Graduate Theses, Dissertations, and Problem Reports 2016 Development and Application of Quantitative Gene Expression Assays in Wild-Caught, Non-Model Fish Species Cassidy Megan Hahn Follow this and additional works at: https://researchrepository.wvu.edu/etd Recommended Citation Hahn, Cassidy Megan, "Development and Application of Quantitative Gene Expression Assays in Wild- Caught, Non-Model Fish Species" (2016). Graduate Theses, Dissertations, and Problem Reports. 5733. https://researchrepository.wvu.edu/etd/5733 This Dissertation is protected by copyright and/or related rights. It has been brought to you by the The Research Repository @ WVU with permission from the rights-holder(s). You are free to use this Dissertation in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you must obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/ or on the work itself. This Dissertation has been accepted for inclusion in WVU Graduate Theses, Dissertations, and Problem Reports collection by an authorized administrator of The Research Repository @ WVU. For more information, please contact [email protected]. Development and Application of Quantitative Gene Expression Assays in Wild-Caught, Non-Model Fish Species Cassidy Megan Hahn Dissertation submitted to the Davis College of Agriculture, Natural Resource and Design at West Virginia University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Forest Resources Science Patricia Mazik, Ph.D., Committee Chair Vicki S. Blazer, Ph.D. Luke Iwanowicz, Ph.D. Amy Welsh, Ph.D. Kyle Hartman, Ph.D. Wildlife and Fisheries Resources Program Morgantown, West Virginia 2016 Keywords: gene expression, non-model species, smallmouth bass, largemouth bass, white sucker, brown bullhead, Great Lakes, transcriptome, fish health, hepatitis B, emerging contaminants, areas of concern, next generation sequencing Copyright 2016 Cassidy Hahn ABSTRACT Development and Application of Quantitative Gene Expression Assays in Non-Model Fish Species Cassidy Megan Hahn The Great Lakes Basin is the most significant freshwater resource in North America. It is a central location for diverse anthropogenic enterprises and consequently has been heavily impacted by the release of toxic substances. Environmental studies increasingly identify the presence of both contaminants of emerging concern (CECs) and legacy contaminants in aquatic environments; however, the biological effects of these compounds on resident fishes remain largely unknown. To address this issue, the U.S. Fish and Wildlife Service Environmental Contaminants Programs initiated a multiagency, collaborative study which included chemical analysis of water and benthic sediment, assessment of caged fathead minnows and assessment of wild fish health using a suite of biological endpoints. In an effort to accurately and efficiently assess the ecological risks of complex chemical mixtures through the integration and synthesis of data from many levels of biological organization, sequence databases for non-model, resident fish species were required. This dissertation contains chapters that describe; 1.) The development of partial transcriptomes for three wild-caught, resident fish species 2.) The utilization of these sequence databases in the development of quantitative gene expression assays 3.) The application of these assays in both laboratory and environmental analyses and 4.) The use of transcriptome sequence databases in the identification of a novel virus. DEDICATION For my Dad. iii ACKNOWLEDGMENTS I would like to thank the Great Lakes Restoration Initiative for funding of this research. I would like to gratefully acknowledge my committee, Vicki Blazer, Patricia Mazik, Luke Iwanowicz, Amy Welsh and Kyle Hartman for their time and guidance throughout my time as a graduate student. Many people at the Leetown Science Center have helped make this research possible. Thank you to Ryan Braham, Heather Walsh, Adam Sperry, Rachel Harrison and Kyra Wood for assistance with field work. Thank you to Kathy Spring, Darlene Bowling, Pam Whittington, Deborah Iwanowicz, Laykn Sanders, Scott Cornman, Tim King, Barb Lubinski and Robin Johnson for assistance in the laboratory. Thank you to my family and friends for sticking with me and supporting me through 11 years of college. Lastly, I’d like to thank my fiancé George for always listening to me and encouraging me. iv FOREWORD This dissertation is comprised of five chapters which have been formatted as manuscripts according to the style of various journals. Chapter two is written in the format of the Journal of Comparative Biochemistry and Physiology: Part D and has been accepted for publication with the following co-authors: Luke Iwanowicz, Robert Cornman, Patricia Mazik, and Vicki Blazer. Chapter three is written in the format of the Journal of Great Lakes Research. Chapter four is written in the format of Environmental Toxicology. Chapter five has been published in the Journal of Virology with the following co-authors: Luke Iwanowicz, Robert Cornman, James Winton. Carla Conway and Vicki Blazer. v TABLE OF CONTENTS ABSTRACT ................................................................................................................................... ii DEDICATION.............................................................................................................................. iii ACKNOWLEDGMENTS ........................................................................................................... iv FOREWORD................................................................................................................................. v Chapter One: Introduction and Literature Review Introduction ................................................................................................................................... 1 Sample Collection.......................................................................................................................... 5 Gene Expression Biomarkers....................................................................................................... 6 Oxidative stress ........................................................................................................................... 7 Anthropogenic stressors ............................................................................................................ 14 Neoplasia ................................................................................................................................... 19 Endocrine disruption ................................................................................................................. 25 Management Implications .......................................................................................................... 32 References .................................................................................................................................... 32 Figures .......................................................................................................................................... 50 Figure 1. ................................................................................................................................. 50 Chapter Two: Transcriptome Discovery in Non-Model Wild Fish Species for the Development of Quantitative Gene Expression Assays Abstract ........................................................................................................................................ 51 1. Introduction ............................................................................................................................. 52 2. Methods .................................................................................................................................... 54 2.1. Transcriptome sequencing.................................................................................................. 54 2.2. Wild fish sampling ............................................................................................................. 56 2.3. Gene expression analysis ................................................................................................... 56 2.4. Cross-species probe hybridization ..................................................................................... 57 2.5. Comparison of nCounter analysis with quantitative real-time PCR .................................. 58 2.6. Normalization of nCounter gene expression data .............................................................. 59 vi 2.7. Statistical analysis .............................................................................................................. 60 3. Results ...................................................................................................................................... 60 3.1. Transcriptome sequencing.................................................................................................. 60 3.2. Cross-species probe hybridization ..................................................................................... 60 3.3. Validation of nCounter codeSet with real-time PCR ......................................................... 61 3.4. Sex-related differences in gene expression ........................................................................ 61 3.5. Seasonal variation in gene