FUNCTIONAL ASPECTS OF THE OSMORESPIRATORY COMPROMISE IN FISHES by Marina Mussoi Giacomin B.Sc. Hon., Federal University of Paraná, 2011 M.Sc., Federal University of Rio Grande, 2013 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Zoology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) February 2019 © Marina Mussoi Giacomin, 2019 The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Functional aspects of the osmorespiratory compromise in fishes submitted by Marina Mussoi Giacomin in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Zoology Examining Committee: Dr. Christopher M. Wood, UBC Zoology Co-supervisor Dr. Patricia M. Schulte, UBC Zoology Co-supervisor Dr. Colin J. Brauner, UBC Zoology Supervisory Committee Member Dr. David J. Randall, UBC Zoology University Examiner Dr. John S. Richardson, UBC Forestry University Examiner Dr. Yoshio Takei, University of Tokyo External Examiner Additional Supervisory Committee Members: Dr. Jeffrey G. Richards, UBC Zoology Supervisory Committee Member ii Abstract The fish gill is a multipurpose organ that plays a central role in gas exchange, ion regulation, acid-base balance and nitrogenous waste excretion. Effective gas transfer requires a large surface area and thin water-to-blood diffusion distance, but such structures also promote diffusive ion and water movements between blood and water that challenge the maintenance of hydromineral balance. Therefore, a functional conflict exists between gas exchange and ionic and osmotic regulation at the gill. The overarching goal of my thesis was to examine the trade-offs associated with the optimization of these different functions (i.e. the osmorespiratory compromise) in species with diverse osmoregulatory strategies, when exposed to environmental stressors such as hypoxia, changes in temperature and salinity. To address this I have used three species of fish that are phylogenetically, ecologically and physiologically diverse, the Atlantic killifish (teleost), the Pacific hagfish (myxine) and the Pacific spiny dogfish (elasmobranch). My results show that salinity influences the capacity to regulate oxygen consumption at low oxygen and hypoxia tolerance in the killifish. Acclimation to fresh water resulted in a lowering of the lamellar respiratory surface area and a higher percentage of the gill lamellae covered by an interlamellar cell mass. These responses could be adaptations to aid survival in hypo-osmotic waters as freshwater-acclimated fish showed a greater ability to downregulate transcellular gill permeability to both ions and water when exposed to hypoxia in comparison to their seawater-acclimated counterparts. However, at salinities ranging from fresh water to 100% sea water, plasma ion concentration and osmolality were unaffected by hypoxia. I also found that there is a strong interaction between gill permeability to gases and to ions and water in hagfish, an osmoconforming marine species in which the osmorespiratory compromise had never been investigated. An increase in gill permeability to urea, ammonia, and water was also seen in the dogfish exposed to elevated temperature, indicating a disruption in the nitrogen conservation mechanisms at the gill. In summary, this thesis has expanded the range of species in which the osmorespiratory compromise has been investigated, and has provided new insights into the mechanisms involved. iii Lay Summary The fish gill is an organ that plays a fundamental role in gas exchange and ion regulation. My thesis investigated how three different fish species deal with the trade-offs associated with optimizing these different functions when exposed to variation in environmental stressors such as salinity, oxygen content and temperature. My results show that there is a strong interaction between permeability of the gill to oxygen and to ions and water in the three species investigated. Adjusting to different salinities differentially influenced how fish handle the lack of oxygen in the water. Having to increase oxygen extraction led to different changes in gill permeability, which were salinity dependent. Understanding how environmental constraints affect fish physiology is of crucial importance as the world’s aquatic environments become increasingly more variable due to human impacts and climate change. iv Preface Chapter 1 is a general introduction providing relevant background information to the research chapters 2 - 6. Chapters 2 – 6 have been written as individual manuscripts. Chapter 7 provides a general discussion, summarizing the major findings of the 5 research chapters. Chapter 2 has been written as a manuscript that is ready for submission as Giacomin, M., Bryant, H., Schulte, P.M. and Wood, C.M. The osmorespiratory compromise: physiological responses and tolerance to hypoxia exposure are affected by salinity acclimation in the euryhaline killifish (Fundulus heteroclitus). H. Bryant analyzed the histological images. I performed all experiments and analyzed all the remaining data. I wrote the manuscript under the supervision of Drs. P.M. Schulte and C.M. Wood. Chapter 3 has been written as a manuscript that is intended for submission as Giacomin, M., Onukwufor, J., Schulte, P.M. and Wood, C.M. The osmorespiratory compromise: the ionoregulatory responses to hypoxia in the euryhaline killifish (Fundulus heteroclitus) acclimated to different salinities. J. Onukwufor performed the diffusive water flux experiments. I performed all remaining experiments and analyzed all the remaining data. I wrote the manuscript under the supervision of Drs. P.M. Schulte and C.M. Wood. A version of Chapter 4 has been published as Giacomin, M., Eom, J., Schulte, P.M. and Wood, C.M. (2018). Acute temperature effects on metabolic rate, ventilation, diffusive water exchange, osmoregulation, and acid-base status in the Pacific hagfish (Eptatretus stoutii). Journal of Comparative Physiology (B). J. Eom collected and analyzed all the ventilatory and heart rate data. I collected and analyzed all the remaining data, with help from C.M. Wood. I wrote the manuscript under the supervision of Drs. P.M. Schulte and C.M. Wood. All co-authors provided editorial feedback to the manuscript. A version of Chapter 5 has been submitted for publication as Giacomin, M., Dal Pont, G., Eom, J., Schulte, P.M. and Wood, C.M. The effects of salinity and hypoxia exposure on oxygen v consumption, ventilation, diffusive water exchange and ionoregulation in the Pacific hagfish (Eptatretus stoutii). Eom collected and analyzed all the ventilatory data. I collected and analyzed all the remaining data with the assistance of G. Dal Pont and C.M Wood. I wrote the manuscript under the supervision of Drs. P.M. Schulte and C.M. Wood. All co-authors provided editorial feedback to the manuscript. A version of Chapter 6 has been published as Giacomin, M., Schulte, P.M. and Wood, C.M. (2017). Differential effects of temperature on oxygen consumption and branchial fluxes of urea, ammonia, and water in the dogfish shark (Squalus acanthias suckleyi). Physiological and Biochemical Zoology 90(6): 627 – 637. C.M. Wood and I designed the experiments. I collected and analyzed all the data, and wrote the manuscript under the supervision of Drs. P.M. Schulte and C.M. Wood. All co-authors provided editorial feedback to the manuscript. All experiments performed for this thesis followed the guidelines of the Canada Council for Animal Care, under joint approval of the animal care committees at the University of British Columbia and Bamfield Marine Science Centre (AUP #: A14-0251 and RS–17-20, respectively). vi Table of Contents Abstract ......................................................................................................................................... iii Lay Summary ............................................................................................................................... iv Preface .............................................................................................................................................v Table of Contents ........................................................................................................................ vii List of Tables .............................................................................................................................. xiv List of Figures ............................................................................................................................ xvii Acknowledgements .................................................................................................................. xxix Chapter 1: Introduction ................................................................................................................1 1.1 Overview ............................................................................................................................ 1 1.2 The fish gill ........................................................................................................................ 1 1.3 The role of the gills in regulating osmotic and ionic balance ............................................ 2 1.4 The role of the gills in regulating oxygen uptake when demand is increased by increases in temperature ............................................................................................................................