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Clonal Expansion and Phenotypic Expression of Pseudogymnoascus CLONAL EXPANSION AND PHENOTYPIC EXPRESSION OF PSEUDOGYMNOASCUS DESTRUCTANS Jordan Khankhet – M. Sc. Thesis McMaster University Biology CLONAL EXPANSION OF THE PSEUDOGYMNOASCUS DESTRUCTANS GENOTYPE IN NORTH AMERICA IS ACCOMPANIED BY SIGNIFICANT VARIATION IN PHENOTYPIC EXPRESSION By: Jordan Khankhet, B. Sc. A Thesis Submitted to the School of Graduate Studies in Partial Fulfillment of the Requirements for the Degree of Master of Science McMaster University © Copyright by Jordan Khankhet, June 2014 Jordan Khankhet – M. Sc. Thesis McMaster University Biology MASTER OF SCIENCE (2014) McMaster University (Biology) Hamilton, Ontario TITLE: Clonal expansion of the Pseudogymnoascus destructans genotype in North America is accompanied by significant variation in phenotypic expression AUTHOR: Jordan Khankhet, B.Sc. (McMaster University) SUPERVISOR: Professor Dr. Jianping Xu NUMBER OF PAGES: ix, 88 ii Jordan Khankhet – M. Sc. Thesis McMaster University Biology ABSTRACT White-nose syndrome is an emerging infectious disease that first appeared in 2006. Since its emergence, it has caused over 5.5 million deaths of bats in North America. Pseudogymnoascus destructans, a psychrophilic fungus, is the causative agent of white- nose syndrome. White-nose syndrome is characterized by the growth of P. destructans on the nose, wings, and/or ears of the bat causing epidermal erosions and ulcers. Previous studies determined that the population of P. destructans in North America is clonal. However, no Canadian isolates of P. destructans were used. In this study, multilocus sequence typing and DNA fingerprinting was used to genotype 112 North American isolates. My results showed that the population in Canada had the same genotype as those from the US and there was also evidence of minor genetic variation in three Canadian isolates. Using 16 P. destructans isolates representative of various geographical locations in North America, significant phenotypic variations based on mycelial growth, production of exudates, and production of pigments and diffusion into agar media were observed. Overall, my results indicate a clonal genotype expansion of P. destructans throughout North America that is accompanied by significant phenotypic variation. iii Jordan Khankhet – M. Sc. Thesis McMaster University Biology ACKNOWLEDGEMENTS First and foremost I would like to thank my supervisor Dr. Jianping Xu for everything he has done for me. I am truly appreciative that I was hired as a research assistant after my second year of undergrad and that he allowed me to be a part of his lab ever since. He was always encouraging and supportive of anything I wanted to do, whether it was in terms of research or my career plans. His door was always open and I felt that our many discussions over the years not only allowed me to become a better scientist but also a better person. He has taught me a great deal and gave me the opportunity to achieve my dreams. For that, I will always be grateful. Next I would like to thank Aaron Vogan for all of his assistance since I started in the lab. He has taught me nearly all of the experimental techniques I have used and has provided valuable guidance along the way. The discussions we often had allowed me to develop my critical thinking and begin developing my own ideas which were instrumental in completing this thesis. He was always available to discuss my classes, research or just about anything else, so for everything he has done for me I am thankful. Finally, I would like to thank my lab colleagues Zixuan Wang, Howard Chang, Nicholas Pun, and Heather Yoell for their support and assistance. They have made my time at McMaster a wonderful experience and something that I will always cherish. iv Jordan Khankhet – M. Sc. Thesis McMaster University Biology THESIS FORMAT This thesis is composed of three chapters. The first chapter provides background information and a brief overview of this study. The second chapter presents my manuscript that has been submitted to a peer-reviewed scientific journal. The third chapter provides an overall conclusion and suggests further research to expand our understanding of this topic. Chapter 1: GENERAL INTRODUCTION Chapter 2: Clonal expansion of the Pseudogymnoascus destructans genotype in North America is accompanied by significant variation in phenotypic expression Authors: Jordan Khankhet, Karen J. Vanderwolf, Donald F. McAlpine, Scott McBurney, David P. Overy, Durda Slavic, Jianping Xu Comments: This study was conducted by JK under the supervision of JX. Isolates used in this study were provided by KJV, DFM, SM, DPO and DS. The manuscript was written by JK and JX. KJV, DFM, SM and DPO provided feedback and editing. Chapter 3: GENERAL CONCLUSIONS AND PERSPECTIVES v Jordan Khankhet – M. Sc. Thesis McMaster University Biology TABLE OF CONTENTS ABSTRACT ...................................................................................................................... iii ACKNOWLEDGEMENTS ............................................................................................. iv THESIS FORMAT ............................................................................................................ v TABLE OF CONTENTS ................................................................................................. vi LIST OF TABLES ......................................................................................................... viii LIST OF FIGURES .......................................................................................................... ix CHAPTER 1: GENERAL INTRODUCTION ................................................................ 1 1.1 Emerging Infectious Disease ................................................................................. 1 1.1.1 The Novel Pathogen Hypothesis ......................................................................... 1 1.1.2 The Endemic Pathogen Hypothesis ..................................................................... 2 1.2 White-Nose Syndrome .......................................................................................... 3 1.2.1 Characteristics of WNS ....................................................................................... 3 1.2.2 Cause of Mortality ............................................................................................... 4 1.2.3 Mortality Rates .................................................................................................... 5 1.2.4 Behavioral Changes ............................................................................................. 6 1.3 Pseudogymnoascus destructans ................................................................................. 6 1.3.1 Phylogenetic Analyses ......................................................................................... 7 1.3.2 Morphology ......................................................................................................... 8 1.3.3 Enzymatic Activity ............................................................................................ 10 1.3.4 Genetics of P. destructans ................................................................................. 12 1.3.5 Potential Treatments .......................................................................................... 13 1.4 Project Objectives .................................................................................................. 15 References ................................................................................................................... 156 CHAPTER 2: CLONAL EXPANSION OF THE PSEUDOGYMNOASCUS DESTRUCTANS GENOTYPE IN NORTH AMERICA IS ACCOMPANIED BY SIGNIFICANT VARIATION IN PHENOTYPIC EXPRESSION ............................. 20 Abstract .......................................................................................................................... 22 Introduction .................................................................................................................... 23 vi Jordan Khankhet – M. Sc. Thesis McMaster University Biology Materials and Methods ................................................................................................... 25 Fungal Isolates ............................................................................................................ 25 DNA Fingerprinting.................................................................................................... 26 Multilocus Sequence Typing ...................................................................................... 27 Morphological Comparisons ...................................................................................... 28 Statistical Analysis...................................................................................................... 29 Results ............................................................................................................................ 29 Genotypes ................................................................................................................... 29 Mycelial growth .......................................................................................................... 30 Exudate production ..................................................................................................... 31 Pigment secretion........................................................................................................ 32 Colony reverse color ..................................................................................................
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