Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2013 Developing methods to mitigate chytridiomycosis, an emerging disease of amphibians Geiger, Corina C Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-86534 Dissertation Published Version Originally published at: Geiger, Corina C. Developing methods to mitigate chytridiomycosis, an emerging disease of amphibians. 2013, University of Zurich, Faculty of Science. ❉❡✈❡❧♦♣✐♥❣ ▼❡❤♦❞ ♦ ▼✐✐❣❛❡ ❈❤②✐❞✐♦♠②❝♦✐✱ ❛♥ ❊♠❡❣✐♥❣ ❉✐❡❛❡ ♦❢ ❆♠♣❤✐❜✐❛♥ Dissertation zur Erlangung der naturwissenschaftlichen Doktorw¨urde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakult¨at der Universit¨atZ¨urich von Corina Claudia Geiger von Chur GR Promotionskomitee Prof. Dr. Lukas Keller (Vorsitz) Prof. Dr. Heinz-Ulrich Reyer Dr. Benedikt R. Schmidt (Leitung der Dissertation) Dr. Matthew C. Fisher (Gutachter) Z¨urich, 2013 ❉❡✈❡❧♦♣✐♥❣ ▼❡❤♦❞ ♦ ▼✐✐❣❛❡ ❈❤②✐❞✐♦♠②❝♦✐✱ ❛♥ ❊♠❡❣✐♥❣ ❉✐❡❛❡ ♦❢ ❆♠♣❤✐❜✐❛♥ Corina Geiger Dissertation Institute of Evolutionary Biology and Environmental Studies University of Zurich Supervisors Dr. Benedikt R. Schmidt Prof. Dr. Heinz-Ulrich Reyer Dr. Matthew C. Fisher Prof. Dr. Lukas Keller Z¨urich, 2013 To all the midwife toads that got sampled during this project ”The least I can do is speak out for those who cannot speak for themselves.” - Jane Goodall Acknowledgements I cordially thank Beni Schmidt for his support which was always so greatly appreciated, be it in fund raising, designing experiments or for his skilled statistical and editorial judgments. He managed to explain the meaning of any complex problem in simple terms and again and again he turned out to be a walking encyclopedia of amphibians, statistical models, Bd and many other common and uncommon topics. My sincere thanks go to Uli Reyer who offered me a warm welcome in his group and gave me the opportunity to use the excellent research facilities in his lab, for the generous financial support and for transmitting his optimism and infectious cheerfulness into the whole group. I’m indebted to Mat Fisher for providing scientific support and throwing a glance on manuscripts before I dared to submit them. I like to express further greatest thanks to Lukas Keller for accepting me as a Ph.D. student and for allowing me access to his laboratory. Further big thanks go to all members of the Ecology Group for contributing scientific discussions, inputs and ideas. My warmest thanks go to Ursina Tobler who freely shared with me the fruits of her experience with mitwife toads, for providing data, knowledge and time to introduce me into the mysteries of real-time PCR and tadpole handling. Deepest thanks go to Jasmin Winkler for always having solutions ready for so many problems I was confronted with and for sharing her ex- periences with common frogs with me. Ursina and Jasmin were always full of passion for the mysteries and unanswered questions related to frogs, ele- phants and especially cryptozoological issues. It was great to have both of you around. I cordially thank Francesco Origgi for providing insights into the se- crets of histological analysis and for his appreciated contribution to the manuscripts. For the fun discussions in the lab I would like to cordially thank Sandy R¨othlisberger. Thanks to her lab work was a pleasure and endless hours of doing PCRs were significantly shorter. Anni M¨ader and Susanne Bischof were the two angels who handled the administrative mat- ters behind the scenes. Special thanks go to Adrian Borgula for providing data on midwife toad populations. The laborious fieldwork would not have been possible without the enor- mous effort of Balthasar Christoffel, Leyla Davis, Marina Eichenberger, i ii ACKNOWLEDGEMENTS Christian Geiger, Verena Geiger, Regina Sch¨ar, Elisabeth Spillmann, Jonas Spillmann, Ursina Tobler and Jasmin Winkler. They were extremely faithful and indispensable helpers. Fieldwork was so much fun with you! Moreover I would like to thank Marina Eichenberger and Regina Sch¨ar for providing their beautyful garden ponds for this research, for always over- whelm me with their seasonal harvest, for their open ears and critical dis- cussions and for having a place to warm up on their cosy soapstone oven after the winter catchments. I also thank Beate W¨uest from the Schloss Schauensee for giving me access to the ponds of the castle and for always being open for a talk about midwife toads. The following foundations and institutions supported my Ph.D. project financially: the Federal Office for the Environment (FOEN), the Swiss Na- ture Conservation Offices of the cantons AG, BE, BS, FR, GE, GR, LU, NE, SZ and ZH, the Institute of Evolutionary Biology and Environmental Stud- ies, the Forschungskredit of the University of Zurich, the MAVA fondation pour la nature, the Promotor Stiftung, the Basler Stiftung f¨ur biologische Forschung, European Union of Aquarium Curators (EUAC) and the World Association of Zoos and Aquariums (WAZA). Collection permits were pro- vided by the cantonal offices for conservation of the cantons BL, LU and ZH. The study was conducted under permit number 75/2009 by the veterinary office of the canton Zurich. I am particularly indebted to my parents and my sister Anina for their never ending encouragement, their ongoing support and for their under- standing when I was too occupied with fieldwork or writing. Last but by no means least I would like to thank Jonas Spillmann for dragging me out to countless climbing trips and for reminding me that there’s a life away from emerging infectious diseases of amphibians. Contents Acknowledgements i Zusammenfassung v Summary xiii General Introduction xix 1 Elevated temperature in laboratory 1 1.1 Introduction............................ 1 1.2 Material and Methods . 3 1.3 Results............................... 4 1.4 Discussion............................. 4 2 Laboratory tests of antifungal agents 7 2.1 Introduction............................ 8 2.2 Material and Methods . 9 2.3 Results............................... 12 2.4 Discussion............................. 14 3 Antifungal agents in mesocosms 19 3.1 Introduction............................ 20 3.2 Material and Methods . 21 3.3 Results............................... 34 3.4 Discussion............................. 43 4 Experiments in natural ponds 55 4.1 Introduction............................ 56 4.2 Material and Methods . 57 4.3 Results............................... 67 4.4 Discussion............................. 70 iii iv CONTENTS 5 Learning from failed trials 79 5.1 Introduction............................ 80 5.2 Material and Methods . 81 5.3 Results............................... 87 5.4 Discussion............................. 92 6 Bd infection on metamorphosing individuals 101 6.1 Introduction . 101 6.2 Material and Methods . 102 6.3 Results...............................103 6.4 Discussion . 103 General Discussion 107 Curriculum vitae 129 Zusammenfassung Amphibien sind st¨arker gef¨ahrdet als irgendeine andere Wirbeltierklasse (Stuart et al., 2004; Hoffmann et al., 2010). Gr¨unde f¨ur diese Gef¨ahrdung sind vor allem Ubernutzung¨ und Habitatsverlust (Stuart et al., 2004). Erst k¨urzlich tauchte eine weitere Bedrohung auf, die zum R¨uckgang von Popu- lationen beitr¨agtund diese sogar zum Aussterben bringt: die sich ausbre- itende infekti¨ose Krankheit Chytridiomykose (Berger et al., 1998; Daszak et al., 2000; Stuart et al., 2004; Skerratt et al., 2007). Der Infektionserreger der Chytridiomykose ist der Chytridpilz Batracho- chytrium dendrobatidis (Bd), welcher 1998 entdeckt und 1999 beschrieben wurde (Longcore et al., 1999). Der Pilz geh¨ort zur Abteilung der Chytrid- iomycota, eine Gruppe von nicht Hyphen bildenden Pilzen (Longcore et al., 1999). Bd kann sich sowohl sexuell als auch asexuell fortpflanzen (Schloegel et al., 2012). W¨ahrend des asexuellen Lebenszyklus bilden sich frei be- wegliche infekti¨ose Zoosporen, welche die Amphibienhaut besiedeln und Thalli bilden, welche sich zu Zoosporangien entwickeln. In diesen Zoospo- rangien werden neue Zoosporen produziert, welche durch einen Schlauch aus der Haut ins Wasser entlassen werden. Hier k¨onnen sie einen neuen Wirt in- fizieren oder ihren bisherigen Wirt wieder infizieren. (Longcore et al., 1999; Berger et al., 2005). Bd infiziert keratinisierte Teile der Amphibienhaut, was zu einer Verdick- ung jener Hautschichten f¨uhrt, die f¨ur viele Stoffe undurchl¨assig sind. Dies beeintr¨achtigt die osmoregulatorischen Hautfunktion des infizierten Indi- viduums, was zu Dehydration, einem Ungleichgewicht im Elektrolythaushalt und letztendlich zum Tod f¨uhren kann (Voyles et al., 2009). Heute ist man sich einig, dass Chytridiomykose Verursacherin ist von raschen Populationsr¨uckg¨angen und lokalem Aussterben. Im “Amphibian Conservation Action Plan“ der Weltnaturschutzorganisation IUCN wir Chy- tridiomykose charakterisiert als “die schlimmste Infektionskrankheit, die je bei Wirbeltieren festgestellt wurde hinsichtlich der Anzahl betroffener Arten und der F¨ahigkeit, diese Arten zum Aussterben zu bringen“ (Gascon et al., 2007). Es ist daher wichtig, dass die Gemeinschaft der Wissenschaftler und Naturschutzbeh¨orden die Bedrohung durch die Chytridiomykose erkennt und Massnehmen dagegen entwickeln. Allerdings fehlen uns Methoden zur Bek¨ampfung dieser noch wenig bekannten Krankheit, was bedeutet, dass v vi ZUSAMMENFASSUNG hunderte von Amphibienarten demn¨achst aussterben werden (Stuart et al., 2004). Der Schl¨ussel