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UNIVERSITY of CALIFORNIA, SAN DIEGO Metabolic Physiology Of UNIVERSITY OF CALIFORNIA, SAN DIEGO Metabolic Physiology of Healthy and Bleached Corals A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Marine Biology by Lauren Linsmayer Committee in charge: Martin Tresguerres, Chair Dimitri D. Deheyn Pieter C. Dorrestein Horst Felbeck William H. Gerwick David I. Kline 2018 Copyright Lauren Linsmayer, 2018 All rights reserved. The Dissertation of Lauren Buckley Linsmayer is approved, and it is acceptable in quality and form for publication on microfilm and electronically: ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ Chair University of California, San Diego 2018 iii DEDICATION This dissertation is dedicated to my beloved parents, Linda and Nick, my brother, Nicholas, and to my Stanford advisor, Dr. George N. Somero. iv TABLE OF CONTENTS Signature Page…………………………………………………………………….. iii Dedication.………………………………………………………………………… iv Table of Contents……………………………………………………………….… v List of Figures…………………..………………………………………………… vii List of Tables……………….…………………………………………………….. x Acknowledgements…………………………………………………………….… xi Vita…………………………………..…………………………………………… xiv Abstract of the Dissertation……………………………………………………… xvi Chapter 1: General Introduction………………………………………………… 1 Chapter 1: References…………………………………………………………… 9 Chapter 2: The Dynamic Oxygen Microenvironment of Corals: Identification of Strombine as a Main End Product of Anaerobic Metabolism………………….… 14 Chapter 2: References……………………………………………………………. 65 Chapter 3: Thermal Bleaching Differentially Affects Host Metabolism in Two Common Caribbean Coral Species, Agaricia agaricites and Orbicella franksi…………….. 72 Chapter 3: References……….……………………………………………………. 111 Chapter 4: The Effects of Menthol-Induced Bleaching on Coral Physiology……. 119 Chapter 4: References……………………………………………………………... 139 Chapter 5: Detecting Diel Patterns in Coral Metabolism Using Untargeted Proteomic and Metabolomic Approaches…………………………..………………………….….. 142 Chapter 5: References………………………………………………………..……. 210 Chapter 6: Discussion……………………………………………………………... 216 v Chapter 6: References………………………………………………………….…. 222 vi LIST OF FIGURES Figure 2.1: Continuous microelectrode measurements of O2 concentrations (µM) on tissue surfaces of Acropora yongei over a diel cycle. ……………………………....……... 50 Figure 2.2: Metabolic enzyme activities in coral tissue homogenates over a diel cycle.………………………………………………………………………….…….. 51 Figure 2.3: Cloud plot of significantly dysregulated ionic features in coral holobiont samples from day and night.……………………………………………………….. 52 Figure 2.4: Predicted metabolic pathways map……………………………………. 53 Figure 2.5: Concentrations (nmol mg protein-1) of strombine and alanopine in corals sampled over diel cycle.…………………………………………………………… 54 Figure 2.6: LC-MS analysis of R-strombine and coral extract.…………………… 55 Figure 2.7: LC-MS analysis of Celite®-filtered S-strombine and coral extract (Experiment A)..………………………………….………………………………… 56 Figure 2.8: LC-MS analysis of S-strombine, coral extract, and coral extract spiked with S-strombine (Experiment B).……………………………………………………… 57 Figure 2.9: LC-MS analysis of HILIC TopTip-filtered S-strombine, coral, normoxic mussel, and anoxic mussel (Experiment C).………………………………………. 58 Figure 2.10: Metabolic enzyme activities in control and bleached coral tissue homogenates.…………………………….………………………………………… 60 Figure 2.11: Picture of the experimental tank used in the diel study.……………………………………………………………………………….. 61 Figure 2.12: Correlations of enzyme activities in A. yongei over diel cycle..……………………………………………………………………………….. 62 Figure 2.13: Potential end-products of anaerobic glycolysis………………………. 63 Figure 3.1: Representative coral bleaching pictures……………………………….. 101 Figure 3.2: Coral color changes during thermal bleaching………………….…….. 102 Figure 3.3: Symbiodinium, chlorophyll a, and coral soluble protein in control and bleached A. agaricites (AA) and O. franksi (OF)………………………………….. 103 vii Figure 3.4: Respiration and photosynthesis rates of corals during thermal bleaching……………………………………………………………………………. 104 Figure 3.5: O2 measurements in healthy and bleached corals in the light and dark.………………………………………..……………………………………….. 106 Figure 3.6: Metabolic enzyme activities (in nmol mg protein-1 min-1) in control and bleached A. agaricities (AA) and O. franksi (OF)………………………………….. 107 Figure 3.7: Photosynthetically active radiation in control and bleaching tanks……. 108 Figure 3.8: Experimental tank photosynthetically active radiation…….…………… 109 Figure 4.1: Coral color score changes during menthol bleaching…………………… 135 Figure 4.2: Coral bleaching parameters in control and menthol bleached A. agaricites (AA) and O. franksi (OF)……………………………………………………….……. 136 Figure 4.3: O2 microsensor measurements on coral tissues during menthol bleaching……………………………………………………………………………… 137 Figure 4.4: Metabolic enzyme activities of control and menthol bleached corals………………………………………………………………………………….. 138 Figure 5.1: Fusion image of all Coomassie and SYPRO Ruby stained 2D gels…………………………………………………………………………………….. 172 Figure 5.2: Fusion image of Coomassie 2D gels (n=30) created in Delta 2D………… 173 Figure 5.3: Heat map of significant proteins from Coomassie (pH 3-10) gels (1000 permutations ANOVA, p<0.02)…………………………………………………….. 174 Figure 5.4: Fusion image of SYPRO 2D gels (n=38) created in Delta 2D……...….. 175 Figure 5.5: Heat map of significant proteins from SYPRO (pH 4-7) gels (1000 permutations ANOVA, p<0.02)…………………………………………………….. 176 Figure 5.6: Coral proteins involved in aerobic metabolism………………………… 177 Figure 5.7: Significant proteins involved in coral cytoskeleton…………………….. 178 Figure 5.8: Other significant coral proteins…………………………………………. 179 Figure 5.9: Significant Symbiodinium photosynthesis-related proteins…………….. 180 Figure 5.10: Other significant Symbiodinium proteins……………………………… 181 viii Figure 5.11: Coral proteins involved in glycolysis………………………………….. 182 Figure 5.12: Symbiodinium proteins involved in aerobic metabolism………………. 184 Figure 5.13: Symbiodinium proteins involved in anaerobic metabolism and glycolysis…………………………………………………………….………………. 185 Figure 5.14: Cloud plot of the significantly dysregulated ionic features in coral holobiont samples from day and night………………………………………………………….. 187 Figure 5.15. Non-metric multidimensional scaling (MDS) plot of the untargeted metabolomics data from “day” and “night” samples………………………………… 188 Figure 5.16: Predicted metabolic pathway map…………………………………….. 189 ix LIST OF TABLES Table 2.1: Percentage of the coral tissues that experienced hypoxia/anoxia at night… 64 Table 3.1: Summary values from Figure 3.3 of bleaching parameters………………. 110 Table 5.1: Protein names and information from SYPRO gels…………………..…… 190 Table 5.2: Protein information from Coomassie gels………………….…………….. 194 Table 5.3: Top metabolite predictions………………………………………..………. 201 Table 5.4: Significant predicted metabolic pathways………………………….…….. 208 x ACKNOWLEDGMENTS There are many people who helped with various aspects of the research presented in this dissertation. I would like to start by thanking my PhD committee members for all of their research mentorship and support. My PhD advisor, Dr. Martin Tresguerres, provided critical help at every stage of the design, development, and interpretation of the experiments outlined in the following chapters, as well as with his help editing the dissertation. I would like to acknowledge Dr. David I. Kline (SIO-UCSD) for his help designing the coral bleaching experiments in Panama and for his thoughtful discussions of coral ecology. Dr. Dimitri D. Deheyn (SIO-UCSD) helped with experimental aquarium setup and oxygen measurements during the diel experiments and interpretation of results. Thanks to Dr. Horst Felbeck (SIO-UCSD) and Dr. William Gerwick (SIO- UCSD) for fruitful discussions about metabolism, enzyme activity, and metabolite identification, respectively. Dr. Gerwick also provided me with the use of laboratory equipment for preparing samples for LC-MS analyses in Chapter 2. Thank you to Dr. Pieter C. Dorrestein (UCSD) for discussions about mass spectrometry and help interpreting the LC-MS data. Dr. Yongxuan Su (UCSD) and Bill Webb (TSRI) developed the liquid chromatography-mass spectrometry (LC-MS) methods used in Chapters 2 and 5, ran the instruments, and went beyond the obligations expected from their paid services. Dr. W. Ross Ellington (Florida State University) donated standards of R-strombine, octopine, and alanopine for the targeted LC-MS analyses in Chapter 2. All of the LC-MS studies conducted at TSRI in Chapters 2 and 5 were done in the laboratory of Dr. Gary Siuzdak. xi I am also grateful to Dr. Forest Rohwer (SDSU) for loaning me O2 microsensors for the preliminary experiments in Chapter 2. Dr. Jennifer Smith (SIO-UCSD) provided me with space in her wet lab for conducting the second set of diel O2 measurements in Chapter 2. Phil Zerofski’s (SIO-UCSD) assistance was essential for the aquarium experiments conducted at SIO, as were Plinio Gondola’s and Deyvis Gonzalez’s help for the experiments at STRI in Panama. I would like to thank Drs. Rachel Collin, Andrew Altieri, and Nancy Knowlton (all STRI),
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