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 Griffith Research Online https://research-repository.griffith.edu.au 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 Clint Chapman i 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. Clint Chapman ii 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. Clint Chapman iii 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. Clint Chapman iv 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 Clint Chapman v 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. Clint Chapman vi 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