epaulette shark increase to maintain oxygen consumption (Routley et al., 2002). However as oxygen saturation decreases and oxygen consumption cannot be maintained, muscular contraction to induce gill ventilation becomes energetically expensive. In response, the epaulette shark undergoes a ventilatory depression to conserve ATP (Renshaw et al., 2002; Routley et al., 2002). In the present study, the transient increase in ventilation in both the epaulette shark and the grey carpet shark
(adults and juveniles) experienced on the transfer of both control and treatment animals into the aquaria, may be explained by handling and confinement stress. Furthermore, anoxia treated epaulette sharks had significantly higher ventilation rates than control animals immediately upon exposure to anoxia. This immediate increase in ventilation in response to reduced oxygen conditions was observed by Renshaw et al. (2002) for anoxic exposure and
Routley et al. (2002) for progressive hypoxia as a strategy to increase gill ventilation to maintain oxygen absorption. However, within five minutes, the mean ventilation rates of the epaulette shark significantly declined below the mean values of control animals, indicating a sensitive and rapid response to anoxic exposure and energy conservation.
During anoxia at 23°C and 25°C, the epaulette shark mean ventilation rates were significantly reduced below mean resting ventilation frequencies, signifying a clear ventilatory depression, supporting findings by Renshaw et al. (2002) and Routley et al.
(2002). At 23°C, ventilatory depression was observed in the epaulette shark after
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Chapter 2: Anoxia tolerance at three different seasonal temperatures approximately 60 minutes in anoxia, while at 25°C ventilatory depression occurred after approximately 30 minutes.
Anoxia treated epaulette sharks at 25°C entered a deeper ventilatory depression significantly earlier than anoxia treated animals at 23°C as revealed by their significantly lower ventilatory frequency. This indicates that during anoxia, changes in temperature influence not only the initiation but also the depth of ventilatory depression in the epaulette shark. This response is similar to other ectothermic vertebrates, in which metabolic rate and subsequently ATP consumption is closely linked to temperature (Tullis and Baillie, 2005).
Exposure to anoxia at elevated temperatures would result in an increase in ATP consumption, the Q10 principle (Lutz and Nilsson, 1997), which in turn would be expected to activate energy conserving mechanisms in an anoxia tolerant animal to a greater extent, as observed in the present study. Therefore it appears that sensitive neurological control of the buccal musculature activity in response to low oxygen conditions may be closely linked to the ATP status in the epaulette shark. This argument can be supported by further analysis of the increase in ventilation rates during re-oxygenation at at each temperature. At 23°C, the epaulette shark had no significant increase in ventilation rates above control animals during re-oxygenation, which may indicate the absence of a significant oxygen debt in response to
1.5 hours of anoxia. However following 1.5 hours of anoxia at 25°C, the epaulette shark had significantly increased ventilation rates above control animals during re-oxygenation, which may be due to the presence and replenishment of an oxygen debt at higher temperatures.
While an increase in ventilation frequencies observed during re-oxygenation following anoxia treatment could signify the presence of an oxygen debt, other ventilatory responses such as buccal pressure, gill vascularisation and blood flow were not measured in this study. The Q10 coefficient calculated in the present study indicates that both the epaulette shark and the grey
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Chapter 2: Anoxia tolerance at three different seasonal temperatures carpet shark had low Q10 values (epaulette shark = 1.3; grey carpet shark = 1.5) in the range from 23°C to 25°C. It should be noted however, that the Q10 coefficient calculated in the present study may not be accurate due to the qualitative nature of ventilation frequency, which does not account for buccal muscular pressure or water exchange in the buccal cavity.
Therefore, oxygen consumption may be significantly increased with no change to ventilation rates. However, the Q10 calculated in the present study observed an increase in metabolism in response to a 2°C increase in temperature, which may significantly affect energy consumption as metabolic rate increased with temperature.
The adult and juvenile grey carpet sharks also displayed a transient increase in ventilation rates in response to handling and confinement in both control and anoxia treatment groups. No significant differences were observed between control and treatment animals in adult grey carpet sharks at any time point throughout the experiment. Mean ventilation rates in adult grey carpet sharks exposed to anoxia at 23°C or 25°C did not fall below mean resting ventilatory values. The mean ventilation frequencies of anoxia treated juvenile grey carpet sharks were significantly lower than control animals, indicating a faster reduction in ventilation back to resting levels in response to anoxia. However, the mean ventilation rates in anoxia treated juvenile grey carpet sharks did not fall below the mean resting ventilatory values. Taken together, these data indicate that while juvenile grey carpet sharks showed some evidence of reducing ventilation rates to resting values, neither juvenile nor adult grey carpet sharks achieved ventilatory depression, below resting levels, in response to anoxia. The absence of ventilatory depression in the grey carpet shark indicates that, unlike the epaulette shark, this species does not conserve energy by decreasing buccal musculature activity during anoxia. Nilsson and Ostlund-Nilsson (2008) discussed the affect of size on anoxia tolerance, in that smaller animals have been reported to possess reduced anoxia tolerance due to i)
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Chapter 2: Anoxia tolerance at three different seasonal temperatures smaller supply of anaerobic fuel, ii) faster accumulation of anaerobic endproducts and iii) higher mass-specific metabolic rate. Further study is required on the anoxia tolerance of the grey carpet shark in comparision to weight and age, to determine whether general body weight or if an ontogenetic shift in lifestyles/habitats is responsible for changes in anoxia tolerance.
During re-oxygenation, following anoxia, grey carpet sharks significantly increased ventilation rates above control animals at both 23°C and 25°C, indicating the presence of a significant oxygen debt. In contrast to the epaulette shark, no significant differences in ventilation rates were observed during re-oxygenation in grey carpet sharks exposed to anoxia at 23°C compared to 25°C, indicating that the grey carpet shark may be already ventilating at its maximum capacity. However it should be noted that these increases in ventilation values were only recorded for 2 hours of re-oxygenation and not for the duration of elevated ventilation frequencies. Therefore, during events of anoxia at higher temperatures, it is not known whether anoxia treated grey carpet sharks would continue to ventilate at these high frequencies following re-oxygenation for a longer duration to replenish a more severe oxygen debt. While the purpose of the present study was to compare anoxia tolerance over three seasonal temperatures, evidence from the present study suggests that an oxygen debt may occur in the grey carpet shark following anoxia and perhaps even in the epaulette shark at high temperatures (25°C). Therefore, further invesitigation is required to determine the presence and magnitude of an oxygen debt experience by the epaulett shark and the grey carpet shark in response to anoxic exposure.
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Chapter 2: Anoxia tolerance at three different seasonal temperatures
From the present study, the duration of anoxia tolerance of both the epaulette shark and the grey carpet shark are temperature dependent, with a significant reduction in the time to LRR with increasing temperatures. During anoxia, the epaulette shark activates energy conserving mechanisms of ventilatory depression and hypometabolism to prolong survival.
There is no evidence that the grey carpet shark activates either of these survival strategies.
While some degree of anoxia tolerance occurs in the juvenile grey carpet shark, longer periods of tolerance to anoxia becomes apparent later in their lifecycle.
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Chapter 2: Anoxia tolerance at three different seasonal temperatures
2.6 Literature cited
Burleson ML, Smatresk NJ. 2000. Branchial chemoreceptors mediate ventilatory responses to hypercapnic acidosis in channel catfish. Comp Biochem Physiol 125:403-414. Butler PJ, Taylor EW. 1975. The effect of progressive hypoxia on the respiration in the dogfish (Scyliorhinus canicula) at different seasonal temperatures. J Exp Biol 63:117- 130. Carlson JK, Parsons GR. 1999. Seasonal differences in routine oxygen consumption rates of the bonnethead shark. J Fish Biol 55:876-879. Carlson JK, Parsons GR. 2003. Respiratory and haematological responses of the bonnethead shark, Sphyrna tiburo, to acute changes in dissolved oxygen. J Exp Mar Biol Ecol 294:15-26. DuPreez HH, McLauchlan A, Marias JFK. 1988. Oxygen consumption of two nearshore elasmobranchs, Rhinobatus annulatus (Muller & Henle, 1841) and Myliobatis aquila (Linnaeus,1758). Comp Biochem Physiol 89A:283-294. Hopkins TE, Cech JJ, Jr. 1994. Effect of temperature on oxygen consumption of the bat ray, Myliobatis californica. Copeia 529–532. Hughes GM, Johnston IA. 1978. Some responses of the electric ray (Torpedo marmorata) to low ambient oxygen tensions. J Exp Biol 73:107-117. Last PR, Stevens JD. 1994. Sharks and Rays of Australia. CSIRO Division of Fisheries. Melbourne, Australia. Lowe CS, Goldman KJ. 2001. Thermal and bioenergetics of elasmobranchs: bridging the gap. Env Biol Fish 60:251-266. Lutz PL, Nilsson GE. 1997. Contrasting strategies for anoxic brain survival-glycolysis up or down. J Exp Biol 200:411-419. Mulvey JM, Renshaw GMC. 2000. Neuronal oxidative hypometabolism in the brainstem of the epaulette shark (Hemiscyllium ocellatum) in response to hypoxic pre-conditioning. Neurosci Let 290:1-4. Nilsson GE. 1996. Brain and body oxygen requirements of Gnathonemus petersii, a fish with an exceptionally large brain. J Exp Biol 199:603-607. Nilsson GE, Ostlund-Nilsson S. 2008. Does size matter for hypoxia tolerance in fish? Biol Rev 83:173-189.
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Nilsson GE, Renshaw GMC. 2004. Hypoxia survival strategies in two fishes: extreme anoxia tolerance in the North European crucian carp and the natural hypoxic preconditioning in a coral-reef shark. J Exp Biol 207:3131-3139. Parsons GR. 1987. Life history and bioenergetics of the bonnethead shark, Sphyrna tiburo: a comparison of two populations. PhD dissertation, University of South Florida, St. Petersburg, FL. Perry SF, Wood CM. 1989. Control and coordination of gas transfer in fishes. Can J Zool 67:2961-2970. Renshaw GMC, Kerrisk CB, Nilsson GE. 2002. The role of adenosine in the anoxic survival of the epaulette shark, Hemiscyllium ocellatum. Comp Biochem Physiol B 131:133-141. Robinson E, Davison W. 2007. The Antarctic notothenioid fish Pagothenia borchgrevinki is thermally flexible: acclimation changes oxygen consumption. Polar Biol 31(3):317-326. Routley MH, Nilsson GE, Renshaw GMC. 2002. Exposure to hypoxia primes the respiratory and metabolic responses of the epaulette shark to progressive hypoxia. Comp Biochem Physiol A 131:313-321. Schmidt-Nielsen K. 1983. Animal Physiology: Adaptation and Environment. 5th ed. University Press, Cambridge. Soderstrom V, Renshaw GMC, Nilsson GE. 1999. Brain blood flow and blood pressure during hypoxia in the epaulette shark Hemiscyllium ocellatum, a hypoxia-tolerant elasmobranch. J Exp Biol 202:829-835. Sundin L, Nilsson S. 2002. Branchial innervations. J Exp Zool 293(3):232-248. Tullis A, Baillie M. 2005. The metabolic and biochemical responses of tropical whitespotted bamboo shark Chiloscyllium plagiosum to alterations in environmental temperature. J Fish Biol 67(4):950-968. Ultsch GR, Jackson DC, Moalli R. 1981. Metabolic oxygen conformity among lower vertebrates: the toadfish revisited. J Comp Physiol 142:439-443. Vulesevic B, McNeill B, Perry SF. 2006. Chemoreceptor plasticity and respiratory acclimation in the zebrafish Danio rerio. J Exp Biol 209(7):1261-1273. Wise G, Mulvey JM, Renshaw GMC. 1998. Hypoxia tolerance in the epaulette shark (Hemiscyllium ocellatum). J Exp Zool 281:1-5.
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CHAPTER THREE
Haematological responses of the grey carpet shark (Chiloscyllium punctatum) and the epaulette shark (Hemiscyllium ocellatum) to anoxia and re-oxygenation exposure
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Chapter 3: Haematological responses to anoxia and re-oxygenation
3.1 Summary
We compared the haematological responses of wild and captive populations of two closely related sharks to a standardised anoxic challenge and during a 12 hour recovery period in normoxia: the epaulette shark (Hemiscyllium ocellatum) and the grey carpet shark
(Chiloscyllium punctatum). Compared to normoxic controls, a significant increase in haematocrit (captive 22.3%; wild 35.9%) coupled with a decline in mean corpuscular haemoglobin concentration occurred in epaulette sharks indicating erythrocyte swelling in response to anoxia. However, the grey carpet shark had a significantly increased haematocrit
(captive 27.2%; wild 29.3%), erythrocyte count (captive 37.6%; wild 46.3%) and haemoglobin concentrations (captive 31.9%; wild 31.5%), suggesting a release of erythrocytes into the circulation and/or haemoconcentration in response to anoxia. Plasma glucose concentrations were maintained in both wild and captive epaulette sharks and in wild grey carpet sharks during anoxia but increased significantly after 2 hours of re-oxygenation
(epaulette: captive 55.8%; wild 50.1%; grey carpet shark: wild 70.3%) and remained elevated for 12 hours. Captive grey carpet sharks had an immediate increase in plasma glucose concentrations after anoxia (96.4%), which was sustained for 12 hours of re-oxygenation.
Lactate concentrations significantly increased in captive and wild animals of both species after anoxia, reaching a peak at 2 hours of re-oxygenation. Both species showed significant, yet divergent, haematological changes in response to anoxia and re-oxygenation, which may not only prolong their survival and assist in recovery, but also reflect their respective ecophysiological adaptations to the extreme environments that they inhabit.
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Chapter 3: Haematological responses to anoxia and re-oxygenation
3.2 Introduction
While the tolerance of most vertebrates to low oxygen conditions is negligible, research into the comparative physiology of species encountering hypoxia and/or anoxia in their natural environment has revealed that some vertebrates have developed a repertoire of specialised adaptive strategies which prolong their tolerance to reduced oxygen conditions. In elasmobranchs, the hypoxia tolerance of the epaulette shark (Hemiscyllium ocellatum) has been well characterised (Wise et al., 1998; Soderstrom et al., 1999; Mulvey and Renshaw,
2000; Routley et al., 2002). During hypoxia, this species up-regulates the level of anaerobic metabolism while conserving ATP usage via adaptive mechanisms such as ventilatory and metabolic depression (Routley et al., 2002), bradycardia (Soderstrom et al., 1999) and neuronal hypometabolism (Mulvey and Renshaw, 2000). More recently the epaulette shark has been observed to tolerate significant periods of anoxia (Renshaw et al., 2002). It has been suggested that tolerance to low oxygen conditions in this species confers an adaptive advantage which enables the epaulette shark to remain active on sheltered coral reef platforms that are cyclically exposed to severe periods of hypoxia during nocturnal low tides (Routley et al., 2002; Nilsson and Renshaw, 2004).
The grey carpet shark (Chiloscyllium punctatum) is a close relative to the epaulette shark, residing in the family Hemiscyllidae. The grey carpet shark has a more varied distribution and is commonly found in seagrass beds, mangrove swamps as well as in tidal pools and on coral reefs (Last and Stevens, 1994), however, it is not commonly found in abundance on the intermittently hypoxic reef flats inhabited by the epaulette shark. While the ability of the grey carpet shark to tolerate conditions of severely reduced oxygen saturation has not previously been examined, our preliminary experiments indicated that this species
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Chapter 3: Haematological responses to anoxia and re-oxygenation may also have the capacity to tolerate prolonged periods of anoxia. We examined the haematological response of these two closely related sharks to anoxia followed by re- oxygenation in normoxia in order to determine whether changes in haematological profiles reflect the ecophysiology of each population.
It is well known that haematological modifications can provide significant advantages in reducing cellular stress and prolonging survival in a range of vertebrates (Soivio et al.,
1974a; Soivio et al., 1974b; Soldatov, 1996; Affonso et al., 2002; Richmond et al., 2005).
Changes in haematological measures such as haematocrit, erythrocyte (RBC) number and haemoglobin concentrations in response to hypoxia have been well characterised in teleost fishes
(Soivio et al., 1974a; Soivio et al., 1974b; Yamamoto et al., 1983; Yamamoto, 1987; Pearson and Stevens, 1991; Affonso et al., 2002; Baker et al., 2005), although a more severe stress, such as anoxia has had little attention. Such adaptive haematological changes have been attributed to a number of different mechanisms, such as fluid shifts out of the blood plasma (Hall et al., 1926;
Kirk, 1974; Swift and Loyd, 1974), cell swelling (Soivio et al., 1974a; Soivio et al., 1974b) and the release of RBCs into the circulating blood from storage organs, such as the spleen (Yamamoto et al., 1983; Yamamoto, 1987; Pearson and Stevens, 1991; Lai et al., 2006). Interestingly, no significant differences in haematocrit and RBC concentrations were observed following hypoxia in the epaulette shark (Routley et al., 2002), the spotted dogfish (Scyliorhinus canicula) (Butler et al., 1979) or the bonnethead shark (Sphyrna tiburo) (Carlson and Parson, 2003), however the haematological responses of an elasmobranch to an anoxic insult had not previously been examined.
Increases in plasma lactate during reduced oxygen conditions are a stereotypical response to an increase in anaerobic metabolism. These increases have been observed following cyclic, acute and progressive hypoxia in elasmobranchs such as the epaulette shark (Wise et al., 1998;
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Chapter 3: Haematological responses to anoxia and re-oxygenation
Mulvey and Renshaw, 2000; Routley et al., 2002) and the electric ray (Torpedo marmorata)
(Hughes and Johnston, 1978), with the increased levels being similar to those observed in hypoxia exposed intolerant teleost fishes (Virani and Rees, 2000; Wells and Baldwin, 2006).
Up-regulation of anaerobic metabolism during anoxia consumes significantly larger quantities of plasma glucose. Elevated plasma glucose concentrations have been reported in anoxia-tolerant vertebrates such as Chrysemys turtles, the crucian carp (Carcassius carassius) and in anoxia intolerant teleosts, to fuel the increases in anaerobic metabolism (McDonald and Milligan, 1992; Lutz and Nilsson, 1997). No changes in plasma glucose concentrations have been observed during hypoxic exposure in either the spotted dogfish (Scyliorhinus canicula) (Butler et al., 1979) or the epaulette shark (Routley et al., 2002).
Results from previous studies concluded that elasmobranchs, in general, may not possess the ability to alter their haematology or plasma glucose concentrations in response to hypoxia or anoxia, a capacity which may be evolutionarily reserved to higher vertebrates
(Lutz and Nilsson, 1997). However, previous studies on hypoxia-intolerant elasmobranchs and the anoxia-tolerant epaulette shark have only investigated the changes in the blood constituents in response to hypoxic challenges. This is the first study to examine the haematological responses of two elasmobranch species to prolonged anoxic exposure followed by re-oxygenation in normoxia.
In general, studies of hypoxia- and anoxia-tolerance have only investigated animals from either wild or captive populations. The hypoxic and anoxic response of the epaulette shark has only been examined in animals caught from their natural environment on coral reef flats. Within these environments, animals are periodically exposed to increasingly severe
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Chapter 3: Haematological responses to anoxia and re-oxygenation periods of hypoxia and it is possible that the anoxia tolerance of this species is enhanced by pre-conditioning as a result of cyclic exposure to hypoxia in their natural environment
(Routley et al., 2002; Nilsson and Renshaw, 2004). Renshaw et al. (2002) observed a pre- emptive induction of hypometabolism due to pre-conditioning 24 hours after an initial anoxic challenge. Since animals raised in a captive environment with a constant oxygen supply have not been pre-exposed to such pre-conditioning events, they provide a useful comparison to the responses of wild sharks that are often exposed to cyclic pre-conditioning provided by the epaulette shark’s natural reef environment. Therefore, the haematological responses to anoxia were examined in animals caught from the wild and compared with those of animals kept under constant environmental conditions in commercial aquaria.
The comparative investigation of hypoxia- and anoxia-tolerant animals offers an enormous potential to provide an understanding of the underlying mechanisms involved in the diverse array of strategies that specialised vertebrates have evolved to deal with oxygen deprivation. This study examined the response of haematocrit, haemoglobin concentrations, erythrocytes, plasma glucose and lactate levels of the epaulette shark and grey carpet shark to a standardised anoxic challenge followed by re-oxygenation in both captive and wild populations.
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Chapter 3: Haematological responses to anoxia and re-oxygenation
3.3 Materials and methods
3.3.1 Study animals and locations
Captive epaulette sharks (n=9) and grey carpet sharks (n=9) were supplied by
UnderWater World (Mooloolaba, Sunshine Coast) and Sea World (Main Beach, Gold Coast) and the experiments were carried out on site. Epaulette sharks held in captivity were supplied
from an accredited commercial collector in the previous year had a mean length of 71.58 ± 7.1
cm and weight of 1.43 ± 0.4 kg. Grey carpet sharks were born in captivity 2 years earlier and
had a mean length of 88.56 ± 8.1 cm and weight of 2.94 ± 0.8 kg. Both species were kept in a constant seawater flow-through aquarium with a normal photoperiod and no additional light sources in their outside habitats.
Wild epaulette sharks (n=10) were collected from Heron Island in the Capricorn bunker group (latitude 23° 27’ S, longitude 151° 55’ E) (GBRMPA Permit # GO4/12675.1) and the experiments were conducted at the Heron Island Research Station. Wild epaulette
sharks had a mean length of 61.77 ± 3.56 cm and weight of 0.55 ± 0.12 kg. Wild grey carpet sharks (n=6) were caught in Moreton Bay (latitude 27º 28’ S, longitude 153º 23’ E) (DPI and
Fisheries Permit #PRM38182I) and experiments were conducted at the Moreton Bay
Research Station. Wild grey carpet sharks had a mean length of 92.62 ± 5.51 cm and weight
of 2.73 ± 0.47 kg. For both wild epaulette sharks and grey carpet sharks, experiments were conducted 24 hours following capture, to allow sufficient time for the ventilatory rate, an indication of stress levels, to return to normal without allowing time for potentially significant changes in biochemical adaptation to captivity. Both captive and wild animals were acclimated in ambient oceanic seawater at 24°C at their respective sites. Food was withheld in
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Chapter 3: Haematological responses to anoxia and re-oxygenation both captive and wild animals, for 24 hours before the first blood samples were taken at the commencement of the experiment.
3.3.2 Anoxic challenge and re-oxygenation
Individual animals were exposed to 1.5 hours of anoxia (experimental groups) or normoxia (normoxic transfer-1 for control groups) in 70 L glass tanks, followed by 12 hours of re-oxygenation after transfer to a separate 200 L normoxic tank at 24°C, re-oxygenation for anoxic treated animals and normoxic transfer-2 for controls. All sharks were maintained in a fasted state. Each treatment tank was filled with normoxic seawater and sealed with plastic wrap and a Perspex lid. Seawater was circulated around the tank using a submersible 228- power head pump (CCC Pty. Ltd., Sydney, New South Wales, Australia) to ensure uniform conditions throughout. In the anoxic tanks, nitrogen gas was bubbled through an air stone to displace dissolved oxygen. The dissolved oxygen concentration [dO2] levels were measured and recorded each minute using TPS WP-90 oxygen probes (TPS Pty. Ltd., Brisbane,
-1 Queensland, Australia). In the anoxic tank the [dO2] was maintained below 0.02 mg L . In the
-1 normoxic tanks, air was bubbled through an air stone to maintain the [dO2] above 7.0 mg L .
Anoxic conditions were maintained within each tank by the moderate adjustment of nitrogen injection into the tank water using brass gange valves. Animals were closely pair-matched for length and one member from each pair was randomly selected for either anoxic exposure or the normoxic control treatment. Animals were weighed and measured at the conclusion of the experiment.
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Chapter 3: Haematological responses to anoxia and re-oxygenation
3.3.3 Blood sampling and haematology
Blood samples were taken rapidly from un-anaesthetised fish immediately prior to the beginning of the experiment (t0) to provide a pre-experimental measurement for each haematological parameter. A 1.5 ml blood sample was taken from the dorsal aorta of each animal using a heparinised syringe via the venipuncture method described in Stoskopf et al. (1984).
Blood samples were then transferred to 3 ml lithium heparin vacutainers (Becton Dickinson,
North Ridge, New South Wales, Australia) and gently mixed. Additional blood samples were collected immediately following control or experimental treatment (post-/ t1.5) then after 2 hours
(t2), 6 hours (t6) and 12 hours (t12) of re-oxygenation in normoxia after anoxic challenge or after normoxic transfer-2 for controls.
Haematocrit was determined using heparinised haematocrit microtubes and centrifuged at
2500 rpm for 5 minutes at 4°C in an Eppendorf 5415D centrifuge. Haematocrit values were determined against a micro-haematocrit grid (Sigma-Aldrich, Newcastle, New South Wales,
Australia). Red blood cell (RBC) concentrations [RBC] were calculated via haemocytometry cell counts using a Neaubaur counting chamber. Blood samples were diluted 1:200 in chilled phosphate buffered saline (pH 7.2) and vortexed gently. The diluted solution (5 μl) was loaded into the Neaubaur counting chamber and examined under 10 x magnification using a compound light microscope. Blood haemoglobin was determined in triplicate, by a cyanmethaemoglobin technique using modified Drabkin’s Reagent (Sigma-Aldrich, Newcastle, New South Wales,
Australia). One millilitre of Drabkin’s reagent and 4 μl of blood were placed into a 1.6 ml cuvette and vortexed gently. Samples were incubated in the dark, at room temperature for 15 minutes before being analysed using a UV Mini 1240 spectrophotometer (Shimadzu, Suzhoa, Republic of
China) at a wavelength of 540 nm. Sample haemoglobin concentrations were compared against
Clint Chapman 86
Chapter 3: Haematological responses to anoxia and re-oxygenation a haemoglobin standard curve using the human haemoglobin standard supplied by Sigma-
Aldrich (Newcastle, New South Wales, Australia).
The RBC indices calculated were: the mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), haematocrit levels and haemoglobin concentrations. The formulae of Stoskopf (1993) were used as follows:
MCV = (Hct / [RBC]) x 10,
MCH = (Hb x 10) / [RBC], and
MCHC = (Hb x 100) / Hct.
Where Hct is haematocrit and Hb is haemoglobin.
Blood plasma was separated from a 0.5 ml blood sample after it was centrifuged at
3000 rpm for 3 minutes at 4°C, then frozen at –80°C for later use. Plasma glucose concentrations were determined using a colorimetric glucose hexokinase assay kit (Sigma-
Aldrich, Newcastle, New South Wales, Australia) following the manufacturer’s directions and read at a wavelength of 340 nm. Briefly, cuvettes containing a 1:500 dilution of plasma to glucose reagent were gently vortexed and incubated at room temperature for 15 minutes before colorimetric analysis. Sample plasma glucose concentrations were compared against a glucose standard curve using the glucose standard solution supplied by Sigma-Aldrich
(Newcastle, New South Wales, Australia).
Plasma lactate concentrations were determined using a colorimetric Lactate Assay kit
(Immuno Diagnostics, St. Peters, New South Wales, Australia) following the manufacturer’s directions and read at a wavelength of 540 nm. Cuvettes containing a 1:100 dilution of plasma
Clint Chapman 87
Chapter 3: Haematological responses to anoxia and re-oxygenation to lactate reagent were gently vortexed and incubated at room temperature for 5-10 minutes before colorimetric analysis. Sample plasma lactate concentrations were compared against a lactate standard curve, using the lactate standard solution supplied by Immuno Diagnostics (St
Peters, New South Wales, Australia).
3.3.4 Statistics
The data was analysed using a Mixed Model Repeated Measures ANOVA so that we could test the main effects and interaction effects of: sample time, population type (wild or captive) and treatment (experiment or normoxic control), followed by a post-hoc Bonferroni adjustment. The alpha level was set at 0.05. The data and error bars are represented as mean ± standard deviation.
3.4 Results
3.4.1 Haematocrit
For epaulette sharks, the mean haematocrit values prior to any treatment (pre-
experimental) were similar for captive (t0 = 19.0 ± 1.4%) and wild (t0 = 22.8 ± 2.8%) animals irrespective of whether they were in control or experimental groups (Figure 3.1A, B).
However, there was a main effect of environment for the grey carpet shark, the mean haematocrit values prior to any treatment were significantly higher (p<0.01) in the in wild
animal group (t0 = 27.3 ± 2.2%) than the captive animal group (t0 = 20.9 ± 2.5%) (Figure
3.1A). No significant differences were observed in pre-experimental mean haematocrit values
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Chapter 3: Haematological responses to anoxia and re-oxygenation of either captive or wild grey carpet sharks allocated to the anoxic challenge group compared to their respective control groups (Figure 3.1B). Furthermore, in the normoxic control groups, there were no significant changes in mean haematocrit values in captive or wild animals for either epaulette sharks or grey carpet sharks at any time points compared to their pre- experimental values, irrespective of sample time (Figure 3.1A).
Immediately following 1.5 hours of anoxic exposure there was a main effect of sample time. Captive and wild epaulette sharks had a significant increase in the mean haematocrit
value (captive, t1.5 = 23.2 ± 1.6%; wild, t1.5 = 30.9 ± 2.4%) above the pre-experimental mean measure (p<0.01), which remained at this elevated plateau for up to 2 hours following re- oxygenation (Figure 3.1B). Similarly, in the grey carpet shark, mean haematocrit values increased significantly from pre-experimental mean values (p<0.001) immediately following
1.5 hours of anoxia in both captive (t1.5 = 26.6 ± 1.5%) and wild (t1.5 = 35.3 ± 2.1%) animals and remained at this elevated plateau for up to 2 hours following re-oxygenation (Figure
3.1B). After 6 hours of normoxic re-oxygenation, mean haematocrit values in wild and captive epaulette sharks as well as grey carpet sharks returned to pre-experimental values and remained there for an additional 6 hours (Figure 3.1B). There were also interactions with the environmental source of the sharks sampled: wild epaulette sharks had significantly higher haematocrit values at the post-anoxia sample point than did captive epaulette sharks
(p<0.001) (Figure 3.1B). However, after 2 hours of re-oxygenation, there were no significant differences in mean haematocrit values between captive and wild epaulette sharks. Wild grey carpet sharks also had significantly higher haematocrit values from captive animals after anoxic exposure and after 2 hours of re-oxygenation.
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Chapter 3: Haematological responses to anoxia and re-oxygenation
A Percent haematocrit in control animals for two species of sharks from two environments 35
30
25
20 Tran 1 Tran 2 15 2hr
Percent (%) 6hr 12hr 10
5
0 Captive Wild Captive Wild
EPAULETTE SHARK GREY CARPET SHARK B Percent haematocrit in response to anoxic challenge and re-oxygenation in two 40 species of shark from two environments * †
35 * †
† † 30 * * 25
20 Pre Post 2hr 6hr Percent Percent (%) 15 12hr
10
5
0 Captive Wild Captive Wild
EPAULETTE SHARK GREY CARPET SHARK
Figure 3.1 Mean haematocrit values for epaulette sharks (H. ocellatum) and grey carpet sharks (C. punctatum) in two different environments. A) Epaulette sharks and grey carpet sharks from captivity and the wild, held in normoxia (controls). Transfer 1 corresponds to the sample before transfer into the control treatment tank and transfer 2 corresponds to the sample immediately before transfer into the normoxia recovery tank. No significant changes were observed within
control groups at any of the sample times. B) Epaulette sharks and grey carpet sharks from captivity and the wild before (pre) and immediately following (post) 1.5 hours of anoxia and at 2 hours, 6 hours and 12 hours of re-oxygenation in normoxia. The symbols indicate significant differences compared to the mean pre-experiment (*) or at 2 hours (†) following anoxic challenge.
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A
Chapter 3: Haematological responses to anoxia and re-oxygenation
3.4.2 Red blood cell count
The mean [RBC] of control animals in either species did not change significantly during the sample time course (Figure 3.2A). In the experimental groups, mean [RBC] prior to treatment revealed no significant differences in captive and wild epaulette sharks (captive,
-6 -1 -6 -1 t0 = 0.34 ± 0.06 x 10 uL ; wild, t0 = 0.34 ± 0.04 x 10 uL ) or grey carpet sharks (captive, t0
-6 -1 -6 -1 = 0.31 ± 0.007 x 10 uL ; wild, t0 = 0.3 ± 0.03 x 10 uL ) (Figure 3.2B). There was an interaction of species by sample time in response to anoxic challenge followed by re- oxygenation. In epaulette sharks, there were no significant changes in mean [RBC] in captive or wild sharks immediately following 1.5 hours of anoxia or throughout the 12 hours of normoxic re-oxygenation (Figure 3.2B) when compared to pre-experimental mean concentrations. Conversely, grey carpet sharks responded to anoxia with a significant increase
-6 -1 in [RBC] in captive animals and wild animals (captive, t1.5 = 0.43 ± 0.03 x 10 uL ; wild, t1.5 =
0.3 ± 0.03 x 10-6 uL-1) from their respective pre-experimental mean concentrations (p<0.001).
Mean [RBC] remained elevated at this plateau for 2 hours following re-oxygenation and then returned back to pre-experimental concentrations by 6 hours of normoxic re-oxygenation
(Figure 3.2B). Mean [RBC] remained at pre-experimental concentrations throughout the remainder of the experiment (12 hours of normoxic re-oxygenation). These results paralleled the significance patterns observed in the haematocrit measures described above and in haemoglobin concentration described below.
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Chapter 3: Haematological responses to anoxia and re-oxygenation
A Red blood cell concentrations in control animals for two species of shark from to environments 0.45
0.4
0.35
) 1 -
uL 0.3
6 - 0.25 Tran 1 Tran 2 0.2 2hr 6hr 0.15 12hr
Concentration Concentration (10 0.1
0.05
0 Captive Wild Captive Wild
EPAULETTE SHARK GREY CARPET SHARK
B Changes in red blood cell concentrations in response to anoxic challenge and re- 0.5 oxygenation in two species of shark from two environments * * 0.45 † †
0.4
) 1
- 0.35
uL 6 - 0.3
0.25 Pre Post 2hr 0.2 6hr 12hr
0.15 Concentration Concentration (10 0.1
0.05
0 Captive Wild Captive Wild
EPAULETTE SHARK GREY CARPET SHARK
Figure 3.2 Mean red blood cell counts for epaulette sharks (H. ocellatum) and grey carpet sharks (C. punctatum) in two different environments. A) Epaulette sharks and grey carpet sharks from captivity and the wild, held in normoxia (controls). Transfer 1 corresponds to the sample before transfer into the control treatment tank and transfer 2 corresponds to the sample immediately before transfer into the normoxia recovery tank. No significant changes were observed within control groups at any of the sample times. B) Epaulette sharks and grey carpet sharks from captivity and the wild, before (pre) and immediately following (post) 1.5 hours of anoxia and at 2 hours, 6 hours and 12 hours of re-oxygenation in normoxia. The symbols indicate significant differences compared to the mean pre-experiment (*) or at 2 hours (†) following anoxic challenge.
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Chapter 3: Haematological responses to anoxia and re-oxygenation
3.4.3 Haemoglobin
Prior to the commencement of the experiment, epaulette sharks from the wild
-1 population had significantly higher haemoglobin concentrations (t0 = 6.24 ± 0.4 g dL ) than
-1 captive animals (t0 = 5.34 ± 0.4 g dL ) (Figure 3.3A). Similarly, the wild grey carpet sharks
-1 had significantly higher mean haemoglobin concentrations (t0 = 7.5 ± 0.5 g dL ) than captive
-1 grey carpet sharks (t0 = 6.27 ± 0.5 g dL ) prior to the commencement of the experiment
(p<0.05) (Figure 3.3A).Throughout the remainder of the normoxic (control) sampling regime, neither epaulette sharks nor grey carpet sharks from either wild or captive populations showed any significant differences in mean haemoglobin concentrations from their respective initial mean concentrations (Figure 3.3A).
With regard to haemoglobin concentration there was a two way interaction of species with time. The mean haemoglobin concentrations of wild and captive epaulette sharks exposed to an anoxic challenge followed by re-oxygenation, did not significantly change from pre-experimental mean concentrations at either the post-anoxia sample point or during the 12 hours of normoxic re-oxygenation (Figure 3.3B). In contrast, captive and wild grey carpet
- sharks had a significant increase in haemoglobin concentrations (captive, t0 = 6.27 ± 0.5 g dL
1 -1 -1 -1 ; t1.5 = 8.27 ± 0.6 g dL ; wild, t0 = 7.5 ± 0.5 g dL ; t1.5 = 9.86 ± 0.4 g dL ) immediately following anoxia treatment (p<0.001), which remained elevated at this plateau for at least 2 hours of normoxic re-oxygenation (Figure 3.3B). Furthermore, wild grey carpet sharks had significantly higher haemoglobin concentrations than captive animals immediately after anoxia and following 2 hours of normoxic re-oxygenation (p<0.001). In both captive and wild grey carpet sharks, mean haemoglobin concentrations had returned to pre-experimental
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Chapter 3: Haematological responses to anoxia and re-oxygenation concentrations by 6 hours of normoxic re-oxygenation, and remained at these concentrations until the last sample point at 12 hours of re-oxygenation.
A Haemoglobin concentrations for control animals in two species of shark from two 100 environments
90
80
) 1
- 70
60 Tran 1 50 Tran 2 2hr 40 6hr 12hr
Concentration Concentration (g dL 30
20
10
0 Captive Wild Captive Wild
EPAULETTE SHARK GREY CARPET SHARK B Changes in haemoglobin concentrations in response to anoxic challenge and re- 120 oxygenation in two species of shark from two environments
* † 100 *
)
1 † - 80
Pre 60 Post 2hr 6hr 40 12hr Concentration (g dL
20
0 Captive Wild Captive Wild
EPAULETTE SHARK GREY CARPET SHARK
Figure 3.3 Mean haemoglobin concentrations for epaulette sharks (H. ocellatum) and grey carpet sharks (C. punctatum)
in two different environments. A) Epaulette sharks and grey carpet sharks from captivity and the wild, held in normoxia (controls). Transfer 1 corresponds to the sample before transfer into the control treatment tank and transfer 2 corresponds to the sample immediately before transfer into the normoxia recovery tank. No significant changes were observed within control groups at any of the sample times. B) Epaulette sharks and grey carpet sharks from captivity and the wild, before (pre) and immediately following (post) 1.5 hours of anoxia and at 2 hours, 6 hours and 12 hours of re-oxygenation in normoxia. The symbols indicate significant differences compared to the mean pre-experiment (*) or at 2 hours (†) following anoxic challenge.
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Chapter 3: Haematological responses to anoxia and re-oxygenation
3.4.4 Calculated erythrocytic indices
There were no significant differences in calculated RBC indices between untreated captive and wild epaulette sharks at pre-experimental mean levels (Table 3.1). However wild and captive epaulette sharks showed a significant increase in mean corpuscular volume
(MCV) immediately following anoxic exposure (p<0.001), which remained elevated for the first 2 hours of normoxic re-oxygenation before returning back to pre-experimental levels by
6 hours of normoxic re-oxygenation. Conversely, a significant decrease in mean corpuscular haemoglobin concentration (MCHC) occurred immediately following anoxia and for 2 hours of normoxic re-oxygenation in both wild (p<0.001) and captive (p<0.01) epaulette sharks, before returning back to pre-experimental levels. These significant differences were not evident when [RBC] rather than haematocrit was used in the calculations to obtain mean corpuscular haemoglobin (MCH) (Table 3.1).
Table 3.1. Erythrocytic indices: mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) calculated for captive (n=9) and wild (n=10) epaulette sharks (H. ocellatum) pre- and post-anoxic challenge and during 12 hours of normoxic re-oxygenation. (± standard deviation).
Captive Wild
Pre Post 2 Hrs 6 Hrs 12 Hrs Pre Post 2 Hrs 6 Hrs 12 Hrs 669.4 663.7 MCV 556.5 563.8 525.7 620.6 772 799.4 688.3 675.4 Anoxia † (ƒL) (±37) (±84)* (±62) (±46) (±67) (±60) (±48) (±80) (±55) (±43)
155 157.9 155.2 159.9 157.9 171.7 167.0 183.6 181.7 173 MCH (±15) (±10) (±19) (±25) (±13) (±30) (±9) (±24) (±19) (±15) 23.8 23.4 MCHC 27.9 27.1 26.4 28.4 21.7 22.9 27.5 28.0 † (g dL-1) (±2) (±2)* (±2) (±1) (±3) (±2) (±2) (±2) (±2) (±2)
MCV 583.7 524.5 632.3 630.5 636.5 722.7 649 632.7 688.1 645.6 Control (ƒL) (±109) (±41) (±41) (±50) (±74) (±85) (±61) (±49) (±8) (±92) 166.7 148.4 171.2 167.0 163.3 197.3 180.3 174.6 214.8 188.1 MCH (±33) (±19) (±15) (±11) (±22) (±21) (±12) (±30) (±13) (±6) MCHC 28.5 28.2 27.1 26.5 25.7 27.4 27.8 27.5 31.2 29.5
(g dL-1) (±1) (±2) (±1) (±2) (±3) (±3) (±1) (±4) (±2) (±3)
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Chapter 3: Haematological responses to anoxia and re-oxygenation
No significant differences in any of the RBC indices were observed in untreated captive or wild grey carpet sharks (Table 3.2) nor in wild or captive grey carpet sharks after
1.5 hours of anoxia and up to 12 hours of normoxic re-oxygenation (Table 3.2).
Table 3.2. Erythrocytic indices: mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean
corpuscular haemoglobin concentration (MCHC) calculated for captive (n=9) and wild (n=6) grey carpet sharks (C. punctatum ) pre- and post-anoxic challenge and during 12 hours of normoxic re-oxygenation. (± standard deviation).
Captive Wild
Pre Post 2 Hrs 6 Hrs 12 Hrs Pre Post 2 Hrs 6 Hrs 12 Hrs MCV 666.3 626.0 691.8 651.8 699.6 910.8 809.5 877.2 930 941.9 Anoxia (ƒL) (±103) (±52) (±78) (±97) (±91) (±118) (±109) (±56) (±60) (±50) 200.8 195.1 196.1 202.2 211.2 246.2 225.6 241.0 255.6 242.9 MCH (±29) (±25) (±14) (±28) (±28) (±23) (±25) (±12) (±33) (±22) MCHC 30.3 31.2 28.5 31.1 30.2 27.1 27.9 27.5 27.7 25.7
(g L-1) (±3) (±3) (±2) (±2) (±2) (±1) (±1) (±2) (±2) (±3) MCV 686.2 687.9 669.3 687.6 643.0 917.4 923.5 997.0 941.3 953.5 Control (ƒL) (±82) (±96) (±74) (±57) (±59) (±40) (±50) (±143) (±49) (±122) 204.7 196.3 190.9 202.1 203.4 254.9 245.2 274.6 252.5 251.2 MCH (±10) (±13) (±13) (±21) (±25) (±13) (±21) (±33) (±7) (±37) MCHC 30 28.8 28.7 29.4 31.6 27.8 26.5 27.6 26.9 26.3
(g L-1) (±2) (±3) (±3) (±1) (±1) (±1) (±2) (±1) (±1) (±1)
3.4.5 Glucose
There was a significant main effect of species. Post-anoxic challenge, the grey carpet sharks had higher plamsa glucose concentrations than did epaulette sharks. This was clarified further by examining the interaction of species, sample time and population type. Throughout the control regime, epaulette sharks (wild and captive) and grey carpet sharks (wild and captive) showed no significant differences in mean plasma glucose concentrations from their respective pre-experimental mean concentrations (Figure 3.4A). The mean plasma glucose concentrations in the epaulette shark and the grey carpet shark prior to treatment were not
-1 significantly different between captive (epaulette shark, t0 = 0.55 ± 0.04 mg dL ; grey carpet
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Chapter 3: Haematological responses to anoxia and re-oxygenation
-1 -1 shark, t0= 0.68 ± 0.04 mg dL ) or wild sharks (epaulette shark, t0 = 0.6 ± 0.08 mg dL ; grey
-1 carpet sharks, t0 = 0.67 ± 0.06 mg dL ) nor in the pre-experimental levels of sharks allocated to anoxic or normoxic groups (Figure 3.4B).
While the response pattern was similar for both species, the grey carpet sharks responded with a plasma glucose level of a higher magnitude. Immediately following anoxic exposure, no significant differences were observed in mean plasma glucose concentrations in
-1 -1 captive (t1.5 = 0.64 ± 0.09 mg dL ) and wild (t1.5 = 0.72 ± 0.1 mg dL ) epaulette sharks or in
-1 the wild grey carpet sharks (t1.5 = 0.77 ± 0.09 mg dL ) compared to their pre-experimental mean concentrations (Figure 3.4B). However, after 2 hours of re-oxygenation, mean plasma
-1 glucose concentrations significantly increased in both captive (t2 = 0.85 ± 0.09 mg dL ) and
-1 wild (t2 = 0.9 ± 0.13 mg dL ) epaulette sharks (p<0.01) compared to pre-experimental concentrations and remained at this elevated plateau throughout the 12 hours of re- oxygenation. Captive grey carpet sharks had a significant increase in mean plasma glucose
concentrations above pre-experimental levels immediately post-anoxia (t1.5 = 1.36 ± 0.09 mg dL-1) (p<0.001). The mean plasma glucose concentrations of captive grey carpet sharks remained at this elevated plateau for up to 12 hours of re-oxygenation.
A further interaction occurred between species, sample time and population revealing that only the captive grey carpet sharks had an immediate elevation in plasma glucose levels post-anoxia (Figure 3.4B). In wild grey carpet sharks, the mean plasma glucose concentrations increased by a significantly greater magnitude than the epaulette shark at 2
-1 hours of re-oxygenation (t2 = 1.14 ± 0.02 mg mL ) (p<0.001).
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Chapter 3: Haematological responses to anoxia and re-oxygenation
A Plasma glucose concentrations for control animals in two species of sharks from 1 two environments
0.9
0.8 1) - 0.7 0.6 Tran 1 0.5 Tran 2 2hr 0.4 6hr 12hr 0.3
Concentration Concentration (mg dL 0.2
0.1
0 Captive Wild Captive Wild
EPAULETTE SHARK GREY CARPET SHARK
B Changes in plasma glucose concentrations in response to anoxic challenge and re-oxygenation in two species of shark from two environments 2
1.8 † #
1.6 ×
* 1) - 1.4 # † × 1.2 † # × † # × Pre 1 Post 2hr 0.8 6hr 12hr
0.6 Concentration Concentration (mg dL 0.4
0.2
0 Captive Wild Captive Wild
EPAULETTE SHARK GREY CARPET SHARK
Figure 3.4 Mean plasma glucose concentrations for the epaulette sharks (H. ocellatum) and grey carpet sharks (C. punctatum ) in two different environments. A) Epaulette sharks and grey carpet sharks from captivity and the wild, held in normoxia (controls). Transfer 1 corresponds to the sample before transfer into the control treatment tank and transfer 2 corresponds to the sample immediately before transfer into the normoxia recovery tank. No significant changes were observed within control groups at any of the sample times. B) Epaulette sharks and grey carpet sharks from captivity and the wild, before (pre) and immediately following (post) 1.5 hours of anoxia and at 2 hours, 6 hours and 12 hours of re- oxygenation in normoxia. Symbols indicate significant differences compared to the mean pre-experiment (*), at 2 hours (†), 6 hours (#), or 12 hours (×) following anoxic challenge.
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Chapter 3: Haematological responses to anoxia and re-oxygenation
3.4.6 Lactate
In control animals, captive and wild epaulette sharks, along with captive grey carpet sharks, showed a significant increase in mean plasma lactate concentrations with a main effect
-1 of species (captive epaulette shark, t1.5 = 121.3 ± 31.9 mg dL ; wild epaulette shark, t1.5 =
-1 -1 145.8 ± 37.3 mg dL ; captive grey carpet shark, t1.5 = 71.9 ± 52.2 mg dL ) immediately following 1.5 hours of confinement in the control aquaria (p<0.001) (Figure 3.5A). Control groups of epaulette sharks had significantly higher plasma lactate levels than grey carpet sharks until 12 hours post-transfer. In both wild and captive epaulette sharks, mean plasma lactate concentrations were sustained at an elevated plateau for 2 hours after transfer-2. Mean plasma lactate concentrations had returned back to mean pre-experimental concentrations 2 hours after transfer-2 in the captive grey carpet sharks or 6 hours after transfer 2 in the captive and wild epaulette sharks.
The pre-experimental mean plasma lactate concentrations in epaulette sharks was
-1 significantly higher in captive animals (t0 = 31.2 ± 13.4 mg dL ) than in wild animals (t0 = 2.3
± 0.5 mg dL-1) (p<0.001) (Figure 3.5A). However, a main effect of sample time was evident in response to 1.5 hours of anoxia: epaulette sharks (captive and wild) had significant increases in mean plasma lactate concentrations immediately following anoxia (captive, t1.5 =
-1 -1 254.1 ± 22.8 mg dL ; wild, t1.5 = 242.1 ± 16.1 mg dL ) (p<0.001) (Figure 3.5B). By 2 hours of normoxic re-oxygenation, mean plasma lactate concentrations continued to increase from
-1 pre-experimental concentrations (captive, t2 = 472.9 ± 37.3 mg dL ; wild, t2 = 359.6 ± 43.2 mg dL-1) (p<0.001). Epaulette sharks maintained these elevated concentrations for at least 6 hours of normoxic re-oxygenation. By 12 hours of normoxic re-oxygenation plasma lactate
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Chapter 3: Haematological responses to anoxia and re-oxygenation
concentrations had returned to 225.1 ± 28.5 mg dL-1 in captive animals and 59 ± 43.3 mg dL-1 in wild animals.
Pre-experimental mean plasma lactate concentrations in grey carpet sharks were not
-1 - significantly different between captive (t0 = 2.5 ± 0.9 mg dL ) and wild (t0 = 3.5 ± 2.3 mg dL
1) animals irrespective of whether they had been allocated to normoxic control groups or anoxia treatment groups (Figure 3.5A and B). The main effect of sample time was evident immediately following 1.5 hours of anoxia, both captive and wild grey carpet sharks had significant increases in mean plasma lactate concentrations post anoxic challenge (captive, t1.5
-1 -1 = 166.9 ± 41 mg dL ; wild, t1.5 = 139.3 ± 34.8 mg dL ) (p<0.001). In wild grey carpet sharks, the mean plasma lactate concentrations were sustained at this elevated plateau for up to 6
-1 hours of re-oxygenation (wild, t6 = 197.2 ± 29.0 mg dL ), while captive grey carpet sharks had an additional significant increase in plasma lactate levels after 2 hours of re-oxygenation
-1 (captive, t2 = 299.1 ± 86.1 mg dL ) and remained at this elevated concentration for up to 6 hours of re-oxygenation. By 12 hours of re-oxygenation, mean plasma lactate concentrations in the captive and wild grey carpet shark had significantly declined (p<0.001), yet they were still significantly elevated above pre-experimental concentrations (captive, t12 = 59.1 ± 16.9
mg dL-1; wild, 92.1 ± 40.7 mg dL-1) (p<0.001).
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Chapter 3: Haematological responses to anoxia and re-oxygenation
A Changes in plasma lactate concentrations for control animals in two species of 300 † shark from two environments
†
250 1)
- 200 * * Tran 1 150 Tran 2 * 2hr 6hr 100 12hr
Concentration (mg dL
50
0 Captive Wild Captive Wild
EPAULETTE SHARK GREY CARPET SHARK
B Changes in plasma lactate concentrations in response to anoxic challenge and re-oxygenation in two species of shark from two environments 600 † # 500
1)
- † † 400 #
# Pre 300 * × * Post # 2hr * † 6hr 12hr 200 *
Concentration Concentration (mg dL × × × 100
0 Captive Wild Captive Wild
EPAULETTE SHARK GREY CARPET SHARK
Figure 3.5 Mean plasma lactate concentrations for the epaulette sharks (H. ocellatum) and grey carpet sharks (C. punctatum) in two different environments. A) Epaulette sharks and grey carpet sharks from captivity and the wild, held in normoxia (controls). Transfer 1 corresponds to the sample before transfer into the control treatment tank and transfer 2 corresponds to the sample immediately before transfer into the normoxia recovery tank. Symbols indicate significant differences compared to the mean post-normoxic transfer 2 (*) and at 2 hours (†), 6 hours (#), or 12 hours (×) after transfer. B) Mean plasma lactate of the epaulette shark and the grey carpet shark. Mean plasma lactate concentrations before and immediately following 1.5 hours of anoxia and at 2 hours, 6 hours and 12 hours of re-oxygenation in normoxia. Symbols indicate significant differences compared to the mean pre-experiment (*), at 2 hours (†), 6 hours (#), or 12 hours (×) following anoxic challenge.
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3.5 Discussion
3.5.1 Haematological differences in control animals from captive and wild populations of two species of shark
Wild epaulette sharks as well as grey carpet sharks are intermittently exposed to hypoxia in their natural environment while captive sharks at UnderWater World or Sea World do not experience hypoxia. The mean haematocrit values and [RBC] were similar for captive and wild epaulette sharks in control groups prior to any treatment. However, haemoglobin concentrations in the wild epaulette sharks were significantly higher. Perhaps wild epaulette sharks maintain higher haemoglobin levels in response to intermittent hypoxic challenge.
In grey carpet sharks, both the mean percentage haematocrit and haemoglobin concentration were significantly higher in animals caught in the wild. It is possible that this haematologically responsive species could have been exposed to the preconditioning effects of low oxygen in their natural environment prior to capture. Last and Stevens (1994) reported that this species can survive out of water for extended periods of time. We have also observed this species in the intertidal zone associated with mangroves. The physiological compensation for such extreme challenges has direct ecological relevance. If, for example, the physiological response of the grey carpet shark to a sudden stranding or decrease in ambient oxygen involves releasing a pool of oxygenated red blood cells, then oxygen delivery would receive a temporary boost.
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3.5.2 Haematological responses to anoxic challenge and re-oxygenation
While [RBC] for epaulette sharks were within the range previously reported by
Baldwin and Wells (1990), both the percentage haematocrit and haemoglobin concentrations were higher. Such variations may be due to seasonality, animal location and/or the effect of confinement and handling stress. Routley et al. (2002) reported no changes in haematocrit levels in response to progressive hypoxia in the epaulette shark, however anoxic exposure and at 2 hours of re-oxygenation in the epaulette shark in the present study resulted in a significant increased in haematocrit. The RBC size (calculated by MCV) significantly increased in both captive and wild epaulette sharks, while the calculated MCHC decreased and no changes in
[RBC] or haemoglobin concentrations were observed. Taken together these data indicate that in the epaulette shark, an increase in RBC volume occurred immediately following anoxia and was still elevated at 2 hours of re-oxygenation. It is generally accepted that such cell swelling occurs in response to reduced oxygen conditions and is predominantly attributed to either a
loss of N+/K+ ATPase channel function, as ATP levels are depleted and/or a fall in extracellular pH, leading to the accumulation of intracellular sodium. In teleost fishes, reduced extracellular pH and PO2 cause a catecholamine-induced activation of β-adrenergic
Na+/H+ exchangers in the RBC membrane (Fievet et al., 1987). The activation of Na+/H+
exchangers removes H+ from the RBC in exchange for extracellular sodium. This influx of sodium is followed by chloride, which causes an osmotic influx of water, effectively alkalizing the intracellular space and resulting in cell swelling (Fievet et al., 1987; Chiocchia and Motais, 1989; Salama and Nikinmaa, 1990).
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Increases in cell volume have been well characterised in response to reduced oxygen conditions in teleost fishes (Fievet et al., 1987; Chiocchia and Motais, 1989; Salama and
Nikinmaa, 1990). Although, both Tufts and Randall (1989) and Wood et al. (1994) reported
the absence of a detectable β-adrenergic Na+/H+ exchanger in the anoxia-intolerant dogfish
(Squalus canicula). However, the presence and activation of H+/Na+ exchangers in an anoxia- tolerant elasmobranch such as the epaulette shark has not been examined.
During hypoxia and anoxia, the accumulation of anaerobic metabolites such as lactate
and H+ can also be responsible for a decrease in extracellular pH (Jensen, 2004). In the present study, increases in RBC volume in the epaulette shark remained elevated at peak levels for 2 hours following anoxia and returned to normal by 6 hours of re-oxygenation in normoxia.
Blood lactate concentrations were significantly higher in the epaulette shark than the lactate levels responsible for inducing RBC swelling in teleosts such as rainbow trout (Oncorhynchus mykiss, Salmo gairdneri) and sea bass (Morone labrax) (Soivio et al., 1980; Thomas and
Hughes, 1982a; Thomas and Hughes, 1982b; Thomas et al., 1986). Therefore, this significant increase in lactate concentrations in the epaulette shark may reflect an extracellular acidosis, which in turn could result in increases in RBC volume. Interestingly, however, a reduction in
RBC volume occurred during re-oxygenation, when ambient oxygen levels were restored to normal, even though plasma lactate concentrations remained elevated. These data suggest that once oxygen is restored, oxygen dependent mechanisms counteract RBC swelling (Renshaw and Nikinmaa, 2007).
Cell swelling also occurs in response to ATP depletion, resulting in a loss of ion homeostasis (Tetens and Lykkeboe, 1981). Erythrocyte ATP depletion causes a loss of
Na+/K+ channel function, leading to the accumulation of intracellular sodium and subsequent
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Chapter 3: Haematological responses to anoxia and re-oxygenation cell swelling. While many authors have discussed the interaction between organo-phosphates and haemoglobin-oxygen binding affinity, controversy over the erythrocyte organo-phosphate concentrations in response to low oxygen conditions exists among different vertebrates
(Marinakis et al., 2003; Wells et al., 2005). While changes in erythrocyte ATP levels in response to anoxia have not been examined in the epaulette shark, depleting erythrocytic ATP concentrations may be responsible for increased RBC volume as a result of a slow loss in ion homeostasis of the RBC, which is reversed by re-oxygenation.
In contrast to the epaulette shark, no evidence of RBC swelling occurred in the grey carpet shark. Plasma lactate levels rose significantly in grey carpet sharks from both captive and wild populations but did not reach the high levels attained in the epaulette shark.
However, there were significant increases in haematocrit, as well as in [RBC] and haemoglobin concentrations in both wild and captive grey carpet sharks immediately following anoxia and at 2 hours of re-oxygenation. These rapid increases in haematocrit, haemoglobin and [RBC] could result from the action of one or more compensatory mechanisms: i) haemoconcentration due to fluid shifts out of the plasma, ii) the accelerated division and maturation of blood cells already in the circulation, iii) an increase in oxygenated
[RBC] due to their release from a storage organ such as the spleen or haemopoetic organs such as the epigonal organ and Leydig organ, and/or iv) an increase in oxygenated [RBC] due to the diversion of blood from muscles and/or the gastrointestinal tract.
Plasma volume shifts per se would not be an evolutionary advantage in low oxygen environments and since they usually occur when the ability to maintain electrolyte homeostasis is lost, one could expect that it heralds slow death rather than anoxia tolerance.
While fluid shifts in response to severe hypoxia or anoxia have not been reported in
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Chapter 3: Haematological responses to anoxia and re-oxygenation
elasmobranchs, Cross et al. (1969) reported that there was no change in renal function and H+ excretion in response to hypercapnia in the dogfish (Squalus acanthias). We are currently examining electrolyte homeostasis in both the epaulette and the grey carpet shark post-anoxic challenge.
Stokes and Firkin (1971) used tritiated thymidine to show that mitosis in the systemic circulation gives rise to new thrombocytes and RBCs in the Port Jackson shark (Heterodontus portusjacksoni). It could be argued that the formation and/or accelerated maturation of RBCs could confer an ecophysiological advantage, provided that the gills remained moist. Since we observed that the [RBC] did return to pre-experimental values in grey carpet sharks during re- oxygenation, it seems unlikely that the elevated [RBC] observed post-anoxic challenge occurred via mitosis in the periphery and disappeared rapidly during re-oxygenation.
However, if stores of oxygenated red blood cells were sequestered in storage organs and then released or if oxygenated blood was diverted from non-essential organs such as muscles and/or the gastrointestinal tract, it is conceivable that these mechanisms would have an ecophysiological relevance for grey carpet sharks experiencing diminished oxygen levels in tidal pools and estuaries. Evidence from teleost fish and some elasmobranchs suggests that the release of red blood cells via splenic contraction does occur in response to elevated catecholamines (Nilsson et al., 1975).
Although splenic contraction has been well characterised in teleost fishes in response to hypoxia (Yamamoto et al., 1983; Pearson and Stevens, 1991; Yamamoto, 1987; Lai et al.,
2006), Butler et al. (1979) reported no changes in RBC concentrations during hypoxia in the dogfish (S. canicula). However, Nilsson et al. (1975) observed a splenic contraction in response to catecholamine and artificial nerve stimulation in both S. acanthias and
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Chapter 3: Haematological responses to anoxia and re-oxygenation
Scyliorhinus canicula in perfused and isolated spleens. In contrast, Opdyke and Opdyke
(1971) observed no release of RBC from the spleen in Squalus acanthias in response to electrical stimulation of the splenic pedicle or catecholamine infusion. Therefore, the function of the elasmobranch spleen to act as a RBC reservoir is currently controversial (Opdyke and
Opdyke, 1971; Nilsson et al., 1975). However, the ability of the spleen to act as a RBC reservoir in an anoxia-tolerant elasmobranch has not been examined.
The elevated [RBC] in the grey carpet shark in response to anoxia could have arisen as an adaptive ecophysiologically relevant response to the low oxygen levels periodically encountered in mangrove swamps, estuaries, coral reefs and tidal pools which are part of the habitat that this species occupies. The increased [RBC] could confer increase oxygen absorption and delivery to vital organs during bouts of naturally occurring hypoxic exposure.
This ability to increase [RBC] may also transfer an adaptive advantage during anoxia, if the haemoglobin in the RBCs is already saturated with oxygen.
3.5.3 Glucose
Pre- and post-experimental blood glucose concentrations in the epaulette shark and wild grey carpet sharks were not significantly higher than other shark species such as the dusky shark (Carcharhinus obscurus) and the nurse hound shark (Scyliorhinus stellaris)
(Piiper et al., 1972; Cliff and Thurman, 1984). However, after 2 hours of re-oxygenation, plasma glucose increased significantly by 55.6% above the post-anoxic levels in both wild and captive epaulette sharks and by 63.9% in wild grey carpet sharks and remained elevated at these levels for at least 12 hours of re-oxygenation. Since all animals were fasted prior to and during the experiment, such increases in plasma glucose concentrations following re-
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Chapter 3: Haematological responses to anoxia and re-oxygenation oxygenation suggests that glycogen stores are involved in the recovery phase following anoxic exposure in both species. A prolonged elevation in glucose levels could potentially replenish an energy debt once re-oxygenation occurred via increased aerobic metabolism.
Elevated plasma glucose concentrations are characteristic of the anoxic response in teleost fishes and higher vertebrates (Hardisty et al., 1976; Wright et al., 1989; MacCormack et al., 2006). However, this strategy relies on glycogen stores during reduced oxygen availability when anaerobic metabolism is the predominant source of ATP (Chippari-Gomes et al., 2005; Baker et al., 2005). In contrast, plasma glucose concentrations were maintained, but not significantly elevated, immediately following anoxic exposure in the wild and captive epaulette sharks and in wild grey carpet sharks.
Routley et al. (2002) observed that plasma glucose levels in wild epaulette sharks remained stable during exposure to progressive hypoxia. Current data confirmed that no changes in plasma glucose concentrations occurred in response to anoxia in either wild or captive epaulette sharks. Similarly, wild grey carpet sharks also showed no significant changes in plasma glucose concentrations immediately after anoxia. Interestingly, captive grey carpet sharks responded with a significant increase in plasma glucose concentrations immediately following anoxic exposure. Even though the pre-experimental levels of glucose were not significantly different in wild and captive grey carpet sharks it was clear that the magnitude and timing of the glucose response in captive grey carpet sharks was different than wild animals. The magnitude of the elevation in plasma glucose levels in response to anoxic challenge differed between the two grey carpet shark populations: after 2 hours of re- oxygenation, wild grey carpet sharks had a significant 63.9% increase while captive grey carpet sharks had a 93.5% increase from pre-experimental levels. Both populations remained
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Chapter 3: Haematological responses to anoxia and re-oxygenation at this concentration for at least 12 hours of re-oxygenation. Since normoxic control groups had no increases in plasma glucose concentrations due to capture or confinement, the significant increase in glucose in response to anoxia followed by re-oxygenation suggest not only that diminished oxygen may act to prime the conversion of glycogen stores to glucose in both captive and wild grey carpet sharks but also that the captive group may have had a greater response because there was a lack of natural periods of hypoxia in captive environments and/or captive grey carpet sharks may have had larger glycogen stores due to increased food availability.
Wild and captive grey carpet sharks also showed a much more pronounced increase in plasma glucose concentrations compared to epaulette sharks. These differences may be due to the magnitude of the energy debt accrued in the grey carpet shark following anoxic challenge resulting in the greater utilisation of glucose following re-oxygenation than that of the epaulette shark.
3.5.4 Lactate
In elasmobranchs, lactate concentrations rise quickly when they are exposed to an acute stressor such as capture and handling (Piiper and Baumgarten, 1968; Piiper et al., 1972;
Cliff and Thurman, 1984; Hoffmayer and Parsons, 2001), confinement (Martini, 1974), transport (Cliff and Thurman, 1984) and diminished oxygen levels (Hughes and Johnston,
1978; Wise et al., 1998; Mulvey and Renshaw, 2000; Routley et al., 2002). In the present study, control epaulette sharks and captive grey carpet sharks showed a significant increase in plasma lactate concentrations following transfer into a normoxic tank. These increases were
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Chapter 3: Haematological responses to anoxia and re-oxygenation most likely in response to handling and confinement as reported by Wise et al. (1998). The pre-experimental plasma lactate concentrations in captive epaulette sharks were significantly higher than those of wild caught epaulette sharks. In a captive environment, these benthic reef sharks may have a lower activity level for two reasons: food is provided and predatory avoidance is not necessary. Such reduced aerobic activity could lead to lactate accumulation.
This phenomenon was not observed in captive grey carpet sharks, which occupy a variety of benthic niches in their natural environment.
Post-anoxic challenge, epaulette shark plasma lactate concentrations peaked after 2 hours of re-oxygenation. Blood lactate concentration reached 52 mmol-1 and 40 mmol-1 in captive and wild epaulette sharks respectively. These values are higher than those previously reported for any other elasmobranchs (Piiper et al., 1972; Cliff and Thurman, 1984;
Hoffmayer and Parsons, 2001). The peak lactate concentrations in grey carpet sharks were not as high (captive, 33 mmol-1; wild, 20 mmol-1) as those reached in the epaulette shark and were comparable to the peak lactate concentrations reported for other elasmobranch species in response to hypoxia (Piiper et al., 1972; Cliff and Thurman, 1984; Hoffmayer and Parsons,
2001). The difference between the plasma lactate response in wild and captive grey carpet sharks may indicate that anaerobic pathways were more responsive in the captive grey carpet shark and/or that the wild population had more active pathways for lactate clearance.
The high plasma lactate levels in the epaulette shark reveal that there was a significant up-regulation of anaerobic metabolism during anoxia which could be expected to prolong survival. Interestingly, both captive epaulette sharks and captive grey carpet sharks had significantly higher plasma lactate concentrations than wild animals following anoxia, revealing that anaerobic pathways were more responsive to anoxia in captive animals than in
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Chapter 3: Haematological responses to anoxia and re-oxygenation wild animals which may have been subject to natural hypoxic pre-conditioning in their own environment. Such differences between wild and captive populations may be explained by differences between the basal/standard aerobic metabolic rate and the maximum aerobic rate, the metabolic scope (Fry, 1947), under specific conditions. In response to anoxia, captive epaulette sharks appeared to activate anaerobic pathways and accumulate lactate much earlier and at a much higher concentration than wild animals that may have already been primed by environmental exposure. In contrast, both species of captive sharks would have had prolonged acclimatisation to a stable captive environment with a constant oxygen supply. In addition, wild animals exposed to continual changes in oxygen saturation within their natural environment may require a larger metabolic scope to delay the accumulation of anaerobic end products.
Delayed peaks in plasma lactate concentrations in both the epaulette shark and the grey carpet shark following anoxia were similar to those observed in the sharpnose shark
(Rhizoprionodon terraenovae) (Hoffmayer and Parsons, 2001), and the electric ray (T. marmorata) (Hughes and Johnston, 1978) in response to hypoxia, and the nurse hound shark
(S. stellaris) (Piiper et al., 1972) and the dusky shark (C. obscurus) (Cliff and Thurman, 1984) after exhaustive exercise. These delayed peaks in lactate concentrations are attributed to slow lactate removal of poorly perfused tissues, such as white muscle fibres, due to vascular shunts and the reduction of circulation to the gut and peripheral tissue (Wells and Baldwin, 2006).
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3.5.5 Conclusions
In its natural environment, on coral reef flats, the anoxia-tolerant epaulette shark is periodically and cyclically exposed to diminished oxygen levels. Previous studies have shown that the epaulette shark initially increases anaerobic metabolism on exposure to hypoxic preconditioning (Wise et al., 1998; Routley et al., 2002) and subsequently enters into a state of neuronal metabolic depression (Mulvey and Renshaw, 2000; Renshaw et al., 2002).
In response to a standardised anoxic challenge, increases in haematocrit and plasma lactate occurred, which paralleled increases in erythrocyte volume and anaerobic metabolism, respectively. Significant increases in plasma lactate is a typical response to anoxic exposure in both teleosts and elasmobranch species, however lactate concentrations in the epaulette shark were the highest ever to be reported in an elasmobranch, indicating not only a high anaerobic capacity but also a high tolerance to the accumulation of metabolic end products such as lactate and associated acidosis. This study is the first to report a change in haematocrit due to erythrocyte swelling in response to anoxia followed by re-oxygenation in an elasmobranch species, the epaulette shark. This is of potential ecophysiological relevance since erythrocyte swelling assists in oxygen offloading.
The grey carpet shark showed significant, but different haematological responses to anoxic exposure compared to the epaulette shark. This study is the first to demonstrate that the grey carpet shark can tolerate an extended anoxic challenge and to report a significant increase in [RBC] in any elasmobranch immediately following anoxia. It is suggested that this increase in [RBC] may be due to a release of erythrocytes into the circulation, due to splenic contraction or a reduction of the blood supply to non-vital organs and the musculature.
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Chapter 3: Haematological responses to anoxia and re-oxygenation
Alternatively, it is possible that haemoconcentration alone or in combination with RBC release results in the significant increase in RBC concentration in grey carpet sharks in response to anoxic challenge.
During anoxia, plasma lactate concentrations significantly increased in both species, indicating the up-regulation of anaerobic metabolism. Additionally, both species maintained plasma glucose concentrations, which indicate a very sensitive regulation of blood glucose levels. Following re-oxygenation, this study clearly shows for the first time, two elasmobranch species entering a phase of hyperglycaemia during normoxic re-oxygenation following anoxia. The hyperglycaemic state during re-oxygenation could potentially reflect an up-regulation of aerobic metabolism, which could serve to rapidly replenish ATP levels.
While the epaulette shark and the grey carpet shark are closely related, they demonstrate different ecophysiological adaptations that would confer an advantage during severe declines in oxygen availability. The epaulette shark significantly up-regulates the level of anaerobic metabolism to generate ATP. In addition, there was an increased RBC volume, which may increase oxygen unloading from the erythrocytes. Similarly, the increase in plasma lactate in the grey carpet shark indicates an up-regulation of anaerobic metabolism. In contrast to the epaulette shark, the grey carpet shark rapidly increased erythrocyte number in response to anoxic challenge, indicating a redirection of oxygen stores during periods of oxygen deficit. Both species demonstrate ecophysiologically relevant adaptations which may prolong survival in the intermittently hypoxic or even briefly anoxic environment encountered in tidal pools, mangrove swamps, and in some estuaries and coral reef environments.
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3.6 Literature cited
Affonso EG, Polez VLP, Correa CF, Mazon AF, Araujo MRR, Moraes G, Rantin FT. 2002. Blood parameters and metabolites in the teleost fish Colossoma macropomum exposed to sulfide or hypoxia. Comp Biochem Physiol C 133:375-382. Baker DW, Wood AM, Kieffer JD. 2005. Juvenile Atlantic and shortnose sturgeons (Family Acipenseridae) have different hematological responses to acute environmental hypoxia. Physiol Biochem Zool 78:916-925. Baldwin J, Wells RMG. 1990. Oxygen transport potential in tropical elasmobranchs from the Great Barrier Reef: Relationship between haematology and blood viscosity. J Exp Mar Bio and Ecol 144:145-155. Butler PJ, Taylor EW, Davison W. 1979. The effect of long term, moderate hypoxia on acid base balance, plasma catecholamines and possible anaerobic end products in the unrestrained dogfish Scyliorhinus canicula. J Comp Physiol B 132:297-303. Carlson JK, Parsons GR. 2003. Respiratory and haematological responses of the bonnethead shark, Sphyrna tiburo, to acute changes in dissolved oxygen. J Exp Mar Biol Ecol 294:15-26. Chiocchia G, Motais R. 1989. Effect of catecholamine on deformability of red cells from trout: relative roles of cyclic AMP and cell volume. J Physiol (Paris) 412:321-332. Chippari-Gomes AR, Gomes LC, Lopes NP, Val AL, Almeida-Val VMF. 2005. Metabolic adjustments in two Amazonian cichlids exposed to hypoxia and anoxia. Comp Biochem Physiol B 141:347-355. Cliff G, Thurman GD. 1984. Pathological and physiological effects of stress during capture and transport in juvenile dusky shark, Carcharhinus obscurus. Comp Biochem. Physiol A 78:167-173. Cross CE, Packer BS, Linta JM, MurdaughV, Robin ED. 1969. H+ buffering and excretion in response to acute hypercapnia in the dogfish Squalus acanthias. Am J Physiol 216:440-452.
Fievet B, Motais R, Thomas S. 1987. Role of adrenergic-dependent H+ release from red cells in acidosis induced by hypoxia in trout. Am J Physiol 252:R269-275. Fry FEJ. 1947. Effects of the environment on animal activity. University of Toronto Studies Biological Series No 55, Publication of the Ontario Fish Research Laboratory 68:1-62.
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Hall FG, Gray IE, Lepkovsky S. 1926. The influence of asphyxiation on the blood constituents of marine fishes. J Biol Chem 67:549-554. Hardisty MW, Zelnik PR, Wright VC. 1976. The effects of hypoxia on blood sugar levels and on the endocrine pancreas, interrenal, and chromaffin tissues of the lamprey, Lampetra fluviatilis (L.). Gen Comp Endo 28(2):184-204. Hoffmayer ER, Parsons GR. 2001. The physiological response to capture and handling stress in Atlantic sharpnose shark, Rhizoprionodon terraenovae. Fish Physiol Biochem 25:277-285. Hughes GM, Johnston IA. 1978. Some responses of the electric ray (Torpedo marmorata) to low ambient oxygen tensions. J Exp Biol 73:107-117. Jensen FB. 2004. Red blood cell pH, the Bohr effect, and other oxygenation-linked
phenomena in blood O2 and CO2 transport. Act Physiol Scand 182:215-227.
Kirk WL. 1974. The effects of hypoxia on certain blood and tissue electrolytes of channel catfish, Ictalurus punctatus (Rafinesque). Trans Am Fish Soc 103:593-600. Lai JC, Kakuta I, Mok HO, Rummer JL, Randall D. 2006. Effects of moderate and substantial hypoxia on erythropoietin levels in rainbow trout kidney and spleen. J Exp Biol 209:2734-27348. Last PR, Stevens JD. 1994. Sharks and Rays of Australia. CSIRO Division of Fisheries. Melbourne, Australia. Lutz PL, Nilsson GE. 1997. Contrasting strategies for anoxic brain survival-glycolysis up or down. J Exp Biol 200:411-419. MacCormack TJ, Lewis JM, Almeida-Val VM, Val AL, Driedzic WR. 2006. Carbohydrate management, anaerobic metabolism, and adenosine levels in the armoured catfish, Liposarcus pardalis (Castelnau), during hypoxia. J Exp Zool 305:363-75. Martini, FF. 1974. Effects of capture and fasting confinement on an elasmobranch, Squalus acanthias. Unpublished Ph.D. Thesis, New York: Cornell University. Marinakis P, Tamburrini M, Carratore V, Di Prisco G. 2003. Unique features of the haemoglobin system of the Antartic notothenioid fish Gobionotothen gibberifrons. Eur J Biochem 270:3981-3987. McDonald DG, Milligan CL. 1992. Chemical properties of the blood. In: Hoar WS, Randall DJ, Farrell AP, editors. Fish Physiology, XIIB. London: Academic Press, p 56–133.
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Mulvey JM, Renshaw GMC. 2000. Neuronal oxidative hypometabolism in the brainstem of the epaulette shark (Hemiscyllium ocellatum) in response to hypoxic pre-conditioning. Neuroscience Letters 290:1-4. Nilsson S, Holmgren S, Grove DJ. 1975. Effects of drugs and nerve stimulation on the spleen and arteries of two species of dogfish, Scyliorhinus canicula and Squalus acanthias. Act Physiol Scand 95:219-230. Nilsson GE, Renshaw GMC. 2004. Hypoxic survival strategies in two fishes: extreme anoxia tolerance in the North European crucian carp and natural hypoxic preconditioning in a coral-reef shark. J Exp Biol 207:3131-3139. Opdyke DF, Opdyke NE. 1971. Splenic responses to stimulation in Squalus acanthias. Am J Physiol 221:623-625. Pearson MP, Stevens ED. 1991. Size and hematological impact of the splenic erythrocyte reservoir in rainbow trout, Oncorhynchus mykiss. Fish Physiol Biochem 9:39-50. Piiper J, Baumgarten D. 1968. Analysis of brachial gaseous exchange in an elasmobranch fish: Scyliorhinus stellaris. J Physiol (Paris) 60:518. Piiper J, Meyer M, Drees, F. 1972. Hydrogen ion balance in the elasmobranch Scyliorhinus stellaris after exhausting activity. Resp Physiol 16:290-303. Renshaw GMC, Kerrisk CB, Nilsson GE. 2002. The role of adenosine in the anoxic survival of the Epaulette shark, Hemiscyllium ocellatum. Comp Biochem Physiol B 131:133- 141. Renshaw GMC, Nikinmaa M. Oxygen sensors of the peripheral and central nervous system. 2007. In: D. Johnson, editor. Handbook of Neurochemistry and Molecular Neurobiology, 3rd edition, Sensory Neurochemisty, Springer, New York, p 272-296. Richmond JP, Burns JM, Rea LD, Mashburn, KL. 2005. Postnatal ontogeny of erythropoietin and hematology in free-ranging steller sea lions (Eumetopias jubatus). Gen Comp Endo 141:240-247. Routley MH, Nilsson GE, Renshaw GMC. 2002. Exposure to hypoxia primes the respiratory and metabolic responses of the epaulette shark to progressive hypoxia. Comp Biochem Physiol A 131:313-321. Salama A, Nikinmaa M. 1990. Effect of oxygen tension on catecholamine-induced formation of cAMP and on swelling of carp red blood cells. Am J Physiol 259:C723-726.
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Soderstrom V, Renshaw GMC, Nilsson GE. 1999. Brain blood flow and blood pressure during hypoxia in the epaulette shark Hemiscyllium ocellatum, a hypoxia-tolerant elasmobranch. J Exp Biol 202:829-835. Soivio A, Nikinmaa M, Westman K. 1980. The blood oxygen binding properties of hypoxic Salmo gairderi. J Comp Physiol 136:83-87. Soivio A, Westman K, Nyholm K. 1974a. Changes in haematocrit values in blood samples treated with and without oxygen; a comparative study with four salmonid species. J Fish Biol 6:763. Soivio A, Westman K, Nyholm K. 1974b. The influence of changes in oxygen tension on the haematocrit value of blood samples from asphyxic rainbow trout (Salmo gairdneri). Aqua 3:395-401. Soldatov AA. 1996. The effect of hypoxia on red blood cells of flounder: a morphologic and autoadiographic study. J Fish Biol 48:321-328. Stokes EE, Firkin BG. 1971. Studies of the Peripheral Blood of the Port Jackson Shark (Heterodontus portusjacksoni) with Particular Reference to the Thrombocyte. Br J Haematol 20:427-435. Stoskopf MK. 1993. Clinical pathology. In Fish Medicine. W.B. Saunders, Philadelphia. Stoskopf MK, Smith B, Klay G. 1984. Clinical note: Blood sampling of captive sharks. J Zool Anat Med 15:116-117. Swift DJ, Lloyd R. 1974. Changes in urine flow rate and haematocrit value of rainbow trout Salmo gairdneri (Richardson) exposed to hypoxia. J Fish Biol 6:379-387. Tetens V, 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. Thomas S, Fievet B, Motais R. 1986. Effect of deep hypoxia on acid-base balance in trout: role of ion transfer processes. Am J Physiol Regul Integr Comp Physiol 250:R319- R327. Thomas S, Hughes GM. 1982a. Effects of hypoxia on blood gas and acid-base parameters of sea bass. J Appl Physiol 53:1336-1341. Thomas S, Hughes GM. 1982b. A study of the effects of hypoxia on acid-base status of rainbow trout blood using an extracorporeal blood circulation. Resp Physiol 49:371- 382.
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Tufts BL, Randall DJ. 1989. The functional significance of adrenergic pH regulation in fish erythrocytes. Can J Zool 67:235-238. Virani NA, Rees BB. 2000. Oxygen consumption, blood lactate and inter-individual variation in the gulf killifish, Fundulus grandis, during hypoxia and recovery. Comp Biochem Physiol A 126:397-405. Wells RMG, Baldwin J. 2006. Plasma lactate and glucose flushes following burst swimming in silver trevally (Pseudocaranx dentex: Carangidae) support the "releaser" hypothesis. Comp Biochem Physiol A 143:347-352. Wells RMG, Baldwin J, Seymour RS, Christian K, Brittain T. 2005. Red blood cell function and haematology in two tropical freshwater fishes from Australia. Comp Biochem Physiol A 141:87-93. Wise G, Mulvey JM, Renshaw GMC. 1998. Hypoxia tolerance in the epaulette shark (Hemiscyllium ocellatum). J Exp Zool 281:1-5.
Wood CM, Perry SF, Walsh PJ, Thomas S. 1994. HCO3- dehydration by the blood of an elasmobranch in the absence of a Haldane effect. Resp Physiol 98:319-37. Wright PA, Perry SF, Moon TW. 1989. Regulation of hepatic glucogenesis and glycogenolysis by catecholamines in rainbow trout during environmental hypoxia. J Exp Biol 147:169-188. Yamamoto K. 1987. Contraction of spleen in exercised cyprinid. Comp Biochem Physiol A 87:1087-1097. Yamamoto K, Itazawa Y, Kobayashi H. 1983. Erythrocyte supply from the spleen and hemoconcentration in hypoxic yellowtail. Mar Biol 73:221-226.
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CHAPTER FOUR
Plasma electrolyte balance of the epaulette shark (Hemiscyllium ocellatum) and the grey carpet shark (Chiloscyllium punctatum) in response to anoxia and normoxic re-oxygenation
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Chapter 4: Plasma electrolyte balance in response to anoxia and re-oxygenation
4.1 Summary
Changes in plasma electrolytes were measured in both the epaulette shark
(Hemiscyllium ocellatum) and the grey carpet shark (Chiloscyllium punctatum) in response to a standardised anoxic challenge (1.5 hours) followed by 12 hours of normoxic re- oxygenation. Compared to normoxic controls and pre-experimental levels, plasma potassium concentrations in the epaulette shark significantly increased by 18.1% at 2 hours of re- oxygenation following anoxic exposure (5.34 ± 0.3 mmol L-1), before slowly declining and returning to pre-experimental levels by 12 hours of re-oxygenation. In contrast, the mean potassium concentrations increased by 45.7% in the plasma of the grey carpet shark immediately following anoxia. At 2 hours of re-oxygenation post anoxia, plasma potassium concentrations remained elevated in the grey carpet shark at 6.95 ± 0.5 mmol L-1, before returning to pre-experimental levels by 6 hours of re-oxygenation. There were no significant differences in chloride concentrations in the epaulette shark, whereas the grey carpet shark had a significant 5.5% decrease in plasma chloride concentrations after 2 hours of re- oxygenation and decreased plasma chloride persisted for the remainder of the experiment (10 hours). Plasma magnesium concentrations significantly increased by 19.9% in the epaulette shark (1.46 ± 0.2 mmol L-1) and by 25.8% in the grey carpet shark (1.57 ± 0.07 mmol L-1) immediately following anoxia and remained elevated for 2 hours of normoxic re-oxygenation.
There was also a 13.5% increase in plasma calcium concentrations in the epaulette shark at 2 hours of re-oxygenation (4.15 ± 0.2 mmol L-1), whereas the grey carpet shark had no significant differences in calcium concentrations over the experimental time course. The small increase in plasma potassium during re-oxygenation and the significant increases in plasma calcium and magnesium in response to anoxia and re-oxygenation in the epaulette shark are similar to anoxia tolerant vertebrates, whereas large increases in potassium following anoxia,
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Chapter 4: Plasma electrolyte balance in response to anoxia and re-oxygenation as observed in the grey carpet shark are similar to vertebrates with intermediate and negilible tolerance to anoxia. The maintenance of plasma calcium and sodium, also observed in the grey carpet shark, correspond to electrolyte changes observed in vertebrates with intermediate anoxia tolerance and indicate ion homeostasis has not be compromised, which was further supported by the maintanence of plasma urea concentrations. The elevation of plasma magnesium in both species and additionally plasma calcium after 2 hours of re-oxygenation in the epaulette shark may reveal a protective mechanism suggesting some degree of non- bicarbonate plasma buffering capacity acting to maintain the acid-base balance in the blood and the conservation of neuronal energy via inhibiting the release of excitatory neurotransmitters.
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Chapter 4: Plasma electrolyte balance in response to anoxia and re-oxygenation
4.2 Introduction
Two closely related benthic elasmobranchs, the epaulette shark (Hemiscyllium ocellatum) and the grey carpet shark (Chiloscyllium punctatum) have been observed to tolerate and survive prolonged periods in low oxygen conditions (Renshaw et al., 2002;
Chapter 2). During hypoxia, the epaulette shark up-regulates the level of anaerobic metabolism (Wise et al., 1998) while conserving ATP usage via adaptive mechanisms such as ventilatory and metabolic depression (Routley et al., 2002), bradycardia (Soderstrom et al.,
1999) and neuronal hypometabolism (Mulvey and Renshaw, 2000). Furthermore, the epaulette shark has been observed to tolerate significant periods of anoxia (Renshaw et al.,
2002; Chapter 2). It has been suggested that this tolerance to low oxygen conditions confers an adaptive advantage which enables the epaulette shark to remain active on sheltered coral reef platforms that are cyclically exposed to severe periods of hypoxia during nocturnal low tides (Routley et al., 2002; Renshaw et al., 2002; Nilsson and Renshaw, 2004).
The grey carpet shark has not been reported to commonly reside in habitats that may expose them to low oxygen saturations, although they have been observed to frequent shallow seagrass areas and are sometimes found over shallow coral reef flats (personal observations).
In both these environments oxygen levels have been reported to reach 2.0 mg. L-1 (Wise et al.,
1998; Carlson and Parson, 2001; Carlson and Parson, 2003). The grey carpet shark has recently been observed to possess a significant tolerance to anoxia, maintaining its righting reflex for approximately 172 11 minutes at 23C, similar to the total time to loss of righting reflex (LRR) in the epaulette shark (Chapter 2). However, as temperature increased, the epaulette shark was observed to tolerate significantly longer periods of anoxia before losing their righting reflex than the grey carpet shark. Therefore, the epaulette shark was concluded
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Chapter 4: Plasma electrolyte balance in response to anoxia and re-oxygenation to possess more tolerance to anoxic exposure than the grey carpet shark (Chapter 2).
Following anoxic exposure in the grey carpet shark, Chapman and Renshaw (in press, Chapter
3) reported (i) significant increases in anaerobic metabolism, indicated by elevated plasma lactate concentrations, (ii) increased erythrocyte concentrations, possibly due to splenic contraction and/or haemoconcentration and (iii) elevated plasma glucose concentrations during re-oxygenation. However, energy conserving mechanisms common in other anoxia tolerant vertebrates, such as neuronal hypometabolism (Mulvey and Renshaw, 2000) and ventilatory depression (Routley et al., 2002) are not activated in the grey carpet shark
(Chapter 2). Therefore while the grey carpet shark possesses a significant ability to survive anoxic exposure, the protective strategies employed by this species are unknown.
In other anoxia tolerant vertebrates, such as the teleosts in the genus Carassius and freshwater turtles in the genera Chrysemys and Trachemys, plasma electrolytes have been observed to be involved in a number of physiological functions during anoxic exposure.
Jackson and Ultsch (1982) and Jackson and Heisler (1982) reported a decrease in plasma sodium and an increase in plasma potassium concentrations following prolonged anoxia in C. picta bellii. Jackson and Heisler (1983) also reported a reduction in chloride concentrations along with an increase in potassium concentrations within the plasma in this species. Taken together, these observations support a loss of transmembrane ion gradients, characterising the disruption of ionic homeostasis. Alternatively, movement of sodium and chloride from the plasma have been observed in many vertebrates exposed to low oxygen concentrations due to the activation of Na+/H+ exchangers inducing cell swelling and subsequently regulating intracellular pH (Fievet et al., 1987; Chiocchia and Motais, 1989; Salama and Nikinmaa,
1990). Jackson and Heisler (1983) also reported an increase in plasma calcium and
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Chapter 4: Plasma electrolyte balance in response to anoxia and re-oxygenation magnesium concentrations, which act as non-bicarbonate buffers responsible for maintaining pH levels and reducing acidification.
In elasmobranchs, the effects of low oxygen saturation on plasma electrolytes have been little studied. Therefore, the present study examined the changes in plasma electrolytes, specifically potassium, sodium, chloride, calcium and magnesium in the epaulette shark and the grey carpet shark in response to a standardised anoxia exposure followed by a re- oxygenation recovery period.
4.3 Materials and methods
4.3.1 Study animals and location
All animals were supplied by UnderWater World (Mooloolaba, Sunshine Coast) and the experiments were carried out on site. All animals had been held in captivity for a period of at least 2 years. Epaulette sharks (n=9) had a mean length of 71.58 + 7.1 cm and weight of
1.43 + 0.3 kg, while grey carpet sharks (n=9) had a mean length of 88.56 + 8.1 cm and weight of 2.94 + 0.8 kg. Animals were kept in a constant seawater flow-through aquarium with a normal photoperiod and no additional light sources in their outside habitats. Food was withheld for 24 hours prior to experimentation and blood sampling.
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Chapter 4: Plasma electrolyte balance in response to anoxia and re-oxygenation
4.3.2 Anoxic exposure
Individual animals were exposed to 1.5 hours of anoxia (experimental treatment) or normoxia (control treatment) followed by 12 hours of re-oxygenation after transfer to a separate normoxic tank, as described in Chapman and Renshaw (in press, Chapter 3). In brief, animals were exposed to anoxic and normoxic control treatments in 70 litre glass tanks.
Each tank was filled with normoxic seawater (24°C) and sealed with plastic wrap and a
Perspex lid. Seawater was circulated around the tank using a submersible 228-power head pump (CCC Pty. Ltd., Sydney, New South Wales, Australia) to ensure uniform conditions throughout. In the anoxic tanks, nitrogen gas was bubbled through an air stone to displace dissolved oxygen. The dissolved oxygen concentrations [dO2] were measured and recorded every minute using TPS WP-90 oxygen probes (TPS Pty. Ltd., Brisbane, Queensland,
-1 Australia). In the anoxic tank the [dO2] was maintained below 0.02 mg L . In the normoxic
-1 tanks, air was bubbled through an air stone to maintain the [dO2] above 7.0 mg L . Control and experimental animals were closely pair-matched for length before being randomly chosen as control or experimental specimens. Animals were weighed and measured at the conclusion of the experiment.
4.3.3 Blood analysis
Blood samples were taken immediately prior to placement in each tank at the beginning of the experiment (pre-/t0) to provide a pre-experimental measurement. A 1.5 ml blood sample was taken from the dorsal aorta of each animal using a heparinised syringe via the venipuncture method described in Stoskopf et al. (1984). Blood samples were then transferred to 3 ml lithium heparin vacutainers (Becton Dickinson, North Ridge, New South
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Chapter 4: Plasma electrolyte balance in response to anoxia and re-oxygenation
Wales, Australia) and gently mixed. Additional blood samples were collected immediately following treatment (post-/t1.5) and then after 2 hours (t2), 6 hours (t6) and 12 hours (t12) of re- oxygenation in normoxia.
Blood plasma was separated from a 0.5 ml blood sample after it was centrifuged at
3000 rpm for 3 minutes at 4C then frozen at –80C for later analysis. Plasma electrolyte concentrations of potassium, sodium, chloride, calcium and magnesium were measured using an Olympus AU400 blood analyser (Olympus America Inc, Diagnostic Systems Divisions,
Two Corporate Centre Drive, Melville, NY, USA) by the staff at the School of Veterinary
Science, The University of Queensland (Brisbane, Australia). Potassium and sodium concentrations were measured using crown ether membrane electrodes and a molecular oriented PVC membrane for plasma chloride concentrations. Plasma calcium and magnesium concentrations were measured via photometric colour analysis. Plasma calcium was incubated in Arsenazo III reagent and measured bichromatically at 660/700 nm, while plasma magnesium concentrations were incubated with Olympus magnesium reagent (Magnesium reagent OSRT6189, Olympus) and measured bichromatically at 520/800 nm.
In addition, plasma urea concentrations were determined using a colorimetric urea
(BUN) assay kit (Sigma Aldrich, Australia) following the manufacturer’s directions and read at a wavelength of 340 nm. Cuvettes containing a 1:100 dilution of plasma to urea reagent was gently vortexed then incubated at 37ºC for 30 seconds before measurement then measured again after an additional 60 second incubation at 37ºC. Plasma urea concentrations were determined by dividing changes in plasma urea absorbances with changes in absorbances of standard solutions, as described by the manufacturer (Sigma Aldrich,
Australia).
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Chapter 4: Plasma electrolyte balance in response to anoxia and re-oxygenation
4.3.4 Statistics
The data was analysed using a Mixed Model Repeated Measures ANOVA with a posthoc adjustment (Bonferroni). Data is presented as the mean standard deviation.
4.4 Results
4.4.1 Plasma potassium levels
The mean plasma potassium concentrations in the epaulette shark prior to treatment were
-1 similar in anoxia treated (t0 = 4.52 ± 0.2 mmol L ) and control groups (t0 = 4.68 ± 0.2 mmol
L-1) (Figure 4.1). No significant differences were observed in mean plasma potassium levels of control epaulette sharks at any time point throughout the experiment. Furthermore, no significant differences in mean plasma potassium concentrations were observed immediately following anoxia in the epaulette shark. However, by 2 hours of normoxic re-oxygenation, mean plasma potassium concentrations increased by approximately 18.1% (p<0.001) from pre-experimental levels. Mean plasma potassium concentrations were not significantly
-1 different from pre-experimental levels at 6 hours (t6 = 4.94 ± 0.3 mmol L ) and 12 hours (t12 =
4.54 ± 0.5 mmol L-1) of re-oxygenation.
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Chapter 4: Plasma electrolyte balance in response to anoxia and re-oxygenation
Changes in plasma potassium concentrations in response to anoxia and re- oxygenation or control treatments in the epaulette shark 6 †
5
) 1 - 4
Pre Post 3 2 hr 6 hr 12 hr
2 Concentration Concentration (mmol L
1
0 Anoxia Control Treatment Figure 4.1. Mean plasma potassium concentrations of the epaulette shark (H. ocellatum). Mean plasma potassium concentrations before and immediately following 1.5 hours of anoxia and at 2 hours, 6 hours and 12 hours of re- oxygenation. Symbols indicate significant differences from pre-experimental mean measures (†).
Changes in plasma potassium concentrations in response to anoxia and re- oxygenation or control treatments in the grey carpet shark 8 *
7 †
) 6 1
-
5 Pre Post 4 2 hr 6 hr 12 hr 3
Concentration (mmol L 2
1
0 Anoxia Control Treatment Figure 4.2. Mean plasma potassium concentrations of the grey carpet shark (C. punctatum). Mean plasma potassium concentrations before and immediately following 1.5 hours of anoxia and at 2 hours, 6 hours and 12 hours of re- oxygenation. Symbols indicate significant differences from pre-experimental mean immediately (*) and 2 hours (†)
following treatment.
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Chapter 4: Plasma electrolyte balance in response to anoxia and re-oxygenation
The mean plasma potassium concentrations in the grey carpet shark prior to treatment
-1 were similar in anoxia treated (t0 = 4.78 ± 0.3 mmol L ) and control groups (t0 = 4.98 ± 0.3 mmol L-1) (Figure 4.2). Furthermore, mean plasma potassium concentrations were not significantly different from pre-experimental levels at any time point throughout the experiment in the grey carpet shark control group. Immediately following anoxia, mean plasma potassium concentrations had significantly increased by approximately 45.7% in the grey carpet shark (p<0.001). Mean plasma potassium concentrations, remained elevated at this plateau for 2 hours following re-oxygenation (p<0.01) and then returned back to pre-experimental concentrations by 6 hours of normoxic re-oxygenation. Mean plasma potassium concentrations remained at pre-experimental concentrations throughout the remainder of the experiment.
4.4.2 Calcium
The mean plasma calcium concentrations in the epaulette shark prior to treatment were
-1 similar in anoxia treated (t0 = 3.66 ± 0.2 mmol L ) and control groups (t0 = 3.76 ± 0.2 mmol
L-1) (Figure 4.3). Immediately following anoxia, the mean plasma calcium concentrations were not significantly different from mean pre-experimental levels. However by 2 hours of re- oxygenation, anoxia treated epaulette sharks had a significant 13.5% increase in mean plasma calcium concentrations from pre-experimental levels (p<0.02). By 6 hours, mean plasma calcium concentrations had returned to pre-experimental levels and remained at these levels for the rest of the re-oxygenation period. No significant differences were observed in mean plasma calcium concentrations in the control treatment animals at any time point throughout the experiment.
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Chapter 4: Plasma electrolyte balance in response to anoxia and re-oxygenation
Changes in plasma calcium concentrations in response to anoxia and re- oxygenation or control treatments in the epaulette shark 5 4.5 †
4
) 1 - 3.5
3 Pre Post 2.5 2 hr 6 hr 2 12 hr
1.5 Concentration Concentration (mmol L 1
0.5 0 Anoxia Control Treatment Figure 4.3. Mean plasma calcium concentrations of the epaulette shark (H. ocellatum). Mean plasma calcium concentrations before and immediately following 1.5 hours of anoxia and at 2 hours, 6 hours and 12 hours of re-
oxygenation. Symbols indicate significant differences from pre-experimental mean immediately (†) following treatment.
Prior to treatment, pre-experimental, the mean plasma calcium concentrations in the
-1 grey carpet shark were not significantly different in either anoxia (t0 = 4.11 ± 0.2 mmol L ) or
-1 control groups (t0 = 3.99 ± 0.2 mmol L ). Furthermore, there were no significant changes in mean plasma calcium concentrations in grey carpet sharks compared to pre-experimental mean concentrations immediately following 1.5 hours of anoxia and throughout the 12 hours of re- oxygenation. In addition, there were no significant differences in mean plasma calcium concentrations in the control treated grey carpet shark compared to their pre-experimental mean concentrations at any of the time points.
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Chapter 4: Plasma electrolyte balance in response to anoxia and re-oxygenation
4.4.3 Sodium
The mean plasma sodium concentrations in the epaulette shark and the grey carpet shark prior to treatment were 267.6 ± 8.2 mmol L-1 and 272.5 ± 5.9 mmol L-1 respectively.
Neither species showed significant differences between experimental or control groups prior to experimental treatment. Immediately following 1.5 hours of anoxia and throughout the 12 hours of re-oxygenation, no significant changes in mean plasma sodium concentrations occurred in either the epaulette shark or the grey carpet shark compared to their respective pre- experimental mean concentrations. Likewise, no significant differences in mean plasma sodium concentrations were observed at any of the time points in the normoxic treated animals, compared to their pre-experimental mean concentrations, in either the epaulette shark or the grey carpet shark.
4.4.4 Chloride
The mean plasma chloride concentrations in the epaulette shark prior to treatment
-1 -1 were t0 = 259.8 ± 7.8 mmol L and t0 = 259.25 ± 5.1 mmol L in the experimental and control groups respectively. No significant differences occurred in the control or anoxic groups at any time point throughout the treatment.
Prior to treatment, the mean plasma chloride concentrations in the grey carpet shark
-1 were not significantly different between anoxia (t0 = 267.25 ± 5.5 mmol L ) and control
-1 groups (t0 = 268 ± 5.7 mmol L ) (Figure 4.4). In addition, no significant differences were observed in mean plasma chloride concentrations immediately following anoxia in the grey carpet shark. However, by 2 hours of re-oxygenation, mean plasma chloride concentrations
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Chapter 4: Plasma electrolyte balance in response to anoxia and re-oxygenation had decreased 5.5% from mean pre-experimental values (p<0.001). Mean plasma chloride concentrations remained at these levels throughout the remainder of the experiment (t6 =
-1 -1 252.75 ± 9 mmol L ; t12 = 254.75 ± 6.2 mmol L ).
Changes in plasma chloride concentrations in response to anoxia and re- oxygenation or control treatments in the grey carpet shark 300 † # × 250
) 1 - 200
Pre Post 150 2 hr 6 hr 12 hr
100 Concentration Concentration (mmol L
50
0
Anoxia Control Treatment
Figure 4.4. Mean plasma chloride concentrations of the grey carpet shark (C. punctatum). Mean plasma chloride concentrations before and immediately following 1.5 hours of anoxia and at 2 hours, 6 hours and 12 hours of re- oxygenation. Symbols indicate significant differences from pre-experimental mean measures immediately (*), 2 hours (†), 6 hours (#) and 12 hours (×) following treatment.
4.4.5 Magnesium
The mean plasma magnesium concentrations in the epaulette shark prior to treatment
-1 were not significantly different between anoxia (t0 = 1.22 ± 0.07 mmol L ) and control (t0 =
1.14 ± 0.04 mmol L-1) groups (Figure 4.5). Immediately following anoxic exposure, epaulette sharks had a significant 19.9% increase in the mean plasma magnesium concentrations (t1.5 =
1.46 ± 0.2 mmol L-1) above the pre-experimental mean measure (p<0.001). Mean plasma magnesium concentrations remained at this elevated plateau for up to 2 hours of re-
-1 oxygenation (t2 = 1.46 ± 0.1 mmol L ) and returned to pre-experimental concentrations by 6
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Chapter 4: Plasma electrolyte balance in response to anoxia and re-oxygenation hours of re-oxygenation. The mean plasma magnesium concentrations of the epaulette shark remained at these concentrations for the remainder of the experiment. No significant differences in mean plasma magnesium concentrations occurred in the epaulette shark control group at any time point throughout the treatment.
Changes in plasma magnesium concentrations in response to anoxia and re- oxygenation or control treatments in the epaulette shark 1.8 1.6 * †
1.4
) 1 - 1.2
1 Pre Post 2 hr 0.8 6 hr 12 hr 0.6
Concentration Concentration (mmol L 0.4
0.2
0 Anoxia Control Treatment Figure 4.5. Mean plasma magnesium concentrations of the epaulette shark (H. ocellatum). Mean plasma magnesium
concentrations before and immediately following 1.5 hours of anoxia and at 2 hours, 6 hours and 12 hours of re- oxygenation. Symbols indicate significant differences from pre-experimental mean immediately (*) and 2 hours (†) following treatment).
The mean plasma magnesium concentrations in the grey carpet sharks were not
-1 significantly different between anoxia groups (t0 = 1.25 ± 0.03 mmol L ) and control groups
-1 (t0 = 1.24 ± 0.1 mmol L ) prior to treatment (Figure 4.6). Immediately following anoxia, the grey carpet shark had a 25.8% increase in mean plasma magnesium concentrations (t1.5 = 1.57
± 0.07 mmol L-1) above pre-experimental levels. Mean plasma magnesium concentrations remained at this elevated plateau for 2 hours following re-oxygenation (t2 = 1.48 ± 0.2 mmol
L-1). By 6 hours, mean plasma magnesium concentrations were not significantly different from pre-experimental or elevated values, remaining at these concentrations until the last
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Chapter 4: Plasma electrolyte balance in response to anoxia and re-oxygenation sample point at 12 hours of re-oxygenation. No changes in mean plasma magnesium concentrations in the grey carpet shark were observed at any time point throughout the control treatment.
Changes in plasma magnesium concentrations in response to anoxia and re- oxygenation or control treatments in the grey carpet shark 1.8 * † 1.6
1.4
) 1 - 1.2
1 Pre Post 2 hr 0.8 6 hr 12 hr 0.6
Concentraion Concentraion (mmolL 0.4
0.2
0 Anoxia Control Treatment Figure 4.6. Mean plasma magnesium concentrations of the grey carpet shark (C. punctatum). Mean plasma magnesium
concentrations before and immediately following 1.5 hours of anoxia and at 2 hours, 6 hours and 12 hours of re- oxygenation. Symbols indicate significant differences from pre-experimental mean immediately (*) and 2 hours (†) following treatment.
Urea
The pre-experimental mean plasma urea concentration in the epaulette shark and the grey carpet shark was 338.81 (±18.3) mg dL-1 and 263.83 (± 10.6) mg dL-1 respectively. No significant differences were observed between mean plasma urea concentrations in response to 1.5 hours of anoxia and 12 hours of normoxic re-oxygenation for either species relative to their respective pre-experimental concentrations in the normoxia or anoxia treated groups.
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Chapter 4: Plasma electrolyte balance in response to anoxia and re-oxygenation
Table 4.1. Percent change in plasma electrolyte and plasma urea concentrations of the epaulette shark (H. ocellatum) and the grey carpet shark (C. punctatum) in response to anoxia and re-oxygenation. (± standard deviation). Symbols indicate significant differences from pre-experimental levels immediately following (post) anoxia (*) and at 2 hours of re-
oxygenation (†) compared to the pre-experimental concentrations.
Epaulette shark Grey carpet shark Pre-Post Pre-2hrs Pre-Post Pre-2hrs Potassium (K+) 9.7 ±3.2 18.1 ± 2.7† 45.7 ± 8.3* 26.8 ± 3.9† Sodium (Na+) -0.8 ± 4.7 3.9 ± 3.6 2.6 ± 2.2 0.4 ± 4.8 Anoxia Chloride (Cl-) -1.2 ± 4.7 -1.5 ± 2.9 2.2 ± 1.7 -5.5 ± 2.5† Calcium (Ca+) 5.7 ± 6.2 13.5 ±5† 3.5 ± 4.7 3.1 ±8 Magnesium (Mg+) 19.9 ± 9.5* 20.2 ± 7.6† 25.8 ± 3.9* 18 ± 11.4† Urea 2.8 ± 2.2 -0.7 ± 1.8 0.3 ± 1.9 -6.0 ± 6.7 Potassium (K+) -5.8 ± 3.5 -7.4 ± 4.9 -4.9 ± 9.9 -6.5 ±7 Sodium (Na+) 1.6 ± 2 1.7 ± 2.2 1.8 ± 4 0.5 ± 4.7 Chloride (Cl-) 0.5 ± 1.2 -1.6 ± 2.2 -1.3 ± 1.9 -1.3 ± 1.9 Control Calcium (Ca+) 1.3 ± 2.1 1.6 ± 3 4.9 ± 8.1 3.7 ± 10.5 Magnesium (Mg+) 3 ± 2.2 1.9 ± 3.4 1.7 ± 6.2 6.4 ± 3 Urea -1.5 ± 2.9 -0.1 ± 4.6 -4.3 ± 8.8 -3.9 ± 6.1
4.5 Discussion
The basal plasma electrolyte concentration can vary significantly among elasmobranch species (Harms et al., 2002; Sulikowski et al., 2003; Mandelman and Farrington, 2007).
These variations have been attributed to differences in electrolyte concentrations in the ambient environment, diet and/or specific physiological characteristics (Harms et al., 2002;
Sulikowski et al., 2003; Mandelman and Farrington, 2007). In the present study, epaulette sharks and grey carpet sharks had similar basal plasma electrolyte concentrations.
Following anoxia, the epaulette shark and the grey carpet shark showed significant changes in plasma electrolyte concentrations. While the epaulette shark has not been observed to undergo a haemococnetrations of the blood in response to anoxia, evidence suggests that this may occur in the grey carpet shark (Chapman and Renshaw, in press, Chapter3). The
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Chapter 4: Plasma electrolyte balance in response to anoxia and re-oxygenation haemoconcentration of the blood may therefore be responsible for changes in plasma electrolyte concentrations in the grey carpet shark in response to anoxia. However, the maintenance of plasma constituents such as sodium, calcium and urea indicates that haemoconcentration of the blood is unlikely to have occurred. Therefore, changes in plasma electrolytes observed in the present study are due to electrolyte movements in response to anoxia exposure and normoxic re-oxygenation.
Following anoxia, the epaulette shark had a significant 18.1% increase in plasma potassium by 2 hours of re-oxygenation following anoxia, while the grey carpet shark had a
45.7% increase in mean plasma potassium concentrations immediately following anoxia treatment, which remained elevated for 2 hours of re-oxygenation before returning to pre- experimental levels. An increase in plasma potassium, hyperkalemia, has been observed in a number of vertebrates in response to hypoxic and anoxic stress (Jackson and Heisler, 1982;
Jackson and Ultsch, 1982; Jackson and Heisler, 1983; Krumschnabel et al., 1998; Hochachka et al., 1996; Warren and Jackson, 2005). However, the cause and mechanisms underlying the movement of potassium in response to anoxia has not been fully characterised.
Hyperkalemia in response to anoxia has been repeatedly observed in anoxia-tolerant and -intolerant vertebrates (Jackson and Heisler, 1982; Jackson and Ultsch, 1982; Jackson and
Heisler, 1983; Krumschnabel et al., 1998). Jackson and Heisler (1983) suggested that increases in plasma potassium in anoxia-tolerant freshwater turtles are attributed to the partial failure of Na+/K+ pumps in the skeletal muscle as a consequence of anoxia induced ATP depletion. Failure of Na+/K+ pumps allows an unopposed influx of sodium and efflux of potassium from the cell. Interestingly, the grey carpet shark and the epaulette shark had no change in mean plasma sodium concentrations at any time point throughout the treatment,
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Chapter 4: Plasma electrolyte balance in response to anoxia and re-oxygenation indicating the functioning of Na+/K+ pumps. This suggests an alternative source of potassium or an influx of sodium from the external environment via the gills or sequestering of sodium excretion from the kidneys and/or rectal gland.
Furthermore, failure or temporary arrest of Na+/K+ pumps is typically accompanied by an intracellular influx in chloride as a consequence of sodium influx from the extracellular fluid. No changes in chloride were observed in the epaulette shark, although the grey carpet shark had a decrease in plasma chloride concentrations at 2 hours of re-oxygenation and throughout the remainder of the experiment. An influx of chloride would result in an increase in cell volume due to osmotically driven water following chloride into the cell. While
Chapman and Renshaw (in press, Chapter 3) revealed there was an increase in haematocrit immediately following anoxia, no evidence of cell swelling was observed in the grey carpet shark.
It appears from the plasma electrolytes that the grey carpet shark is unlikely to be experiencing complete energy failure because of the maintenance of plasma sodium and calcium and urea concentrations. The rise in extracellular potassium may be due to the activation of ATP sensitive potassium channels (KATP channels), indicating that ATP levels may not be depleted but are very low in this animal. Jonas et al. (1991) reported evidence of
KATP channels opening in the clawed frog (Xenopus laevis) in response to severely low ATP levels. The opening of KATP channels clamp membrane potential and counteract neuronal excitation (Lutz and Nilsson, 1997). Thus, these channels directly respond to the intracellular energy charge and may be intimately involved in maintaining energy homeostasis.
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Chapter 4: Plasma electrolyte balance in response to anoxia and re-oxygenation
Immediate increases in plasma potassium concentrations were not observed in the epaulette shark during the 1.5 hours of anoxia treatment given in this study, supporting the findings of Chapter 2, that the epaulette shark may possess significantly greater tolerance to anoxia due to the activation of energy conserving mechanisms, such as ventilatory depression
(Routley et al., 2002), bradycardia (Soderstrom et al., 1999) and neuronal hypometabolism
(Mulvey and Renshaw, 2000). Therefore, the small increase in plasma potassium concentrations observed in the epaulette shark may be due to the presence of these energy conserving strategies delaying the opening of the KATP channels.
In contrast, Chapter 2 reported the absence of these energy conserving strategies in the grey carpet shark in response to anoxia. Therefore, ATP levels may be depleted faster in the grey carpet shark, resulting in opening of KATP channels earlier and to a greater magnitude, supporting observations made in the present study.
A number of other mechanisms not directly related to the energetic state of the cell may also contribute to the loss of potassium homeostasis under anoxic conditions observed in the grey carpet shark. Bianchini et al. (1988) reported that cell swelling in anoxia-intolerant trout hepatocytes led to a rise in potassium membrane permeability. While no changes in RBC volume were observed during anoxia in the grey carpet shark, increased potassium permeability of other cell membranes have not been investigated.
Similar to the grey carpet shark, Warren and Jackson (2005) observed a significant increase in plasma potassium, with no changes in sodium concentrations in anoxia treated leopard frogs (Rana pipiens). Interestingly, the plasma chloride concentrations in the grey carpet shark decreased during re-oxygenation. Reductions in plasma chloride concentrations
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Chapter 4: Plasma electrolyte balance in response to anoxia and re-oxygenation may be explained via movement of extracellular chloride i) into the intracellular compartments (Chiocchia and Motais, 1989), ii) through the gills into the external water
(Liber et al., 2005), iii) by urinary excretion (Hunn, 1969) or iv) a combination of these mechanisms. However, movement of chloride is often closely associated with sodium concentrations, which appeared to be maintained following anoxia and re-oxygenation in the grey carpet shark. Therefore, further investigation of the movements of sodium and chloride ions in the grey carpet shark during anoxic exposure are required to explain the results observed in the present study.
In contrast, the epaulette shark displayed a significant increase in RBC volume in response to anoxia and 2 hours of re-oxygenation (Chapman and Renshaw, in press, Chapter
3), which may have resulted in an increase in RBC potassium permeability, causing the 18.1% increase in plasma potassium observed in the present study. Chapman and Renshaw (in press,
Chapter 3) also suggested that cell swelling may be due to the activation of Na+/H+ exchangers in the RBC membrane. However, the present study observed no significant differences in plasma sodium or chloride concentrations in response to anoxia and re-
oxygenation in the epaulette shark. This therefore indicates that the activation of Na+/H+ exchangers in the RBC membrane are not responsible for RBC swelling in the epaulette shark following anoxic exposure as previously suggested (Chapman and Renshaw, in press, Chapter
3).
While plasma bicarbonate acts as a significant pH buffer within the blood, non- bicarbonate buffers, such as magnesium and calcium have also been observed to be heavily involved in maintaining the acid-base balance during events of anoxia. The anoxia-tolerant freshwater turtle (T. scripta) and the leopard frog (R. pipiens) that shows and intermediate
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Chapter 4: Plasma electrolyte balance in response to anoxia and re-oxygenation degree of anoxia-tolerance, have been observed to steadily increase plasma concentrations of both magnesium and calcium throughout anoxia, providing a significant buffering capacity against high plasma lactate (Jackson and Heisler, 1982; Warren and Jackson, 2005; Warren et al., 2006). Although other tissues may serve as a source for magnesium and calcium, Jackson and Heisler (1983) concluded that the common mobilisation of these elements was from bone and/or shell. Elasmobranch skeletons however, are absent of bone and are constructed of calcified cartilage (Dingerkus et al., 1991). Cartilaginous tissue is also known to store magnesium and calcium, although at much lower concentrations than bone. However, calcium and magnesium concentrations of calcified cartilage are species specific within elasmobranchs (Dingerkus et al., 1991). The calcified cartilaginous skeleton of the epaulette shark and the grey carpet shark may therefore be a source of magnesium and calcium that may be mobilised during events of anoxic stress, although other tissues such as skeletal muscle could potentially serve the same function during anoxic exposure, by releasing stored calcium and magnesium.
A significant increase in plasma lactate concentrations occurred after 1.5 hours of anoxia in the epaulette shark, and to a lesser extent the grey carpet shark (Chapman and
Renshaw, in press, Chapter 3). However, changes in blood pH associated with these high plasma lactate concentrations have not been examined in either species. Bicarbonate and non- bicarbonate buffers, such as magnesium and calcium, act to maintain the acid-base balance within the blood in most vertebrates. However the buffering capacity of the epaulette shark and the grey carpet shark needs to be examined. The present study found an increase in plasma magnesium concentrations in both species and a significant increase in plasma calcium concentrations in the epaulette shark. These increases however, were significantly smaller than those observed in T. scripta and R. pipiens (Jackson and Heisler, 1982; Warren
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Chapter 4: Plasma electrolyte balance in response to anoxia and re-oxygenation and Jackson, 2005). While the epaulette shark and the grey carpet shark were exposed to 1.5 hours of anoxia, T. scripta and R. pipiens were exposed to anoxia for 12 weeks at temperatures close to 0C and 2.5 hours at 15C, respectively. Therefore, such differences in plasma magnesium and calcium concentrations may be due to the prolonged anoxic exposure time of both T. scripta and R. pipiens compared to the sharks in the present study. It is likely that bicarbonate buffers maintain the acid-base balance during short term anoxia with only small changes in non-bicarbonate buffers and that greater mobilisation of non-bicarbonate buffers, such as magnesium and calcium, may occur during prolonged anoxic challenges. The small increase in both magnesium and calcium in the epaulette and the grey carpet shark may also be due to limited stores of magnesium and calcium, which may be mobilised and used as non-bicarbonate buffers during anoxic exposure. Both Jackson and Heisler (1982) and Warren and Jackson (2005) concluded that mobilised magnesium and calcium were sourced from bone and shell stores during anoxic exposure. Therefore, cartilaginous fish like the epaulette shark and the grey carpet shark may have significantly reduced stores to mobilise during anoxia, which could explain the smaller increases observed in these species in response to anoxia compared to bony vertebrates. Another explanation for the small increase in both magnesium and calcium in the epaulette and the grey carpet shark may be due to a limited capacity of the epaulette shark and the grey carpet shark to mobilise magnesium and calcium as non-bicarbonate buffers during anoxic exposure.
Magnesium also appears to play a protective role in anoxia-tolerant vertebrates.
During anoxia, the loss of ion gradients does not occur in anoxia-tolerant vertebrates despite a reduction in ion pump activity. This reduction in activity (ion channel arrest) is suggested to play a vital role in ATP conservation during anoxic exposure (Hochachka et al., 1996).
Magnesium has been observed to be involved in binding to the neuronal ligand-gated ion
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Chapter 4: Plasma electrolyte balance in response to anoxia and re-oxygenation channels such as the N-methyl-D-aspartate receptor (NMDAR; glutamate receptor subtype).
The involvement of magnesium blocking of NMDAR significantly reduces the release of extracellular excitatory neurotransmitters such as aspartate and glutamate (Benveniste et al.,
1984). These neurotransmitters can bind to their corresponding receptors and cause further ionic imbalance via receptor-mediated influx of sodium and calcium. Therefore elevated plasma magnesium concentrations observed in the epaulette shark in response to anoxia may aid in NMDAR blockage and subsequently reduce the release of excitatory neurotransmitters, maintain ionic homeostasis and conserve cellular ATP levels.
Following anoxia and re-oxygenation, the plasma electrolyte responses in the anoxia- tolerant epaulette shark suggest maintenance of ion homeostasis and mobilisation of electrolytes that may assist in maintaining the acid-base balance and inhibiting the release of excitatory neurotransmitters and subsequently conserving neuronal ATP levels. In contrast, the grey carpet shark had a significant increase in plasma potassium and maintenance of plasma sodium, similar to vertebrates with intermediate anoxia tolerance. However, further investigation is required to determine whether the grey carpet shark is conserving ATP levels similar to anoxia-tolerant vertebrates, slowly consuming ATP levels similar to vertebrates with intermediate anoxia tolerance or is surviving non-fatal incidences of necrosis and apoptosis during anoxic exposure.
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4.6 Literature cited
Benveniste H, Drejer J, Schousboe, A Diemer NH. 1984. Elevation of the extracellular concentrations of glutamate and aspartate in the rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis. J Neurochem 43:1369– 1374. Bianchini L, Fossat B, Porthé-Nibelle J, Ellory JC, Lahlou B. 1998. Effects of hyposmotic shock on ion fluxes in isolated trout hepatocytes. J Exp Biol 137:303-18. Carlson JK, Parsons GR. 2001. The effects of hypoxia on three sympatric shark species: Physiological and behavioural responses. Enviro Biol Fish 61:427-433. Carlson JK, Parsons GR. 2003. Respiratory and hematological responses of the bonnethead shark, Sphyrna tiburo, to acute changes in dissolved oxygen. J Exp Biol 294:15-26. Chiocchia G, Motais R. 1989. Effect of catecholamine on deformability of red cells from trout: relative roles of cyclic AMP and cell volume. J Physiol (Paris) 412:321-332. Dingerkus G, Séret B, Guilbert E. 1991. Multiple prismatic calcium phosphate layers in the jaws of present-day sharks (Chondrichthyes; Selachii). Experientia 47(1):38-40. Fievet B, Motais R, Thomas S. 1987. Role of adrenergic-dependent H+ release from red cells in acidosis induced by hypoxia in trout. Am J Physiol 252:R269-275. Harms C, Ross T, Segars A. 2002. Plasma biochemistry reference values of wild bonnethead sharks, Sphyrna tiburo. Vet Clin Path 31(3):111-115. Hochachka PW, Buck LT, Doll CJ, Land SC. 1996. Unifying theory of hypoxia tolerance: molecular/metabolic defense and rescue mechanisms for surviving oxygen lack. Proc Natl Acad Sci USA 93:9493–9498. Hunn, JP. 1969. Chemical composition of rainbow trout urine following acute hypoxic stress. Trans Am Fish Soc 98:20–22. Jackson DC, Heisler N. 1982. Plasma ion balance of submerged anoxic turtles at 3 degrees C: the role of calcium lactate formation. Respir Physiol 49(2):159-174. Jackson DC, Heisler N. 1983. Intracellular and extracellular acid-base and electrolyte status of submerged anoxic turtles at 3 degrees C. Respir Physiol 53(2):187-201. Jackson DC, Ultsch GR. 1982. Long-term submergence at 3 C of the turtle, Chrysemys picta bellii, in normoxic and severely hypoxic water. II. Extracellular ionic responses to extreme lactic acidosis. J Exp Biol 96:29-43.
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Jonas P, Koh DS, Kampe K. 1991. ATP-sensitive and Ca-activated K channels in vertebrate axons: Novel links between metabolism and excitability. Pfluger Arch 418:68-73. Krumschnabel G, Frischmann ME, Schwarzbaum PJ, Wieser W. 1998. Loss of K+ homeostasis in trout hepatocytes during chemical anoxia: a screening study for potential causes and mechanisms. Arch Biochem Biophys. 353(2):199-206. Mandelman JW, Farrington MA. 2007. The estimated short-term discard mortality of a trawled elasmobranch, the spiny dogfish (Squalus acanthias). Fish Res 83(2-3):238- 245. Liber K, Weber L, Levesque C. 2005. Sublethal toxicity of two wastewater treatment polymers to lake trout fry (Salvelinus namaycush). Chemo 61(8):1123-1133. Lutz PL, Nilsson GE. 1997. Contrasting strategies for anoxic brain survival-glycolysis up or down. J Exp Biol 200:411-419. Mulvey JM, Renshaw GMC. 2000. Neuronal oxidative hypometabolism in the brainstem of the epaulette shark (Hemiscyllium ocellatum) in response to hypoxic pre-conditioning. Neurosci Let 290:1-4. Nilsson GE, Renshaw GMC. 2004. Hypoxic survival strategies in two fishes: extreme anoxia tolerance in the North European crucian carp and natural hypoxic preconditioning in a coral-reef shark. J Exp Biol 207:3131-3139. Renshaw GMC, Kerrisk CB, Nilsson GE. 2002. The role of adenosine in the anoxic survival of the epaulette shark, Hemiscyllium ocellatum. Comp Biochem Physiol B 131:133- 141. Routley MH, Nilsson GE, Renshaw GMC. 2002. Exposure to hypoxia primes the respiratory and metabolic responses of the epaulette shark to progressive hypoxia. Comp Biochem Physiol A 131:313-321. Salama A, Nikinmaa, M. 1990. Effect of oxygen tension on catecholamine-induced formation of cAMP and on swelling of carp red blood cells. Am J Physiol 259:C723-726. Soderstrom V, Renshaw GMC, Nilsson GE. 1999. Brain blood flow and blood pressure during hypoxia in the epaulette shark Hemiscyllium ocellatum, a hypoxia-tolerant elasmobranch. J Exp Biol 202:829-835. Stoskopf MK, Smith B, Klay G. 1984. Clinical note: Blood sampling of captive sharks. J Zool Anat Med 15:116-117.
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Sulikowski JA, Treberg JR, Hunting Howell W. 2003. Fluid regulation and physiological adjustments in the winter skate, Leucoraja ocellata, following exposure to reduced environmental salinities. Enviro Biol Fish 66:339-348. Warren DE, Jackson DC. 2005. The role of mineralized tissue in the buffering of lactic acid during anoxia and exercise in the leopard frog Rana pipiens. J Exp Biol 208:1117- 1124. Warren DE, Reese SA, Jackson DC. 2006. Tissue glycogen and extracellular buffering limit the survival of red-eared slider turtles during anoxic submergence at 3 degrees C. Physiol Biochem Zool 79(4):736-744. Wise G, Mulvey JM, Renshaw GMC. 1998. Hypoxia tolerance in the epaulette shark (Hemiscyllium ocellatum). J Exp Zool 281:1-5.
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CHAPTER FIVE
The absence of splenic and hepatic contraction to release erythrocytes into the circulation of the grey carpet shark (Chiloscyllium punctatum) in response to anoxia and re-oxygenation
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Chapter 5: Absence of erthyoctye release in response to anoxia
5.1 Summary
A transient elevation in red blood cell number in response to anoxic exposure has previously been measured in the grey carpet shark. The presence of a splenic and hepatic release of red blood cells as a cause for the changes in haematological parameters previously was investigated in the grey carpet shark (Chiloscyllium punctatum) in response to a standardised anoxic challenge. Compared to normoxic controls the present study observed significant increases in haematological values, such as haematocrit (anoxia, 24.67 ± 1.2%; control, 18.2 ± 3.4%), haemoglobin (anoxia, 8.65 ± 1.1 g dL-1; control, 5.58 ± 0.5 g dL-1) and erythrocyte (RBC) (anoxia, 0.34 ± 0.03 x 10-6 uL-1; control, 0.24 ± 0.04 x 10-6 uL-1) concentrations in the grey carpet shark following 1.5 hours of anoxia. However no significant differences were observed in either the spleen somatic index (SSI) or the splenic haemoglobin concentration (SpHb) of anoxia treated animals (SSI, 0.43 ± 0.03% body weight; SpHb, 0.28
± 0.05 g kg-1) compared to normoxic controls (SSI, 0.42 ± 0.07% body weight; SpHb, 0.27 ±
0.05 g kg-1). Likewise, the hepatic somatic index (HSI) and hepatic haemoglobin concentrations (HHb) showed no significant difference between anoxia (HSI, 2.08 ± 0.5% body weight; HHb, 0.13 ± 0.05 g kg-1) and normoxic (HSI, 2.27 ± 0.4% body weight; HHb,
0.16 ± 0.06 g kg-1) treated animals. Therefore, the present study concludes that changes in haematological values in the grey carpet shark following anoxia are not due to the release of
RBCs from a storage reservoir and are therefore possibly due to an increase in mitotic division of RBCs and/or haemoconcentration of the blood.
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5.2 Introduction
In response to a number of stressful stimuli many vertebrates show an increase in the number of erythrocytes (RBCs) per unit volume of blood. This can be caused by (i) an increase in the production of RBCs via erythropoietic pathways (Soldatov, 1996; Fandrey,
2004), (ii) a decrease in plasma volume (haemoconcentration) due to fluid shifts and/or increased diuresis (Swift and Lloyd, 1974; Nikinmaa and Tervonen, 2004; Tervonen et al.,
2006), and/or (iii) the liberation of RBCs from storage organs into the circulation (Yamamoto et al., 1980; Kita and Itazawa, 1989; Pearson and Stevens, 1991). In response to periods of reduced oxygen availability, many vertebrates have been observed to show an increase in the number of RBC per unit volume of blood via one, or a combination of mechanisms mentioned above.
In teleosts, the spleen acts as the predominant blood reservoir. During periods of metabolic stress, teleosts have been observed to increase RBC concentrations in the peripheral circulation (Yamamoto et al., 1980; Yamamoto et al., 1983; Yamamoto, 1987), via the contraction of splenic muscle tissue, which release stored RBCs into the circulatory system.
The adaptive significance of the splenic reservoir is to fine tune the blood respiratory system to short-term variations in metabolic rate via increasing the oxygen carrying capacity of the blood.
Mautner and Pick (1915) as well as Grab et al. (1929) suggested the liver as a possible secondary organ that acts as a RBC reservoir. Guntheroth and Mullins (1962) disagreed and found no evidence of RBC storage in the liver of the dog. In elasmobranchs, the liver is a highly developed structure unique from other vertebrates. The elasmobranch liver is involved
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Chapter 5: Absence of erthyoctye release in response to anoxia in metabolism and has a number of functions in the body including detoxification, glycogen and lipid storage, plasma protein synthesis and buoyancy within the water column. From the literature, no erythropoietic activity occurs in the liver of elasmobranchs (Yoffey, 1929), however the role of the liver to store RBCs had not previously been examined in this group of animals.
The ability of the spleen to act as a RBC storage reservoir has now been well characterised in many teleosts in response to hypoxia. During hypoxic exposure the contraction of the splenic muscles results in a reduction in spleen size (Pearson and Stevens,
1991) and causes a corresponding release of RBCs into the peripheral circulation noted by significant increases in haematocrit, haemoglobin and RBC concentrations (Yamamoto et al.,
1980; Yamamoto et al., 1983; Yamamoto, 1987; Yamamoto, 1988; Kita and Itazawa, 1989;
Yamamoto and Itazawa, 1989).
The role of the spleen to act as a RBC reservoir in elasmobranchs is still currently controversial. Nilsson et al. (1975) observed a clear contraction of the spleen accompanied by
RBC release in response to catecholamines and artificial nerve stimulation in both Squalus acanthias and Scyliorhinus canicula, while Butler et al. (1979) observed no contraction of the spleen in response to hypoxia in the hypoxia-intolerant dogfish (S. canicula). Nilsson et al.
(1975) specifically investigated the ability of the spleen to contract in response to a chemically induced stimulation in S. canicula, while Butler et al. (1979) canulated the afferent branchial blood vessel and observed no increase in haematocrit in response to hypoxic stress in S. canicula. Due to the different methodologies used by these authors, further study is required to determine the presence of a splenic contraction in response to
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Chapter 5: Absence of erthyoctye release in response to anoxia metabolically demanding stimuli such as hypoxia. The role of the spleen to act as a RBC reservoir in an anoxia-tolerant elasmobranch had not been examined.
Most elasmobranchs are intolerant to prolonged periods of reduced oxygen saturation, however the grey carpet shark has recently been characterised as possessing a significant tolerance for up to approximately 2-3 hours in anoxia at 23C (Chapter 2). At this temperature the grey carpet shark has been observed to tolerate similar periods of anoxia exposure to the anoxia-tolerant epaulette shark (Chapter 2). During hypoxia and anoxia, the epaulette shark activates energy conserving mechanisms such as ventilatory depression (Routley et al., 2002), bradycardia (Soderstrom et al., 1999) and neuronal hypometabolsim (Mulvey and Renshaw,
2000), characteristic of other anoxia-tolerant vertebrates. However, the grey carpet shark was not observed to possess these energy conserving mechanisms (Chapter 2) and its physiological strategies are more similar to anoxia-intolerant vertebrates.
Recently, Chapman and Renshaw (in press, Chapter 3), observed an increase in haematocrit, haemoglobin and RBC concentrations in the grey carpet shark following anoxic exposure. The present study therefore aims to investigate two of the most likely sources of
RBC stores by determining the presence of a splenic and/or hepatic contraction in the grey carpet shark in response to anoxia.
The presence of splenic and hepatic blood reseviors within the grey carpet shark were investigated by measuring changes in the organ weight (somatic index) and the organ haemoglobin concentration. Yamamoto et al. (1980) observed that the release of RBCs due to a splenic contraction resulted in a reduction in spleen weight and haemoglobin concentrations.
Pearson and Stevens (1991) also reported that decreases in both spleen somatic index (SSI)
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Chapter 5: Absence of erthyoctye release in response to anoxia and spleen haemoglobin concentrations (SpHb) corresponded to a physical reduction in the size of the spleen in air exposed rainbow trout (Oncorhynchus mykiss). While other methods, such as the surgical isolation of the spleen via the ligation of splenic arteries and veins have been used in teleost fishes (Gallaugher et al., 1992) this method could not be used in elasmobranchs due to the fusion of splenic veins with the pancreas. Therefore, calculation of the somatic index and haemoglobin concentration of both the spleen and the liver were the most accurate method to use in vivo to determine the activation of RBC reservoirs in the grey carpet shark in response to anoxia.
5.3 Materials and methods
5.3.1 Study animals and location
Twelve grey carpet sharks (n=12) were supplied by UnderWater World Aquarium
(Mooloolaba, Sunshine coast). Grey carpet sharks had a mean length of 48.23 ± 3 cm and weight of 368.87 ± 77.6 g. Animals were kept in constant flow through aquaria with no additional light sources in their outside habitats, relying on a normal photoperiod. Food was withheld from the animals for at least 24-48 hours before experiments were performed.
5.3.2 Anoxic exposure
Animals were exposed to anoxic treatment and normoxic control conditions as previously described by Chapman and Renshaw (in press, Chapter 3). Briefly, animals were exposed to anoxia or control treatment in 70 litre glass tanks, sealed with plastic wrap and
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Chapter 5: Absence of erthyoctye release in response to anoxia custom fitted Perspex lids. Tanks were filled with normoxic seawater (24°C), which was circulated around the tank using a 228-power head pump (CCC Pty Ltd., Australia) to ensure uniform conditions throughout. Nitrogen gas was injected through air stones within the tanks to displace dissolved oxygen from the water (anoxic treatment), while air was bubbled through air stones to maintain normal oxygen saturation (control treatment). Dissolved oxygen concentrations [dO2] were measured and recorded each minute using TPS WP-90 oxygen probes (TPS Pty. Ltd., Australia). In the anoxic tanks, the [dO2] was maintained
-1 -1 below 0.02 mg L and normoxic [dO2] was maintained above 7.0 mg L . Animals were pair matched for length and then randomly selected for either 1.5 hours of anoxic exposure or 1.5 hours of normoxic control treatment. Animals were weighed and measured on conclusion of the experimental treatments.
5.3.3 Blood sampling and haematology
Blood samples were taken from each grey carpet shark immediately prior to placement
(pre) in each tank to provide a pre-experimental measurement for each haematological parameter and immediately following treatment (post). A 1.5 ml blood sample was taken from the dorsal aorta of each animal using a heparinised syringe via the venipuncture method described in Stoskopf et al. (1984). Blood samples were then transferred to 3 ml lithium heparin vacutainers (Becton Dickinson, North Ridge, New South Wales, Australia) and gently mixed.
Haematocrit was determined using heparinised haematocrit microtubes and centrifuged at 2500 rpm for 5 minutes at 4°C in an Eppendorf 5415D centrifuge. Haematocrit values were determined against a micro-haematocrit grid (Sigma-Aldrich, Newcastle, New
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Chapter 5: Absence of erthyoctye release in response to anoxia
South Wales, Australia). Erythrocyte (RBC), concentrations were calculated via haemocytometry cell counts using a Neaubaur counting chamber. Blood samples were diluted
1:200 in chilled phosphate buffered saline (pH 7.2) and vortexed gently. The diluted solution
(5 l) was loaded into the Neaubaur counting chamber and examined under 10x magnification using a compound light microscope. Blood haemoglobin was determined in triplicate, by a cyanmethemoglobin technique using modified Drabkin’s Reagent (Sigma-Aldrich, Newcastle,
New South Wales, Australia). One millilitre of Drabkin’s reagent and 4 l of blood were placed into a 1.6 ml cuvette and vortexed gently. Samples were incubated in the dark, at room temperature for 15 minutes before being analysed using a UV Mini 1240 spectrophotometer
(Shimadzu, Suzhoa, Republic of China) at a wavelength of 540 nm. Sample haemoglobin concentrations were compared against a haemoglobin standard curve using the human haemoglobin standard supplied by Sigma-Aldrich (Newcastle, New South Wales, Australia).
The RBC indices calculated were: the mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) which were derived from RBC, haematocrit, and haemoglobin concentrations using the formulae of Stoskopf (1993) as follows:
MCV = (Hct / RBC) x 10,
MCH = (Hb x 10) / RBC, and
MCHC = (Hb x 100) / Hct.
5.3.4 Tissue analysis
Following treatment, animals were anaesthetised in a 70 L glass aquarium containing anoxic seawater diluted with a 1.5ml L-1 benzocaine dilution (5% benzocaine in ethanol).
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Chapter 5: Absence of erthyoctye release in response to anoxia
Animals were immediately weighed on a PB3002-L Delta Range precision balance (Mettler
Toledo, Brisbane Australia). An incision was made into the abdominal cavity to expose the spleen and the liver. Haemostats were used to isolate the spleen and liver from efferent and afferent blood supply and the organs were quickly excised. Organs were weighed on a
Sartorius BP310P analytical scale (Data weighing systems, Chicago, USA). Spleen and hepatic somatic index (SSI and HSI) was calculated as (organ weight/body weight) x 100 as described in Pearson and Stevens (1991). The accuracy of this method is however reliant on the sensitivity of the scales used to measure animal and organ weights. Pearson and Stevens
(1991) reported a 38% decrease in SSI and a 33% decrease in spleen Hb in rainbow trout (O. mykiss). The scales used in the present study had an accuracy of 0.001 g and therefore were sensitive to changes in organ weight as low as 0.01% in the grey carpet shark.
Haemoglobin content of the spleen and liver was determined by applying the cyanmethemoglobin method. Tissue was homogenised for 5 minutes in modified Drabkins solution as described by Yamamoto et al. (1980). From the homogenate, 20 l was gently vortexed with 5 ml of Drabkin’s solution and allowed to incubate in the dark, at room temperature for 15 minutes before being analysed using a UV Mini 1240 spectrophotometer
(Shimadzu, Suzhoa, Republic of China) at a wavelength of 540 nm. Sample haemoglobin concentrations were compared against a haemoglobin standard curve using the human haemoglobin standard supplied by Sigma-Aldrich (Newcastle, New South Wales, Australia).
5.3.5 Statistics
Data was analysed using a two tailed student-t test, assuming equal variance, with a
95% confidence interval. Data and error bars are represented as mean ± standard deviation.
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Chapter 5: Absence of erthyoctye release in response to anoxia
5.4 Results
5.4.1 Haematology of blood samples
The mean haematocrit levels prior to treatment were not significantly different in experimental (anoxia) and control (normoxia) groups (18.2 ± 3.4%) (Figure 5.1). Immediately following 1.5 hours of anoxic exposure, mean hematocrit levels significantly increased (24.67
± 1.2%) above the mean pre experimental measure (p<0.01) (Figure 5.1). No significant differences in mean haematocrit levels were observed before or immediately follong control treatments.
Changes in percent hematocrit in response to anoxia and re-oxygenation or control treatments in the grey carpet shark 30
* 25
20
15 Pre
Post Percentage Percentage (%) 10
5
0 Anoxia Control Treatment Figure 5. 1. Mean hematocrit levels before (pre) and immediately following (post) 1.5 hours of anoxia exposure or normoxic control treatment in the grey carpet shark (C. punctatum). Symbols indicate significant differences compared to the mean pre-experimental levels (*).
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Chapter 5: Absence of erthyoctye release in response to anoxia
Prior to treatment, the mean haemoglobin concentration and mean RBC concentration was 5.58 ± 0.5 g dL-1 and 0.24 ± 0.04 x 10-6 uL-1 respectively, with no significant difference between experimental (anoxia) and control (normoxia) animals (Figure 5.2 and 5.3).
Following anoxic treatment, a significant increase (p<0.001) was observed in mean haemoglobin levels (8.65 ± 1.1 g dL-1) (Figure 5.2) and mean RBC concentrations (0.34 ±
0.03 x 10-6 uL-1) (Figure 5.3) from their mean pre-experimental levels. No significant changes were observed in mean haemoglobin levels or mean RBC concentrations in the control animals. No significant differences in MCH, MCV and MCHC were observed following anoxic or control treatments from their respective pre-experimental levels.
Changes in haemoglobin concentration in response to anoxia and re-oxygenation or control treatments in the grey carpet shark 12
10 *
) 1 - 8
6 Pre Post
4
Concentration Concentration (g dL
2
0 Anoxia Control Treatment Figure 5. 2. Mean haemoglobin levels before (pre) and immediately following (post) 1.5 hours of anoxia exposure or normoxic control treatment in the grey carpet sharks (C. punctatum). Symbols indicate significant differences compared to the mean pre-experimental levels (*).
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Chapter 5: Absence of erthyoctye release in response to anoxia
Changes in red blood cell concentration in response to anoxia and re-oxygenation or control treatments in the grey carpet shark 0.4 * 0.35
0.3
) 1
-
uL 6 - 0.25
0.2 Pre Post
0.15
Concentration Concentration (10 0.1
0.05
0 Anoxia Control Treatment Figure 5.3. Mean RBC concentrations before (pre) and immediately following (post) 1.5 hours of anoxia exposure or normoxic control treatment in the grey carpet sharks (C. punctatum). Symbols indicate significant differences compared
to the mean pre-experimental levels (*).
5.4.2 Splenic and hepatic tissue analysis
The response of the spleen to anoxia indicated no significant differences in mean spleen somatic index (SSI) or mean spleen haemoglobin (SpHb) concentrations compared to control animals. The mean SSI of anoxia treated animals was 0.43 ± 0.03% body weight and control animals had a mean SSI of 0.42 ± 0.07% body weight. The mean splenic haemoglobin concentration in anoxia treated animals was 0.28 ± 0.05 g kg-1 body weight and 0.27 ± 0.05 g kg-1 body weight in control animals.
Similarly, the liver had no significant difference in mean hepatic somatic index (HSI) or mean hepatic haemoglobin (HHb) concentration between anoxia treated and control animals. Anoxia treated animals had a mean HSI of 2.08 ± 0.5% body weight, while control
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Chapter 5: Absence of erthyoctye release in response to anoxia animals had a mean HSI of 2.27 ± 0.4% body weight. The mean haemoglobin concentration of the liver was 0.13 ± 0.05 g kg-1 body weight in anoxia treated animals and 0.16 ± 0.06 g kg-
1 body weight in control animals.
5.5 Discussion
The present study observed increased haematological values, such as haematocrit, haemoglobin and RBC concentrations in blood samples taken from the grey carpet shark following anoxic insult. These increases in haematological values indicate an increase in RBC numbers, an increase in RBC mitotic maturations and/or a fluid shift from the blood plasma
(haemoconcentration). Elevated haematological values caused by increased RBC concentrations are often attributed to a release of sequestered RBCs from a storage organ such as the spleen into the blood circulation. Many teleost fishes have been reported to undergo a catecholamine induced splenic contraction in response to hypoxia, air exposure, animal handling, trauma, physical constraint, confinement or exercise (Yamamoto et al., 1980;
Yamamoto et al., 1983; Yamamoto, 1987; Yamamoto, 1988; Kita and Itazawa, 1989;
Yamamoto and Itazawa, 1989; Pearson and Stevens, 1991; Gallaugher et al., 1992; Lai et al.,
2006). The adaptive significance of the splenic reservoir is to fine-tune the blood respiratory system to short-term variations in metabolic rate via increasing the oxygen carrying capacity of the blood.
While it is known that the spleen of elasmobranchs has a role in RBC production
(Fange and Johansson-Sjobeck, 1975), the present study observed no changes in spleen somatic index or splenic haemoglobin concentrations in the grey carpet shark following anoxic exposure. While re-absorption of RBCs back into the spleen occurs following stress,
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Chapter 5: Absence of erthyoctye release in response to anoxia
Chapman and Renshaw (in press, Chapter 3), reported that RBC concentrations remained elevated for 2 hours following anoxic exposure. Therefore, it is unlikely that RBCs would have been re-absorbed back into the spleen immediately following the anoxic stress used in this study. Butler et al. (1979) also reported no changes in erythrocyte concentrations in response to hypoxia in the dogfish (S. canicula). Interestingly, Nilsson et al. (1975) observed a splenic contraction by catecholamines and artificial nerve stimulation in both Squalus acanthias and Scyliorhinus canicula in perfused and isolated spleens. However, Opdyke and
Opdyke (1971) observed no release of RBC from the spleen in Squalus acanthias in response to electrical stimulation of the splenic pedicle or catecholamine infusion, supporting findings by Butler et al. (1979). Therefore, it appears that while both these species possess the mechanisms to induce a splenic contraction by administering electrical and chemical stimulation, the ability of the elasmobranch spleen to act as a RBC reservoir or to contract in response to metabolically demanding stimuli, such as hypoxia, anoxia or exercise, has not been observed.
Controversy over the presence or absence of the spleen to act as a RBC reservoir in elasmobranchs may in some part be due to the methodology used between authors. The present study investigated changes in spleen somatic index and spleen haemoglobin concentrations as reported by Yamamoto et al. (1980), Yamamoto et al. (1983), Yamamoto
(1987) and Pearson and Stevens (1991). Yamamoto et al. (1980) observed that the release of
RBCs due to a splenic contraction resulted in a 46% reduction in spleen weight following mild hypoxia and an 80% reduction in spleen weight following severe hypoxia in the yellowtail (Seriola quinqueradiata). The duplication of this method in the present study observed no changes in spleen somatic index or haemoglobin concentrations in the grey carpet shark following anoxic exposure, suggesting the absence of a splenic contraction.
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However, some controversy exists over the use of this method to conclusively determine whether the spleen acts as a RBC reservoir and the presence of a splenic contraction. Other methods such as the surgical isolation of the spleen via the ligation of splenic arteries and veins have been used in teleost fishes (Gallaugher et al., 1992). However, this method could not be used in elasmobranchs due to the fusion of splenic veins with the pancreas. Isolation of the spleen via ligation is therefore impossible in these sharks.
While the liver of elasmobranchs has not been observed to have any haemopoietic role, this organ is highly developed in these vertebrates. The liver is involved in metabolism and has a number of functions in the body including detoxification, glycogen and lipid storage, plasma protein synthesis and buoyancy within the water column. However, additional functions of this organ in elasmobranchs have not been thoughly examined. From the present study, no changes in liver somatic index or hepatic haemoglobin concentrations were observed in the grey carpet shark in response to anoxia. The release of RBCs from a storage reservoir has been repeatedly shown to increase the oxygen carry capacity of the blood during events of hypoxia and/or metabolic stress. During events of anoxia when no oxygen is available, increasing RBCs for the sake of increasing the oxygen carry capacity of the blood would not increase survival in the grey carpet shark. However, a release of oxygenated RBCs from a storage organ may reduce the affects of anoxic stress by providing additional small doses of stored oxygen. Therefore, increases in RBC concentrations in the grey carpet shark may maintain oxygen absorption during hypoxic exposure, as observed in teleosts, however the oxygen holding capabilities of stored RBCs and their potential benefit to supply small doses of oxygen during anoxia has not been investigated.
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Another explanation for the increase in haematological values is due to fluid shifts from the blood plasma. Tervonen et al. (2006) along with Swift and Lloyd (1974) reported an increase in urine flow in the rainbow trout (Salmo gairdneri) following hypoxic exposure, while Kirk (1974) observed a fluid shift from the blood plasma into the tissues following hypoxia in the channel catfish (Ictalurus punctatus). While such fluid shifts in the grey carpet shark may be present, the methodologies used to test the presence of these changes have not been investigated. While, the insertion of a urinary catheter into the bladder of teleost has been repeatedly used to determine changes in urinary excretion during events of stress (Curtis and Wood, 1992; Erikson et al., 2006; Janech et al., 2006), limited data is available on urinary catheterisation in elasmobranchs. While a number of researchers have used these methods of inserting catheters for urine collection (Smith, 1931; Janech and Piermarini, 2002; Janech et al., 2006), a significant amount of controversy exists on the accuracy of urinary catheterisation techniques due to the variation in elasmobranch urinary anatomy. Extending from the kidneys, elasmobranchs have two urinary sinuses on the left and right side of the body. The urinary sinuses are separated by a thin body wall before merging together and exiting through the urinary pore (Whitehouse and Grove, 1949). Therefore the insertion of a urinary catheter into the grey carpet shark must ensure the consistent placement and positioning of the catheter through the urinary pore but not into either of the urinary sinuses to generate correct replication and collection of urine from the animal. The position and length of each of these structures varies significantly between elasmobranch species and intensive anatomical dissections are required for each species before correct urinary catheterisation can be determined. A pilot study was performed on urine collection in 10 grey carpet sharks with each shark having a urinary catheter inserted through its urinary pore. However, results were unreliable due to the difficulty in consistent placement of the catheter.
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It has also been suggested that increased RBC concentration in response to hypoxia could be due to an increase in the rate of mature RBC division. Teleost fish have been observed to increase the rate of mature RBC division in response to prolonged mild hypoxia
(Soldatov, 1996). Soldatov (1996) reported an increase in RBC division after 2 hours, and observed a 37% increase in RBC concentrations after 6 hours of hypoxia. However, in elasmobranchs, the production site of blood is not fully understood and the production of blood cells has been observed in a number of different tissues (Zapata, 1981). Haematopoietic sites in elasmobranchs include the thymus, the spleen (Fange and Johansson-Sjobeck, 1975) and two unique organs called the Leydig and epigonal organs (Zapata, 1981; Mattisson and
Fange, 1982). More recently, Walsh and Luer (2004) observed frequent mitotic activity in elasmobranch peripheral blood, supporting mitotic replication as well as maturation of erythrocytes in the peripheral blood of elasmobranchs (Saunders, 1966; Stokes and Firkin,
1971; Glomski et al., 1992).
The division of mature nucleated RBCs during prolong hypoxia increases the oxygen carrying capacity of the blood in order to maintain oxygen absorption from the ambient hypoxic water. The presence of mitotic division of RBCs in the grey carpet shark during anoxia was not investigated in the present study however, in the absence of oxygen, elevated
RBC concentrations due to mitotic division would not increase the oxygen carry capacity of the blood due to the absence of available oxygen, from the ambient water. In addition, mitotic division of RBCs is an energetically expensive process which would further deplete ATP levels and reduce survival time during an anoxic event.
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While the present study observed significant increases in haematological values including haematocrit, haemoglobin and RBC concentrations, no evidence of a contraction and/or release of RBCs from two potential storage organs, the spleen and the liver, were observed in the grey carpet shark following anoxic exposure. While limitations to the investigation of a splenic contraction in elasmobranchs may be present, due to the sensitivity of scales to changes in organ weight and the difficulty in isolating or removing potential storage organs, this study concludes that there is no evidence that changes in haematological values are due to the release of RBC from a storage reservoir in the spleen or liver. Such changes may possibly be due to haemoconcentration of the blood and/or mitotic division of
RBCs in the circulation. Further investigation on the presence of a haemoconcentration and mitotic division of RBCs in the periphery is required. This can be achieved via the Evans blue plasma volume test and radioactive RBC labelling (RBC-Cr51) technique described by Conte et al., (1963).
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5.6 Literature cited
Butler PJ, Taylor EW, Davison W. 1979. The effect of long term, moderate hypoxia on acid base balance, plasma catecholamines and possible anaerobic end products in the unrestrained dogfish Scyliorhinus canicula. J Comp Physiol B 132:297-303. Conte FP, Wagner HH, Harris TO. 1963. Measurement of blood volume in the fish (Salmo gairdneri gairdneri). Am J Physiol 205:533-540. Curtis BJ, Wood CM. 1992. Kidney and urinary bladder responses of freshwater rainbow
trout to isosmotic NaCl and NaHCO3 infusion. J Exp Biol 173:181-203. Erickson RJ, McKim JM, Lien GJ, Hoffman AD, Batterman SL. 2006. Uptake and elimination of ionisable organic chemicals at fish gills: II. Observed and predicted effects of pH, alkalinity, and chemical properties. Enviro Tox Chem 25:1522-1532. Fandrey J. 2004. A cordial affair – erythropoietin and cardioprotection. Cardio Res 72(1):1-2. Fange R, Johansson-Sjobeck ML. 1975. The effect of splenectomy on the hematology and on the activity of delta-aminolevulinic acid dehydratase (ALA-D) in hemopoietic tissues of the dogfish, Scyliorhinus canicula (Elasmobranchii). Comp Biochem Physiol A 52:577-580. Gallaugher P, Axelsson M, Farrell AP. 1992. Swimming performance and haematological variables in splenectomised rainbow trout, Oncorhynchus mykiss. J Exp Biol 171:301- 314. Glomski CA, Tamburlin J, Chainani M. 1992. The phylogenetic odyssey of the erythrocyte. III. Fish, the lower vertebrate experience. Histol Histopathol 7(3):501-528. Grab W, Janssen S, Rein H. 1929. Zoo Biol. 89:324. In: Guntheroth WG, Mullins GL. 1962. Liver and spleen as venous reservoirs. Am J Physiol 204(1):35-41. Guntheroth WG, Mullins GL. 1962. Liver and spleen as venous reservoirs. Am J Physiol 204(1):35-41. Janech MG, Piermarini PM. 2002. Renal water and solute excretion in the Atlantic stingray in freshwater. J Fish Biol 61:1053-1057. Janech MG, Fitzgibbon WR, Ploth DW, Lacy ER, Miller DH. 2006. Effect of low environmental salinity on plasma composition and renal function of the Atlantic stingray, a euryhaline elasmobranch. Am J Renal Physiol 291:770-780. Kirk WL. 1974. The effects of hypoxia on certain blood and tissue electrolytes of channel catfish, Ictalurus punctatus (Rafinesque). Trans Am Fish Soc 103:593-600.
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Kita J, Itazawa Y. 1989. Release of erythrocytes from the spleen during exercise and splenic constriction by adrenaline infusion in the rainbow trout. Jap J Ichthyol 36:48-52. Kuwahira I, Kamiya U, Iwamoto T, Moue Y, Urano T, Ohta Y, Gonzalez NC. 1999. Splenic contraction-induced reversible increase in haemoglobin concentration in intermittent hypoxia. J Appl Physiol 86(1):181-187. Lai JC, Kakuta I, Mok HO, Rummer JL, Randall D. 2006. Effects of moderate and substantial hypoxia on erythropoietin levels in rainbow trout kidney and spleen. J Exp Biol 209:2734-8. Mautner H, Pick EP. 1915. Munch Med Wochschr 2:1141. In: Guntheroth WG, Mullins GL. 1962. Liver and spleen as venous reservoirs. Am J Physiol 204(1):35-41. Mattisson A, Fange R. 1982. The cellular structure of the Leydig organ in the shark, Etmopterus spinax (L.). Biol Bull 162:182-194. Mulvey JM, Renshaw GMC. 2000. Neuronal oxidative hypometabolism in the brainstem of the epaulette shark (Hemiscyllium ocellatum) in response to hypoxic pre-conditioning. Neurosci Let 290:1-4. Nikinmaa M, Tervonen V. 2004. Regulation of blood haemoglobin concentration in hypoxic fish: Proceedings of the 7th International Symposium on Fish Physiology, Toxicology, and Water Quality. U.S. Environmental Protection Agency, Ecosystems Research Division. 243-252. Nilsson S, Holmgren S, Grove DJ. 1975. Effects of drugs and nerve stimulation on the spleen and arteries of two species of dogfish, Scyliorhinus canicula and Squalus acanthias. Act Physiol Scand 95:219-230. Opdyke DF, Opdyke NE. 1971. Splenic responses to stimulation in Squalus acanthias. Am J Physiol 221:623-625. Pearson MP, Stevens ED. 1991. Size and hematological impact of the splenic erythrocyte reservoir in rainbow trout, Oncorhynchus mykiss. Fish Physiol Biochem 9:39-50. Routley MH, Nilsson GE, Renshaw GMC. 2002. Exposure to hypoxia primes the respiratory and metabolic responses of the epaulette shark to progressive hypoxia. Comp Biochem Physiol A 131:313-321. Saunders DC. 1966. Elasmobranch blood cells. Copeia 66(2):348-351. Smith HW. 1931. The absorption and excretion of water and salts by elasmobranch fishes. I. Fresh water elasmobranchs. Am J Physiol 98:279-295.
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Soderstrom V, Renshaw GMC, Nilsson GE. 1999. Brain blood flow and blood pressure during hypoxia in the epaulette shark Hemiscyllium ocellatum, a hypoxia-tolerant elasmobranch. J Exp Biol 202:829-835. Soldatov AA. 1996. The effect of hypoxia on red blood cells of flounder: a morphologic and autoadiographic study. J Fish Biol 48:321-328. Stokes EE, Firkin BG. 1971. Studies of the peripheral blood of the Port Jackson shark (Heterodontus portusjacksoni) with particular reference to the thrombocyte. Br J Haematol 20:427-435. Stoskopf MK. 1993. Clinical pathology. In Fish Medicine. W.B. Saunders, Philadelphia p:882. Stoskopf MK, Smith B, Klay G. 1984. Clinical note: Blood sampling of captive sharks. J Zool Anat Med 15:116-117. Swift DJ, Lloyd R. 1974. Changes in urine flow rate and haematocrit value of rainbow trout Salmo gairdneri (Richardson) exposed to hypoxia. J. Fish Biol 6:379-387. Tervonen V, Vuolteenaho O, Nikinmaa, M. 2006. Haemoconcentration via diuresis in short- term hypoxia: A possible role for cardiac natriuretic peptide in rainbow trout. Comp Biochem Physiol A 144:86-92. Walsh CJ, Luer CA. 2004. Elasmobranch hematology: Indentification of cell types and practical applications. In: Elasmobranch Husbandry Manual. Ed. Smith M, Warmolts D, Thoney D, Hueter R. Ohio Biological Survey, Columbus Ohio. Whitehouse RH, Grove AJ. 1949. Dissection of the Dogfish. London, University Tutorial Press Reprint. Yamamoto K. 1987. Contraction of spleen in exercised cyprinid. Comp Biochem Physiol A 87:1083-1087. Yamamoto K. 1988. Contraction of spleen in exercised freshwater teleosts. Comp Biochem Physiol A 89:65-66. Yamamoto K, Itazawa I. 1989. Erythrocyte supply from the spleen of exercised carp. Comp Biochem Physiol A 92:139-144. Yamamoto K, Itazawa Y, Kobayashi H. 1980. Supply of erythrocytes into the circulating blood from the spleen of exercised fish. Comp Biochem Physiol A 65:5-11. Yamamoto K, Itazawa Y, Kobayashi H. 1983. Erythrocyte supply from the spleen and hemoconcentration in hypoxic yellowtail. Mar Biol 73:221-226.
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Yoffey JM. 1929. A contribution to the study of the comparative histology and physiology of the spleen, with reference chiefly to its cellular constituents. J Anatomy 88(3):314- 346. Zapata A. 1981. Ultrastructure of elasmorbranch lymphoid tissue. 2. Leydig’s and epigoneal organs. Devel Comp Immun 5:43-52.
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CHAPTER SIX
Characterisation of the heat shock protein (Hsp70) response in the brain, heart and blood following anoxia and re-oxygenation in the grey carpet shark (Chiloscyllium punctatum) and the epaulette shark (Hemiscyllium ocellatum)
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6.1 Summary
Heat shock protein 70 kDa (Hsp70) plays a significant protective role during events of cellular stress. This study characterises the Hsp70 response to anoxic stress in the grey carpet shark (Chiloscyllium punctatum) and the epaulette shark (Hemiscyllium ocellatum) using two different protocols.
In the first series of experiments, grey carpet sharks were exposed to two episodes of anoxia, 24 hours apart, until the animal’s righting reflex was lost. Total time to loss of righting reflex (LRR) was recorded for each anoxic exposure. The Hsp70 concentrations were determined via western blotting on the cerebellum, the ventricle of the heart and plasma separated blood (whole blood pack) samples. The grey carpet shark took significantly longer to loose its righting reflex in the second anoxic episode compared to the first. No significant differences were observed in Hsp70 concentrations in the cerebellum, the ventricle or the blood following two episodes of anoxia, until LRR, in the grey carpet shark.
In a second series of experiments, both the grey carpet shark and the epaulette shark were exposed to a single standardised episode of 1.5 hours of anoxia followed by 12 hours of re-oxygenation in normoxia. Blood samples were taken before anoxia and immediately following anoxic exposure, then at 2, 6 and 12 hours of re-oxygenation in normoxia. No significant differences were observed in Hsp70 concentrations of whole blood packs above constitutive levels in the grey carpet shark or the epaulette shark following a standardised episode of anoxia and 12 hours of re-oxygenation. These data reveal that Hsp70 is not induced above consituative levels in either the grey carpet shark or the epaulette shark in response to limited anoxic stress.
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6.2 Introduction
Heat shock proteins (HSPs) are synthesised in response to a wide range of physiological and environmental stressors within a wide range of animals and tissues
(Latchman, 1999). HSPs exist at constitutive levels at all times and are involved in maintaining cellular homeostasis by assisting in protein folding, cellular repair and protein transport (Latchman, 1998; 1999). Following stress, HSPs play a protective role by acting as molecular chaperones, which are involved in recognising, repairing and binding to damaged proteins, preventing abnormal protein interaction, inhibiting protein synthesis and protecting
DNA and RNA within the nucleus (Marx, 1983; Airaksinen et al., 1998; Feder and Hofmann,
1999). HSPs have been reported to respond to a variety of stressors, including pollutants, pH, temperature, salinity, ischemia, hypoxia, and cellular energy depletion, as well as to psychological stress such as exposure to predators, pathogens, and overcrowding (Misra et al.,
1989; Kohler et al., 1996; McCully et al., 1996; Vijayan et al., 1997; Bierkens et al., 1998;
Feder and Hofmann, 1999).
Heat Shock Protein 70 kDa (Hsp70) is the most widely understood family of the HSPs and its production is significantly up-regulated in response to a broad array of stressors.
Hsp70 has been identified in a wide range of organs and tissues (Marber et al., 1995; Currie et al., 2000; Murtha and Keller, 2003; Delaney and Klesius, 2004), however tissue specific differences in constitutive and inducible Hsp70 concentrations do occur (Koban et al., 1991;
Dietz and Somero, 1993; Currie et al., 2000). Species-specific differences in constitutive and inducible Hsp70 levels have also been observed. Delaney and Klesius (2004) observed an increase in Hsp70 in juvenile hypoxia-tolerant Nile tilapia (Oreochromis niloticus) in response to hypoxia, while no significant differences were found in hypoxia-intolerant species
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Chapter 6: Hsp70 response to anoxia and re-oxygenation such as rainbow trout (Oncorhynchus mykiss) (Airaksinen et al., 1998) and Chinook salmon
(O. tshawytscha) (Currie and Tufts, 1997). It has therefore been suggested that species and tissue specific differences in Hsp70 levels are a consequence of the type and severity of stress imposed and the animal’s physiological protective response to these events.
In elasmobranchs, the HSP response to reduced oxygen conditions has been little studied. The epaulette shark has been characterised as an anoxia-tolerant elasmobranch that is periodically exposed to cyclic events of severe hypoxia on coral reef flats. The epaulette shark utilises energy conserving mechanisms such as ventilatory depression (Routley et al., 2002), bradycardia (Soderstrom et al., 1999) and neuronal hypometabolism (Mulvey and Renshaw,
2000), along with the up-regulation of anaerobic pathways (Wise et al., 1998) in order to survive these periods of low oxygen availability. In the anoxia-tolerant epaulette shark,
Renshaw et al. (2002) observed significant increases in inducible Hsp70 concentrations above constitutive levels in the cerebellum, but not the brain stem following two episodes of anoxia.
It is possible that the activation of protective proteins, such as Hsp70, in specific areas of the brain may be involved in reducing cellular damage during events of anoxic stress.
Recently, anoxia tolerance in another tropical elasmobranch, the grey carpet shark
(Chiloscyllium punctatum) has been reported (Chapman and Renshaw, in press, Chapter 3).
This species is a close relative to the epaulette shark inhabiting intertidal areas, including sandy and rocky substrates, seagrass areas as well as coral reefs (Last and Stevens, 1994).
This species was observed to tolerate anoxia for approximately 3 hours at 23C (Chapter 2) with protective responses including the up-regulation of anaerobic pathways and changes in blood physiology following anoxia (Chapman and Renshaw, in press, Chapter 3). These
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Chapter 6: Hsp70 response to anoxia and re-oxygenation changes in blood physiology were not observed in the epaulette shark and indicate the presence of different adaptive responses to anoxic stress.
Since these two closely related species respond with different protective mechanisms, this study used two different anoxic regimes to characterise the Hsp70 response in the metabolically active cerebellum and the heart ventricle in the grey carpet shark following anoxic stress. This study also investigated the Hsp70 response in whole blood cell packs, in both the grey carpet shark and the epaulette shark following a standardised anoxic stress to determine whether the blood cells could be used as a less invasive measure of characterising
Hsp70 activation following anoxic stress.
6.3 Materials and methods
6.3.1 Study animals and location
Grey carpet sharks and epaulette sharks were supplied by UnderWater World
Aquarium (Mooloolaba, Sunshine Coast) and Sea World Aquarium (Main Beach, Gold
Coast). All experiments were performed at UnderWater World Aquarium. Animals were kept in outdoor, constant flow through aquaria with natural seawater and no additional light sources, thus relying on a normal photoperiod. Food was withheld from the animals for at least 24 hours before experiments were performed.
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6.3.2 Anoxic exposure
Animals were exposed to experimental treatment (anoxia) and control treatment
(normoxia) as reported in Chapter 2. Briefly, each tank was equipped with semi-permeable lids and filled with normoxic seawater (24°C), which was circulated around the tank using a
228-power head pump (CCC Pty Ltd. Australia). In the anoxic tanks, nitrogen gas was bubbled through an air stone to displace dissolved oxygen so that the dissolved oxygen
-1 concentration [dO2] remained below 0.02 mg L . In the normoxic tanks, air was bubbled
-1 through an air stone to maintain the [dO2] above 7.0 mg L . The [dO2] levels were measured and recorded each minute using TPS WP-90 oxygen probes (TPS Pty. Ltd., Australia).
6.3.3 Blood sampling and haematology
Blood samples were taken from grey carpet sharks and epaulette sharks exposed to either the anoxia or control treatment. A 0.5 ml blood sample was taken from each animal using the Stoskopf et al. (1984) method of venipuncture into the dorsal aorta with heparinised syringes. Blood samples were then heparinised in 3 ml lithium heparin vacutainers (Becton
Dickinson, North Ridge, New South Wales, Australia) and gently mixed. Blood plasma was separated from the blood sample by centrifugation at 3000 rpm for 5 minutes at 4C, then frozen at –80C for later analysis. Blood samples with the plasma removed contain all cellular components of the blood and will be referred to as a whole blood pack throughout this study.
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Chapter 6: Hsp70 response to anoxia and re-oxygenation
6.3.4 Two anoxic episodes
Twelve grey carpet sharks (n=12) were pair-matched for length and randomly selected for experimental or control treatment. Grey carpet sharks had a mean length of 40.86 ± 3.0 cm and weight of 193 ± 49.7 g. Animals were weighed and measured at the conclusion of the experiment. Animals were exposed to a double episode of anoxia following the procedures described previously by Renshaw et al. (2002). In short, six animals were exposed to anoxia and timed until they lost their righting reflex (E1) then allowed a re-oxygenation period in a separate normoxic tank. Twenty-four hours later, animals were exposed to a second anoxic episode until they lost their righting reflex (E2). Concurrently, six animals were exposed to normoxia in identical closed tanks for the same amount of time as their pair-matched experimental counterparts, to act as untreated normoxic controls. On the conclusion of the treatment, animals were immediately anaesthetised (5% benzocaine in ethanol solution, diluted 1.5ml L-1 in seawater) and blood samples were taken, along with brain and heart samples. The cerebellum of the brain and the ventricle of the heart were removed and all tissue was frozen separately at –80°C. Total time to loss of righting reflex (LRR) following two anoxic episodes was analysed using a paired student t-test. Tissue samples were analysed at Griffith University laboratories and data was analysed using a two tailed student t-test, assuming equal variance and two-way ANOVA with a 95% confidence interval.
6.3.5 Standardised anoxic episode
Eight grey carpet sharks (n=8) and eight epaulette sharks (n=8) were selected by closely pair-matching the animals for length and then randomly selecting for either anoxic exposure or the normoxic control treatment. Grey carpet sharks had a mean length of 88.56
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Chapter 6: Hsp70 response to anoxia and re-oxygenation
±8.1 cm and weight of 2.94 ± 0.8 kg. Epaulette sharks had a mean length of 71.58 ± 7.1 cm and weight of 1.43 ± 0.4 kg. Animals were weighed and measured at the conclusion of the experiment.
Animals were exposed to 1.5 hours of anoxia (treatment) or normoxia (control) followed by 12 hours of re-oxygenation in a separate normoxic tank. Blood samples were taken before anoxia (pre) and immediately following anoxia (post). Blood samples were also taken at 2 hours, 6 hours and 12 hours of re-oxygenation in normoxia. The blood samples were analysed at Griffith University laboratories and data was analysed using a Mixed Model
Repeated Measures ANOVA with a post hoc adjustment (Bonferroni).
6.3.6 SDS Page electrophoresis
Each tissue sample was homogenised using a glass homogeniser in 200 l cold lysis buffer (4°C) (50mM Tris pH 7.2, 150mM NaCl, 1% Nonident P-40, 0.5% Deoxycholate,
1mM Phenylmethylsulfonyl fluoride; PMSF) with 5 l of protease inhibitor (Bio-Rad laboratories, Australia). Samples were centrifuged at 8000rpm for 5 minutes at 4°C in an
Eppendorf 5415D centrifuge and the supernatant was removed for analysis. Total protein concentrations were measured using a Bio-Rad protein assay kit (Bio-Rad laboratories,
Australia) according to the manufacturer’s directions. Samples were incubated in the dark, at room temperature for 15 minutes, before being analysed using a UV Mini 1240 spectrophotometer at a wavelength of 540 nm. Protein standard curves were calculated using bovine serum albumin (Pierce Albumin Standard). A standard quantity of protein was mixed gently with 5 l of loading dye (0.5M Tris pH 6.8, 20% Glycerol, 4% Sodium dodecyl
(laurel) sulfate (SDS), 2% -Mercaptolyethanol, 0.01% Bromophenol blue) and heated in a
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Chapter 6: Hsp70 response to anoxia and re-oxygenation water bath at 100°C for 3 minutes to denature the protein. Samples were loaded into a well of
5% acrylamide stacking gel over an 8% resolving gel. Additionally, one well contained 7 l of kaleidoscope molecular weight marker (Bio-Rad laboratories, Australia), and another contained 2 l of Hela cell lysate (BD Biosciences, Transduction Laboratories, Australia) as a positive control and served as a common calibration point. Gels were immersed in SDS running buffer (1.4% Glycine, 0.3% Tris, 0.1% SDS).
Electrophoresis was applied at a constant voltage of 80 volts to fractionate the proteins until the loading dye reached the bottom of the gel. Gels were removed and loaded for western blotting on nitrocellulose paper (Bio-Rad laboratories, Australia) and submerged in transfer buffer (0.1% NaHCO3, 0.04% Na2CO3, 25% Methanol). Protein transfer occurred over 10-12 hours at 10 volts at 4°C. Gels were stained with Ponceau red to confirm protein transfer and subsequently washed off before immuno-detection.
6.3.7 Immuno-detection
Nitrocellulose membranes were immersed in blocking buffer (5% skim milk powder,
1M Tris, 3M NaCl, 0.05% Tween 20, pH 8.0) three times for 15 minutes each on an orbital shaker. Mouse anti-Hsp70 (BD Biosciences, Transduction Laboratories, Australia) was diluted at 1:1000 in blocking buffer for one hour at room temperature. Membranes were rinsed in blocking buffer three times for 15 minutes each and incubated with Goat Anti-mouse
IgG conjugate (BD Biosciences, Transduction Laboratories, Australia) diluted 1:2000 in blocking buffer for 30 minutes at room temperature. Membranes were again rinsed three times in blocking buffer for 15 minutes each and finally washed in cold Alkaline phosphatase buffer (APB) (100mM Tris, 100mM NaCl, 50mM MgCl26H20) for 15 minutes prior to
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Chapter 6: Hsp70 response to anoxia and re-oxygenation incubation in substrate buffer (APB, 6.6% Nitro Blue Tetrazolium, 3.3% 5-Bromo-4-Chloro-
3-Indoyl Phosphate). Incubation occurred in the dark and at room temperature. Membranes were then scanned using a Bio-rad densitometer and band intensity was analysed using
Quantum One software.
6.4 Results
6.4.1 Two anoxic episodes
In response to the first episode of anoxia, mean times to LRR in the grey carpet shark were 49.39 (± 22.4) min. Following 24 hours of recovery in normoxia, the mean time to LRR during the second anoxic episodes was significantly longer (83.2 ± 14.9 min)(p<0.01) (Figure
6.1).
Examination and analysis of the densitometry of western blots showed that the positive controls, Hela cell lysate, had clear banding and density for Hsp70. However, examination and analysis of the densitometry of western blots showed no significant differences in mean Hsp70 concetrtations in the cerebellum of the grey carpet shark immediately following two episodes of anoxia (86800.48 ± 11448.82 Int.mm2) or the control groups (80803.42 ± 12068.89 Int.mm2) (Figure 6.2). Similarly, the ventricle of the grey carpet shark had no significant differences in mean Hsp70 concentrations between anoxia treated (70351.1 ± 21789.55 Int.mm2) and control treatment animals (71407.44 ± 19584.59
Int.mm2). Furthermore Hsp70 concentrations of the whole blood pack showed no significant differences in mean Hsp70 concentration levels from control treatment animals (20006.61 ±
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Chapter 6: Hsp70 response to anoxia and re-oxygenation
3076.28 Int.mm2) and anoxia treatment animals (18376.61 ± 5670.48 Int.mm2) following two episodes of anoxia.
Time to loss of righting reflex in the grey carpet shark during two episodes of anoxia 120
100
80
60 E1
E2 Time(min)
40
20
0 1 2 3 4 5 6 Animals Figure 6.1. Total time to loss of righting reflex in the grey carpet sharks (C. punctatum) following two episodes of anoxia. Episode one (E1) and episode two (E2).
Hsp70 concentration in the cerebellum, ventricle and the blood following two anoxic exposures in the grey carpet shark 120000
100000
80000
60000 Anoxia Control
Absorbance 40000
20000
0 Cerebellum Ventricle Blood Tissue type Figure 6.2. Mean Hsp70 concentrations in the cerebellum, ventricle and whole blood of the grey carpet sharks (C. punctatum) following two episodes of anoxia until loss of righting reflex.
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Chapter 6: Hsp70 response to anoxia and re-oxygenation
In the grey carpet shark, no significant differences were observed between the brain cerebellum and the heart ventricle in either control animals or anoxia treated animals exposed to two anoxic episodes (Figure 6.2). However, whole blood had significantly smaller concentrations of Hsp70 (p<0.001) than the cerebellum and the ventricle in both control and anoxia treated animals.
6.4.2 Standardised anoxic episode
The grey carpet shark and the epaulette shark were exposed to a standard 1.5 hours of anoxia followed by 12 hours of normoxic re-oxygenation. No significant differences were observed in pre-experimental mean Hsp70 concentrations between experimental treated
(anoxia) and control treated (normoxia) grey carpet sharks (Figure 6.3). Immediately following 1.5 hours of anoxia, no significant difference in mean Hsp70 concentrations were observed in the blood from pre-experimental measures. Furthermore, no significant differences occurred at 2, 6 and 12 hours of normoxic re-oxygenation from pre-experimental mean levels.
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Chapter 6: Hsp70 response to anoxia and re-oxygenation
Changes in Hsp70 concentration in the blood of the grey carpet shark in response to anoxia and re-oxygenation 160000
140000
120000
100000 Pre Post 80000 2hr 6hr 12hr Absorbance 60000
40000
20000
0 Anoxia Control Treatment Figure 6.3. Mean Hsp70 concentrations in the whole blood of the grey carpet sharks (C. punctatum) before (pre) and immediatelty following (post) 1.5 hours anoxia and at 2 hours, 6 hours and 12 hours of normoxic re-oxygenation.
Likewise, the mean Hsp70 concentration in the epaulette shark prior to treatment (pre) showed no significant differences between either experimental (anoxia) or control (normoxia) groups (Figure 6.4). No significant differences were observed in mean Hsp70 concentrations immediately following anoxic exposure. Furthermore, no significant differences occurred at 2,
6 and 12 hours of normoxic re-oxygenation from pre-experimental mean levels, although the large variance of Hsp70 concentrations in the whole blood pack of anoxia treated epaulette sharks may have masked smaller changes in Hsp70.
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Chapter 6: Hsp70 response to anoxia and re-oxygenation
Changes in Hsp70 concentration in the blood of the grey carpet shark in response to anoxia and re-oxygenation 80000
70000
60000
50000 Pre Post 40000 2hr 6hr 12hr Absorbance 30000
20000
10000
0 Anoxia Control Treatment Figure 6. 4. Mean Hsp70 concentrations in the whole blood of the epaulette sharks (H. ocellatum) before (pre) and immediately following (post) 1.5 hours anoxia and at 2 hours, 6 hours and 12 hours of normoxic re-oxygenation.
6.5 Discussion
6.5.1 Two anoxic episodes
From previous studies, the LRR during anoxia in the epaulette shark indicates the onset of an energy conserving hypometabolism (Mulvey and Renshaw, 2000). Following repeated anoxic exposure, Renshaw et al. (2002) observed that the epaulette shark lost its righting reflex significantly earlier in the second anoxic insult compared to the first, indicating the activation of a preconditioning mechanism. This preconditioning mechanism allows the epaulette shark to enter a protective state earlier during repeated periods of oxygen deprivation. Conversely, the time to LRR in the grey carpet shark during repeated anoxia was significantly longer than the original anoxic insult. Previous studies suggest that the grey carpet shark does not undergo an energy conserving state of neuronal hypometabolism and the
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Chapter 6: Hsp70 response to anoxia and re-oxygenation
LRR in this species may be due to the exhaustion of neuronal energy levels bringing the animal very close to death (Chapter 2). Similarly, the leopard frog has been reported to slowly deplete cellular energy during anoxia and is essentially slowly dying (Milton et al., 2003).
Alternatively, in the absence of a neuronal hypometabolism in the grey carpet shark, the delayed time to LRR during the second episode of anoxia could indicate that a pre- conditioning response has activated other protective mechanisms following the first anoxic episode, however this warrants further investigation. The neuronal ATP levels in the grey carpet shark have not been examined in response to anoxia and further study is required to determine whether the grey carpet shark is slowly depleting its ATP levels similar to the leopard frog or activating energy conserving mechanisms to prolong survival during anoxic exposure.
In the anoxia-tolerant epaulette shark, significant differences in Hsp70 concentrations in the cerebellum occurred after two episodes of anoxia (Renshaw et al., 2004). Renshaw et al. (2004) concluded that the induction of these protective mechanisms may be a preconditioning effect to cyclic periods of oxygen deprivation stress experienced in this shark’s natural environment. The present study observed the presence of constitutive levels of
Hsp70 within the cerebellum of the brain, the ventricle of the heart and the whole blood in the grey carpet shark. However, following two episodes of anoxia, no significant differences in
Hsp70 concentrations were observed. The delayed LRR in the grey carpet shark is therefore not explained by changes in protective Hsp70 concentrations above constitutive levels.
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Chapter 6: Hsp70 response to anoxia and re-oxygenation
The mode of action to induce the up-regulation of Hsp70 above constitutive levels appears to be stressors that are proteotoxic (Latchman, 1998; 1999). Hsp70 induction occurs when increasing levels of denatured proteins overwhelm constitutive Hsp70 concentrations.
The proportion of denatured proteins therefore depends on the severity of the stressor and the physiological response to deal with these conditions. Anoxia-tolerant species that activate energy conserving mechanisms during anoxic exposure have a delayed Hsp70 up-regulation and thus have a larger Hsp70 induction threshold due to the protein protection mechanisms that are activated (Renshaw et al., 2002). Renshaw et al. (2002) concluded that due to the energy conserving strategies utilised by the epaulette shark during anoxia, this species possesses a significantly larger damage threshold to induce Hsp70 than anoxia-intolerant species and the Hsp70 response in the cerebellum of the epaulette shark was increased further if metabolic sensors and oxygen sensors were both activated.
Alternatively, a lack of Hsp70 induction has also been reported in response to lethal stress doses. Vijayan et al. (1998) observed that the lack of Hsp70 expression in lethal doses of sodium dodecylsulphate was correlated to fish mortality, suggesting that there is a relationship between Hsp70 induction, cellular protection and consequently, cellular survival.
Lewis et al. (2001) agreed and proposed that variable induction and down-regulation of the
Hsp70 response is due to lethal exposure and reflects a complete breakdown of protein metabolism including HSPs.
The anoxia tolerance and the presence of energy conserving mechanisms have yet to be determined in the grey carpet shark. While the grey carpet shark has been observed to tolerate significant periods of anoxia, similar to the epaulette shark, Chapter 2 suggests that the grey carpet shark does not undergo an energy conserving state of neuronal
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Chapter 6: Hsp70 response to anoxia and re-oxygenation hypometabolism and the LRR in this species may be due to the exhaustion of neuronal energy levels bringing the animal very close to death. Interestingly, no changes in Hsp70 concentrations within the cerebellum, ventricle or the blood occurred in the grey carpet shark in response to two episodes of anoxia in the present study. The absence of an inducible up- regulation Hsp70 in the grey carpet shark following two anoxic exposures indicates a complete absence of Hsp70 activation due to a lethal anoxic exposure, possibly reflecting a complete breakdown of protein metabolism.
Recent studies have reported tissue specific differences in Hsp70 levels. Dietz and
Somero (1993) found differences in constitutive Hsp70 levels to vary widely between the brain, liver and gill tissue in four marine teleosts exposed to temperature stress. Currie et al.
(2000) found similar results in the hypoxia- and anoxia-intolerant rainbow trout (O. mykiss), however concluded that the blood accumulated the greatest amount of mRNA Hsp70 compared with liver and gill tissue. In elasmobranchs, Renshaw et al. (2002) observed similar differences in constitutive Hsp70 concentrations. The epaulette shark had significantly lower constitutive levels of Hsp70 in the cerebellum than in the brainstem. The grey carpet shark displayed similar tissue specific differences in constitutive Hsp70 concentrations, with the cerebellum and the heart ventricle showing significantly higher concentrations than the blood.
6.5.2 Standardised anoxic episode
The activating mechanisms and response time for an up-regulation in Hsp70 concentrations following an anoxia stress event and re-oxygenation have not been thoroughly investigated in either the epaulette shark or the grey carpet shark. The Hsp70 concentration of the blood in the epaulette shark and the grey carpet shark following an anoxic insult and
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Chapter 6: Hsp70 response to anoxia and re-oxygenation throughout a re-oxygenation recovery period was therefore used to determine the time course to induce Hsp70 above constitutive levels. No changes in Hsp70 concentrations were observed in the blood of the grey carpet shark following anoxia or at any time point throughout 12 hours of re-oxygenation. Also, no changes in Hsp70 within the blood of the epaulette shark were observed in this study. This provides evidence that the Hsp70 response to anoxia could also be tissue specific in the epaulette shark.
6.5.3 Conclusion
Repeated anoxic stress until LRR or a standardised anoxic stress followed by re- oxygenation in the grey carpet shark does not induce an Hsp70 response above constitutive levels in the blood or in highly metabolic organs such as the cerebellum and ventricle of the heart. Similarly, the blood of the epaulette shark had no changes in Hsp70 levels in response to a standardised episode of anoxia and re-oxygenation. This indicates either a tissue specific absence of Hsp70 induction in the blood, or that one episode of anoxia was not sufficient to induce Hsp70 above constitutive levels in this anoxia-tolerant species.
These data add to the body of knowledge about tissue specific responses of Hsp70 to stress and eliminate the possibility that blood samples could be used as non-invasive indicators of anoxic stress in the epaulette shark or the grey carpet shark. While a significant increase in time to LRR in response to anoxia in the grey carpet shark demonstrates the presence of a preconditioning effect and/or activation of protective mechanisms to increase survival, further study is required to identify the cause of this increased time until LRR.
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Chapter 6: Hsp70 response to anoxia and re-oxygenation
6.6 Literature cited
Airaksinen S, Råbergh CM, Sistonen L, Nikinmaa M. 1998. Effects of heat shock and hypoxia on protein synthesis in rainbow trout (Oncorhynchus mykiss) cells. J Exp Biol 201(17):2543-2551. Bierkens J, Van de Perre W, Maes J. 1998. Effect of different environmental variables on the synthesis of Hsp70 in Raphidocelis subcapitata. Comp Biochem Physiol A 120:29-34. Currie S, Moyes CD, Tufts BL. 2000. The effects of heat shock and acclimation temperature on Hsp70 and Hsp30 mRNA expression in rainbow trout: in vivo and in vitro comparisons. J Fish Biol 56:398-408. Currie S, Tufts B. 1997. Synthesis of stress protein 70 (Hsp70) in rainbow trout (Oncorhynchus mykiss) red blood cells. J Exp Biol 200(3):607-614. Delaney MA, Klesius PH. 2004. Hypoxic conditions induce Hsp70 production in blood, brain and head kidney of juvenile Nile tilapia Oreochromis niloticus (L.). Aqua 236:633- 644. Dietz TJ, Somero GN. 1993. Species- and tissue-specific synthesis patterns for heat-shock proteins HSP70 and HSP90 in several marine teleosts fishes. Physiol Zool 66(6):863- 880. Feder ME, Hofmann GE. 1999. Heat-shock proteins, molecular chaperones, and the stress response. Annu Rev Physiol 61:243-282. Koban M, Yup AA, Agellon LB, Powers DA. 1991. Molecular adaptation to environmental temperature: heat shock response of the eurythermal teleosts Fundulus heteroclitus. Mol Mar Biol Biotech 1:1-17. Kohler HR, Rahman B, Graff S, Berkus M, Triebskorn R. 1996. Expression of the stress-70 protein family (HSP70) due to heavy metal contamination in the slug, Deroceras reticulatum: an approach to monitor sublethal stress conditions. Chemo 33:1327-1340. Last PR, Stevens JD. 1994. Sharks and Rays of Australia. CSIRO Division of Fisheries. Melbourne, Australia. Latchman DS. 1998. Heat shock proteins: protective effect and potential therapeutic use. Int J Mol Med 2:375- 381. Latchman DS. 1999. Stress proteins an overview. In: Stress Proteins. Handbook of Experimental Pharmacology. Ed Latchman, DS. Springer-Verlag. Berlin.
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Lewis S, Donkin ME, Depledge MH. 2001. Hsp70 expression in Enteromorpha intestinalis (Chlorophyta) exposed to environmental stressors. Aqua Toxicol 51:277-291. Marber MS, Mestril R, Chi S, Sayen MR, Yellon DM, Dillmann WH. 1995. Overexpression of the rat inducible 70kD heat stress protein in a transgenic mouse increases the resistance of the heart to ischemic injury. J Clin Invest 95:1446-1456. Marx JL. 1983. Surviving heat shock and other stresses. Heat Shock: From Bacteria to Man, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 251-253. McCully JD, Lotz MM, Krukenkamp IB, Levitsky S. 1996. A brief period of retrograde hyperthermic perfusion enhances myocardial protection from global ischemia: Assication with accumulation of Hsp70 mRNA and protein. J Mol Cell Cardiol 28:231-241. Milton SL, Manuel L, Lutz PL. 2003. Slow death in the leopard frog Rana pipiens: neurotransmitters and anoxia tolerance. J Exp Biol 206:4021-4028. Misra S, Zafarullah M, Price-Haughey J, Gedamu L. 1989. Analysis of stress-induced gene expression in fish cell lines exposed to heavy metals and heat shock. Biochem Biophys Acta 1007(3):325-333. Mulvey JM, Renshaw GMC. 2000. Neuronal oxidative hypometabolism in the brainstem of the epaulette shark (Hemiscyllium ocellatum) in response to hypoxic pre-conditioning. Neurosci Let 290:1-4. Murtha JM, Keller ET. 2003. Characterisation of the heat shock response in mature zebrafish (Danio rerio). Exp Gerontol 18:1-9. Renshaw GMC, Kerrisk CB, Nilsson GE. 2002. The role of adenosine in the anoxic survival of the Epaulette shark, Hemiscyllium ocellatum. Comp Biochem Physiol B 131:133- 141. Renshaw GM, Warburton J, Girjes A. 2004. Oxygen sensors and energy sensors act synergistically to achieve a graded alteration in gene expression: consequences for assessing the level of neuroprotection in response to stressors. Front Biosci 9:110-116. Routley MH, Nilsson GE, Renshaw GMC. 2002. Exposure to hypoxia primes the respiratory and metabolic responses of the epaulette shark to progressive hypoxia. Comp Biochem Physiol A 131:313-321. Soderstrom V, Renshaw GMC, Nilsson GE. 1999. Brain blood flow and blood pressure during hypoxia in the epaulette shark Hemiscyllium ocellatum, a hypoxia-tolerant elasmobranch. J Exp Biol 202:829-835.
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Stoskopf MK, Smith B, Klay G. 1984. Clinical note: Blood sampling of captive sharks. J Zool Anat Med 15:116-117. Vijayan MM, Pereira C, Forsyth RB, Kennedy CJ, Iwama GK. 1997. Handling stress does not affect the expression of hepatic heat shock protein 70 and conjugation enzymes in rainbow trout treated with β-naphthoflavone. Life Sci 61(2):117-127. Vijayan MM, Pereira C, Kruzynsky G, Iwama GK. 1998. Sublethal concentrations of contaminant induced expression of hepatic heat shock protein 70 in two salmonids. Aqua Toxicol 40:101-108. Wise G, Mulvey JM, Renshaw GMC. 1998. Hypoxia tolerance in the epaulette shark (Hemiscyllium ocellatum). J Exp Zool 281:1-5.
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GENERAL DISCUSSION
The epaulette shark is tolerant to cyclic episodes of severe hypoxia, which may be an adaptation to the repeated occurrence of severely reduced dissolved oxygen in its natural habitat (Wise et al., 1998; Routley et al., 2002; Nilsson and Renshaw, 2004). While some elasmobranchs have strategies to compensate for dissolved oxygen levels that fall to 50% of normal saturation in sea water (Hughes and Johnston, 1978; Carlson and Parsons, 2001), sharks are not tolerant to extreme hypoxia or anoxia (Butler et al., 1975; Butler et al., 1979;
Short et al., 1979; Metcalfe and Butler, 1984). Until recently, the epaulette shark
(Hemiscyllium ocellatum) was the only elasmobranch species reported to possess a significant tolerance to severe hypoxia and even anoxia at temperatures up to 28C (Renshaw et al.,
2002). However, data collected for this thesis provided evidence that the grey carpet shark
(Chiloscyllium punctatum) can also tolerate 172.0 ( 11.0) minutes of anoxia at 23°C
(Chapter 2). The grey carpet shark is a close relative of the epaulette shark, residing in the same family, Hemiscyllidae, and has been reported to inhabit a wide range of habitats that may expose these animals to ambient hypoxia, including sea grass beds, mangrove swamps and occasionally coral reef flats (Last and Stevens, 1994).
In ectothermic vertebrates, seasonal change in temperature is a key environmental variable that plays a major role in controlling physiological functions, such as oxygen consumption and metabolic rate (Schmidt-Neilsen, 1983; Lutz and Nilsson, 1997; Carlson and
Parsons, 1999). This is the first study to examine the affect of temperature on the anoxia tolerance in two species of elasmobranch. The present study revealed that the duration of anoxia tolerance of both the epaulette shark and the grey carpet shark was temperature dependent. A significant reduction in the time to loss of righting reflex (LRR) occurred at the
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higher end of the temperature range examined in both species. Both the epaulette shark and the grey carpet shark withstood significantly longer periods of anoxia at lower temperatures possibly due to a temperature induced decrease in metabolic rate. Anoxia-tolerant vertebrates such as teleosts in the genus Carassius and freshwater turtles in the genera Chrysemys and
Trachemys have also been observed to have reduced anoxia tolerance times with elevated temperatures (Lutz and Nilsson, 1997). The crucian carp (Carassius carassius) and freshwater turtle can survive for several months in anoxia at temperatures of less than 10C. However, as temperature increases (20-25C), the anoxic survival time is significantly reduced to a couple of days due to a subsequent increase in metabolic rate (Lutz and Nilsson, 1997). This thesis observed that a temperature change of 2°C at seasonal temperatures commonly encountered by both species in their natural habitats (23C -27C), had a significant affect on anoxia tolerance times in both the epaulette shark and the grey carpet shark.
While the anoxia tolerance was similar in the epaulette shark and the grey carpet shark at 23°C, as the ambient temperature increased to 25°C and then 27°C, the epaulette shark was able to tolerate significantly longer periods of anoxia than the grey carpet shark. The Q10 analysis found ventilation rate sensitivity was lower in the epaulette shark compared to the grey carpet shark throughout the seasonal temperature range used in this thesis (23°C-27°C).
While it has been noted that ventilation rates are not an accurate indication of oxygen consumption (Chapter 2), the higher Q10 coefficient recorded in the grey carpet shark may explain the reduced anoxia tolerance times observed in this species. It is concluded that this increase in anoxia tolerance with elevated temperature is due to the epaulette shark’s ability to reversibly activate energy conserving mechanisms, such as ventilatory depression (Routley et al., 2002), bradycardia (Soderstrom et al., 1999) and neuronal hypometabolism (Mulvey and
Renshaw, 2000). The grey carpet shark does not activate ventilatory depression in response to
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anoxia and the LRR in the grey carpet shark was followed by an almost immediate cessation of gill ventilation. This suggests that the LRR in the grey carpet shark is not a result of metabolic depression to conserve ATP, as observed in the epaulette shark (Renshaw et al.,
2002), but may be due to severely low/depleted neuronal energy levels. This depletion of energy levels in the grey carpet shark was supported by significant increases in plasma potassium.
Hyperkalemia has been repeatedly observed in response to anoxia in vertebrate animals (Jackson and Heisler, 1982; Jackson and Ultsch, 1982; Jackson and Heisler, 1983;
Krumschnabel et al., 1998; Warren and Jackson, 2005). However, the exact mechanisms responsible for potassium movements into the plasma are not fully understood. From the current literature, anoxia induced hyperkalemia is due to i) the failure of ion pumps due to depleted ATP levels (Jackson and Heisler, 1983), ii) ion channel arrest to conserve ATP
(Hochachka et al., 1996) and iii) the opening of ATP sensitive potassium channels (Jonas et al., 1991).
Following anoxia and during re-oxygenation, no changes in plasma sodium occurred in either shark species and a minor decrease in plasma chloride occurred during re- oxygenation only in the grey carpet shark. Hyperkalemia and a lack of change in plasma sodium concentrations were observed in anoxia treated leopard frogs (Rana pipiens)
(Knickerbocker and Lutz, 2001; Warren and Jackson, 2005), similar to the grey carpet shark.
Knickerbocker and Lutz (2001) observed that the anoxia-intermediate leopard frog initially maintained neuronal intracellular potassium as a mechanism to delay the onset of neuronal depolarisation. This prolonged maintenance of intracellular potassium has been attributed to a retarded leakage of potassium pumps (50% decrease in potassium leakage), which reduces the
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rate of extracellular potassium accumulation, prolonging depolarisation and conserving energy expenditure from the reduced activity of ion pumps. When ATP levels fall to 35% of normoxic values, a threshold is reached and potassium homeostasis is lost (Knickerbocker and
Lutz, 2001), resulting in a massive release of inhibitory and excitatory neurotransmitters such as GABA and glutamate, respectively (Lutz and Reiners, 1997), similar to the anoxic catastrophe of the mammalian brain.
The exact mechanisms that cause a loss of potassium homeostasis in response to hypoxia or anoxia are not fully understood. Jonas et al. (1991) reported evidence of the opening of ATP sensitive potassium channels in response to severely low ATP levels in the axons of the clawed toad (Xenopus laevis), which may explain the hyperkalemia accompanied by constant plasma sodium concentrations observed in the leopard frog and the grey carpet shark in response to anoxia. Further study is required to determine the source and cause of hyperkalemia, along with the ATP levels of the grey carpet shark in response to anoxia.
In addition, a complete loss in ion homeostasis cannot be ruled out in either shark species following anoxia and during re-oxygenation. While the disruption of ions in response to anoxia may provide a protective mechanism to conserve ATP, they may also indicate a detrimental breakdown of ionic homeostasis due to severely low/depleted ATP levels.
Jackson and Heisler (1983) suggested that increases in plasma potassium in anoxia-tolerant freshwater turtles are attributed to the partial failure of Na+/K+ pumps in the skeletal muscle as a consequence of anoxia induced ATP depletion. Failure of Na+/K+ pumps allows an unopposed influx of sodium and chloride, along with an efflux of potassium from the cell.
Interestingly, the grey carpet shark and the epaulette shark had no change in mean plasma sodium concentrations at any time point throughout the treatment, indicating the functioning
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of Na+/K+ pumps. However an influx of sodium and chloride from the external environment via the gills or sequestering of ions such as sodium, chloride and potassium excretion from the kidneys and/or rectal gland has not been examined in elasmobranchs in response to anoxia.
The grey carpet shark showed significant haematological responses to anoxic exposure and re-oxygenation. In particular, there was a significant increase in RBC concentrations immediately following anoxia. An increase in RBC concentrations in response to metabolically demanding stimuli have been reported due to i) the release of RBCs from a storage reservoir such as the spleen (Yamamoto et al., 1980; Kita and Itazawa, 1989; Pearson and Stevens, 1991), ii) haemoconcentration of the blood (Swift and Lloyd, 1974; Nikinmaa and Tervonen, 2004; Tervonen et al., 2006) and iii) mitotic division within the peripheral blood (Soldatov, 1996; Fandrey, 2004). In response to hypoxia, these three mechanisms have been reported to increase the RBC concentration per unit volume of blood and subsequently act to fine tune the oxygen carrying capacity of the blood in unit time (Swift and Lloyd, 1974;
Yamamoto et al., 1980; Pearson and Stevens, 1991; Soldatov, 1996).
In response to hypoxia, the release of RBCs into the circulation via a splenic contraction has been well documented in teleost fishes (Yamamoto et al., 1980; Pearson and
Stevens, 1991). However, no studies have investigated the release of RBCs in response to anoxia. This thesis also examined the possibility that the spleen and the highly developed liver act as RBC reservoirs. The grey carpet shark showed a significant increase in RBCs immediately following anoxic exposure. Rapid re-absorption of RBCs upon exposure to normoxic conditions following anaesthesia in the grey carpet shark is unlikely as RBCs remained elevated in the blood for 2 hours following similar anoxic episodes before returning to pre-experimental levels (Chapman and Renshaw, in press, Chapter 3). However, it is
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possible that RBCs may have rapidly moved back into the spleen or the liver during anaesthesia or before the organs were excised. While other studies used a cephalic blow to eliminate this concern (Yamamoto et al., 1983; Yamamoto, 1987; Pearson and Stevens,
1991), it is possible that relaxation of the splenic/hepatic musculature during anaesthesia may have allowed RBCs to be re-absorbed. Therefore, the absence of changes in organ weight and haemoglobin concentrations in the spleen and liver immediately following anoxia suggests either no release of RBCs from these two potential storage organs or the rapid re-absorption of RBCs during anaesthesia in the grey carpet shark. If the release of RBCs from a storage organ is masked due to re-absorption, increased RBC concentrations into the circulation would not increase the oxygen uptake in an anoxic environment. However, the release of stored oxygenated RBCs via a splenic/hepatic contraction would act to increase the oxygen delivery to vital organs such as the heart and brain.
While hypoxia has been observed to increase the mitotic division of RBCs (Soldatov,
1996; Fandrey, 2004), no studies have investigated the presence of RBC division during anoxia. Soldatov (1996) reported the onset of RBC division during the initial stages of mild hypoxia. During mild hypoxia, at 30% oxygen saturation, Soldatov (1996) observed the presence of mitotic RBC division after 2 hours and noted a 37% increase in RBC concentrations after 6 hours of hypoxia in the flounder (Pleuronectes flesus luscus). The transient increase in RBC concentrations in this thesis following anoxia suggests that maturation of RBCs is unlikely due to their rapid disappearance during re-oxygenation. In addition, the production of RBCs during anoxia is also unlikely due to the energy expenditure required to produce RBCs.
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Swift and Lloyd (1974), along with Tervonen et al. (2006) observed haemoconcentration of the blood in response to hypoxia in rainbow trout (Salmo gairdneri and Oncorhynchus mykiss). While the present study did not measure haemoconcentration of the blood in the grey carpet shark, maintenance of plasma urea concentrations may suggest the absence of haemoconcetration as a cause for elevated RBC concentrations in the grey carpet shark in response to anoxia. Increased RBC concentrations due to haemoconcentration of the blood has been reported to confer significant advantages to increase the oxygen carrying capacity of the blood in unit time during bouts of naturally occurring hypoxic exposure (Swift and Lloyd, 1974; Tervonen et al., 2006). However, haemoconcentration of the blood during anoxia has little known physiological advantages.
During anoxia, the grey carpet shark appeared to increase its anaerobic metabolism, which was reflected by a significant increase in plasma lactate concentrations. Unlike anoxia- tolerant vertebrates that can significantly up-regulate anaerobic metabolism and tolerate the toxic accumulation of its end-products (Lutz and Nilsson, 1997; Jackson, 2000), the peak plasma lactate concentrations in the grey carpet shark were comparable to hypoxic exposure in anoxia-intolerant elasmobranch and teleost species (Piiper et al., 1972; Cliff and Thurman,
1984; Hoffmayer and Parsons, 2001). This indicates that the grey carpet shark may have reached an anaerobic limit of up-regulation during anoxia.
Anoxia-tolerant turtles and the anoxia-intermediate leopard frog significantly increase bicarbonate and non-bicarbonate buffers, such as magnesium and calcium, to assist in maintaining the blood pH during events of anoxia (Jackson, 2000; Warren and Jackson,
2005). No significant changes in plasma calcium concentrations were observed in the grey carpet shark, however a significant increase in magnesium occurred during re-oxygenation
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following anoxic exposure. This increase in plasma magnesium may indicate the mobilisation of non-bicarbonate buffers in the blood as the accumulative concentrations of lactate diffuse into the blood during re-oxygenation.
The epaulette shark also showed significant haematological responses to anoxic exposure that were different from those of the grey carpet shark. This study is the first to report a change in haematocrit due to erythrocyte swelling in response to anoxia followed by re-oxygenation in an elasmobranch species, the epaulette shark. Hypoxia and anoxia induced cell swelling has been well documented in teleosts due to the activation of Na+/H+ channels aiding intracellular alkalisation and thereby protecting intracellular pH (Soivio et al., 1974a, b). However, plasma sodium and chloride concentrations were maintained during anoxia and re-oxygenation in the epaulette shark, indicating the absence of cell swelling due to the activation of Na+/H+ channels. These results support findings from both Tufts and Randall
(1989) and Wood et al. (1994) who reported the absence of a detectable β-adrenergic Na+/H+ exchanger in the anoxia-intolerant dogfish (Squalus canicula). Therefore further investigation is required into the acid-base balance of the epaulette shark during anoxia and re-oxygenation to fully understand how the epaulette shark tolerates high plasma lactate levels and induces
RBC swelling observed in the present study.
Significant increases in haematocrit and plasma lactate paralleled increases in erythrocyte volume and anaerobic metabolism respectively. Significant increases in plasma lactate is a typical response to anoxic exposure due to an increase in anaerobic metabolism.
However, lactate concentrations in the epaulette shark were the highest ever to be reported in an elasmobranch, indicating not only a high anaerobic capacity but also a high tolerance to the accumulation of metabolic end products such as lactate, which can result in an associated
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acidosis. During anoxia and re-oxygenation, elevated concentrations of magnesium and calcium increased in the plasma of the epaulette shark, which may act as non-bicarbonate buffers against anaerobic acidosis similar to other anoxia tolerant vertebrates (Jackson and
Heisler, 1983).
The oxygen affinity of haemoglobin and the unloading of oxygen from RBCs can be significantly affected by declines in pH, known as the Bohr Effect. The presence of a Bohr shift in elasmobranchs appears to be species specific (Wells and Weber, 1983; Wells and
Davie, 1985; Cooper and Morris, 2004), with significant Bohr and Haldane Effects present in some species (Wells and Weber, 1983) and absent in others (Wells and Davie, 1985). Cooper and Morris (2004) reported a low Bohr shift in the benthic Port Jackson shark (Heterodontus portjacksoni), however the Bohr Effect has not been reported in either the epaulette shark or the grey carpet shark. If a Bohr Effect is present, the significant increase in plasma lactate concentrations may reduce plasma pH levels and be responsible for RBC swelling, which may increase oxygen unloading during anoxic exposure in the epaulette shark.
During anoxia, anoxia-tolerant vertebrates have been observed to significantly increase blood glucose concentrations. The plasma glucose of the crucian carp has been observed to increase by 5-fold during anoxia (Walker and Johansen, 1977), while the freshwater turtle has an 8-fold increase in plasma glucose concentrations (Penney, 1974). In this thesis, both the epaulette shark and the grey carpet shark maintained plasma glucose concentrations during anoxic exposure, which indicates a very sensitive regulation of blood glucose levels. Following re-oxygenation, this study clearly shows for the first time two elasmobranch species entering a phase of hyperglycaemia during normoxic re-oxygenation following anoxia. The hyperglycaemic state during re-oxygenation could potentially reflect an
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up-regulation of aerobic metabolism, which could serve to rapidly replenish ATP levels lost during anoxia. Interestingly the epaulette shark had a significantly smaller increase in plasma glucose concentrations following re-oxygenation than the grey carpet shark, which may indicate a smaller energy deficit to replenish upon re-oxygenation due to the activation of energy conserving mechanisms and up-regulation of anaerobic metabolism during anoxia.
The release of glycogen stores and the conversion of lactate into glucose through the
Cori cycle in the liver may explain the increase in plasma glucose concentrations during re- oxygenation observed in both species. Gluconeogenesis from lactate, via the Cori cycle, would supply metabolic fuel for increases in aerobic metabolism that may be required to replenish oxygen debts experienced during anoxia in both species. The activation of the Cori cycle during re-oxygenation would also remove accumulated lactic acid in the plasma and subsequently relieve the disruption to the acid-base balance and reduce the incident of acidosis. Further study is required to determine the cause and source of increased plasma glucose in response to anoxia and normoxic re-oxygenation in the epaulette shark and the grey carpet shark.
While the epaulette shark and the grey carpet shark are closely related, they have adapted different strategies to deal with severe declines in oxygen availability. In contrast to the epaulette shark, the grey carpet shark rapidly increased erythrocyte number, indicating a redirection of oxygen stores during periods of oxygen deficit, which may subsequently prolong survival in a fluctuating inter-tidal estuarine environment. The epaulette shark possesses physiological strategies similar to other anoxia-tolerant vertebrates to conserve ATP levels, which in turn enable it to tolerate anoxic exposure. The epaulette shark also displayed a significant increase in anaerobic pathways, which indicated a high anaerobic capacity during
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anoxia, also present in other anoxia-tolerant vertebrates (Lutz and Nilsson, 1997; Jackson,
2000). In contrast, the grey carpet shark does not appear to possess mechanisms to conserve
ATP expenditure during anoxia, such as reversible metabolic and ventilatory depression found in both the epaulette shark and other anoxia-tolerant vertebrates such as the crucian carp and fresh water turtles. Unlike anoxia-intolerant vertebrates that rapidly deplete ATP levels during the first few minutes of anoxia, the grey carpet shark is able to survive prolonged periods of anoxia at high temperatures. The characteristics displayed by the grey carpet shark appear to indicate that this species is slowly depleting its ATP levels during an anoxic insult and therefore may be classified as having an intermediate tolerance to anoxic exposure. The intermediate anoxia tolerance displayed by the grey carpet shark demonstrates the presence of ecophysiologically relevant adaptations which may prolong survival in the intermittently hypoxic or even briefly anoxic environments encountered in tidal pools, mangrove swamps, and in some estuaries and coral reef environments. Further study is therefore required to understand how the grey carpet shark protects ATP levels from being rapidly depleted to allow this species to tolerate substantial anoxic challenges at high temperatures.
This thesis reveals ecophysiological adaptations possessed by two benthic elasmorbranchs. The epaulette shark and the grey carpet shark inhabit many coast habitats throughout subtropical and tropical waters surrounding Australia. Investigation of the physiological responses of these animals to environmental stressors such as hypoxia and anoxia provides indicators of how these species may respond to changes in ambient water quality and environmental stressors. Furthermore, the physiological response of the epaulette shark and the grey carpet shark to environmental stressors provides critical information on the husbandry requirements of these species, which are commonly held and bred in captivity.
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The epaulette shark and the grey carpet shark are also valuable in biomedical research.
Vertebrate animals that possess cellular protective mechanisms against hypoxic and anoxic stress events may be used as clinical models. Due to the differences in anoxia tolerance possessed by the epaulette shark and the grey carpet shark, both species may offer novel protective mechanisms activated during hypoxic and anoxic stress. Cellular protective mechanism found in these animals may subsequently be manipulated to assist in cellular protection during degenerative events such as heart attack and stroke.
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Literature cited
Butler PJ, Taylor EW. 1975. The effect of progressive hypoxia on respiration in the dogfish (Scyliorhinus canicula) at different seasonal temperatures. J Exp Biol 63:117-130. Butler PJ, Taylor EW, Davison W. 1979. The effect of long term, moderate hypoxia on acid base balance, plasma catecholamines and possible anaerobic end products in the unrestrained dogfish Scyliorhinus canicula. J Comp Physiol B 132:297-303. Carlson JK, Parsons GR. 2001. The effects of hypoxia on three sympatric shark species: Physiological and behavioural responses. Environ Biol Fish 61:427-433. Carlson JK, Parsons GR. 1999. Seasonal differences in routine oxygen consumption rates of the bonnethead shark. J Fish Biol 55:876-879. Carlson JK, Palmer CL, Parsons GR. 1999. Oxygen consumption rate and swimming efficiency of the blacknose shark Carcharhinus acronotus. Copeia 1:34-39. Cliff G, Thurman GD. 1984. Pathological and physiological effects of stress during capture and transport in juvenile dusky shark, Carcharhinus obscurus. Comp Biochem Physiol A 78:167-173. Cooper AR, Morris S. 2004. Haemoglobin function and respiratory status of the Port Jackson shark, Heterodontus portjacksoni, in response to lowered salinity. J Comp Physiol B 174:23-236. Fandrey J. 2004. A cordial affair – erythropoietin and cardioprotection. Cardio Res 72(1):1-2. Hochachka PW, Buck LT, Doll CJ, Land SC. 1996. Unifying theory of hypoxia tolerance: molecular/metabolic defense and rescue mechanisms for surviving oxygen lack. Proc Natl Acad Sci USA 93:9493–9498. Hoffmayer ER, Parsons GR. 2001. The physiological response to capture and handling stress in Atlantic sharpnose shark, Rhizoprionodon terraenovae. Fish Physiol Biochem 25:277-285. Hughes GM, Johnston IA. 1978. Some responses of the electric ray (Torpedo marmorata) to low ambient oxygen tensions. J Exp Biol 73:107-117. Jonas P, Koh DS, Kampe K. 1991. ATP-sensitive and Ca-activated K channels in vertebrate axons: Novel links between metabolism and excitability. Pfluger Arch 418:68-73. Jackson DC. 2000. Living without oxygen: Lessons from the freshwater turtle. Comp Biochem Physiol 125A:299-315.
Clint Chapman 201
Jackson DC, Heisler N. 1982. Plasma ion balance of submerged anoxic turtles at 3C: the role of calcium lactate formation. Respir Physiol 49(2):159-174. Jackson DC, Heisler N. 1983. Intracellular and extracellular acid-base and electrolyte status of submerged anoxic turtles at 3 degrees C. Respir Physiol 53(2):187-201. Jackson DC, Ultsch GR. 1982. Long-term submergence at 3ºC of the turtle, Chrysemys picta bellii, in normoxic and severely hypoxic water. II. Extracellular ionic responses to extreme lactic acidosis. J Exp Biol 96:29-43. Kita J, Itazawa Y. 1989. Release of erythrocytes from the spleen during exercise and splenic constriction by adrenaline infusion in the rainbow trout. Jap J Ichthyol 36:48-52. Knickerbocker DL, Lutz PL. 2001. Slow ATP loss and the defense of ion homeostasis in the anoxic frog brain. J Exp Biol 204:3547-3551. Krumschnabel G, Frischmann ME, Schwarzbaum PJ, Wieser W. 1998. Loss of K+ homeostasis in trout hepatocytes during chemical anoxia: a screening study for potential causes and mechanisms. Arch Biochem Biophys. 353(2):199-206. Last PR, Stevens JD. 1994. Sharks and Rays of Australia. CSIRO Division of Fisheries. Melbourne, Australia. Lutz PL, Nilsson GE. 1997. Contrasting strategies for anoxic brain survival - glycolysis up or down. J Exp Biol 200:411-419. Lutz PL, Reiners R. 1997. Survival of energy failure in the anoxic frog brain: delayed release of glutamate. J Exp Biol 200:2913-2917. Metcalfe JD, Butler PJ. 1984. Changes in activity and ventilation in response to hypoxia in unrestrained, unoperated dogfish (Scyliorhinus canicula L.). J Exp Biol 108:411-418. Mulvey JM, Renshaw GMC. 2000. Neuronal oxidative hypometabolism in the brainstem of the epaulette shark (Hemiscyllium ocellatum) in response to hypoxic pre-conditioning. Neuroscience Letters 290:1-4. Nikinmaa M, Tervonen V. 2004. Regulation of blood haemoglobin concentration in hypoxic fish: Proceedings of the 7th International Symposium on Fish Physiology, Toxicology, and Water Quality. U.S. Environmental Protection Agency, Ecosystems Research Division. 243-252. Nilsson GE, Renshaw GMC. 2004. Hypoxic survival strategies in two fishes: extreme anoxia tolerance in the North European crucian carp and natural hypoxic preconditioning in a coral-reef shark. J Exp Biol 207:3131-9.
Clint Chapman 202
Pearson MP, Stevens ED. 1991. Size and hematological impact of the splenic erythrocyte reservoir in rainbow trout, Oncorhynchus mykiss. Fish Physiol Biochem 9:39-50. Penny D. 1974. Effects of prolonged diving anoxia on the turtle, Pseudemys scripta elegans. Comp Biochem Physiol 47A:933-941. Piiper J, Meyer M, Drees F. 1972. Hydrogen ion balance in the elasmobranch Scyliorhinus stellaris after exhausting activity. Resp Physiol 16:290-303. Renshaw GMC, Kerrisk CB, Nilsson GE. 2002. The role of adenosine in the anoxic survival of the epaulette shark, Hemiscyllium ocellatum. Comp Biochem Physiol B 131:133- 141. Routley MH, Nilsson GE, Renshaw GMC. 2002. Exposure to hypoxia primes the respiratory and metabolic responses of the epaulette shark to progressive hypoxia. Comp Biochem Physiol A 131:313-321. Schmidt-Nielsen K. 1983. Animal Physiology: Adaptation and Environment. 5th ed. University Press, Cambridge. Short S, Taylor EW, Butler PJ. 1979. The effectiveness of oxygen transfer during normoxia and hypoxia in the dogfish (Scyliorhinus canicula) before and after cardiac vagotomy. J Comp Physiol 132:289-295. Soderstrom V, Renshaw GMC, Nilsson GE. 1999. Brain blood flow and blood pressure during hypoxia in the epaulette shark Hemiscyllium ocellatum, a hypoxia-tolerant elasmobranch. J Exp Biol 202:829-835. Soldatov AA. 1996. The effect of hypoxia on red blood cells of flounder: a morphologic and autoadiographic study. J Fish Biol 48:321-328. Soivio A, Westman K, Nyholm K. 1974a. Changes in haematocrit values in blood samples treated with and without oxygen; a comparative study with four salmonid species. J Fish Biol 6:763. Soivio A, Westman K, Nyholm K. 1974b. The influence of changes in oxygen tension on the haematocrit value of blood samples from asphyxic rainbow trout (Salmo gairdneri). Aqua 3:395-401. Swift DJ, Lloyd R. 1974. Changes in urine flow rate and haematocrit value of rainbow trout Salmo gairdneri (Richardson) exposed to hypoxia. J Fish Biol 6:379-387. Tervonen V, Vuolteenaho O, Nikinmaa M. 2006. Haemoconcentration via diuresis in short- term hypoxia: A possible role for cardiac natriuretic peptide in rainbow trout. Comp Biochem Physiol A 144:86-92.
Clint Chapman 203
Tufts BL, Randall DJ. 1989. The functional significance of adrenergic pH regulation in fish erythrocytes. Can J Zool 67:235-238. Walker RM, Johansen PH. 1977. Anaerobic metabolism in goldfish (Carcassius auratus). Can J Zool 55:1304-1311. Warren DE, Jackson DC. 2005. The role of mineralized tissue in the buffering of lactic acid during anoxia and exercise in the leopard frog Rana pipiens. J Exp Biol 208:1117- 1124. Wells RMG, Davie PS. 1985. Oxygen binding by the blood and haematological effects of capture stress in two big game-fish: Mako shark and striped marlin. Comp Biochem Physiol A 81:643-646. Wells RMG, Weber RE. 1983. Oxygenation properties and phosphorylated metabolic intermediates in blood and erythrocytes of the dogfish, Squalus acanthias. J Exp Biol 103:95-108. Wise G, Mulvey JM, Renshaw GMC. 1998. Hypoxia tolerance in the epaulette shark (Hemiscyllium ocellatum). J Exp Zool 281:1-5.
Wood CM, Perry SF, Walsh PJ, Thomas S. 1994. HCO3- dehydration by the blood of an elasmobranch in the absence of a Haldane effect. Resp Physiol 98:319-37. Yamamoto K. 1987. Contraction of spleen in exercised cyprinid. Comp Biochem Physiol A 87:1087-1097. Yamamoto K, Itazawa Y, Kobayashi H. 1980. Supply of erythrocytes into the circulating blood from the spleen of exercised fish. Comp Biochem Physiol A 65:5-11. Yamamoto K, Itazawa Y, Kobayashi H. 1983. Erythrocyte supply from the spleen and hemoconcentration in hypoxic yellowtail. Mar Biol 73:221-226.
Clint Chapman 204