TENROlD HORMONES AND GOITROGEN-INDUCED METAMORPHOSIS IN THE SEA LAMPREY (Pett-omyzonmarinus). Richard Giuseppe Manzon A thesis submitted in confodty with the requirements for the degree of Doctor of Philosophy Graduate Department of Zoology University of Toronto 8 Copyright by Richard Giuseppe Manzon, 2ûûû. National Library Bibliotfieque nationale: du Canada Acquisitions and AcquisitTons et BiMiogmphE Services services bibliographiques The author has granted a non- L'auteur a accorde une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, 10- distribue or sen reproduire, prêter, distn%uerou copies of this thesis in microforni, vendre des copies de cette thèse sous paper or electronic formats. la fonne de micxofiche/nlm, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être ïmpthés reproduced without the author's ou autrement reproduits sans son permission. autorisation. ABSTRACT THYROlD HORMONES AND GOLTROGEN-INDUCED METAMORPHOSIS IN THE SEA LAMPREY (Petromyzon mariBus). Richard Giuseppe Manzon Doctor of Philosophy, 2000 Department of Zoology, University of Toronto The larval sea lamprey undergoes a tme metamorphosis fiom a sedentary, filter- feeding larva to a fiee-swimming, parasitic juvenile that feeds on the blooà and body fluids of teleost fishes, As is the case with arnphibians and teleosts, thyroid hormones (TH) are believed to be involved in lamprey metamorphosis. However, the onset of lamprey metamorphosis is characterized by a sharp decline in semm TH titers rather than the typical increase observed in ail other vertebrates known to undergo this type of postembryonic developmental event. Furthermore, the goitrogen potassium perchlorate (KC104), which lowers serum TH titers and prevents metamorphosis in most vertebrates, can induce precocious metamorphosis in Iarval lampreys. The primary goals of this thesis were to elucidate the role of depressed serum TH titers in the goitrogen-induced metamorphosis of lampreys and to provide some insight into the mechanisms involved in this induction. 1have shown that KCI04-induced Iamprey metamorphosis cmbe bIocked by using exogenous TH to prevent the decline in serum TH titers associateci with induced metamorphosis. The ability to induce metamorphosis was not unique to KC104. The goitrogens sodium perchlorateTpotassium thiocyanate and methimmole aiso induced metamorphosis in Iarval sea lampreys, and the incidence of metamorphosis was positively correlated to the magnitude of the decline in serum TH concentrations, These data are consistent with the proposed ideas that either a decline in serum TH concentrations is essentiai for the onset of metamorphosis in Iampreys, or elevated serum TH concentrations are inhibitory to metamorphosis (Le., high TH titers have an anti-metamorphic effect in lampreys). In addition, 1have shown that KC104 adrninistered to lamal Iamprey endostyles in vim reduces iodide uptake and organification, as weli as totai endostylar thyroglobuiin. The fact that the goitrogen KC104 acts directly on the endostyle to inhibit thyroidai activity Mersupports the notion that goitrogens induce metamorphosis via their effects on TH synthesis. In summary, 1have shown that goitrogens act directly on the larval lamprey endostyle to inhibit thyroidal activity, thus resulting in a decline in serum TH titers that is Luiked to and essential for the onset of metamorphosis in larnpreys. As 1approached the completion of my doctoral thesis, 1came to the conclusion that this was the most ciifficuit and rewarding endeavor that I have ever tackled. However, my definition of "ciifficuit'' changed when 1set out to acknowledge the many people that helped me along the way, without these individuals 1would not have succeeded at completing thïs thesis. 1think 1may have procrastinated in writing these acknowledgments as much as 1did with any section of this thesis. In fact, atone point 1thought 1 wouid provide myself with a restriction in the length of these acknowledgments. My acknowledgments with this approach would have been: 1thank aLl those peopIe kaown and unknown to me which have crossed my path during my doctoral studies and did not intentionally act to impede my progress. However, 1 realized that I was just trying to avoid the inevitable, which is kind of what 1am doing now, and that certain people in my Life really do deserve to be thanked and acknowledged for helping; so here goes (as you will see 1did not impose a length restriction), 1will begin by thanking the many members, past and present, of Dr. J.H. Youson's laboratory who slogged it out in the trenches with me. These postdoctoral fellows, technicians, graduate students and undergraduates provided many hours of stimulating, intellectual discussion as well as nonsensical banter, that helped me get through the day. Most days in the lab were truly enjoyable, and this was due to the people 1worked with. in particular 1 would to thank IIya Adam, Mehmet Danis, JulieeHeinig, Bojan Lipicnïk, Rod Roopsingh and Paul Robson, who left their work behind to help me collect the thousands of larval larnpreys used dunng the course of this thesis, and more importantly, were the reason 1 managed to collect the serum fiom all these lampreys before my retirement* 1wodd also like to thank four @ed undergraduate students: QUOCHuynh, Nancy Manjovski, Bnan Peck and Preshi Shanmugathasan; a11 were a pleasure to teach and train, 1learned something from each of you and in exchange for training, you put up with me and helped me get through the many radioimmunoassays that always seemed to fail on my Iab bench- Thanks also to Sevana Yaghoubian for introducing me to the art of poiyacrylamide gel electrophoresis and Western blotting- A special thanks to DE. John Holmes, Mike Wiïeand Luciano Marra, al1 of you taught me dot about being a graduate student and biologist. John, you introduced me to the lamprey and taught me a great deai about their biology, ecology and how to catch and care for them. You tnily were "The Source"- It was a rare occasion when you did not have an answer to my question and you always helped me find my way back to the cabin when we were out in the field. Lu, you tdywere a fkiend and mentor to me; you were aiways there to help whether it was with my lab work, grant applications, schoobng, teaching duties, or life in generai. Thanks to all the Sea Lamprey Control personnel in both Canada and the USA, and the US Fish and WildLSe Service, Essex Junction, Vermont for your help with larval lamprey collections. Whether you were answering my questions, providing me with survey data, sending me survey maps or electrofistiing dongside me, you were always an excellent resource. John Gersmehl, Jeff Slade and EueKoon frequently went above and beyond the cal1 of duty to ensure that my iamprey hunting was successfiil. Thanks to Dr. John Leatheriand for my introduction to the radioimmunoassay technique and Dr. Geoffrey Edes and Audrey Waytiuk for performing the deiodinase assays presented in Chapter 2 of this thesis. Thanks to Drs. Rudy Boonstra and Mark Sheridan for being my mentors; even though you were not necessary dÏrectLy involved in this thesis, you always provided me with sound advice- 1would LÏke to thank the members of my advisory committee, Drs. Rick Elinson and Mike Filosa for providing me with guidance throughout this thesis and keeping me on the right track. Speciai thanks to Dr. Mike Filosa, you were a teacher in the tmest sense of the word befitting of the titLe "SENSEI"that so few deserve- The contribution you made to my thesis in the final year was invaluable, You not only helped me with the technical and academic aspects of my doctoral thesis, but you taught me about culture, the arts, history, and life in general. Thanks to Dr. John Youson, my doctoral supervisor, for providing me with the atmosphere and guidance to excel in my graduate studies. John, you saw abiiities in me that 1did not know 1 had and you always knew when to push and challenge me to bring my work to the next level. You were teacher, supervisor, and mentor, but over the years you have also become my fiend. Last, but not least, 1would like to acknowtedge the four people who have provided me with the love, support, £iiendship and encouragement that fueled the completion of this thesis: my parents Giuseppe and Teresa Manzon, my wife Lori, and my second mother, Sally Wood. To my parents Giuseppe and Teresa, what can 1Say but thanks, you sacrificed so much to help me both financidy and emotionally. Thanks for putting up with my me through al1 the rough spots and for ignoring me when 1was out of fine. You both showed a tremendous interest in my schooLing and my research, and you were always anxious to know how things were going and what I was up to- You were there for me when 1needed you, supported me in all my decisions and provided me with this opportunity. SalIy, th& for continually pestering me with questions about my work, providing me with encouragement when 1needed it, and barking at me to get back to work when 1was taking tmmany breaks- Most of A,thanks forjust putting up with me, giving me space when 1needed it and being my friend- To my wife, best fiiend, and colleague, Lori, from the bottom of my heart, THANKS! You are the one person without whom 1reaily could not have finished thïs thesis and words could never describe my appreciation. You provided me with help in ali aspects ofthis work frorn the field, aquatics room and laboratory to the editing of endless manuscn'pts- More importantly, you gave me your unconditional Love, support and encouragement, You helped me to find the confidence in myself that 1needed to succeed; we really do make a good team- The research presented in this thesis was supported by operating grants fiom the Naturd Sciences and Engineering Research Council of Canada @TSERC) to J.H.