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Analysis of Seasonal Changes in Thermal Stress Resilience and Innate Immunity in the Temperate Coral, Astrangia Poculata, from Future Climate Impacts

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Analysis of Seasonal Changes in Thermal Stress Resilience and Innate Immunity in the Temperate Coral, Astrangia Poculata, from Future Climate Impacts Grand Valley State University ScholarWorks@GVSU Masters Theses Graduate Research and Creative Practice 12-2020 Analysis of seasonal changes in thermal stress resilience and innate immunity in the temperate coral, Astrangia poculata, from future climate impacts Tyler Eugene Harman Grand Valley State University Follow this and additional works at: https://scholarworks.gvsu.edu/theses Part of the Biology Commons, and the Terrestrial and Aquatic Ecology Commons ScholarWorks Citation Harman, Tyler Eugene, "Analysis of seasonal changes in thermal stress resilience and innate immunity in the temperate coral, Astrangia poculata, from future climate impacts" (2020). Masters Theses. 998. https://scholarworks.gvsu.edu/theses/998 This Thesis is brought to you for free and open access by the Graduate Research and Creative Practice at ScholarWorks@GVSU. It has been accepted for inclusion in Masters Theses by an authorized administrator of ScholarWorks@GVSU. For more information, please contact [email protected]. Analysis of seasonal changes in thermal stress resilience and innate immunity in the temperate coral, Astrangia poculata, from future climate impacts Tyler Eugene Harman A Thesis Submitted to the Graduate Faculty of GRAND VALLEY STATE UNIVERSITY In Partial Fulfillment of the Requirements For the Degree of Master of Biology Department of Biology December 2020 Acknowledgements The work detailed in this thesis research would not have been possible without the help of many mentors, colleagues, friends, and family. I thank my committee members, Dr. Daniel Barshis, Dr. Sarah Hamsher, and Dr. Briana Salas for their help structuring this research, providing their input, and the help of editing this document. Additional thanks to Dr. Barshis for continuing to be a great mentor in coral research throughout my entire scientific career thus far. Special thanks to Dr. Sean Grace for his help in field work/coral collections in Rhode Island, Dr. Daniel Nielsen for his help in creating the ROS methodology and input on data analysis, Dr. Caroline Palmer and Dr. Laura Mydlarz for her help with structuring the immunity methodology, and Dr. James Cervino for providing use of the DIVING-PAM. I thank both of my lab mates, Cassidy Gilmore and Darrick Gates for their help in assisting with these experiments and general coral husbandry, for without them I could not have achieved this. I also extend appreciation to Dr. Koty Sharp, Dr. Randi Rotjan, Dr. Sean Grace, and the annual Astrangia Research Workshop hosted by Roger Williams University and Southern Connecticut State University for fostering creative conversations and collaborations leading to this work. I thank our funding sources, The Graduate School at Grand Valley State University and NASA’s Michigan Space Grant Consortium, for their generous opportunity for me to conduct this research. I also thank my family, friends, and loved ones for their support throughout my entire graduate school career - it has meant so much over these two years. Lastly, I express my sincere thanks and appreciation to my graduate mentor, Dr. Kevin Strychar. I thank you for the opportunity to be a graduate student in your lab, your mentorship on doing great science, and the continued support you provide to support me currently and beyond as I continue my journey in scientific research. 3 Abstract Over the years, global warming has had a devastating effect on coral reef ecosystems. Anthropogenic influences have caused significant increases in greenhouse gases, with a subsequent increase in solar radiation held within Earth’s atmosphere leading to increasing global temperatures. The increasing temperatures from concurrent increases in greenhouse gases impact fragile marine ecosystems such as coral reefs, which require particular environmental parameters such as temperature in order to survive and maintain a diverse ecosystem in which many marine species rely on. These increases in temperature exacerbate phenomena such as bleaching events and coral disease, drastically impacting coral on a global scale and with the threat of extinction. However, most research has been focused on corals in tropical/subtropical systems. Corals within temperate systems have been studied less-so in terms of how global warming will impact their physiology and future survivorship. This thesis focuses on the temperate coral, Astrangia poculata, with colonies collected from Narragansett Bay in Rhode Island, USA, to understand how this species will respond to increased temperatures and disease exposure. This thesis will focus on two separate experiments, one primarily on heat stress, and the other on understanding disease impacts and its relation to elevated temperatures. The heat stress experiment subjected colonies of A. poculata to treatments of ambient and increased temperatures over a period of ten days to understand the accumulation of reactive oxygen species (ROS), a toxic chemical byproduct of bleaching mechanisms within Photosystem II (PSII) in symbiotic algae. Measurements of maximum quantum yield via pulse amplitude modulation fluorometer techniques (i.e. to assess photosynthetic health of A. poculata’s algal symbiont) and photo quantification via Winters et al. (2009) (i.e. to determine symbiont density) were taken to compare to ROS concentrations measured using imaging flow cytometry (IFCM). Results from 4 this experiment found that ROS concentrations from elevated temperature treatments were lower compared to ambient temperature treatments, albeit no statistical significance was found. No statistical differences between elevated and ambient temperature treatments were found within maximum quantum yield, indicating the possible influence of increased nitrogen exposure and endolithic algae. In addition, differences between treatments found in pixel intensity results (i.e., symbiont density via photo quantification) suggest influence by seasonality and endolithic algae. The results from this experiment suggest that A. poculata be considered a resilient coral species to future elevated temperatures. The second experiment was to determine the influence of temperature on the baseline immunity of symbiotic and aposymbiotic A. poculata, as no previous studies have identified immune responses within A. poculata. The use of lipopolysaccharide (LPS) provide a general understanding of immunity within this species as a substitute for a pathogen. The exposure of LPS was set to measure the signaling protein prophenoloxidase (PPO) and melanin within the melanin-synthesis pathway to determine an immune response. Astrangia poculata fragments were exposed to LPS for a 12-hour period at two different temperatures, ambient (18 °C) and elevated (26 °C). Melanin was significantly higher within symbiotic corals compared to aposymbiotic corals and no statistical difference was found with regard to PPO concentration, suggesting that this species is susceptible to disease at elevated temperatures. The difference in response based on symbiotic state suggests the influence of other potential immune responses, such as the complement pathway and the coral microbiome. With the lack of differences found in PPO and response differences found between symbiotic state, this research recommends future projects into other immune responses to determine the holistic immune system within A. poculata. 5 Table of Contents: 1. Title Page 1 2. Approval Page 2 3. Acknowledgements 3 4. Abstract 4 5. Table of Contents 6 6. Abbreviations 8 7. List of Tables/Figure 9 8. Chapter 1 – Introduction to Corals and Climate Change 11 a. Introduction 11 b. Purpose 16 c. Scope 18 d. Assumptions 19 e. Hypothesis 20 f. Research Questions 23 g. Significance 24 h. Literature Cited 25 9. Chapter 2 (Manuscript – Coral Reefs) 29 a. Title Page 29 b. Abstract 30 c. Introduction 32 d. Methodology 36 e. Results 41 6 f. Discussion 44 g. Acknowledgements 52 h. Literature Cited 53 i. Figures 60 j. Supplementary Material 70 10. Chapter 3 (Manuscript – Journal of Experimental Biology) 75 a. Title Page 75 b. Summary Statement 76 c. Abstract 77 d. Introduction 78 e. Methodology 81 f. Results 84 g. Discussion 85 h. Acknowledgements 88 i. Literature Cited 89 j. Figures 96 k. Supplementary Material 104 11. Chapter 4 109 a. Extended Literature Review 109 b. Extended Methodology 121 c. Literature Cited 128 7 Abbreviations AWRI Annis Water Resource Institute IFCM Imaging Flow Cytometer LPS Lipopolysaccharide PAM Pulse Amplitude Modulation PAMP Pathogen Associated Molecular Pattern PAR Photosynthetically Active Radiation PBS Phosphate Buffer Solution PSII Photosystem II PPO Prophenoloxidase ROS Reactive Oxygen Species SCUBA Self-contained underwater breathing apparatus 8 List of Tables Chapter 2 Table 1: Three-way ANOVA results of aposymbiotic ROS fluorescence 64 Table 2: Three-way ANOVA results of symbiotic ROS fluorescence 65 Table 3: Three-way ANOVA results of symbiotic state ROS fluorescence 66 Table S1: Post-hoc Wilcoxon comparisons with Fv/Fm and pixel intensity 73 Table S2: Tukey HSD post-hoc results of aposymbiotic ROS fluorescence 74 Table S3: Tukey HSD post-hoc results of symbiotic ROS fluorescence 75 Chapter 3 Table 1: Three-way ANOVA results of melanin concentrations 102 Table 2: Three-way ANOVA results of PPO concentrations 103 Table S1: Tukey HSD post-hoc results of melanin concentrations 106 List of Figures Chapter 2 Figure 1: Map of Narragansett Bay, RI, USA 57 Figure 2: Schematic representation of experimental aquarium system 58 Figure 3: Images of individual cells from A. poculata stained with
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