THE PHYSIOLOGICAL RESPONSES OF SURF SMELT (HYPOMESUS PRETIOSUS) TO ENVIRONMENTAL STRESS IN PUGET SOUND, WA, USA A Thesis Presented to the Faculty of California State University, Stanislaus In Partial Fulfillment of the Requirements for the Degree of Master of Science in Ecology and Sustainability By Leah Marie Mellinger August 2018 CERTIFICATION OF APPROVAL THE PHYSIOLOGICAL RESPONSES OF SURF SMELT (HYPOMESUS PRETIOSUS) TO ENVIRONMENTAL STRESS IN PUGET SOUND, WA, USA by Leah Marie Mellinger Signed Certification of Approval page is on file with the University Library. Dr. Brian Sardella Date Professor of Biological Sciences Dr. Scott Hamilton Date Professor of Ichthyology Dr. Jeffrey Scales Date Professor of Biological Sciences © 2018 Leah Marie Mellinger ALL RIGHTS RESERVED DEDICATION I dedicate this thesis to my little family. I am so grateful for their support. Without my husband, Craig Mellinger, helping with my daughter, holding down the fort when I went on my research trips and being my rock, this thesis would not have been possible. Also, the additional support of my mother, Lisa Andreae Holmes, helping with watching my daughter and animals as well as helping editing was so crucial to my success. Thank you, I love you and couldn’t have done this without you. iv ACKNOWLEDGEMENTS This thesis was a huge undertaking and was only made possible by all those involved. Thank you to Dr. Brian Sardella for being my advisor and supporting my ambitious research ideas. Thanks to Dr. Scott Hamilton for being my committee member as well as helping set up my flow through system at Moss Landing Marine Lab. Thank you to Dr. Jeffrey Scales for joining my committee at the last minute and helping with the statistics and image J along with Dr. Kenneth Schoenly. Thanks to Dr. Matt Cover and Dr. Terry Jones for additional assistance with the microtome and microscope camera. Thank you to the BRC committee for my funding and Dr. Dietmar Kueltz and Leslie Oberholtzer for use of the UC Davis Histology Lab. Also, I thank Jerry Mayberry of Elk Grove Milling, Inc. for donating barrels for my flow through system at Moss Landing Marine Lab. A special thank you to all those involved in my Puget Sound and California collections, specifically: Karl Menard of the UC Davis Bodega Bay Marine Lab, Anna Kagely, Kurt Fresh, Joshua Chamberlin, and Jason Hall of NOAA NWFSC Seattle, WA, Phil Dionne and James Losee of Washington Department of Fish and Wildlife, Walker Duval, Tom Freidrich, Tim Wilson, and Sayre Hodgson of the Nisqually Tribe, Maxim Lundquist and Todd Zackey of the Tulalip Tribe, and Davey’s Live Bait. v TABLE OF CONTENTS PAGE Dedication ............................................................................................................... iv Acknowledgements ................................................................................................. v List of Tables .......................................................................................................... vii List of Figures ......................................................................................................... viii Abstract ................................................................................................................... ix Introduction ............................................................................................................. 1 Objective and Hypothesis ........................................................................... 9 Methods................................................................................................................... 11 Collection Sites ........................................................................................... 11 Assessment of Regional Water Quality ...................................................... 13 Fish Sampling Protocol ............................................................................... 14 Tissue Microarray Construction ................................................................. 14 Quantification of Representative Stress Proteins ........................................ 15 Statistical Analysis ...................................................................................... 17 Results ..................................................................................................................... 18 Regional Water Quality .............................................................................. 18 Marine Parasite Prevalence and Intensity ................................................... 18 Stress Protein Abundance based on Location and Season .......................... 22 Stress Protein Abundance in the Brain based on Temperature, Salinity and Parasite Intensity .................................................................................. 29 Stress Protein Abundance in the Heart based on Temperature, Salinity and Parasite Intensity .................................................................................. 31 Stress Protein Abundance in the Liver based on Temperature, Salinity and Parasite Intensity .................................................................................. 33 Discussion ............................................................................................................... 37 Conclusions ............................................................................................................. 42 Future Directions .................................................................................................... 44 vi References ............................................................................................................... 46 Appendices A. T-Test: Caspase 3, Glutathione S-Transferase, Heat Shock Protein 70, and Ubiquitin Florescence in the Brain and Parasite Intensity ..................................................................................................................... 57 B. T-Test: Caspase 3, Glutathione S-Transferase, Heat Shock Protein 70, and Ubiquitin Florescence in the Heart and Parasite Intensity ..................................................................................................................... 59 C. T-Test: Caspase 3, Glutathione S-Transferase, Heat Shock Protein 70, and Ubiquitin Florescence in the Liver and Parasite Intensity ..................................................................................................................... 61 D. Stepwise Backwards Linear Regression of Caspase 3 Florescence in the Liver ..................................................................................................................... 63 E. Stepwise Backwards Linear Regression of Glutathione S-Transferase Florescence in the Brain ..................................................................................................................... 65 F. Stepwise Backwards Linear Regression of Glutathione S-Transferase Florescence in the Heart ..................................................................................................................... 67 G. Stepwise Backwards Linear Regression of Heat Shock Protein 70 Florescence in the Liver ..................................................................................................................... 69 H. Stepwise Backwards Linear Regression of Ubiquitin Florescence in the Liver ..................................................................................................................... 71 vii LIST OF TABLES TABLE PAGE 1. Temperature and Salinity Data ......................................................................... 18 2. Two Way ANOVA Table GST in the Brain versus Season and Location ....... 24 3. Two Way ANOVA Table HSP70 in the Brain versus Season and Location ... 24 4. Two Way ANOVA Table GST in the Heart versus Season and Location ....... 26 5. Two Way ANOVA Table Caspase 3 in the Liver versus Season and Location 28 6. Two Way ANOVA Table HSP70 in the Liver versus Season and Location .... 28 7. Two Way ANOVA Table Ubiquitin in the Brain versus Season and Location 28 viii LIST OF FIGURES FIGURE PAGE 1. North Puget Sound, Washington Historical Water Quality Data ..................... 6 2. South Puget Sound, Washington Historical Water Quality Data ..................... 7 3. Wild Surf Smelt Collection Sites ...................................................................... 12 4. Summer and Winter Parasite Prevalence .......................................................... 20 5. Parasite Intensity versus Temperature and Salinity .......................................... 21 6. Mean Brain Abundances versus Location and Season ..................................... 23 7. Mean Heart Abundances versus Location and Season ..................................... 25 8. Mean Liver Abundances versus Location and Season ..................................... 27 9. The Effect of Temperature on Raw and Mean Brain GST Abundance ............ 30 10. The Effect of Temperature on Raw and Mean Heart GST Abundance ............ 32 11. The Effect of Temperature on Raw and Mean Liver Caspase 3 Abundance .... 34 12. The Effect of Temperature on Raw and Mean Liver HSP70 Abundance ........ 35 13. The Effect of Temperature on Raw and Mean Liver Ubiquitin Abundance .... 36 ix ABSTRACT Drastic environmental changes can severely affect a fish’s metabolism, growth, reproduction and overall survivability. There is a concern that the synergy of multiple stressors, like the threat of climate change, will lead to population collapses of environmentally sensitive