MULTI-STRESS PROTEOMICS: THE GLOBAL PROTEIN RESPONSE TO MULTIPLE ENVIRONMENTAL STRESSORS IN THE PORCELAIN CRAB PETROLISTHES CINCTIPES A Thesis presented to the Faculty of California Polytechnic State University San Luis Obispo In partial fulfilment of the Requirements for the Degree Master of Science in Biological Sciences by Michael A. Garland August 2015 © 2015 Michael A. Garland ALL RIGHTS RESERVED ii COMMITTEE MEMBERSHIP TITLE: Multi-stress proteomics: The global protein response to multiple environmental stressors in the porcelain crab Petrolisthes cinctipes AUTHOR: Michael A. Garland DATE SUBMITTED: August 2015 COMMITTEE CHAIR: Lars Tomanek, Ph.D. Associate Professor of Biological Sciences COMMITTEE MEMBER: Nikki Adams, Ph.D. Professor of Biological Sciences COMMITTEE MEMBER: Kristin Hardy, Ph.D. Assistant Professor of Biological Sciences iii ABSTRACT Multi-stress proteomics: The global protein response to multiple environmental stressors in the porcelain crab Petrolisthes cinctipes Michael A. Garland Global climate change is increasing the number of hot days along the California coast as well as increasing the incidence of off-shore upwelling events that lower the pH of intertidal seawater; thus, intertidal organisms are experiencing an increase in more than one stress simultaneously. This study seeks to characterize the global protein response of the eurythermal porcelain crab Petrolisthes cinctipes to changes in thermal, pH, and tidal regime treatments, either combined or individually. The first experiment examined temperature stress alone and sought to determine the effect of chronic temperature acclimation on the acute heat shock response. We compared the proteomic response of cheliped muscle tissue following a month-long acclimation to either (1) constant 10°C, (2) daily fluctuation from 10-20°C, or (3) daily fluctuation from 10-30°C, all followed by either a 30°C acute heat shock or 10°C control. We found that ATP supply via the phosphagen system, changes in glycolytic enzymes, muscle fiber restructuring, respiratory protein fragmentation, and immunity were primarily affected by acclimation and subsequent heat shock. Acclimation to the “extreme” regimes (10°C and 10-30°C) resulted in the greatest proteomic changes, while acclimation to the moderate regime (10-20°C) resulted in a more mild response to heat shock (i.e., fewer adjustments to relative protein abundance). The second experiment sought to determine the proteomic response of gill tissue following a 17 d acclimation to daily changes in pH (ambient pH 8.1 vs low pH 7.6), tidal regime (constant immersion vs 6 h emersion), and temperature (ambient 11°C vs 22-31°C heat shock during emersion). Low pH alone reduced expression of molecular chaperones of the endoplasmic reticulum, lectins, and serine proteases involved in activating the prophenoloxidase cascade. It also increased the abundance of Na+/K+-ATPase, nitrogen metabolism enzymes, and induced changes in tubulin expression, all suggesting an increase in ammonium excretion. Addition of emersion during low pH reduced the abundance of several metabolic proteins including those involved in the proposed ammonium excretion mechanism, suggesting a decrease in metabolic function in part to prevent toxic accumulation of ammonium in the branchial chambers. Combined pH, emersion, and thermal stress increased the abundance of proteins involved in cuticle binding and crosslinking. These results indicate that the responses to pH, tidal cycle, and temperature are highly dependent on one another and that changes in ER protein maturation, ion transport, immunity, and cuticle structure are the primary biochemical systems impacted by these environmental stressors in crustacean gill. Keywords: proteomics, crustacean, gill, muscle, climate change, thermal stress, pH, hypercapnia, emersion, multi-stress, immunity, Petrolisthes iv ACKNOWLEDGMENTS I would like to thank Dr. Lars Tomanek for giving me an opportunity to grow as a scientist. His mentorship, encouragement, and support will not be forgotten. I would also like to thank my committee, Dr. Nikki Adams and Dr. Kristin Hardy, for their support and feedback, as well as Dr. Christina Vasquez for her thoughtful input into these manuscripts. A special thanks to Marcus Zuzow for his technical help throughout the years and for interesting conversations about philosophy and society. I especially want to thank Holland Elder who shared in the ups and downs inherent in the endeavor of science (and academics in general). Thanks to all of my local friends and fellow lab members including Loredana Serafini, James Koman, Josh Mier, Hayley Chilton, Aubrie Fowler, Mark Hamer, Arlo White, Jessica Griffiths, Neha Patel, Alex Barbella, Michael Maples, Avery Cromwell, Molly Pendley, Kory Heiken, Christopher Sakoda, and all graduate students, faculty, and staff in the Biological Sciences department. My gratitude goes to the Stillman Lab for their invaluable collaboration as this research would not have been possible without them. I also want to thank my family for their support and encouragement throughout my years as a student. This research was supported by National Science Foundation (NSF) grants MCB-1041225 and EF-1041227, and by the Council on Ocean Affairs, Science, and Technology (COAST). v TABLE OF CONTENTS LIST OF TABLES ........................................................................................................... ix LIST OF FIGURES .......................................................................................................... x 1.0 INTRODUCTION ...................................................................................................... 1 1.1 Physical Science: Global climate change .............................................................. 3 Atmospheric and oceanic warming .......................................................................... 3 Ocean acidification .................................................................................................. 6 Temperature and pH along the California coast ......................................................11 1.2 Biological Science: Physiological adaptation ........................................................14 Chronic and acute thermal stress ...........................................................................14 Additional note on molecular chaperones ...............................................................20 Hypercapnia ...........................................................................................................21 1.3 The porcelain crab Petrolisthes cinctipes .............................................................26 Eastern pacific Petrolisthes congeners ...................................................................26 Physiology of Petrolisthes cinctipes ........................................................................28 Demographics ........................................................................................................30 Population genetics and evolution ..........................................................................32 Social Systems and Behavioral Ecology .................................................................35 2.0 THERMAL ACCLIMATION MANUSCRIPT ..............................................................42 2.1 Abstract ................................................................................................................43 2.2 Introduction ..........................................................................................................45 2.3 Materials and Methods .........................................................................................49 Animal collection, maintenance, and experimental design ......................................49 Homogenization .....................................................................................................50 Two-dimensional gel electrophoresis (2D-GE)........................................................50 Gel image analysis .................................................................................................51 Mass Spectrometry ................................................................................................52 Exploratory statistical analysis ................................................................................53 2.4 Results and discussion ........................................................................................54 Principal component analysis .................................................................................54 Energy metabolism .................................................................................................58 vi Phosphotransfer proteins ....................................................................................58 Glycolysis ...........................................................................................................61 Energy metabolism - conclusion .........................................................................62 Hemocyanin ...........................................................................................................63 Cytoskeletal proteins ..............................................................................................64 Thick filament (myosins) .....................................................................................64 Thin filament (actins) ...........................................................................................66 Actin-binding proteins