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

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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.

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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

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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.

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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

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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

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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

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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 CM-H2DCFDA 59

Figure 4: Time series boxplots of maximum quantum yield (Fv/Fm) 60

Figure 5: Time series boxplots of pixel intensity 61

Figure 6: Time series boxplots of ROS fluorescence in aposymbiotic fragments 62

Figure 7: Time series boxplots of ROS fluorescence in symbiotic fragments 63

Figure S1: Preliminary pixel intensity analysis 68

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Figure S2: IFCM plot comparison between channel 2 and channel 5 intensities 69

Figure S3: Linear model between pixel intensities and maximum quantum yield 70

Figure S4: Linear model between maximum quantum yield and ROS fluorescence 71

Figure S5: Linear model between ROS fluorescence and pixel intensity 72

Chapter 3

Figure 1: Visual representation of the melanin-synthesis pathway in coral immunity 96

Figure 2: Map of field site location in Narragansett Bay, RI, USA 97

Figure 3: Visual representation of experimental aquarium system 98

Figure 4: Boxplot comparison of melanin concentrations between treatments 99

Figure 5: Boxplot comparison of melanin concentrations between seasons 100

Figure 6: Boxplot comparison of prophenoloxidase concentrations 101

Figure S1: Preliminary pixel intensity analysis 105

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Chapter 1 – Introduction to Corals and Climate Change

Introduction

Environments globally are suffering from many aspects of climate change. Since the beginning of the industrial revolution, global atmospheric concentration of carbon dioxide (CO2) has increased at an exponential rate and is believed to have been caused by the use of fossil fuels for energy, transportation, and construction. In addition, humans have reduced and burned millions of acres of forests for agriculture and livestock purposes. Further to this, even livestock contributions by expelling methane contribute to the accumulation of greenhouse gas emissions

(Crowley 2000; Höök and Tang 2013). Carbon dioxide is categorized as a greenhouse gas and it is one of the primary gases which contribute to global warming; CO2 helps trap solar radiation emanated from the sun to help encapsulate heat to warm the Earth. An overabundance of CO2 from anthropogenic sources causes more solar radiation to be trapped in our atmosphere, causing catastrophic impacts around the world including sea level rise and changes in precipitation. With increasing temperatures from increasing CO2, more severe warming occurs in polar regions, such as within the Arctic Circle. The rapid melting of glaciers drains into the ocean, causing increases in sea level rise as much as 3 mm per year (Rahmstorf 2010). Changes in precipitation occur concurrently with sea level rise; warming causes increased evaporation rates, prolonging droughts, in addition to increased capacity to store water vapor (Trenberth 2011). As sea level rise occurs, millions of coastal communities are potentially affected due to property damage and public health issues, which is compounded by changes in precipitation leading to more frequent floods and droughts (Rockstrom et al. 2009; Hallegatte et al. 2011; Trenberth 2011).

Anthropogenic increases in CO2 indirectly exacerbate the problem by causing permafrost in high

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latitude environments to melt faster, releasing trapped methane into the atmosphere, which is more potent compared to CO2 (Shuur et al. 2015).

Climate change is having a profound impact on many ecosystems either directly or indirectly. For instance, terrestrial ecosystems such as forests and grasslands suffer from alterations in precipitation, either in the form of frequent flooding or droughts (Trenberth 2011).

Flooding from intense storms can disrupt riparian zones that terrestrial organisms use, while droughts can impact access to water resources for terrestrial plant and multi-cellular organisms, reducing their ability to reproduce and survive, reducing their overall abundances (Rosenzweig et al. 2008; Brodribb et al. 2019). Similarly, migration patterns of many species have been affected by warming, with many species moving northward toward the poles (Rosenzweig et al.

2008). In addition, entire ecosystems have been or are beginning to change as endemic organisms are being replaced by invasive species, outcompeting local populations for food

(Mainka et al. 2010). Some specialized ecosystems such as tropical rainforests, wetlands, and polar ecosystems are especially vulnerable. Polar regions appear to be warming faster than expected and when coupled to less sea ice accumulation over winter months, some species habitats become reduced (e.g., penguins, walruses) resulting in less nursing and breeding habitats

(Turner and Overland 2009; Descamps et al 2017). Wetlands may also be affected by elevated temperatures due to changes in water input from precipitation, potentially drying out these ecosystems and reducing plant biomass.

The effects of climate change on the marine environment is broad and includes issues such as warming, sea level rise, ocean acidification, and eutrophication, to name a few. Thermal impacts from climate change can cause shifts in phytoplankton communities, as well as reduction in populations in some areas, directly reducing the overall primary production in the upper photic

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zone and causing trophic cascades on larger organisms (Beaugrand et al. 2003; Doney 2006;

Hays et al. 2006; Barton et al. 2016). Increases in precipitation in coastal areas can increase the amount of nutrient runoff from nearby agricultural fields, carrying remnants of nitrogen from fertilizers causing eutrophication and plankton blooms. Harmful algal blooms can be so abundant in concentration that reduced sunlight and dissolved oxygen levels can lead to suffocation

(Beman et al. 2005; Howarth et al. 2011; Le Moal et al. 2019). Sea level rise is indirectly caused by the melting of glaciers and the thermal expansion of ocean waters from climate change

(Brodribb et al. 2019). This can impact ecosystems such as coral reefs by reducing the amount of light needed for photosynthesis (Chow et al. 2019). In addition, warmer waters cause heat stress for many organisms who may already be living at their maximum tolerance levels. As a consequence, such animals either move to cooler habitats or if unable to move, adapt or die.

Coral reefs are affected by climate change in many ways, mainly by warming ocean temperatures and increasing acidification by carbon dioxide (CO2). Warmer ocean temperatures cause coral to bleach, either by reductions in the pigmentation of their symbiotic algae or by expelling their symbiotic algae from their tissues (Douglas 2003; Camp et al. 2020). These algae are vital for the coral host’s survival as algal symbionts provide up to 90% of their nutritional needs (Muscatine and Porter 1977; Hoegh-Guldberg et al. 2008). Coral bleaching events have increased in frequency over the past decade, which increases the overall mortality of coral individuals, only to be outcompeted by dense, thick algal mats (Pandolfi et al. 2011). Coral mortality can reduce the species diversity of coral ecosystems, where up to 25% of marine organisms utilize coral reefs for reproductive habitat and feeding purposes (Moberg and Folke

1999; Woodhead et al 2019). Increased CO2 levels can reduce overall growth rates in coral organisms (Anderson and Gledhill 2013). Atmospheric CO2 dissolves in the ocean near the water

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surface, reacting with water forming carbonic acid (H2CO3). Further reductions of carbonic acid eventually transition to bicarbonate and carbonate, and in time reacts with calcium to form calcium carbonate which corals utilize to build their skeletal structure. However, increases in

CO2 yields increases in carbonate ions, increasing the amount of hydrogen ions in the water and causing the pH of the ocean to become more acidic (i.e. ocean acidification; OA). Reductions in pH instigates the breakdown of calcium carbonate skeletal structures, causing low growth rates and reduced ability to use calcium carbonate (Hoegh-Guldberg et al. 2007; Rodolfo-Metalpa et al. 2010). Coral disease has increasingly become a threat over the past few decades due to increases in ocean temperatures (Sweet and Bythell, 2017) and OA; thermal extremes and pH may weaken various immunity pathways causing organisms to be more susceptible to infection.

However, disease resistance can differ between species and some are more disease resistant than others; this is from the upregulation of suppressants that benefit disease mitigation. The microbial composition in the mucus layer of corals provide a first defense against disease, however, its composition can be altered due to various temperature anomalies (Nguyen-Kim et al. 2015). In addition, thermal stress reduces important components such as prophenoloxidase and melanin, which stimulate immune pathways necessary to deter various pathogens (Palmer et al. 2011).

Much research has been focused on tropical coral reefs to understand their susceptibility to thermal extremes, disease, and OA (Palmer et al. 2010, Palumbi et al. 2014, Mollica et al.

2018), however, temperate corals have been studied less-so regarding these impacts. This study examines the effects of both temperature and disease on the temperate coral species, Astrangia poculata (Ellis and Solander, 1786). Knowing temperatures are likely to continue to increase over the next few decades (IPCC, 2019), few studies have examined how this coral will respond

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to heat stress and/or other cellular components that cause bleaching (i.e., reactive oxygen species). This species also exists in areas of southern Florida ravaged by coral diseases such as yellow-band disease, as well as potential exposure of diseases in its northern range from microplastic ingestion, which may harbor harmful bacteria. It is currently not known how this species responds to disease.

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Purpose

The purpose of this research is to understand the resilience of the temperate coral

Astrangia poculata from thermal stress events and disease exposure. Although this species inhabits multiple areas along the east coast, few studies exist that describe how A. poculata responds to heat stress or disease exposure. For example, it is not known if A. poculata can survive as oceans warm and/or whether the added detriment, i.e. disease, will affect its long-term survivorship.

There are a limited number of studies that examine thermal stress resilience of A. poculata (Jacques et al. 1983; Aichelman et al. 2019). To my knowledge, however, other cellular functions that can be used to categorize its resilience have not been studied. One such example is reactive oxygen species (ROS), which are by-products of photosynthetic processes that can increase in concentration from high light and high temperatures and become toxic. These products have been correlated to trigger bleaching mechanisms in many symbiotic tropical coral species (Kristiansen et al. 2009; McGinty et al. 2012; Nielsen et al. 2018). In temperate species, less is known regarding the bleaching mechanism. It is well known, however, that many temperate species are facultatively symbiotic (i.e. living with and without algal symbionts) and those that are symbiotic should show symptoms of bleaching and relative ROS concentrations similarly. I hypothesize that aposymbiotic species (those lacking symbionts) may be more resilient to elevated temperatures compared to symbiotic individuals.

The effects of pathogen infection and disease is unknown in A. poculata. In particular, no association of any coral disease exists with this species in the scientific literature. In southern

Florida where many corals are dying due to disease A. poculata appears not to be affected.

Rotjan et al. (2019) hypothesized that the ingestion of pathogens by A. poculata occurs and in

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their study, they observed microplastics covered in E. coli were favored among traditional organic matter. Astrangia poculata populations in Rhode Island Bay are surrounded by heavy urbanization and likely microplastics, which can be the potential source of disease with this population (Chapron et al. 2018; Lamb et al. 2018; Rotjan et al. 2019). However, there are no reports describing disease presence in A. poculata in Rhode Island. Hence, there is a need to better understand whether A. poculata is susceptible to pathogen infection or whether they have an immune system that prevents disease.

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Scope

This thesis work utilizes both aposymbiotic and symbiotic A. poculata individuals from

Narragansett Bay, RI. This project focuses on understanding the response of symbiotic and aposymbiotic individuals collected from two seasons. Individuals from Narragansett Bay already inhabit waters with summer temperatures of ~24 °C and winter temperatures as low as 4 °C.

Since the International Panel of Climate Change (IPCC, 2019) reports an increase of ocean temperatures by 2 °C by the end of the century, this study will expose A. poculata to an experimental elevated temperature of 26 °C. In addition, considering A. poculata is likely exposed to numerous pathogens such as yellow-band and stony coral tissue loss disease in southern Florida and harmful bacteria associated with microplastics in its northern range (i.e.

Rhode Island), this species will be exposed to disease by using a lipopolysaccharide (LPS; isolated from E. coli O127:B8) associated with a known pathogen to better understand how disease impacts this coral species.

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Assumptions

This study expands on a multitude of in-situ studies that examined ROS, photosynthetic health, and bleaching events regarding heat stress, as well as simulated disease impacts (Palmer et al. 2011, Nielsen et al. 2018). Environmental conditions (temperature, light intensity, water flow) were mimicked from my field site located in Rhode Island as closely as possible in all experimental conditions prior to beginning any studies. In addition, multiple water changes were undertaken to maintain adequate nutrient concentrations and mimic the collection site as best as possible. Genotypic changes were not assessed, but both coral collections (i.e. summer and winter) were from the same field location. All measurements within this thesis correspond to differences between symbiotic and aposymbiotic A. poculata colonies based on experimental treatments and season collected.

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Hypotheses

The hypotheses in this thesis are all related to Astrangia poculata. Hypotheses related to thermal stress (chapter 2) are split into two categories, symbiotic A. poculata individuals and aposymbiotic A. poculata.

Thermal stress experiments:

 Symbiotic individuals

o Null: Astrangia poculata exposed to heat stress will show no significant

difference between ambient and elevated temperatures regarding reactive oxygen

species (ROS), photosynthetic efficiency, and pixel intensity. In addition, no

significant increases or decreases will be found for these measurements over time.

No significant difference in these measured responses (e.g., ROS, etc.) will be

found between individuals collected from summer and winter collection times.

o Alternative: Astrangia poculata exposed to heat stress will show significant

differences in reactive oxygen species (ROS) between temperature treatments as

well as over time, with elevated temperature treatments having higher ROS

fluorescence compared to ambient treatments. In addition, symbiotic individuals

will result in more ROS fluorescence compared to aposymbiotic individuals.

Significant differences in photosynthetic efficiency will be found between

elevated treatments and ambient treatments, with lower photosynthetic efficiency

found in elevated temperature treatments. In addition, significant decreases over

time in all measured variables will be found in elevated temperature treatments.

Significant differences will be observed with pixel intensity between elevated

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treatments and ambient treatments, with higher pixel intensities found in elevated

temperature treatments. In addition, significant increases in pixel intensity will be

found in elevated temperature treatments over time. Significant differences will

be found between individual colonies collected in winter and summer, with higher

ROS fluorescence, higher photosynthetic efficiency, and higher symbiont density

found in summer collections compared to winter collections.

 Aposymbiotic individuals

o Null: Astrangia poculata exposed to heat stress will show no significant

difference between ambient and elevated temperatures regarding ROS,

photosynthetic efficiency, and pixel intensity. In addition, no significant increases

or decreases will be found for these measurements over time. No significant

difference will be found between individual colonies collected in winter and

summer.

o Alternative: Astrangia poculata exposed to heat stress will show significant

differences in reactive oxygen species (ROS) between temperature treatments,

with elevated temperature treatments having higher ROS fluorescence compared

to ambient treatments. In addition, aposymbiotic corals in elevated temperatures

will have significant increases in ROS fluorescence over time. Aposymbiotic

individuals will have less ROS fluorescence compared to symbiotic individuals.

Significant differences in photosynthetic efficiency will be found between

elevated treatments and ambient treatments, with lower photosynthetic efficiency

found in elevated temperature treatments. In addition, significant decreases over

time in all measured variables will be found in elevated temperature treatments.

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Significant differences will be observed with pixel intensity between elevated

treatments and ambient treatments, with higher pixel intensities found in elevated

temperature treatments. In addition, significant increases in pixel intensity will be

found in elevated temperature treatments over time. Significant differences will

be found between individual colonies collected in winter and summer, with higher

ROS fluorescence, higher photosynthetic efficiency, and higher symbiont density

found in summer collections compared to winter collections.

LPS exposure experiments:

 Null: Astrangia poculata (symbiotic and aposymbitoic forms) exposed to LPS will show

no differences of prophenoloxidase and melanin concentrations with the melanin

synthesis pathway, regardless of symbiotic state or temperature treatments.

 Alternative: Astrangia poculata (symbiotic and aposymbitoic forms) placed in ambient

temperatures with exposure to LPS will show lower amounts of prophenoloxidase and

melanin while colonies in elevated temperatures with LPS exposure will have higher

amounts of both prophenoloxidase and melanin, indicating immunological stress from

LPS exposure. Aposymbiotic colonies will have higher amounts of prophenoloxidase and

melanin in both thermal treatments compared to symbiotic colonies, indicating that

symbiosis, or lectin-glycan interactions from the complement pathway, performs

immunity functions within A. poculata.

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Research Questions

 How will increased temperatures impact Astrangia poculata individuals, both symbiotic

and aposymbiotic, regarding reactive oxygen species (ROS) concentrations,

photosynthetic efficiency, and symbiotic density? How does this compare to individuals

in ambient treatments?

 How will exposure to LPS impact the concentrations of prophenoloxidase and melanin in

symbiotic and aposymbiotic Astrangia poculata in ambient temperature treatments versus

elevated temperature treatments?

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Significance

This study fulfills the requirements for the Master of Science degree at Grand Valley

State University. Firstly, A. poculata is understudied regarding climate change impacts and will serve as additional evidence to potentially provide federal protection. Secondly, A. poculata is facultatively symbiotic, which can provide additional knowledge of tropical coral reefs by understanding the impacts of thermal stress and disease without the influence of symbiosis.

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Chapter 2 – Manuscript Submission – Coral Reefs

Full Title:

Seasonal thermal resilience of Astrangia poculata based on future thermal extremes

Tyler E. Harman1, Daniel Barshis2, Briana Hauff Salas3, Sarah E. Hamsher1,4, Kevin B. Strychar1

1Annis Water Resource Institute – Grand Valley State University

740 West Shoreline Dr, Muskegon, MI 49441

2Department of Biology – Old Dominion University

5115 Hampton Blvd, Norfolk, VA 23529

3Department of Math and Science – Our Lady of the Lake University

411 SW 24th St, San Antonio, TX 78207

4Department of Biology – Grand Valley State University

1 Campus Drive, Allendale Charter Twp, MI 49401

Communicating author email address: [email protected]

Keywords: reactive oxygen species, thermal stress, Astrangia poculata, climate change, imaging

flow cytometry

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Abstract

Climate change has had a devastating effect on coral reef ecosystems, with most research focused on corals in tropical and subtropical systems. Corals in temperate systems have been less studied, particularly regarding how climate change will impact their physiology and future survivorship. This research focuses on the temperate coral, Astrangia poculata, and how it will respond to increased temperatures. Colonies were collected from Fort Wetherill State Park, RI

(41°28'40.8"N, 71°21'45.8"W) during summer and winter seasons and exposed to experimental treatments of ambient (18 °C) and elevated temperatures (26 °C) to simulate future heat stress.

We measured photosynthetic efficiency (maximum quantum yield; Fv/Fm), pixel intensity

(inverse relationship with symbiont density), and reactive oxygen species (ROS) concentrations within symbiotic and aposymbiotic A. poculata to determine the influence of temperature and seasonal collection. We observed higher Fv/Fm ratios in summer corals compared to winter corals (p ≤ 0.05). For pixel intensity, we observed lower intensities (i.e., higher symbiont density) within symbiotic and aposymbiotic A. poculata in elevated temperature treatments, and higher intensities (i.e. lower symbiont density) in symbiotic coral from winter collections compared to summer (p ≤ 0.05). No differences in ROS were found in host tissue cells with any experiment, suggesting that ROS is produced intracellularly in algal symbionts and did not produce enough to leak into host tissue cells to produce significance differences. Overall, higher

ROS fluorescence was found in summer corals compared to winter corals (p ≤ 0.05) in both symbiotic and aposymbitoic colonies. Significantly higher ROS fluorescence was found in symbiotic individuals compared to aposymbitoic colonies (p ≤ 0.05). These results indicate that the thermal threshold for toxic ROS production in A. poculata is higher than 26 °C. Therefore, future studies should examine thermal thresholds at higher temperatures. Further, we suggest that

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symbiotic A. poculata individuals are more influenced by oxidative stress compared to aposymbiotic individuals due to symbiotic density. Our study also indicates that seasonal changes may have a direct impact on the physiology of A. poculata. Experiments involving colonies collected from the winter showed significantly lower concentrations of ROS, indicating that during the winter a quiescent state may significantly reduce ROS concentrations, in addition to growth rates, metabolism, and symbiont density.

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Introduction

Climate change studies have been at the forefront of marine science research for the past several decades due to increases in atmospheric carbon dioxide (CO2) (i.e., deforestation, energy sources, transportation, etc.) (Mac Dowell et al. 2017; Giannakis et al. 2020). These activities exacerbate the greenhouse effect, trapping more solar radiation and increasing global atmospheric and oceanic temperatures (Carpenter et al. 2008, Hoegh-Guldberg and Bruno 2010,

Cheng et al. 2019). Increases in ocean temperature impact a multitude of organisms, including corals. Coral reefs are considered fragile ecosystems, establishing themselves in environments with specific temperature, nutrient concentrations, salinity, and light availability (Hoegh-

Guldberg 2011). When exposed to increased temperatures, corals undergo a process called bleaching in which they expel their mutualistic photosynthetic dinoflagellates, Symbiodiniaceae, and/or lose color. This leaves the coral under threat of mortality, as corals rely on these symbionts to supply most of their energy requirements (Muscatine and Porter 1977; Douglas

2003; Pandolfi et al. 2011). Not all coral populations suffer the same level of bleaching and mortality due to climate change, as different coral species can house different Symbiodiniaceae species, making some more resilient to climate change than others in terms of acclimation to stressors (Brown 1996; Douglas 2003; Berkelmans and van Oppen 2006, Camp et al., 2020).

Over time, bleaching events can lead to an adapted population of Symbiodiniaceae in heat stressed corals, giving these corals a better physiological advantage to rising sea temperatures

(Douglas 2003; Buerger et al. 2020).

Another physiological impact from bleaching events includes decreasing photosynthetic efficiency (Brown 1996, Warner et al. 1999, Cziesielski et al. 2019, Camp et al. 2020). High temperatures can damage of thylakoid membranes and Photosystem II (PSII) and interrupt the

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Calvin cycle in Symbiodiniaceae, resulting in reduced rates of photosynthesis causing increases in the irradiance absorbed and an over-accumulation of reactive oxygen species (ROS)

(Fridovich 1978; Brown 1996; Warner et al. 1999; Douglas 2003; Krieger-Liszkay et al. 2008;

Tolleter et al. 2013; Hillyer et al. 2016; Wietheger et al. 2018). Reactive oxygen species are used for various cellular functions such as cellular defense and apoptosis (Kristiansen et al. 2009;

McGinty et al. 2012), but the accumulation of ROS can be responsible for the expulsion of algal symbionts (Downs et al. 2002; Tolleter et al. 2013; Gardner et al. 2017). Increased levels of ROS may cause membrane oxidation, protein denaturation, DNA chain breaks, and the degradation of

PSII (Fridovich 1978; Warner et al. 1999; Krieger-Liszkay et al. 2008; Kristiansen et al. 2009;

McGinty et al. 2012; Toledo-Hernandez and Ruiz-Diaz 2014; Roberty et al. 2016; Wietheger et al. 2018). Studies have shown correlations between the decrease in photosynthetic efficiency and increased ROS concentrations, leading to the concept of ROS negatively impacting coral-algal mutualism (Tchernov et al. 2004; Hillyer et al. 2016; Gardner et al. 2017). Accumulation of ROS can leak into the host tissues, causing damage to the mitochondria and triggering additional immune responses (McGinty et al. 2012; Roberty et al. 2016; Wietheger et al. 2018).

The rise in ocean temperatures are more relevant to corals in tropical environments, where bleaching frequently occurs (Brown 1996, Garzón-Ferreira et al. 2001, Claar et al. 2018,

Morgans et al. 2019). As reported by the IPCC (IPCC 2019), oceanic temperatures are hypothesized to increase over the next several decades, causing other coral such as temperate species to similarly experience stress and perhaps bleaching. The effects of climate change on these organisms are much less studied but have gained popularity in recent years. Examples of previously studied environmental conditions includes, high-light tolerance (Miller 1995), responses to increased CO2 (Maier et al. 2013), and heterotrophy mitigating the impacts of

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thermal stress (Aichelman et al. 2016). Species such as Cladocora caespitosa (Rodolfo-Metalpa et al. 2010) and Oculina arbuscula (Aichelman et al. 2016) have been studied, but few studies have targeted Astrangia poculata.

Astrangia poculata is a facultatively symbiotic coral defined by existing as both symbiotic and aposymbiotic (symbiotic densities less than 105 cm-2; Cummings 1983). This coral has a mutualistic relationship with Breviolum psygmophilum (J.E. Parkinson & LaJeunesse), its only algal symbiont. In contrast to obligately symbiotic tropical corals, this species of coral uses heterotrophic feeding, rather than fully relying on its algal symbionts (Szmant-Froelich and

Pilson 1980; Miller 1995; Sharp et al. 2017). However, symbionts do provide an advantage over aposymbiotic colonies regarding thermal adaptation and higher growth rates (Dimond and

Carrington 2007; Aichelman et al. 2019). The distribution of A. poculata ranges from its northern limit in Cape Cod, Massachusetts (Dimond et al. 2013) to as far south as the Atlantic coast of Florida and the northern coast of the Gulf of Mexico (Thornhill et al. 2008; Jaap et al.

2015). Aichelman et al. (2019) described thermal stress on photosynthetic and respiration outputs from two populations of A. poculata observing that local adaptation is an important component to thermal resistance. Despite such studies, other cellular functions have yet to be examined when these corals are experiencing thermal stress.

This study investigated how A. poculata responded to future temperatures predicted for the year 2100 (+2°C; IPCC 2019) by comparing symbiotic and aposymbiotic colonies from

Narragansett Bay in Rhode Island collected in summer and winter conditions. We assessed resistance to thermal stress by measuring photosynthetic efficiency (Fv/Fm), pixel intensity

(inverse of relative symbiont density), and ROS concentrations. We hypothesize that increased temperatures will decrease photosynthetic efficiency and symbiont density in A. poculata over

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time, but that ROS concentrations will increase over time at higher temperatures. This implies that increased ROS will negatively impact photosynthetic efficiency and symbiont densities within A. poculata.

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Methods

Coral Collection and Husbandry

Colonies of A. poculata were collected at Fort Wetherill State Park in Jamestown, Rhode

Island (Fig 1; 41°28'40.8"N, 71°21'45.8"W) using SCUBA on 14th July 2019 and 20th February

2020 (RIDEM permit #429, Type 1). Symbiotic and aposymbiotic coral colonies were collected by using a hammer and chisel to pry colonies from the substrate which were then placed in mesh bags. These colonies were collected at multiple sites at depths ranging from 5-9 meters. At the surface after the dive, the coral colonies were immediately transferred to plastic bags filled with seawater collected from the dive site and sealed with rubber bands. All samples were chilled on ice while transported back to the mesocosm facility located at the Annis Water Resource Institute in Muskegon, MI. Upon arrival to the mesocosm facility, all coral colonies were then placed into a custom-built recirculating aquarium system and acclimated to control conditions at rates of +1

°C day-1 (Fig. 2). Each system was filled with artificial seawater (deionized water and Instant

Ocean Reef Salt) and maintained at 18 °C. Full-spectrum LED lights (Bozily, Inc., Beijing,

China) were programmed to simulate diurnal patterns (07:00 to 19:00 light) with a maximum intensity of ~60 μmol m-2 s-1 using an Apogee SQ-420 Smart Quantum Sensor (Apogee

Instruments, Logan, UT). Corals were maintained by feeding with brine shrimp (Artemia nauplii) three days per week, 40% water changes twice per week, and water chemistry measurements (nitrate, phosphate, magnesium, alkalinity, and calcium) assessed once per week to maintain parameters. In addition, all sampled corals were placed briefly into other tanks for approximately one hour once per week for cleaning filamentous algae.

Experimental design

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Corals were fragmented using a Gryphon C-40 bandsaw (Gryphon Corporation, Sylmar,

CA) and then glued to acrylic glass discs using IC-gel (Bob Smith Industries, Atascadero, CA).

The fragmented corals were then evenly and randomly spread between the two tank systems.

Overall, 77 fragments resulted from the summer collected coral (44 symbiotic and 33 aposymbiotic) versus 40 fragments from the winter collection (20 symbiotic and 20 aposymbiotic). Each fragment was given an ID based on location, collection date, symbiotic state, colony number, and fragment number for easier identification. A detailed description on reducing symbiont density in aposymbiotic A. poculata prior to experiments can be found in the provided supplemental material (Fig S1).

Coral fragments were given two weeks to acclimate after fragmentation before any experimental analysis. Two identical recirculating aquarium systems were set-up to test the effects of heat stress on A. poculata. Corals in system 1 were exposed to ambient temperatures

(18 °C) and system 2 had elevated temperatures at 26 °C (Fig. 2). Temperature ramping occurred over a period of two weeks at a rate of +0.5 °C per day. The experimental study occurred over a period of 10 days. Sampling occurred every other day for a total of six sampling periods. Corals were sampled to measure (1) photosynthetic efficiency (maximum quantum yield; Fv/Fm) in

Photosystem II (PSII) using a DIVING-PAM (Walz, Germany), (2) symbiotic density via photo quantification (Winters et al., 2009), and (3) ROS concentrations via imaging flow cytometry

(IFCM).

Maximum quantum yield (Fv/Fm)

Measurements of photosynthetic health (i.e. maximum quantum yield; Fv/Fm) throughout the thermal stress treatments were taken using a DIVING-PAM (Walz, Germany) to

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identify the efficiency of photosystem II (PSII) reaction centers, which correlates to overall photosynthetic performance (Maxwell and Johnson, 2000). To assess the state of PSII for each fragment throughout the experiment, measurements of Fv/Fm were taken in the dark after corals had been dark-adapted for 30-minutes. Coral fragments were placed into a plastic container filled with artificial seawater at treatment temperature, with the distance between the cable and coral surface maintained ~10 mm following the DIVING-PAM manual. Measurements of Fv/Fm were taken in triplicate (summer symbiotic: n = 132, winter symbiotic: n = 60, summer aposymbiotic: n = 99, winter aposymbiotic: n = 60) for each coral fragment to obtain average Fv/Fm values.

Symbiotic density via photo quantification

Photo quantification measurements followed the methodology described by Winters et al.

(2009) to non-invasively quantify algal symbiont density in each fragment; this method identifies an inverse relationship with pixel intensity against symbiont density. A Kodak grayscale was placed inside a 30-gallon aquarium tank filled with artificial seawater while photographs were taken in triplicate with a GoPro HeroTM camera. Photographs were uploaded into custom

MATLAB files (Alex Blekhman 2005©) and were calibrated to a KodakTM grayscale to make photographic corrections. Subsequent analyses of these photographs were done by selecting ten points on the fragment to generate red intensity values (summer symbiotic: n = 440, winter symbiotic: n = 200, summer aposymbiotic: n = 330, winter aposymbiotic: n = 200) which correspond to symbiotic density (i.e. lower red intensity values correspond to higher symbiotic density).

ROS concentrations via imaging flow cytometry (IFCM)

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Subsequently after PAM and photographic measurements, fragments were carefully removed from their acrylic glass discs and placed into 50 mL Falcon tubes with 3.5 mL of 0.22

µm-filtered artificial seawater. Fragments were vortexed for 30 seconds to remove symbionts and coral tissue. The tissue slurry was filtered through a 70 µm cell strainer to remove any coral skeleton debris, filamentous algae, and mucus in the samples. Aliquots (1 mL) of the remaining slurry were pipetted into 1.5 mL Eppendorf tubes. Samples were then washed with 0.75 mL filtered artificial seawater three times at 1,000 RCF. ROS dye (CM-H2DCFDA; 2.9 µL) was added to samples to produce a 10 µM concentration, per manufacturer recommendations. These samples were incubated for 40 mins in the dark at the experimental treatment temperature along with rhythmic agitation at 300 rpm using a Thermomixer R (Eppendorf, Hamburg, Germany) to ensure homogenization of the stain. After incubation, samples were washed twice with 0.75 mL of 0.01 M phosphate buffered saline (PBS; pH 7.4) before analysis.

Processed samples were analyzed with an Amnis Imagestream X Mark II imaging flow cytometer (IFCM; Luminex, Seattle, WA). IFCM settings were as follows: 40× magnification,

60 µm field of view, 0.5 mW 488 nm laser intensity, and a low-flow rate/high sensitivity setting for higher quality imaging. Two collection gates were generated based on preliminary analyses to separate algal and host tissue congregations (Fig. S2). Algal collections were associated with high intensities in channel 5 corresponding to autofluorescence (640-745 nm) and host tissue collections were associated with high intensities in channel 2 corresponding to FITC fluorescence (505-560 nm). In addition, individual pictures of cells within these gates confirmed whether these cells were associated with symbiotic algae or with host tissue cells (Fig. 3).

Aliquots (30 µL) of samples were placed into 1.5 mL Eppendorf tubes, with each sample analyzed for 100,000 cell events to keep every analysis uniform. Following sample processing,

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FITC intensities were exported using the IDEAS analysis software (Luminex, Seattle, WA) from their respective collection gates. All cell fluorescent events per fragment were averaged to acquire a mean fluorescence value (summer symbiotic: n = 87, winter symbiotic: n = 40, summer aposymbiotic: n = 55, winter aposymbiotic: n = 38).

Statistical analyses

All data were recorded using Microsoft Excel (Microsoft Corporation 2020) and all statistical analyses were done with RStudio (RStudio Team 2020) in R Version 4.0.0 (R Core

Team 2020). The data were tested for normality using Shapiro-Wilk tests, where maximum quantum yield and pixel intensity data were non-parametric while the IFCM data were parametric after log-transformation. General linear models were used to detect differences between days with each variable, to which all data for each variable were pooled to determine significant differences, as no differences between days were detected. Maximum quantum yield and pixel intensity data were analyzed using Kruskal-Wallis statistical tests to determine significant differences between treatments, seasons, and symbiotic state. Post-hoc pairwise

Wilcoxon comparisons were run to determine the statistical significance between targeted comparisons. IFCM data were analyzed using a three-way ANOVA to detect significant differences between treatments, season, and cell-type, along with interactions between these dependent variables of both aposymbiotic and symbiotic fragments. A final three-way ANOVA was run on algal cells by analyzing summarized ROS fluorescent values to include the influence of density; this analysis sought to determine significant differences between treatment, season, symbiotic state, and their interaction terms.

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Results

Photosynthetic health

Significant differences in Fv/Fm rates were found overall between temperatures, symbiotic states, and seasons (X 2 = 159; p ≤ 0.05). Between temperatures, Fv/Fm rates were statistically similar regarding summer aposymbiotic (means: 18°C = 0.383; 26°C = 0.330), summer symbiotic (means: 18°C = 0.771; 26°C = 0.753), winter aposymbiotic (means: 18°C =

0.387; 26°C = 0.372), and winter symbiotic fragments (means: 18°C = 0.537; 26°C = 0.588) (Fig.

4; Table 1). Between symbiotic state, symbiotic fragments had significantly higher maximum quantum yield ratios (µ = 0.73) compared to aposymbiotic fragments (µ = 0.361) (p ≤ 0.05), except for comparisons between winter aposymbiotic and winter symbiotic fragments in ambient temperature conditions. Between seasons, no statistical differences were found with aposymbiotic fragments, while summer symbiotic fragments were significantly higher compared to winter symbiotic fragments (p ≤ 0.05).

Pixel Intensity (Symbiotic Density)

Significant differences in pixel intensity (i.e. relative symbiont density) were found overall between temperatures, symbiotic states, and seasons (X 2 = 590.88; p ≤ 0.05). Between temperatures, elevated temperatures had significantly lower pixel intensities with summer and winter aposymbiotic fragments and winter symbiotic fragments (µ = 224.6, µ = 207.3, µ = 149.3, respectively) compared to ambient temperatures (µ = 230.1, µ = 227.1, µ = 178, respectively), implying that these fragments in elevated temperatures had higher symbiotic algae densities (p ≤

0.05) (Figure 5; Table 1). Between symbiotic state, symbiotic fragments (µ = 153.3) had significantly lower pixel intensities compared to aposymbiotic fragments (µ = 223.6) (p ≤ 0.05).

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Between seasons, summer symbiotic fragments had lower average pixel intensities compared to winter symbiotic fragments from ambient treatments (p ≤ 0.05). Pixel intensities with symbiotic fragments in elevated temperatures and between both temperatures with aposymbiotic fragments found no influence of season.

Imaging Flow Cytometry (IFCM)

When comparing the ROS fluorescence of aposymbiotic fragments across all the conditions tested, temperature, season of collection, cell type, and the interaction between season and cell type were significant factors (Table 2). Algal symbionts had significantly higher ROS fluorescence (µ = 22264) than host tissue cells (µ = 4089.2) between all seasons and treatments

(Fig. 6; p ≤ 0.05). Differences in ROS fluorescence regarding temperatures were similar.

Between seasons, summer had significantly higher ROS fluorescence in algal cells (µ = 25647.7) compared to winter aposymbiotic individuals (µ = 17752.5) (p ≤ 0.05). Between the interaction term of all three dependent variables, algal cells from summer aposymbiotic individuals had significantly higher ROS concentrations in ambient temperature treatments (µ = 27257.3) compared to algal cells from winter ambient individuals (µ = 17973.1) (p ≤ 0.05).

When comparing the ROS fluorescence of symbiotic fragments across all the conditions tested, temperature, season of collection, cell type, and the interaction terms of treatment and cell type and between season and cell type influenced ROS fluorescence (Table 2). Algal symbionts had significantly higher ROS fluorescence (µ = 26723.3) compared to host tissue cells (µ =

3900.8) (Fig. 7; p ≤ 0.05). Between temperatures, algal cells within ambient treatments (µ =

27867.5) were significantly higher compared to elevated treatments (µ = 25614.9) (p ≤ 0.05).

Between seasons, summer algal cell fluorescence (µ = 27949.3) was significantly higher

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compared to winter algal cell fluorescence (µ = 24087.4) (p ≤ 0.05). The interaction term between all three dependent variables, summer algal cell fluorescence (means: 18°C = 28901.7,

26°C = 27040.1) was significantly higher compared to winter algal cells (means: 18°C =

25695.5, 26°C = 22479.4) in both temperatures (p ≤ 0.05).

Comparisons of algal cell ROS fluorescence between symbiotic and aposymbiotic individuals (fluorescent values summed) found that season, symbiotic state, and the interaction term between treatment and season significantly influenced ROS fluorescence in algal cells

(Table 2). Targeted comparisons found that season significantly influenced ROS fluorescence in both aposymbiotic and symbiotic algal cells in ambient temperature treatments (p ≤ 0.05).

Symbiotic algal cell ROS fluorescence was significantly higher compared to aposymbiotic ROS fluorescence overall, from both ambient and elevated treatments (p ≤ 0.05). ROS fluorescence between symbiotic state when comparing summer and winter corals found significantly higher

ROS fluorescence in symbiotic algal cells compared to aposymbiotic algal cells (p ≤ 0.05). In addition, the interaction term between all dependent variables found significantly higher ROS fluorescence in summer aposymbiotic algal cells in ambient and elevated treatments compared to winter aposymbiotic algal cells (p ≤ 0.05) and significantly higher fluorescence in winter symbiotic algal cells in ambient and elevated treatments compared to winter aposymbiotic algal cells (p ≤ 0.05).

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Discussion

Photosynthetic health (Fv/Fm)

Maximum quantum yield (Fv/Fm) within PSII yielded no significant differences of symbiotic corals between ambient and elevated temperature treatments in both collections, challenging our initial hypothesis (Fig. 4; Table 1). Our summer Fv/Fm data compared to A. poculata data from Aichelman et al. (2019) were much higher but show similar values over the winter months. We surmise that our Fv/Fm values may be higher as a consequence of consistent feeding. Borell and Bischof (2008) observed that feeding corals during an experimental treatment mitigates the negative consequences associated with thermal stress on

Symbiodiniaceae; maximum potential quantum yield was 50-70% higher in individuals that were on a fed diet compared to starved individuals (Borell and Bischof 2008). Another possible explanation is potentially high nitrogen exposure. Weekly water chemistry results confirmed

- high NO3 levels (>4 ppm), most likely influenced by consistent feeding of Artemia nauplii which occurred on a weekly basis. Shantz and Burkepike (2014) and Marubini and Davies

(1996) showed that constant nitrate concentrations benefits the photobiology and density in symbiotic coralline algae, implying the density of B. psygmophilum may have influenced overall

Fv/Fm rates (Warner et al. 2002). In addition, a final explanation is surmised from the low irradiance during experiments (~60 µmol photons m-2 s-1) mimicking in situ conditions. In

Aichelman et al. (2019), a photosynthesis vs. irradiance curve (P-I curve) was used to determine the saturating irradiance for photosynthesis in A. poculata; Aichelman’s study found that 400

µmol photons m-2 s-1 was considered to be the saturating irradiance for A. poculata. Ralph and

Gademann (2005) identify the correlation of irradiance and Fv/Fm and furthermore, Gorbunov et al. (2001) identified that Fv/Fm ratios were influenced by irradiance adaptation. In this case, our

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low irradiance exposure could have potentially caused our Fv/Fm ratios from symbiotic A. poculata to be significantly higher compared to other Fv/Fm data with symbiotic A. poculata such as data in Aichelman et al. (2019).

Maximum quantum yield (Fv/Fm) values from summer vs. winter collections were higher in the summer, indicating differences in seasonality with maximum quantum yield associated with photosystem II (PSII). Values of PSII ranging from 0.6 to 0.7 indicate healthy physiological function (Kemp et al. 2014); lower PSII values suggest stress. Due to the quiescent state of A. poculata in the winter months, reductions in symbiont density are common (Jacques et al. 1983;

Dimond and Carrington 2008), resulting in lower Fv/Fm rates (Higuchi et al. 2015) and not inherently correlated to stress. We postulate that the presence of endolithic algae, such as

Ostreobium species (Bornet & Flahault, 1889), associated with our coral, may have contributed to the observed higher Fv/Fm values compared to those reported Aichelman et al. (2019).

Jacques et al. (1983) observed influence of endolithic algae with photosynthetic rate regardless of symbiont density since these populate near the corallum surface (i.e. the calcium carbonate skeleton). On a seasonal basis, Hassenrück et al. (2013) suggests that the presence of endolithic algae within Desmophyllum dianthus (Esper, 1794), an azooxanthellate cold-water coral, showed higher proportions of Fv/Fm (i.e. healthier photobiology in PSII) values in summer months compared to winter months. Yamazaki et al. (2008), who studied endolithic-infected and non- infected Acropora digitifera, also observed infected colonies having significantly higher Fv/Fm rates compared to non-infected colonies. To further postulate the influence of endolithic algae, we compare our total pixel intensity and maximum quantum yield data (Fig. S3) and observed a negative relationship indicating that low pixel intensity (high symbiont density) relates to high maximum quantum yield ratios. However, this relationship does not align when comparing

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maximum quantum yield (Fig. 4) and pixel intensity values (Fig. 5) of winter symbiotic A. poculata, suggesting the influence of endolithic algae in winter A. poculata.

The Fv/Fm values (i.e. PSII) within our aposymbiotic corals remained much lower compared to the symbiotic corals, but were similar to results in Aichelman et al. (2019) and

DeFilippo et al. (2016), with Fv/Fm values between 0.3 and 0.5, which is considered normal with symbiotic densities below 105 cm-2 (Cummings 1983). When we compare maximum quantum yield ratios in temperatures of 26 °C, no changes occurred within our Fv/Fm data, but found decreases in Fv/Fm from Aichelman et al. 2019. This suggests that Ostreobium endolithic algae contributed to overall Fv/Fm rates, as similarly seen with our symbiotic A. poculata.

Pixel Intensity (Symbiotic Density)

Aposymbiotic fragments within elevated temperature treatments resulted in lower pixel intensities compared to their ambient treatment counterparts in both collections, with these findings potentially influenced by Osterobium endolithic algae. The photo-quantification method by Winters et al. (2009) compared chlorophyll a and c2 pigment measurements to pixel intensities from a photo quantification method; this comparison found a negative relationship

2 between these data (r =0.82) indicating higher pixel intensities had reduced chlorophyll a and c2 pigment. Chlorophyll a pigments are indistinguishable between symbiotic coralline algae and endolithic algae (Kleppel et al. 1989). The concept of indistinguishable chlorophyll a correlate to endolithic algae influencing lower pixel intensities within elevated temperature treated aposymbiotic fragments by means of chlorophyll a pigments. Additionally, endolithic algae tend to be more prominent in coral colonies associated with reduced Symbiodiniaceae concentrations

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allowing for such algae to congregate within the coral skeletons, as well as increased temperature environments (Carilli et al. 2010; Reyes-Nivia et al. 2013).

Elevated temperatures from summer symbiotic coral have a higher median pixel intensity value overall compared to ambient treatments, albeit no statistical difference was found. The lack of differences between pixel intensities (i.e. relative symbiont density) could be answered by feeding events. Aichelman et al. (2016) demonstrated this, where any amount of feeding within a heterotrophic temperate coral significantly influences algal symbiont density. In addition, other factors such as antioxidants (i.e. catalase and superoxide) are beneficial to reducing bleaching events in coral organisms from increases in ROS (Krueger et al. 2015; Marty-Rivera et al. 2018).

McGinty et al. (2012) further argues that Breviolum psygmophilum, A. poculata’s algal symbiont, is capable of increasing antioxidant concentrations in higher temperatures.

Differences in mean pixel intensity between ambient and elevated temperatures in winter symbiotic corals compared to the summer symbiotic corals shows the influence of extreme temperatures on symbiont density. Dimond and Carrington (2007) showed significant differences in growth rates and chlorophyll densities between seasons, with lower values found in individuals during the winter. Knowing the seasonal changes in symbiont densities, and growth rates (Jacques et al. 1983; Dimond and Carrington 2007; Grace 2017), the exposure to rapid elevated temperatures within a period of weeks rather than months may have caused a severe increase in symbiont density over time in some colonies. When analyzing changes over time, increases in pixel intensity for both temperature treatments in symbiotic fragments from the winter collection are observed. Elevated temperatures may have provided a temporary boost in symbiont densities in the short-term, but eventually succumbed to reduced densities nearing the end of the experimental treatment from thermal stress.

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Reactive oxygen species (ROS) via imaging flow cytometry (IFCM)

In our study, the ROS fluorescence was analyzed between algal and host cells. No statistical differences were found between temperature treatments in host tissue cells. The lack of significance in host tissue comparisons indicate that these temperature treatments were not severe enough to over-produce ROS and detect significant concentrations in host tissues. CM-

H2DCFDA binds to ROS constituents such as hydrogen peroxide (H2O2) and singlet oxygen

1 ( O2). These products occur via photosystem II (PSII) and can be detected in host tissues if large concentrations of ROS are produced from thermal stress events (McGinty et al. 2012).

Comparisons with algal cell ROS fluorescence in summer collections show higher amounts of fluorescence from ambient temperatures compared to elevated temperatures in both aposymbiotic and symbiotic individuals, albeit no significance was found. Similarities between our algal cell responses and those observed in McGinty et al. (2012) are apparent, such that higher levels of ROS fluorescence were observed in ambient temperature treatments compared to elevated temperatures. This may suggest the influence of antioxidants reducing ROS levels in algal symbionts (Gardner et al. 2017; Wietherger et al. 2018). McGinty et al. (2012) described the influence of antioxidants such as super-oxide dismutase (SOD) and catalase (CAT) help mitigate the negative effects of ROS exposure and reduce the overall amount of ROS within algal symbiont cells. They additionally found lower levels of ROS within B. psygmophilum algal cells in higher temperatures, implying the influence of antioxidants as temperatures increase to more stressful scenarios. This leads to an assumption that this coral species had not surpassed its thermal tolerance threshold and is resilient at these higher temperatures (Jacques et al. 1983;

McGinty et al. 2012). In addition, Lesser et al. (1990) explained the relationship of ROS produced intracellularly in Symbiodiniaceae to impact host tissue and their antioxidant

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production to combat the toxicity of ROS (Rehman et al. 2016; Lopes et al. 2018). With our host tissue fluorescence unchanged throughout the entire study, we suggest that host tissue antioxidants were not affected by this short-term temperature anomaly. Host tissues likely acquired antioxidants which provided additional support to mitigate photosynthetically produced

ROS (Lopes et al. 2018).

We observed inversed trends of our winter aposymbiotic algal cell data, which had higher median ROS fluorescence from elevated temperatures compared to ambient temperatures. The high fluorescence from elevated temperatures can imply the generation of antioxidant components could be seasonally driven. Seasonal changes were observed within A. poculata’s microbiome (Sharp et al. 2017) as well as changes in metabolic rates in A. poculata. We suggest a state of quiescence from A. poculata collected during winter months, resulting in less feeding and growth and ultimately less energy available for multiple repair mechanisms to mitigate environmental stressors (Dimond and Carrington 2007). Studies such as Wall et al. (2018) and

Palmer et al. (2011) indicate the reduction of antioxidant production from thermal stress events in some species (i.e. higher ROS levels), but our results are most likely influenced by reduced energy repositories coming from a state of quiescence. We surmise that the seasonal differences in ROS fluorescence may be related to the quiescent state in A. poculata from winter. Dimond and Carrington (2007), Grace (2017), and Sharp et al. (2017) similarly suggest the likelihood of quiescence for A. poculata in colder temperatures, resulting in less growth, symbiont density, and shifts in its microbiome. Comparisons between our maximum quantum yield data and ROS fluorescence show a distinctive negative trend (Fig. S4), where our results with winter collections do not appear to have higher Fv/Fm values (Fig. 4) along with lower ROS fluorescence (Fig. 6). We suggest that quiescence reduces the overall photosynthetic processes

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from less incoming energy (i.e. reduced feeding and light exposure), resulting in lower production of ROS.

Differences in ROS fluorescence between aposymbiotic and symbiotic state were analyzed as a function of cell density with ROS fluorescence compared to comparisons of mean

ROS fluorescence in algal cells in total. Summed ROS fluorescence was used to properly measure statistical significance as comparisons of mean ROS fluorescent values may be statistically similar even though symbiotic A. poculata have higher symbiotic algal densities compared to aposymbiotic individuals. Summed ROS fluorescence corresponds to significantly more ROS in symbiotic individuals compared to aposymbiotic individuals. Higher ROS fluorescence in symbiotic individuals suggests that symbiotic A. poculata will be more susceptible to the stressors of ROS than compared to aposymbiotic A. poculata. However, with

18 °C as the mean temperature Rhode Island populations are exposed to, concentrations of ROS in these ambient treatments should be considered normal. A. poculata are then assumed to be resilient to ROS toxicity at elevated temperatures predicted in the foreseeable future.

Conclusions and implications for future work

Many temperate coral species are understudied regarding fundamental aspects of the coral holobiont, in addition to how climate change will impact them in the future. Our studies suggest B. psygmophilum is a thermally tolerant symbiotic dinoflagellate to temperatures as high as 26 °C and perhaps higher. Future studies should examine the upper thermal limit of A. poculata, even at temperatures that today may seem irrelevant but, in the future, may very well be “normal”. We suggest that A. poculata is a thermally tolerant coral species capable of maintaining consistent maximum quantum yield ratios, symbiont densities, and maintaining low

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ROS concentrations from thermal stress exposure. Intracellular and extracellular antioxidants, such as CAT and SOD, from algal symbionts and host tissue we surmise are the primary driver(s) in mitigating oxidative stress in this species in thermal stress events. With elevated temperature treatments, lower ROS fluorescence was found when compared to ambient temperature. Ambient temperatures (18 °C) are the mean temperature that Rhode Island populations are exposed to, suggesting that such conditions are not instigating oxidative stress.

Dimond and Carrington (2007) have suggested that this symbiotic relationship with B. psygmophilum only provides partial benefits to this heterotrophic, facultative symbiotic coral species suggesting that oxidative stress in ROS is not as impactful compared to tropical coral species with obligate symbiosis. This study also helps to support the concept of quiescence associated with seasonal changes within A. poculata, where reduced metabolism, symbiont density, and feeding occur during the winter months. However, our study further expands such work by showing reductions in ROS concentrations are directly influenced by quiescence. This study helps to provide a foundation to focus future work on aspects such as host and symbiont antioxidant concentrations as well as host derived reactive nitrogen species (RNS; NO- via

Hawkins et al. 2014) to explore other avenues of thermal stress impacts on A. poculata. In addition, future work pertaining to multiple thermal extremes such as 30 °C, 32 °C, and 34 °C may help clarify the limits of thermal resilience in this species and how ROS and antioxidant concentrations change over time.

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Acknowledgements

We acknowledge our funding resources, NASA’s Michigan Space Grant Consortium

(Grant #223872-40826-200), Grand Valley State University (Presidential Grant), and the Annis

Water Resource Institute (Graduate Student Fellowship) for making this research possible.

Special thanks to S. Grace, D. Nielsen, and J. Cervino for their insight and help throughout this research. Additional thanks for D. Gates and C. Gilmore for their help with experiments. The authors extend appreciation to K. Sharp, R. Rotjan, S. 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.

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Figures

Figure 1: Map of Narragansett Bay, RI, USA. Red triangle indicates Astrangia poculata collection site in Narragansett Bay (41°28'40.8"N, 71°21'45.8"W).

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Figure 2: Schematic representation of experimental aquarium system. Each temperature treatment consisted of a recirculating system with eight tanks and a shared sump. Astrangia poculata colonies were evenly spread throughout all eight tanks.

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Figure 3: Images of individual cells from Astrangia poculata stained with CM-H2DCFDA. (A) Brightfield image dictating a photographed Breviolum psygmophilum cluster. (B) FITC channel fluorescence of B. psygmophilum. (C)

Auto-fluorescence of B. psygmophilum in channel 5. (D) Brightfield image dictating a photographed host cell of A. poculata. (E) FITC channel fluorescence of A. poculata host cell. (F) Auto-fluorescence is not shown within channel

5 indicating that it is a cluster of host cell tissue.

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Figure 4: Boxplots of maximum quantum yield ratios (Fv/Fm) in aposymbiotic/symbiotic fragments between temperature treatments and seasonal collection (top) and a pairwise comparison table depicting statistical differences in Fv/Fm between each treatment (bottom). Shades of blue indicate statistical comparisons between symbiotic state, shades of red indicate comparisons between season of collection, and significant p-values are depicted in bold text

(bottom). “NR” depicts non-relevant comparisons. Boxplots include maximum/minimum values, inner/outer quartiles, and the median value, in addition to outlier data shown as circles. A Kruskal-Wallis test on all Fv/Fm values found significance between treatments, seasons, and symbiotic state (X 2 = 159; p ≤ 0.05).

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Figure 5: Boxplots of pixel intensity in aposymbiotic/symbiotic fragments between temperature treatments and seasonal collection (top) and a pairwise comparison table depicting statistical differences in pixel intensity between each treatment (bottom). Shades of blue indicate statistical comparisons between symbiotic state, shades of red indicate comparisons between season of collection, shades of green indicate comparisons between temperature treatment, and significant p-values are depicted in bold (bottom). “NR” depicts non-relevant comparisons. Boxplots include maximum/minimum values, inner/outer quartiles, and the median value, in addition to outlier data shown as circles. A Kruskal-Wallis test on all pixel intensity values found significance between treatments, seasons, and symbiotic state (X 2 = 590.88; p ≤ 0.05).

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Figure 6: Boxplots of ROS fluorescence in symbiotic algae and host tissue cells from aposymbiotic fragments between temperature treatments seasonal collection (top) and a pairwise comparison table depicting statistical differences in ROS fluorescence between each treatment (bottom). Shades of blue indicate statistical comparisons between symbiotic state, shades of red indicate comparisons between season of collection, and significant p-values are depicted in bold (bottom). “NR” depicts non-relevant comparisons. Boxplots include maximum/minimum values, inner/outer quartiles, and the median value, in addition to outlier data shown as circles.

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Figure 7: Boxplots of ROS fluorescence in symbiotic algae and host tissue cells from symbiotic fragments between temperature treatments seasonal collection (top) and a pairwise comparison table depicting statistical differences in

ROS fluorescence between each treatment (bottom). Shades of blue indicate statistical comparisons between symbiotic state, shades of red indicate comparisons between season of collection, and significant p-values are depicted in bold (bottom). “NR” depicts non-relevant comparisons. Boxplots include maximum/minimum values, inner/outer quartiles, and the median value, in addition to outlier data shown as circles.

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Three-way ANOVA: Aposymbiotic ROS Df Sum sq Mean sq F-value p-value Treatment 1 1.07E+08 1.07E+08 4.04 0.0476 Season 1 2.81E+08 2.81E+08 10.619 0.0016 Cell Type 1 7.68E+09 7.68E+09 290.318 2.00E-16 Treatment:Season 1 3.65E+07 3.65E+07 1.38 0.2434 Treatment:Cell Type 1 3.95E+07 3.95E+07 1.492 0.2252 Season:Cell Type 1 2.23E+08 2.23E+08 8.444 0.0047 Treatment:Season:Cell Type 1 4.17E+06 4.17E+06 0.158 0.6922 Residuals 85 2.25E+09 2.64E+07

Table 1: Three-way ANOVA results of ROS fluorescence data from aposymbiotic Astrangia poculata fragments analyzing treatments, seasons, cell type, and the interaction terms. Bolded p- values indicate significance.

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Three-way ANOVA: Symbiotic ROS

Df Sum sq Mean sq F-value p-value

Treatment 1 3.18E+07 3.18E+07 4.459 0.0368

Season 1 1.51E+08 1.51E+08 21.17 1.06E-05

Cell Type 1 1.66E+10 1.66E+10 2324.307 2.00E-16

Treatment:Season 1 2.53E+05 2.53E+05 0.035 0.851

Treatment:Cell Type 1 3.66E+07 3.66E+07 5.14 0.0252

Season:Cell Type 1 5.35E+07 5.35E+07 7.51 0.0071

Treatment:Season:Cell Type 1 9.55E+06 9.55E+06 1.341 0.2493

Residuals 85 8.48E+08 7.13E+06

Table 2: Three-way ANOVA results of ROS fluorescence data from symbiotic Astrangia poculata fragments analyzing treatments, seasons, cell type, and the interaction terms. Bolded p- values indicate significance.

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Three-way ANOVA: Algal cell ROS

Df Sum sq Mean sq F-value p-value

Treatment 1 1.40E+09 1.40E+09 5.32 0.0232

Season 1 8.69E+08 8.69E+08 33.1281 1.01E-07

Symbiotic State 1 3.93E+08 3.93E+08 14.9591 1.99E-04

Treatment:Season 1 3.78E+06 3.78E+06 0.1442 0.705

Treatment:Symbiotic State 1 7.16E+05 7.16E+05 0.0273 0.8691

Season:Symbiotic State 1 8.34E+07 8.34E+07 3.1787 0.0777

Treatment:Season:Symbiotic State 1 3.32E+07 3.32E+07 1.2659 0.2633

Residuals 97 2.55E+09 2.62E+07

Table 3: Three-way ANOVA results of summed ROS fluorescence data from algal cells analyzing treatments, seasons, symbiotic state, and their interaction terms. Bolded p-values indicate significance.

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Supplemental Material

A visual analysis of the summer collected fragments revealed that some coral, originally aposymbiotic, had developed significant concentrations of B. psygmophilum while acclimating to aquarium conditions over a five-month period. A preliminary photographic analysis of pixel intensities was run to separate aposymbiotic-like colonies from symbiotic-like colonies (Fig. S1).

Any aposymbiotic fragments below the mean line (see Fig. S1) were assumed to be symbiotic- like colonies, while those above the line were likely aposymbiotic-like colonies. These

“symbiotic-like” colonies were placed under low/no-light conditions between fragmentation and experiment periods to allow them to expel as many symbionts as possible. No logistical errors occurred with winter coral experiments; accumulations of symbionts were not noticed with the aposymbiotic corals from winter collections before any experimental analysis.

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Supplemental Figures

Figure S1: Preliminary pixel intensity analysis of aposymbiotic (A) and symbiotic (B) A. poculata fragments. This analysis identified aposymbiotic corals that were considered

“symbiotic” (i.e. within the 2±SE region or below) and placed these fragments under no-light conditions prior to experiments to reduce B. psygmophilum concentrations. The black line represents the mean pixel intensity from all fragments, the red lines represent 2±SE of the mean, and the blue asterisks represent these aposymbiotic fragments needed for no-light conditions.

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Figure S2: Plot comparing fluorescent intensities of cellular material between Channel 2 (FITC) and Channel 5 (auto-fluorescence) of a processed A. poculata sample. Preliminary analyses helped determine the spatial selection of the “Algal collection” and “Host collection” gates from which Channel 2 intensity data was derived.

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Figure S3: Linear model comparison between mean pixel intensity and mean maximum quantum yield. This model helps explain the negative relationship seen between these two measurements

(Correlation coefficient: -0.71; p≤0.05).

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Figure S4: Linear model comparison between mean maximum quantum yield and mean ROS fluorescence. This model helps explain the negative relationship seen between these two measurements (Correlation coefficient: -0.70; p≤0.05).

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Chapter 3 – Manuscript Submission – Journal of Experimental Biology

Full Title: Ecological simulation of baseline immunity indicates potential disease susceptibility in Astrangia poculata

Running Title: Baseline immunity in Astrangia poculata

Tyler E. Harman1, Daniel J. Barshis2, Briana Hauff Salas3, Sarah E. Hamsher1,4, Kevin B. Strychar1

1Annis Water Resource Institute – Grand Valley State University 740 West Shoreline Dr, Muskegon, MI 49441

2Department of Biology – Old Dominion University 5115 Hampton Blvd, Norfolk, VA 23529

3 Department of Math and Science – Our Lady of the Lake University 411 SW 24th St, San Antonio, TX 78207

4Department of Biology– Grand Valley State University 1 Campus Drive, Allendale Charter Twp, MI 49401

Communicating author email address: [email protected]

Keywords: immunity, thermal stress, melanin synthesis, disease, Astrangia poculata

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Summary statement For the first time, this research aimed to understand immunity within a facultatively symbiotic temperate coral species by simulating disease exposure and the influence of temperature on these immune responses.

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Abstract Global warming currently devastates corals by increasing ocean temperatures resulting in large-scale bleaching events. Coral diseases have risen alongside bleaching in the past few decades, severely increasing mortality in tropical corals. As these continue, the response of corals in temperate systems are unknown. This research focuses on Astrangia poculata and how it will respond to increased temperature and disease exposure. This study examined colonies collected from Narragansett Bay located in Rhode Island to comparatively assess ambient (18 °C) versus elevated temperatures (26 °C) in the presence of disease (i.e. lipopolysaccharide isolated from E. coli O127:B8). This study assessed prophenoloxidase and melanin via absorbance to determine A. poculata’s immune response. No significant differences were found in prophenoloxidase between symbiotic state, treatments, or season. Melanin had higher concentrations in symbiotic compared to winter aposymbiotic coral (p≤0.05). This study is the first to report an immune response in A. poculata. Overall, we observed low melanization across treatments indicating potential susceptibility to disease. It is plausible that the surface mucus layer may contribute to protection where the upregulation of a melanin-synthesis pathway is not necessary. Also, we suggest that coral lectins within the complement pathway play a larger role in immunity in A. poculata, where higher melanin concentrations in symbiotic individuals were observed. Although this study introduces the plausibility of disease susceptibility in A. poculata, future studies should investigate additional parameters such as lectins to further understand the entirety of innate immunity in this temperate species.

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Introduction Oceanic temperatures have been impacted worldwide due to increases in atmospheric carbon dioxide (CO2) from anthropogenic inputs such as fossil fuel combustion (Carpenter et al., 2008; Cheng et al., 2019). Increases in ocean temperatures affect many organisms including corals, which require specific temperatures, nutrient concentrations, salinity, and light availability in order to maintain the productive reef ecosystems (Hoegh-Guldberg, 2011). Global warming has increased ocean temperatures, causing coral bleaching events which expel their symbiotic algae. While global warming impacts corals by bleaching (Douglas, 2003; Pandolfi et al., 2011), global warming also exacerbates the presence of disease on coral reefs. Previous studies have shown that physiological stressors such as temperature allow opportunistic pathogens to more easily infect thermally stressed corals, further increasing mortality in these organisms (Sokolow, 2009; Hoegh-Guldberg and Bruno, 2010; Palmer et al., 2010; Maynard et al., 2015; van Woesik and Randall, 2017). Several components of the coral holobiont can provide disease resistance such as the microbial composition within the surface mucus layer (SML) (Krediet et al., 2013; Nguyen-Kim et al., 2015), coral lectin from the complement pathway (Palmer et al., 2012), and immune components such as prophenoloxidase (PPO) and melanin in the melanin-synthesis pathway (Palmer et al., 2011a,b). The melanin-synthesis pathway is a signaling pathway in invertebrates that is initialized by a recognition protein (i.e. peptidoglycan recognition protein; PGRP) that cascades other signaling proteins such as PPO and phenoloxidase (PO), leading to the production of melanin (Fig. 1). Melanin is utilized in cnidarian immunity by encapsulating foreign objects and pathogens and triggering responses such as antimicrobial defense (Mydlarz et al., 2009; Palmer et al., 2012; Mansfield and Gilmore, 2019). The complement pathway is another signaling pathway observed in cnidarian immunity, which tags invading pathogens for opsonization (i.e. cell death) by using lectins from coral host cells (Zhou et al., 2018; Mansfield and Gilmore, 2019). This pathway is also involved in symbiotic interactions; symbionts bypass this opsonization by binding to lectins using cell-surface glycans (Kvennefors et al., 2010). Increases in ocean temperatures can impact disease resistance by reducing immune responses of the coral holobiont (Palmer et al., 2008, 2010; Mydlarz and Palmer, 2011). As such, disease resistance associated with the coral host shows that some species are more disease resistant than others; differences between species can be found by the differences in the

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upregulation of signaling proteins within pathways like the melanin-synthesis pathway (Mydlarz et al., 2009; Merselis et al., 2018; Muller et al., 2018). Increases in disease are more immediately relevant to tropical coral species where most of these impacts occur (Brown, 1996; Garzón- Ferreira et al., 2001; Muller et al., 2020). With increasing ocean temperatures hypothesized to continue to occur over the next several decades from anthropogenic influence, impacts from diseases are suggested to increase in tandem. The prevalence of diseases from future global warming could cause corals in temperate environments to also experience these events in the future, if not already in some areas (Wada et al., 2018). Astrangia poculata (Ellis and Solander, 1786) is a coral species that lives off the east coast of the United States of America within temperate systems as far north as Massachusetts (Dimond et al., 2013) and sub-tropical regions as far south as southern Florida (Thornhill et al., 2008; Jaap et al., 2015). This species maintains an exclusive symbiotic relationship with Breviolum psygmophilum (J.E. Parkinson & LaJeunesse, 2018), a symbiotic dinoflagellate from the family Symbiodiniaceae. A. poculata is unique as it demonstrates a facultative symbiosis, either as a symbiotic colony or an aposymbiotic colony (i.e. symbiont density less than 105 cm-2), and utilizes heterotrophy as its main source of energy. To the best of our knowledge, there are no reports characterizing disease associated with A. poculata despite southern populations of this coral potentially being exposed to diseases such as yellow-band disease (YBD), white-band disease (WBD), and stony coral tissue loss disease (SCTLD) that continue to severely impact many tropical coral species in southern Florida (Cervino et al., 2004, 2008; Gignoux-Wolfsohn et al., 2012; Gignoux-Wolfsohn and Vollmer, 2015; Aeby et al., 2019; Muller et al., 2020). In the northern range of this coral species, Rotjan et al. (2019) suggests that disease could be introduced by way of microplastics. Their study suggests A. poculata populations in urban coastal waters prefer E. coli colonized microplastics over other food sources (Rotjan et al., 2019). As such, it is plausible that such pathogens (i.e. E. coli) have been ingested by A. poculata (Jiang et al., 2018; Lamb et al., 2018; Tang et al., 2018). This study investigates the influence of temperature and season on disease susceptibility in A. poculata. We compared symbiotic and aposymbiotic colonies from Narragansett Bay in Rhode Island to understand its potential disease resilience by exposing these corals to lipopolysaccharide (LPS), an important pathogen associated molecular pattern (PAMP) associated with gram-negative bacteria that is involved with triggering multiple pathways in

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cnidarian immunity (Palmer et al., 2011b; Hauff et al., 2014). Colonies were subjected to two treatments: ambient (18 °C) and LPS exposure or heat stress (26 °C) and LPS exposure from corals collected in the summer and winter seasons. The exposure of LPS simulates disease exposure (Palmer et al., 2011b) and we evaluated disease susceptibility by measuring melanin and PPO to identify resilience in this coral species. We hypothesize that LPS exposure coupled with elevated temperatures will significantly increase the coral hosts PPO and melanin concentrations compared to ambient temperature treatments. In addition, we hypothesize that symbiotic temperate coral colonies will have higher PPO and melanin concentrations compared to aposymbiotic colonies. Lastly, we hypothesize that winter corals will have significantly higher concentrations of PPO and melanin compared to summer corals.

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Methods

Coral Collection and Husbandry

Symbiotic and aposymbiotic A. poculata were collected at Fort Wetherill State Park in Jamestown, Rhode Island (Fig. 2; 41°28'40.8"N, 71°21'45.8"W) using SCUBA on 14 July 2019 and 20 February 2020 (RIDEM permit #429; Type 1). Collected colonies were transferred to plastic bags filled with seawater and kept on ice while transported to the Annis Water Resource Institute in Muskegon, MI within 48 hours. Colonies were maintained in custom-built, recirculating aquarium systems (Fig. 3) using artificial seawater (deionized water and Instant Ocean Reef Salt) kept at 18°C, with light intensities at a maximum of ~60 μmol m-2 s-1. Full- spectrum LED lights (Bozily, Inc., Beijing, China) were programmed to simulate diurnal patterns (07:00 to 19:00 light) and calibrated using an Apogee SQ-420 Smart Quantum Sensor (Apogee Instruments, Logan, UT). Hatched brine shrimp (Artemia nauplii) were used to feed corals three days per week, in addition to biweekly water changes and weekly water chemistry measurements (i.e. nitrate, phosphate, magnesium, alkalinity, and calcium).

Experimental design

Corals from both seasonal collections were divided using a Gryphon C-40 bandsaw (Gryphon Corporation, Sylmar, CA) to generate fragments that were glued to acrylic glass discs using IC-gel (Bob Smith Industries, Atascadero, CA). A total of 51 symbiotic and 34 aposymbiotic fragments were made from summer collections, whilst winter collections resulted in 19 symbiotic and 22 aposymbiotic fragments. A longer acclimation period occurred for summer collections and resulted in aposymbiotic coral developing substantial concentrations of symbionts (B. psygmophilum). A detailed description on reducing symbiont density in aposymbiotic A. poculata prior to experiments can be found in the provided supplemental material (Fig S1).

Fragments had a two-week acclimation period before experiments began. Two identical recirculating systems were used to test the combined influences of heat stress and disease impacts by exposure to ambient temperatures (18°C) with LPS or to elevated temperatures (26°C) and LPS (Fig. 3). Temperature ramps were completed over a period of two weeks at a

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rate of +0.5 °C per day until the desired temperature of 26 °C was met. Fragments were placed into smaller plastic containers filled with artificial seawater with added LPS (5µg mL-1; Simga- Aldrich, St. Louis, MO) that were placed inside of the tanks to maintain treatment temperatures, but above the water surface to prevent LPS contamination to the rest of the aquarium system (Palmer et al., 2011b). These fragments were exposed to LPS for a total of 12 hours (08:00 to 20:00). After exposure, fragments were then flash-frozen in liquid nitrogen, wrapped in foil, and stored in a -80 °C freezer until further analysis.

Immunity parameters via microplate assays

Methods for melanin and prohpenoloxidase (PPO) analysis followed Palmer et al. (2011b). Briefly, flash frozen coral fragments were allowed to thaw and then air-blasted to remove tissue into plastic bags filled with 25mL of phosphate buffered saline (PBS) solution (50 mmol L-1 at pH 7.8; 50µmol L-1 dithiothreitol). Tissue was homogenized in a pestle and mortar and placed into 50mL Falcon tubes after homogenization. Three aliquots (0.5mL) were immediately separated in 1.5mL Eppendorf tubes for melanin analysis. All microplate assays were done with a Tecan SunriseTM microplate reader (Tecan Group Ltd., Männedorf, Switzerland) with MagellanTM Software (Tecan Group Ltd., Männedorf, Switzerland).

Melanin concentrations were determined by taking triplicates of the tissue slurry and adding 0.3mL of 10M NaOH, gently vortexed with a Thermomixer R (Eppendorf, Hamburg, Germany) for 40 minutes at 300rpm, and dissolved overnight for twelve hours. Samples were then vortexed, followed by three aliquots (200µL) of each sample added to 96-well plates, with known concentrations of commercial melanin dissolved in 10M NaOH to create a standard curve on each analyzed plate. Samples were measured at 410nm to determine absorbance concentrations, and all absorbance data was averaged for each fragment (summer symbiotic: n = 49, winter symbiotic: n = 18, summer aposymbiotic: n = 33, winter aposymbiotic: n = 22). Data were converted from absorbance values to milligrams of melanin cm-2 (Palmer et al., 2011b). Surface areas were calculated using a modified paraffin wax technique following Holmes (2008). Coral fragment skeletons were dipped in paraffin wax, allowed to cool, and weighed twice to obtain a difference between the first and second wax covering. This difference between the first and second wax coverings was then used to estimate surface area as follows (Holmes, 2008):

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푐푚 푆푢푟푓푎푐푒 푎푟푒푎 (푐푚) = 34.32 × 푑푖푓푓푒푟푒푛푐푒 푖푛 푚푎푠푠 (푔) 푔

The remaining tissue slurry in the 50mL Falcon tubes were then flash-frozen in liquid nitrogen, re-thawed, and homogenized using a pestle and mortar. This slurry was placed on ice after homogenization for 5 minutes allowing any proteins become resuspend in solution. PPO samples were subsequently vortexed after homogenization with steel beads, placed on ice for five minutes, and centrifuged for five minutes (2400g at 4°C) using an Allegra 25R centrifuge (Beckman Coulter Life Sciences, Indianapolis, IN). Triplicate aliquots of the supernatant (1mL) were separated from the remaining tissue slurry and samples were frozen at -80 °C until ready for use. Each sample (20µL) was added into 96-well plates, in addition to PBS (40µL at 50mM; pH 7.5), Trypsin (25µL at 0.1mg mL-1), and L-DOPA (30µL at 10mM) (Sigma Aldrich, St. Louis, MO) added to each well. Absorbance was measured at 490nm over a 15-minute period to observe change in absorbance, and all absorbance data was averaged for each fragment (summer symbiotic: n = 47, winter symbiotic: n = 19, summer aposymbiotic: n = 31, winter aposymbiotic: n = 21). Protein extracts for PPO analysis were standardized using a Bradford Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA), with change in absorbance normalized to mg of protein-1 (Palmer et al., 2011b).

Statistical analyses

All data were recorded using Microsoft Excel (Microsoft Corporation 2020) and all data manipulation and statistical analyses were done within RStudio (RStudio Team, 2020) in R Version 4.0.0 (R Core Team, 2020). All data were tested for normality using Shapiro-Wilk tests and transformed if needed to fulfill normality requirements. Three-way ANOVA analyses were used to detect differences in both melanin and PPO concentrations between temperature treatments, season, symbiotic state, and their interaction terms. Tukey’s HSD post-hoc tests were used to further understand the significance of targeted comparisons.

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Results

Symbiotic state primarily influenced melanin concentrations (F = 11.169; p ≤ 0.05), whereas no significance was found between treatments or seasons (Table 1). Symbiotic fragments in ambient temperatures had significantly higher melanin concentrations (µ = 0.0133 mg melanin cm-2) (p ≤ 0.05) compared to aposymbiotic fragments in ambient temperatures (µ = 0.0081 mg melanin cm-2), regardless of season (Fig. 4). In winter collections, symbiotic fragments had significantly higher melanin concentrations (µ = 0.0137 mg melanin cm-2) compared to aposymbiotic fragments (µ = 0.008 mg melanin cm-2) (p ≤ 0.05) regardless of temperature treatment (Fig. 5; Table S1). No significance was found between temperatures (18 °C and 26 °C) and we did not observe any significant difference in targeted comparisons between all three dependent variables.

No significant differences were influenced by temperature treatments, season, and symbiotic state with PPO in A. poculata (Table 2; Fig. 6). However, symbiotic state was considered marginally significant (p = 0.0586), implying a potential trend albeit no statistical significance being found.

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Discussion

In response to exposure of the pathogen associated molecular pattern (PAMP), lipopolysaccharide (LPS), significant differences of PPO in A. poculata were not observed. Signaling proteins in the melanin-synthesis pathway have been suggested to be model components to measure baseline immunity in corals (Palmer et al., 2010). Generally, coral organisms with upregulations in phenoloxidase (PO), subsequently activated from prophenoloxidase (PPO), are suggested to be less susceptible to disease impacts (Palmer et al. 2011a). In comparison of our PPO results to Palmer et al. (2011b), they observed a similar lack of differences in PPO in Orbicella faveolata Ellis and Solander (formerly Montastraea faveolata). The lack in significant differences from Palmer et al. (2011b) suggested that other immunity parameters such as the complement pathway may have priority over melanin-synthesis pathway responses, or that this species did not upregulate PPO due to aquarium conditions (i.e. stability in environmental parameters) (Palmer et al., 2011a; Wall et al., 2018). Treatment and/or duration in this study may not be significantly adverse to detect change in early components in the melanin-synthesis pathway, as previously noted when detecting differences in reactive oxygen species (ROS) in A. poculata (Chapter 2), as other studies have indicated elevated temperatures upregulate melanin-synthesis components (Mydlarz et al., 2009a). Hauff et al. (2014) observed the rapid nature of this signaling pathway when exposed to LPS, suggesting that periods longer than eight hours may not produce any significance when analyzing early signaling proteins in immune responses.

Higher melanin concentrations in symbiotic compared to aposymbiotic individuals indicate an influence of symbiosis on immune responses in A. poculata. Coral lectins may also be involved in immune responses (Wood-Charlson et al., 2006; Kvennefors et al., 2008, 2010; Zhou et al., 2017, 2018; Mansfield and Gilmore, 2019). The function of lectins as an immunity role in A. poculata could explain the lack of differences in PPO concentrations and the differences in melanin between symbiotic state. Palmer et al. (2011b) suggests that the lack of differences in PPO in M. faveolata could favor other parameters (i.e. coral lectins) compared to melanin- synthesis components. Zhou et al. (2018) hypothesize that the complement pathway may be involved in cnidarian immunity and be triggered by PAMPs such as LPS. Mansfield and Gilmore (2019) also suggested the downregulation of the complement pathway when mutualistic

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Synbiodiniaceae interact with the coral host. Coral lectin may be associated with cell-surface glycans on B. psygmophilum and are unable to activate opsonization within the complement pathway and rely on the melanin-synthesis pathway as a secondary immune response. The higher melanin concentrations in symbiotic individuals compared to aposymbitoic individuals may suggest this melanin-synthesis pathway as a secondary immune response.

In addition, higher concentrations of melanin were observed in the symbiotic versus aposymbiotic colonies from winter collections, indicating an influence associated with season. The surface mucus layer (SML) in corals have been suggested to be the first line of defense when exposed to pathogenic bacteria, but the SML can be affected by environmental parameters such as high temperatures, degrading their protective properties (Krediet et al., 2013; Nguyen- Kim et al., 2015; Merselis et al., 2018). In addition, SML in tropical corals are largely influenced by energetic reserves from algal symbionts, suggesting that bleaching may also influence the structure of the SML (Merselis et al., 2018). With the use of heterotrophy in A. poculata, the SML may not be as affected by higher temperatures as tropical corals and may be an important defense against pathogenic bacteria, preventing the need to utilize components in the melanin- synthesis pathway. However, the microbiome in A. poculata changes on a seasonal basis, where winter significantly effects the microbiome between aposymbiotic and symbiotic A. poculata, but spring, summer, and fall microbiomes resulted in no significant differences between symbiotic state (Sharp et al., 2017). Melanin concentrations were significantly higher in winter symbiotic Astrangia compared to winter aposymbitoic individuals. The difference in microbiomes may be influential in the upregulation of melanin-synthesis components, where symbiotic individuals may be more reliant on the melanin-synthesis pathway to mitigate disease impacts than aposymbiotic individuals.

To the author’s knowledge, this is the first time that immunity has been identified in a facultative, temperate coral species. Based on the results in prophenoloxidase (PPO) and melanin concentrations in A. poculata, it is presumed that this species is disease susceptible as some studies have identified low concentrations in melanin as being susceptible to disease (Palmer et al., 2010, 2011a; Toledo-Hernandez and Ruiz-Diaz, 2014). Future studies should subject A. poculata to particular diseases to understand disease susceptibility in-vivo rather than lab- controlled E. coli PAMP exposure, as Narragansett Bay in Rhode Island, USA is an urbanized

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coastal marine system, where microplastics constantly pollute the bay area, are favored by A. poculata (Rotjan et al., 2019), and are typically associated with various bacteria (Jiang et al., 2018; Lamb et al., 2018; Tang et al., 2018). In addition, this study was unable to compare non- LPS infected A. poculata individuals, and future studies should incorporate the comparison of these controls to identify upregulation or downregulation of immune responses. We speculate that other functions such as coral lectins and the surface mucus layer have more importance in disease resistance compared to the melanin-synthesis pathway. Understanding lectin-glycan interactions and their role in disease mitigation as well as the influence on the SML in A. poculata may help us further understand the increase and decrease of components in the melanin synthesis pathway, and ultimately the immune functions in facultative symbiotic corals.

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Acknowledgements

Special thanks to S. Grace, C. Palmer, and L. Mydlarz for their insight and help throughout this research. The authors extend appreciation to K. Sharp, R. Rotjan, S. 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.

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Competing interests

The author(s) declare(s) that there is no conflict of interest.

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Funding

We acknowledge our funding resources, NASA’s Michigan Space Grant Consortium (Grant #223872-40826-200), Grand Valley State University (Presidential Grant), and the Annis Water Resource Institute (Graduate Student Fellowship) for making this research possible.

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Figures

Figure 1: Visual representation of signaling within the melanin-synthesis pathway in coral immunity. Pattern recognition proteins (PRRs) initiate these signaling pathways by reacting with pathogen associated molecular patterns (PAMPs) on invading pathogens (i.e. gram-negative bacteria). The association of these two components triggers the production of prophenoloxidase (PPO) and phenoloxidase (PO) within the pathway, ultimately generating melanin, which provides protein from foreign objects via encapsulation and generation of antimicrobial compounds.

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Figure 2: Map of field site location in Narragansett Bay, RI, USA. Red triangle indicates location of field site in Narragansett Bay (41°28'40.8"N, 71°21'45.8"W).

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Figure 3: Visual representation of experimental aquarium system. Each temperature treatment consisted of a recirculating system with eight tanks and a shared sump. Inside each individual tank, a smaller plastic container was placed inside with the artificial seawater under the exposure of lipopolysaccharide (LPS; 5µg mL-1). A. poculata colonies were evenly spread throughout all eight tanks.

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Figure 4: Boxplot comparison of aposymbiotic and symbiotic melanin concentrations (mg melanin per surface area (cm2)) between treatments, including both summer and winter collections. Boxplots include maximum/minimum values, inner/outer quartiles, and the median value, in addition to outlier data shown as circles. The letters above indicate significance difference from one another (p ≤ 0.05).

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Figure 5: Boxplot comparison of aposymbiotic and symbiotic melanin concentrations (mg melanin per surface area (cm2)) between seasons, including both ambient and elevated temperature treatments. Boxplots include maximum/minimum values, inner/outer quartiles, and the median value, in addition to outlier data shown as circles. The letters above indicate significance difference from one another (p ≤ 0.05).

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Figure 6: Boxplot comparison of aposymbiotic and symbiotic prophenoloxidase (PPO; ΔAbs (490nm) mg protein-1) concentrations between treatments, including both summer and winter collections. Boxplots include maximum/minimum values, inner/outer quartiles, and the median value, in addition to outlier data shown as circles. No statistical differences were observed. The y-axis was adjusted in order to visualize the boxplots.

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Three-way ANOVA: Melanin Df Sum sq Mean sq F-value p-value Treatment 1 0.17 0.166 0.391 0.53326 Sym State 1 4.76 4.759 11.169 0.00113 Collection 1 0.24 0.239 0.561 0.45559 Treatment:Sym State 1 0.4 0.399 0.937 0.33501 Treatment:Collection 1 0.11 0.112 0.264 0.60849 Sym State:Collection 1 0.31 0.314 0.736 0.39271 Treatment:Sym State:Collection 1 0.14 0.135 0.318 0.57418 Residuals 107 48.57 0.426

Table 1: A three-way ANOVA analysis of melanin in A. poculata individuals between temperature treatments, symbiotic state, season, and the interaction terms between the three dependent variables. Bolded text indicates statistical significance (p≤0.05).

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Three-way ANOVA: PPO Df Sum sq Mean sq F-value p-value Treatment 1 2.02 2.02 1.603 0.2082 Sym State 1 4.6 4.602 3.652 0.0586 Collection 1 0.52 0.524 0.416 0.5205 Treatment:Sym State 1 0.07 0.075 0.059 0.8077 Treatment:Collection 1 1.13 1.133 0.899 0.3451 Sym State:Collection 1 0.7 0.696 0.552 0.459 Treatment:Sym State:Collection 1 0.22 0.22 0.174 0.6772 Residuals 110 138.63 1.26

Table 2: A three-way ANOVA analysis of prophenoloxidase (PPO) in A. poculata individuals between temperature treatments, symbiotic state, season, and the interaction terms between the three dependent variables.

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Supplemental Material

A visual analysis of the summer collected fragments revealed that some coral, originally aposymbiotic, had developed significant concentrations of B. psygmophilum while acclimating to aquarium conditions over a five-month period. A preliminary photographic analysis of pixel intensities was run to separate aposymbiotic-like colonies from symbiotic-like colonies (Fig. S1). Any aposymbiotic fragments below the mean line (see Fig. S1) were assumed to be symbiotic- like colonies, while those above the line were likely aposymbiotic-like colonies. These “symbiotic-like” colonies were placed under low/no-light conditions between fragmentation and experiment periods to allow them to expel as many symbionts as possible. No logistical errors occurred with winter coral experiments; accumulations of symbionts were not noticed with the aposymbiotic corals from winter collections before any experimental analysis.

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Supplemental Figures

Figure S1: Preliminary pixel intensity analysis of aposymbiotic (A) and symbiotic (B) A. poculata fragments. This analysis identified aposymbiotic corals that were considered “symbiotic” (i.e. within the 2±SE region or below) and placed these fragments under no-light conditions prior to experiments to reduce B. psygmophilum concentrations. The black line represents the mean pixel intensity from all fragments, the red lines represent 2±SE of the mean, and the blue asterisks represent these aposymbiotic fragments needed for no-light conditions.

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Tukey's HSD comparisons: Melanin Treatment:Sym State p-value Elevated Aposymbiotic Ambient Aposymbiotic 0.9937 Ambient Symbiotic Ambient Aposymbiotic 0.0149 Elevated Symbiotic Elevated Aposymbiotic 0.3423 Elevated Symbiotic Ambient Symbiotic 0.657

Sym State:Season p-value Symbiotic Summer Aposymbiotic Summer 0.1067 Aposymbiotic Winter Aposymbiotic Summer 0.9998 Symbiotic Winter Symbiotic Summer 0.6678 Symbiotic Winter Aposymbiotic Winter 0.0422

Table S1: Tukey’s HSD post-hoc results between the interaction terms of treatment and symbiotic state and between symbiotic state and season. Bolded p-values indicate statistical difference (p≤0.05).

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Chapter 4

Extended Literature Review

Introduction

Coral reefs are some of the most biodiverse ecosystems on the planet, similar to tropical rainforests in terrestrial ecosystems. Although corals cover a small percentage of the total ocean, coral reefs provide habitat, breeding grounds, juvenile development, and feeding grounds to roughly 25% of all marine species (Moberg and Folke 1999; Karleskint et al. 2013). Hard corals

(scleractinian) make up most coral reefs and stand as the base of these reef ecosystems; scleractinian corals secrete calcium carbonate and utilize this for building their skeletons

(Karleskint et al. 2013). These strong structures help establish the reef environment, but also assist in shoreline protection from storms and hurricanes in the form of fringing reefs (Karleskint et al. 2013). Socio-economic criteria identify coral reefs as important economic structures for the various ecosystems services they provide. Coral reefs generate an estimated $2.7 trillion per year from ecosystem services on a global scale (Spalding et al. 2017). While the ecosystem services of coral reefs are extensive, some include carbonate budget, species richness, water quality control, nutrient cycling, pharmaceuticals, and so forth (Mumby et al. 2007; Rogers et al. 2015).

Taxonomically, corals are characterized within the Phylum Cnidaria and are invertebrate organisms. These organisms form congregations of thousands of individual polyps that function as one large organism (Karleskint et al. 2013). These reef habitats are typically within shallow, tropical environments, consisting of oligotrophic water chemistry characteristics, but other reefs exist in mesophotic regions in the ocean and even exist at higher latitudes (Karleskint et al.

2013). An important mutualistic relationship exists between coral organisms and a family of

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dinoflagellate algae called Symbiodiniaceae that live within the tissue of coral organisms

(Rowan 1998; Karleskint et al. 2013). These algal symbionts are important for the survival of coral organisms as these algae provide up to 90% of nutrients for corals to consume through the photosynthetic process. Many species of Symbiodiniaceae exist, such as Symbiodinium,

Breviolum, Cladiocopum, and Durisdinium (LaJeunesse et al. 2018). Each of these species evolved to symbiotically associate with a species of coral and typically have one primary algal association but some can have small populations of other symbiont species within their tissues.

Coral reefs have been on the decline from various anthropogenic impacts such as overfishing, pollution and coastal development (Karleskint et al. 2013). However, climate change continues to be the primary driver of coral reef demise on a global scale. Current technologies relating to energy, transportation, and agricultural and livestock practices generate

CO2 from the burning of fossil fuels, a potent greenhouse gas (GHG). GHGs are molecules that retain solar radiation from the sun, which drives the climate globally. The increase in GHGs from human-induced activities has caused global atmospheric and oceanic temperatures to increase in accordance with CO2 emissions by approximately 1 °C and over 100ppm, respectively, since the industrial age (Hoegh-Guldberg and Bruno 2010; Cheng et al. 2019).

Impacts on coral caused by climate change are primarily split between three events (1) thermal stress events and bleaching, (2) coral disease impacts, and (3) ocean acidification (OA).

As OA is not involved in this research work, the following sections will be related specifically to thermal stress and disease. In addition, this research work investigates reactive oxygen species involved in thermal stress/bleaching events and innate immunity associated with disease exposure and will go into detail in their respective general section. Finally, this research work involves the understanding of temperate corals in the future of climate change and will

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investigate the current status of Astrangia poculata research and potential routes for future scientific research.

Thermal Stress

With the rise of GHGs from human-induced emissions, oceanic temperatures have been rising since the dawn of the industrial age. Increased oceanic temperatures cause a phenomenon known as bleaching within coral reefs, defined as the subsequent expulsion of a coral’s algal symbionts when exposed to thermal stress events (Douglas 2003). Bleaching causes coral to lose their colorful pigments and through their translucent tissue, exposes the white skeleton below.

This can result in mortality of the organism as coral acquire up to 90% of their needed nutrients from this symbiotic relationship. However, bleaching is considered a natural occurrence in environmental disturbance events such as hurricanes or from El Nino cycles. Corals can regain their algal symbionts after the environmental disturbance has subsided; however, prolonged thermal stress events result in more severe and frequent bleaching events. In the past few decades, multiple mass bleaching events have occurred in areas such as the Great Barrier Reef in

Australia, resulting in massive die offs of various coral species and the subsequent congregation of macroalgae on coral reef skeletons (Pandolfi et al. 2011).

Bleaching events are primarily indicated by the increased concentration of reactive oxygen species. The input of elevated temperatures elicits the damage of photosynthetic pathways within the symbiotic algae of the coral, specifically photosystem II (PSII) (Warner et al. 1999; Kristiansen et al. 2009). In particular, the D1 polypeptide is central to the photosynthetic process of PSII; lower turnover rates and eventual loss of the D1 polypeptide occur regarding thermal stress and prevent electron transport into the thylakoid membrane (Long

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et al. 1994; Warner et al. 1999). Toxic singlet oxygen can be generated from the reaction between O2 and P680 (primary electron donor in PSII) from the quinone acceptor (Qa) region in

PSII, as these electrons will be pushed into an “electron sink” without the D1 polypeptide to initiate electron transport (Long et al. 1994; Krieger-Liszkay et al. 2008; Hillyer et al. 2016).

Singlet oxygen subsequently reacts with various cellular materials such as proteins and nucleic acids and can result in D1 polypeptide degradation and reduced PSII activity (Krieger-Liszkay et al. 2008). Ultimately, the excess energy buildup from degraded electron transport within

Symbiodiniaceae causes the imbalance in normal cellular function, resulting in high ROS concentrations (Roberty et al. 2016). Other forms of ROS that are generated in both photosystem

- I (PSI) and PSII are hydrogen peroxide (H2O2), superoxide (O2 ), and hydroxyl radicals (OH); these can instill similar damages such as singlet oxygen as they are highly reactive products

(Krieger-Liszkay et al. 2008; McGinty et al. 2012; Hillyer et al. 2015; Gardner et al. 2017; Lopes et al. 2018). ROS is generally formed within these photosynthetic processes in Symbiodiniaceae and are able to leech out into host tissue cells (McGinty et al. 2012), making ROS a primary indicator of Symbiodiniaceae expulsion (McGinty et al. 2012; Hillyer et al. 2016; Gardner et al.

2017). Additionally, corals can produce ROS from the mitochondria within the host tissue cells, producing forms of ROS such as nitric oxide that is prevalent in thermal stress events

(Kristiansen et al. 2009; Hawkins et al. 2014).

With thermal stress being an important factor in climate change impacts on corals, this becomes an unpractical issue for corals all over the globe. Notably, however, various species of

Symbiodiniaceae produce different levels of ROS and can partially drive thermal resilience

(McGinty et al. 2012; Wietheger et al. 2018). Both McGinty et al. (2012) and Wietheger et al.

(2018) have produced data that reflects these findings. McGinty et al. (2012) subjected multiple

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clades of Symbiodiniaceae to various thermal stress treatments and identified the amount of ROS generated after periods of 12 days. Clade C1 (now Cladocopium spp.) was identified as the most significantly impacted by higher temperatures (31 °C), while B2 (now Breviolum spp.) and E1

(now Effrenium spp.) had significant negative net ROS production when exposed to higher temperatures. In addition, catalase (CAT) and superoxide dismutase (SOD) concentrations were analyzed in parallel with ROS, indicating that clade B2 had significant increases in CAT activity with increased temperatures, while clades C1 and E1 resulted in no significant difference; implying many species of Symbiodiniaceae have various thermal sensitivities and complementing factors that mitigate temperature stress (McGinty et al. 2012). Similarly,

Wietheger et al. (2018) examined Symbiodiniaceae cultures exposed to both thermal stress and oxidative stress (H2O2) treatments to understand ROS concentrations over time. Thermal stress treatments showed significant increase in fluorescent levels with clades A1, B2, and F1 regarding general ROS and superoxides. Clade B2 (Breviolum psygmophilum) showed increases in ROS in both thermal and oxidative stress contrary to findings described by McGinty et al.

(2012). It should be noted, however, that the difference in thermal treatments of McGinty et al.

(2012) and Wietheger et al. (2018) (31°C vs. 35°C, respectively) identify a potential thermal stress cap for B. psygmophilum. Clade E showed no difference in thermal stress treatments, indicating mechanisms could interact to place this genus in another league of thermal tolerance

(Wietheger et al. 2018).

Multiple studies have investigated the thermal tolerance niche between various species of

Symbiodiniaceae (Berkelmans and van Oppen 2006; Roberty et al. 2016), but local adaptation is another primary driver in thermal tolerance, shown in studies such as Aichelman et al. (2019) and Palumbi et al. (2014). Palumbi et al. (2014) examined Acropora hyacinthus colonies within

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Ofu Island in American Samoa and the differences in their thermal resilience in areas of highly variable (HV) vs. moderately variable (MV) temperature environments. A. hyacinthus from each habitat were transplanted to opposite sites to understand its resilience, finding that HV translocated corals increased in resilience from growth rates, survivorship, and symbiont densities compared to MV natives. Numerous contigs such as transcription factors, heat shock proteins, and tumor necrosis factor receptors were involved in thermal acclimation. In contrast to previous studies investigating ROS concentrations in thermally stressed corals, Symbiodiniaceae had little to no influence to bleaching resistance and resulted in developments in thermal resilience without symbiont changes (Palumbi et al. 2014). Aichelman et al. (2019) examined the local adaptation of two populations of Astrangia poculata regarding thermal stress event effects on photosynthetic efficiency, respiration, and symbiont density. Thermal stress caused less efficiency within photosynthesis, and less symbiont density and reduced respiration rates within this facultative coral. These results indicate the differences between Rhode Island and Virginia populations, such that VA populations are more thermally tolerant due to its local adaptation to warmer temperatures (Aichelman et al. 2019).

Disease

Coral diseases have been on the rise since the early 1980s, including numerous diseases such as white plague, black band, yellow band, and rapid tissue necrosis (Garzon-Ferreira et al.

2001; Nugues et al. 2004; Luna et al. 2007). Factors such as pollution, nutrient runoff, and aeolian dust from the Saharan region in Africa have been suggested to cause disease, primarily within the Caribbean region (considered the “disease hotspot” of the world’s oceans) (Nugues et al. 2004; Ruiz-Moreno et al. 2012). The primary driver for an increase in disease associated with increases in thermal ocean temperatures due to anthropogenic warming. Thermal stress events

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give opportunistic pathogens the ability to affect coral individuals, as high temperatures inhibit the coral’s effort to prevent infections (Cervino et al. 2008; Bourne et al. 2009; Ruiz-Moreno et al. 2012; Sweet and Bythell 2017; Thurber et al. 2017; Wall et al. 2018). As coral disease becomes more understood, many ecological interactions with coral disease have yet to be characterized (e.g. prokaryotic, eukaryotic, and viral pathogens) (Bourne et al. 2009).

One of the many factors associated with disease resistance and protection in coral from pathogenic bacteria involves the surface mucus layer (SML). The SML is considered a primary component within the coral holobiont that contains a diverse bacteria and virus community within the layer immediately above the host tissue (Bourne et al. 2009; Nguyen-Kim et al. 2015;

Sweet and Bythell 2017; Thurber et al. 2017). The SML contains congregations of beneficial symbiotic bacteria that help produce anti-microbial compounds in order to mitigate pathogenic bacteria infection, but thermal stress events may be reducing the effectiveness (Gochfeld and

Aeby 2008; Krediet et al. 2013). In addition, the microbial community within the SML can undergo radical shifts regarding composition caused by thermal stress (Sweet and Bythell 2017;

Thurber et al. 2017). Virus-like particles (VLPs) within these microbial assemblages are maintained by lysis processes under normal conditions, and are hypothesized to defend bacterial pathogens within the SML (Nguyen-Kim et al. 2015; Sweet and Bythell 2017; Thurber et al.

2017). Thermal stress events often exhibit an increase of VLPs, exacerbating their initial infectious properties due to an increase in VLP population and invade the coral host and trigger various immunological pathways (Sweet and Bythell 2017; Thurber et al. 2017), in addition to some VLPs particularly targeting their algal symbionts (Cervino et al. 2004).

Microbiomes are incredibly important for overall disease resistance within coral organisms. This, however, varies between genotypes and over time as their environment changes

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(Rosenberg and Zilber-Rosenberg 2018). Rosado et al. (2018) showed the importance of beneficial microorganisms in corals (BMCs) by partially reducing bleaching caused by Vibrio corallylliticus, as well as demonstrating the ability of BMCs to improve microbiome communities when exposed to Vibrio spp. (Rosado et al. 2018). Various components of the microbiome in corals exist, such as a stable core microbiome, a microbiome influenced by spatial parameters, and a microbiome influenced by abiotic and biotic factors such as temperature. In addition, life histories are another important aspect in coral microbiomes, where some coral species transfer microbiomes between parent and offspring while others acquire their microbiomes from the surrounding seawater, which can be negative influenced by climate change (Sweet and Bulling 2017). Establishments of microbiomes in corals help dictate symbiosis interactions with Symbiodiniaceae, avoid the establishment of pathogenic bacteria, and potentially help trigger various immunity pathways by chemical cues such as the melanin synthesis pathway (Krediet et al. 2013).

Innate immunity in corals are similar to other invertebrates, consisting of receptors, signaling pathways, and effector responses. Receptors, such as lectins, integrins, and toll-like receptors are caused from interactions with foreign materials such as gram-negative bacteria, fungi, and cell injury. These contain pathogen associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), that interact with host receptors. PAMPs trigger one or multiple signaling pathways like melanin synthesis and complement pathways which cause effector responses to occur (i.e. antimicrobial defenses and phagocytosis target foreign materials) (Palmer et al. 2012; Toledo-Hernandez and Ruiz-Diaz 2014; Mansfield and Gilmore 2019).

Two pathways in particular have received recent attention and include melanin synthesis and complement pathways. The melanin synthesis pathways are primarily triggered by protein

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receptors from PAMPs associated with foreign bacteria, triggering components such as prophenoloxidase (PPO) and subsequent phenoloxidase (PO), to eventually generate melanin and

ROS, which correspond to the production of antimicrobial compounds to help protect against foreign bacteria and pathogens. The melanin synthesis pathway has been categorized as an important pathway in coral immunity, and is considered vital for general disease susceptibility in various coral species (Palmer et al. 2010, 2011; Toledo-Hernandez and Ruiz-Diaz 2014).

Mydlarz et al. (2009) and Palmer et al. (2011) have explored the components of the melanin synthesis pathway associated with thermal stress and disease exposure treatments to understand various coral species susceptibility. Palmer et al. (2011) measured melanin and PPO activity of corals exposed to both thermal stress and LPS exposed treatments, finding that the interaction of

LPS and elevated temperature induces more PPO in some corals species such as Porites astreoides, while others had less generated from simulated disease exposure. This implies the importance of genotypes regarding disease resistance, where Palmer et al. (2010) suggests more

PPO corresponds to more disease resistance. Mydlarz et al. (2009) found complex results of PPO concentrations between thermally stressed corals compared to yellow-band diseased corals. More

PPO was generated from thermal stress events (i.e. bleached corals) rather than YBD disease exposure, suggesting that the complexity of symbiosis may stimulate immune responses

(Mydlarz et al. 2009). In addition, Muller et al. (2018) found similar results of bleaching being an important trigger for increased susceptibility with disease. Findings from Mydlarz et al.

(2009) and Muller et al. (2018) could be related to studies shown in Zhou et al. (2018) and Zhou et al. (2020), which involves the support of the complement system with disease resistance.

The complement system involves C-type lectins that tag foreign bacteria and pathogens for opsonization/phagocytosis (Palmer et al. 2012; Mansfield and Gilmore 2019). Additionally,

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C-type lectins have been discovered to play an important role in symbiosis, binding with surface glycans on Symbiodiniaceae. According to Zhou et al. (2018), this pathway also provides a mechanism for disease resistance. By exposing Pocillopora damicornis symbionts to its specific lectin gene, Zhou et al. (2018) observed similar treatments of LPS and thermal extremes reduced the association of symbionts with lectin-binding properties, implying that the dissociation of symbiosis helps lectins tag foreign invaders for opsonization. Similar studies (see Kvennefors et al. (2008; 2010)) observed similar results in Acropora millepora, distinguishing C-type lectins involved in immunity. These results, in addition to Mydlarz et al. (2009) and Muller et al.

(2018), suggest the complement pathway may initially provide disease resistance prior to the occurrence of the melanin synthesis pathway, in which subsequent production of antimicrobial components take place.

In addition to factors regarding disease on coral host immunity, coral diseases such as yellow-band disease particularly infect Symbiodiniceae within corals (Wilson et al. 2001;

Cervino et al. 2004, 2008). Cervino et al. (2004, 2008) investigated the impacts of YBD Vibrio spp. on Symbiodiniaceae of various coral species and found that YBD was a congregation of

Vibrio spp., rather than a single species, working in consortium to physically degrade

Symbiodiniaceae, damaging thylakoid membranes and inducing reductions in photosynthetic efficiency (Cervino et al. 2004, 2008). These results highlight potential exacerbation of thermal stress events from the degradation of Symbiodiniaceae, reducing the input of energy from photosynthetic processes (Cervino et al. 2004; Merselis et al. 2018). Wilson et al. (2001) observed similar findings but suggested that while not all Symbiodiniaceae species were infected, some species did not contain VLPs within those algal symbionts. Their results imply that some species may be resistant to algal symbiont targeted diseases (Wilson et al. 2001).

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Astrangia poculata

Astrangia poculata is a temperate coral species located in the western Atlantic, off the east coast of the United States. This species has a wide geographic distribution from its northern range in Massachusetts to its southern range of southern Florida and the Gulf of Mexico, occurring anywhere from 0 to 263m in depth. Few studies have shown its distribution into the

Caribbean, northern South America, or Western Africa (Peters et al. 1988; Dimond et al. 2013).

Considering this species and its wide range of distribution, it is considered an extremely tolerant coral species, withstanding various temperatures, turbidity levels, salinity levels, and light intensities. A. poculata is an ahermatypic coral species (non-reef building) and is facultatively symbiotic. Individuals can display non-symbiotic, symbiotic, or mixed concentrations of symbiosis with Breviolum psygmophilum (J.E. Parkinson & LaJeunesse 2018). In addition to their facultative characteristic, A. poculata also utilize heterotrophy for their metabolic needs

(Szmant-Froelich and Pilson 1980; Peters et al. 1988; Aichelman et al. 2019). Phenotypes between individuals based on location have been visually identified; southern individuals of A. poculata are more branching in structure compared to northern individuals which form small, encrusting colonies (Peters et al. 1988). Seasonality is an important factor associated with this species, as time of year influences its symbiosis, metabolic rate, growth, microbiome structure, and a quiescent state (Jacques et al. 1983; Dimond and Carrington 2007; Dimond and Carrington

2008; Grace 2017; Sharp et al. 2017). Few studies have examined A. poculata and its interactions within its localized food web. Some interactions between the carpet tunicate (Didemnum vellixum) and the red boring sponge (Cliona celata) have caused competition with A. poculata, particularly in areas of its northern range (Grace 2017).

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Astrangia poculata has grown in popularity over the past few years, becoming a staple for environmental simulation experiments. Many papers have begun to understand this coral’s ability to withstand various temperature treatments, examining components such as metabolic rates and calcification (Jacques et al. 1983), changes in its microbiome based on seasonality

(Sharp et al. 2017), understanding differences in physical damage (Burmester et al. 2017), and characterizing differences in photosynthetic efficiency and respiration (Aichelman et al. 2019).

Other papers have investigated growth impacts from elevated CO2 and nutrient levels (Holcomb et al. 2010), physical damage recovery (DeFilippo et al. 2016), and most recently, impacts from microplastic ingestion (Rotjan et al. 2019). Many of these papers are fundamentally investigating this understudied coral to reveal its resilience and susceptibility to various abiotic stressors. More notably, this species may help provide information from such studies as it can help identify important inner workings that may not be identified easily in tropical species, mainly due to its facultative symbiotic properties. Amongst the scientific community, A. poculata species are being compared to Aiptasia (anemone), which is used to understand symbiotic properties.

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Extended Methodology

Coral Sampling and Husbandry

Astrangia poculata colonies were collected offshore in Fort Wetherill State Park

(Jamestown, RI), located within Narragansett Bay (41°28'40.8"N, 71°21'45.8"W). Collections were done using SCUBA, with dives conducted by Tyler Harman, Dr. Sean Grace, Brendan

DeGrim, and Melisa Beecher on July 14th, 2019 and by Tyler Harman and Dr. Sean Grace on

February 21st, 2020. This collection location has been identified by Dr. Sean Grace prior to the scheduled dives and has been sampled numerously in other scientific research projects. All coral colonies were carefully removed by using a hammer and chisel to pry colonies from the rocky substrate; fragmented colonies were considered single colonies for laboratory analyses. Colonies were sampled at least one meter apart to ensure distinct individuals and were placed in mesh bags throughout the duration of collections. Upon surfacing, colonies were placed in buckets of seawater and subsequently transferred into plastic bags filled with seawater, sealed with rubber bands, and placed into coolers filled with ice.

Colonies were transported to the Annis Water Resource Institute at Grand Valley State

University in Muskegon, MI within 48 hours from collections at the field sampling site and placed into custom recirculating aquarium systems. These systems consisted of eight separate tanks (3L), which were connected by 1.27 cm PVC piping to drain through filter socks and into a sump container, with which Bubble Magnus NAC3.5 protein skimmers (Jiangmen Jiyang

Aquarium Equipment Co., Jiangmen, Guangdong, China) filtered the water to reduce nitrate and phosphate buildup. An additional aquarium foam filter was attached to the outflow of the sump to filter large particulates. The outflow from the sump was powered by a Blueline HD 30 non-

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submersible pump (Champion Lighting & Supply, Ambler, PA) at 2233.39 liters per hour and ran through an Aqua Euro Max Chill chiller to maintain field site temperatures (AquaEuro

Systems, Los Angeles, CA). All water was dispersed evenly throughout the eight tanks and aerated by the inflow ball valves to promote oxygenation in the aquarium system.

With a recirculating system, frequent water changes (i.e. twice per week) were done in order to avoid buildup of nitrates, phosphates, and other marine chemicals. Drain piping was integrated into all sumps to easily remove water for these frequent changes. New artificial seawater was made by using de-ionized water (DI) and Instant Ocean reef salt (Spectrum Brands,

Blacksburg, VA) to achieve a salinity concentration of 35 parts per thousand (ppt). Water chemistry measurements were completed once per week to monitor concentrations nitrate and phosphate, so as to correct these concentrations by an additional water change if needed. All water measurements were done by using RedSea kits (Red Sea U.S.A., Houston, TX) and measured by following manufacturer instructions. Additionally, all coral colonies were fed with brine shrimp eggs (Artemia nauplli) to maintain healthy coral colonies for experiments. Brine shrimp were hatched in a separate system consisting of two plastic containers, fitted with aeration stones powered by a small air pump. Artificial seawater (25ppt; 1L) was placed in the containers, followed by five grams of brine shrimp eggs and left to hatch for two days. Hatched brine shrimp were extracted from the containers and placed into a beaker. The aquarium system pumps were shut off, and each individual tank was given 1mL of brine shrimp slurry and left for one hour for the corals to feed. Small submersible pumps inside each tank promoted homogenization of the brine shrimp so coral colonies could feed as evenly as possible.

Fragmentation/Randomization techniques

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All Astrangia colonies were fragmented for experiments using a Gryphon C-40 bandsaw

(Gryphon Corporation, Sylmar, CA). All colonies were first rid of additional skeleton sections where no tissue was present, along with large aggregations of filamentous algae and red boring sponges (Cliona celata). Colonies were then cut into several fragments depending on the size of the original colony but were fragmented into 2-3 cm2 pieces. These fragments were glued onto acrylic glass discs using IC-gel (Bob Smith Industries, Atascadero, CA) and given a unique ID based on colony number, fragment number, and symbiotic state at the time of fragmentation.

A randomization process was applied all fragments to be assigned to a temperature treatment/LPS experiment. A custom randomization program was made in Microsoft Excel

(Microsoft Corporation, Redmond, WA). All fragment IDs were placed into individual Excel cells in a single column, and randomly selected by using the following function:

=@퐼푁퐷퐸푋(퐴1: 퐴76, 푅퐴푁퐷퐵퐸푇푊퐸퐸푁(1,76))

Where “A1:A76” represents the column number (A) and first row through the last row numbers

(1 and 76, respectively); these can be changed depending on the location of cells within the spreadsheet. The other numbers in the RANDBETWEEN function represents the amount of cells that the function randomly selects from.

Coral Tissue Extraction Protocol (Chapter 2)

Coral fragment tissues were extracted for reactive oxygen species (ROS) analysis via imaging flow cytometry (IFCM) using a modified method from Nielsen et al. (2018). Fragments were peeled gently from their acrylic glass discs and placed into a 50mL Falcon tube filled with

0.22µm filtered artificial seawater (3.5mL). Fragments were agitated for 30 seconds by vortexing

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to remove host tissue and algal symbiont cells to produce a “slurry”. After agitation, the fragments were removed and discarded, and the tissue slurry mix was filtered through a 70µm cell strainer (Thermo Fisher Scientific, Waltham, MA) to remove filamentous algae, skeleton pieces, and coral mucus from the samples. Aliquots (1mL) of tissue slurry were transferred into

1.5mL Eppendorf tubes and was washed three times with 0.22µm filtered seawater. Samples were centrifuged for 30 seconds at 1000 RPM, 0.75mL of the supernatant was removed, then washed with 0.75mL of filtered seawater, and repeated three times. After washing the samples,

0.5mL of supernatant was removed and general oxidative dye (CM-H2DCFDA @ 50µg per vial)

(Thermo Fisher Scientific, Waltham, MA) was added to each sample (2.9µL; 10mM concentration). Oxidative dye was reconstituted in 50µL of 100% ethanol per manufacturer instructions. To achieve a 10µM concentration, the following calculation was made to identify the volume of reconstituted dye added to 0.5mL:

휇푔 50µ푔 + 50µ퐿 = 1000 푚퐿

Calculating the micromolar concentration within the reconstituted sample using the molar mass of the dye (577.8013 grams/mol) then followed:

1000휇푔 1푚표푙 1.73 × 10 푚표푙 × = 푚퐿 577801300휇푔 푚퐿

1.73 × 10푚표푙 1000푚퐿 1.73 × 10푚표푙 × = 푚퐿 퐿 퐿

1.73 × 10푚표푙 1000000휇푚표푙 × = 1731휇푀 퐿 푚표푙

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Then using the M1V1 = M2V2 equation and multiplying by 1000, the needed volume of reconstituted dye for each sample to achieve a 10µM concentration occurred as follows:

(10휇푀) × (0.5푚퐿) 1000휇퐿 × = 2.9휇퐿 (1731휇푀) 푚퐿

The volume of dye was added to each sample destined for oxidative dye additions and incubated for 40 minutes under treatment temperatures and agitated at 300RPM to ensure even distribution of dye in each sample. After incubation, all samples had 0.5mL of phosphate buffer saline (PBS) added and then were subsequently washed twice with 0.75mL of PBS to prepare for analysis via

IFCM.

Lipopolysaccharide exposure (Chapter 3)

Lipopolysaccharide (LPS) exposure was done to trigger signaling proteins within the melanin-synthesis pathway, specifically prophenoloxidase (PPO) and melanin. In order to expose

Astrangia fragments to LPS, plastic bins were placed inside of each individual tank in our custom aquarium systems; this was done to contain the LPS from contaminating the other parts of the aquarium system. First, seawater was vacuum-filtered through a 0.22µm filter to remove any particulates and other microbes. Water was maintained at temperature treatments (18 °C and

26 °C) in separate incubation chambers. Prior to the treatment, 1420mL of filtered seawater was placed into 2L plastic containers, followed by the addition of 7.1mg of LPS extracted from E. coli (O127:B8; Sigma Aldrich, St. Louis, MO). The solution was homogenized using a magnetic stir bar. On the day of experiments at 0800 hours, the plastic containers containing LPS were carefully placed inside each individual tank, with the lip of the plastic container 5cm above the water surface to avoid contamination into the remaining aquarium system. Coral fragments were

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then added to each container and incubated in the solution for 12 hours until extraction and subsequent liquid nitrogen flash-freezing.

Tissue extraction (Chapter 3)

Tissue extractions were a modified protocol from Palmer et al. (2011b). Each flash- frozen fragment was thawed before dry tissue extractions for 15 minutes. Fragments were placed into small plastic bags and airbrushed until all tissue was removed from the skeleton. A coral extraction buffer (25mL of PBS solution at 50mM at 7.8pH with 50µM dithiothreitol) was poured into the plastic bag, sealed, and shaken vigorously to ensure all coral tissue was within the extraction buffer solution. Holes were cut into the plastic bag with the tissue slurry poured into pestle and mortars and homogenized for one minute. Homogenized tissue slurries were then poured into 50mL Falcon tubes for subsequent microplate assay analysis. All pestle and mortars were rinsed thoroughly with DI water in-between each homogenization to remove any remaining buffer solution and coral tissues. Airbrush tips were cleaned with Kim-wipes and 70% ethanol and allowed to dry between every tissue extraction.

Exploratory statistical analysis

All statistical analysis was done within R (Version 4.0.0; R Foundation for Statistical

Computing, Vienna, Austria). A work directory was selected using the “setwd” function, and organized data within that directory was uploaded into R by using the “read.csv” function to obtain a data frame. All exploratory data analysis used the “shapiro.test” function to determine normality within all data sets. Additional linear model functions and “ggqqplot(residuals())” function visually assessed the data to complement normality tests. All data with a p-value greater than 0.05 was considered normal (parametric), while p-values lower than 0.05 were considered

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not-normal (non-parametric). Non-parametric data would utilize transformations such as log and square-root transformation to achieve normality if possible.

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