Wilfrid Laurier University Scholars Commons @ Laurier Theses and Dissertations (Comprehensive) 2018 Gill Structure & Function in Parasitic and Non-parasitic Lampreys: The Effects of Metamorphosis and Freshwater-Seawater Transfer Julia Sunga [email protected] Follow this and additional works at: https://scholars.wlu.ca/etd Part of the Comparative and Evolutionary Physiology Commons, Integrative Biology Commons, and the Zoology Commons Recommended Citation Sunga, Julia, "Gill Structure & Function in Parasitic and Non-parasitic Lampreys: The Effects of Metamorphosis and Freshwater-Seawater Transfer" (2018). Theses and Dissertations (Comprehensive). 2083. https://scholars.wlu.ca/etd/2083 This Thesis is brought to you for free and open access by Scholars Commons @ Laurier. It has been accepted for inclusion in Theses and Dissertations (Comprehensive) by an authorized administrator of Scholars Commons @ Laurier. For more information, please contact [email protected]. Gill Structure & Function in Parasitic and Non-parasitic Lampreys: The Effects of Metamorphosis and Freshwater-Seawater Transfer by Julia Sunga Bachelor of Science Honours Zoology, University of Guelph, 2016 A thesis presented to the Department of Biology, Faculty of Science Wilfrid Laurier University, Waterloo, Ontario, N2L 3C5, CANADA in partial fulfillment of the requirements for the degree of Master of Science in Integrative Biology 2018 ©Julia Sunga 2018 AUTHOR'S DECLARATION I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including any required final revisions, as accepted by my examiners. I understand that my thesis may be made electronically available to the public. ii Abstract Lamprey (Petromyzontiformes) are a phylogenetically ancient group of jawless fishes that begin their lives as filter-feeding larvae (ammocoetes) before undergoing a complex metamorphosis into juvenile lamprey that involves major changes to their internal and external body plan. Some parasitic species, such as the sea lamprey (Petromyzon marinus), migrate to sea following metamorphosis, where they use their oral discs and rasping tongue to attach to and ingest vast quantities of blood from fishes. Thus, sea lamprey have to counter the simultaneous challenges of hyposmoregulation in sea water and the generation of large quantities of ammonia due to the catabolism of protein-rich blood. A goal of this study was to characterize how changes in the structure and function of the gills facilitated osmoregulation and nitrogenous waste (N- waste) excretion by sea lamprey following metamorphosis, particularly after acclimation to sea water and the ingestion of blood from teleost fishes. Accordingly, key features of the lamprey gill including the distribution and abundance of Na+/K+-ATPase (NKA) and H+-ATPase (V- ATPase) pumps involved in ion regulation, and ammonia transporting Rhesus glycoproteins and urea transporting proteins, were investigated using through immunohistochemical staining and Western blotting techniques. In contrast to the sea lamprey, there are other species of lamprey that remain in fresh water following metamorphosis. Many of these species are non-parasitic including the northern brook lamprey (Ichthyomyzon fossor), but some such as the closely related silver lamprey (Ichthymyzon unicuspis) are parasitic. To learn more about how an exclusively FW existence affected ion transport and ammonia excretion by lampreys, the gills of post-metamorphic (juvenile) northern and silver lamprey were compared to those of larval and juvenile sea iii lamprey. As in sea lamprey, the gills of both species were characterized by the presence of Rhesus c-like glycoprotein (Rhcg-like) and urea transport (UT) protein but, the distribution of these proteins more closely resembled those of larval sea lamprey than juvenile sea lamprey. In both the silver and northern brook lamprey, Rhcg-like protein co-localized with V-ATPase, suggesting that H+ excretion was coupled with Rhcg-like protein mediated diffusion trapping of NH3. Similarly, UT abundance in both species was comparable to that of the larval sea lamprey. I conclude that in freshwater lampreys, NH3 extrusion via apical Rhcg-like proteins is coupled to V-ATPase mediated H+ excretion, which maintains favourable diffusion gradients by trapping + NH3 as NH4 . Given that the lampreys and teleosts have evolved along separate lineages for at least 360 million years, I propose that this method of ammonia excretion is an ancient strategy used by aquatic organisms to facilitate ammonia excretion across the gills in fresh water. In + contrast, the need for V-ATPase trapping of NH3 as NH4 is not required in sea water, in which the Rhcg-like proteins were restricted to the basolateral membrane and co-localized with NKA in sea water mitochondrion-rich cells (SW MRCs). These findings suggest that Rhcg-like protein may mediate ammonia excretion by loading the SW MRC with ammonia, with the resulting + + + + NH4 pumped out of the cell via substitution for H on an apical Na /H exchanger, or via an + outwardly directed NH4 electrochemical gradient that favours excretion via paracellular junctions. iv Acknowledgements I would like to thank my supervisor Dr. Michael P. Wilkie for his patience, support and guidance throughout my thesis. I would also like to thank Dr. Jonathan Wilson for the use of his lab space, technical guidance and support during this research process. Your input has been invaluable. Additionally, I would like to thank the entire Wilkie lab past, present, and transient (Dr. Oana Birceanu, Laura Tessier, Adrian Ionescu, Phillip Pham-Ho, Chris White, Scott Hepditch, Darren Foubister, Heather Bauman, Vedya Ramsaran, and Josh Sutherby), for the many hours spent by these individuals assisting in animal collection, care, dissections and more. Many thanks must also be extended to the labs of Dr. Steve Perry and Dr. S. Hirose for their donation of the necessary antibodies which were so crucial to this project. Additional thanks to Dr. Margaret Docker and her lab for the invaluable assistance and input into this project. Finally, I must thank my friends, family, teammates, and my partner Geoff, who have helped me maintain perspective throughout this project. Without your support, patience, and even your distractions, I truly would not have made it through this process. v Table of Contents AUTHOR'S DECLARATION ....................................................................................................... ii Abstract .......................................................................................................................................... iii Acknowledgements ......................................................................................................................... v Table of Contents ........................................................................................................................... vi List of Figures ................................................................................................................................ ix List of Abbreviations ................................................................................................................... xiii Chapter 1 General Introduction ...................................................................................................... 1 1.1 Lamprey Life Cycle and Metamorphosis .............................................................................. 2 1.2 Ammonia Toxicity and the Effects of Feeding on Nitrogenous Waste Excretion ................ 5 1.3 Freshwater Nitrogenous Waste Excretion Mechanisms in Other Fish ................................. 7 1.4 Effects of Salinity on Gill Function .................................................................................... 10 1.5 Evolution of Petromyzontiformes and ‘Paired’ Lamprey Species ...................................... 12 1.6 Hypotheses and Objectives ................................................................................................. 14 Figures ....................................................................................................................................... 17 Chapter 2 ....................................................................................................................................... 21 Re-organization of the pathways of branchial ammonia and urea excretion during metamorphosis and the freshwater-seawater transition of the anadromous sea lamprey (Petromyzon marinus) .. 21 Introduction ............................................................................................................................... 22 Materials and Methods .............................................................................................................. 24 Experimental Animals and Holding ...................................................................................... 24 Experimental Protocols ......................................................................................................... 26 Analytical Methods ................................................................................................................ 29 Results ....................................................................................................................................... 35 Differences in Mass Loss Between Metamorphosing and Non-metamorphosing Sea Lamprey ..............................................................................................................................................