Using TEM to Evaluate the Importance of the Microbiome and Virome Between Fire and Scleractinian Corals

by William Duke

A THESIS

submitted to

Oregon State University

Honors College

in partial fulfillment of the requirements for the degree of

Honors Baccalaureate of Science in Earth Sciences (Honors Scholar)

Presented June 2, 2020 Commencement June 2020

AN ABSTRACT OF THE THESIS OF

William Duke for the degree of Honors Baccalaureate of Science in Earth Sciences on June 2, 2020. Title: Using TEM to Evaluate the Importance of the Microbiome and Virome Between Fire Corals and Scleractinian Corals.

Abstract approved:______Rebecca Vega Thurber

As important ecological cornerstones, reefs face threats from a myriad of sources, such as global climate change, and importantly, disease, the latter often as a result of microbial pathogens. An understudied group of major corals, fire corals, and their even less understood microbiome present an opportunity to learn more about the coral ecosystem and lead to a better understanding of their decline in recent decades. Here, through the use of Transmission

Electron Microscopy, we evaluate fire corals from the Pacific Ocean and scleractinian corals from the Atlantic Ocean at the microscopic level, with a focus on the viruses and bacteria present. The discovery of a novel giant virus-like-particle (VLP) in the fire coral tissue shows that further study of fire corals may add to the field of virology, while a morphologically distinct bacteria-like-object (BLO), likely Rickettsiales, in both fire coral and stony coral suggests this bacteria may not be the sole cause of , a specific disease of a sub group of scleractinian coral. Finally, the presence of similar VLPs and BLOs across both fire corals and stony corals indicate that the microbiomes of these two groups are equally complex, even when compared across ocean basins.

Key Words: Coral, virus, bacteria, Rickettsiales

Corresponding e-mail address: [email protected]

©Copyright by William Duke June 2, 2020

Using TEM to Evaluate the Importance of the Microbiome and Virome Between Fire Corals and Scleractinian Corals

by William Duke

A THESIS

submitted to

Oregon State University

Honors College

in partial fulfillment of the requirements for the degree of

Honors Baccalaureate of Science in Earth Sciences (Honors Scholar)

Presented June 2, 2020 Commencement June 2020

Honors Baccalaureate of Science in Earth Sciences project of William Duke presented on June 2, 2020.

APPROVED:

______Rebecca Vega Thurber, Mentor, representing Microbiology

______Grace Klinges, Committee Member, representing Microbiology

______Andrew Thurber, Committee Member, representing the College of Earth, Ocean, and Atmospheric Sciences

______Toni Doolen, Dean, Oregon State University Honors College

I understand that my project will become part of the permanent collection of Oregon State University, Honors College. My signature below authorizes release of my project to any reader upon request.

______William Duke, Author

ACKNOWLEDGEMENTS

I would like to thank my mentor, Dr. Rebecca Vega Thurber, for continuously guiding me through this project, introducing me to this field, and inspiring me to work diligently throughout my undergraduate career; Grace Klinges, Ph.D. candidate, for asking me to join her research in the Florida Keys and her unending leadership in the field; Teresa Sawyer for lending her expertise with electron microscopy; Dr. Erinn Muller for providing insight on the experiment in Floridan as well as facilities at the Mote Marine Laboratory; TARA Pacific for providing fire coral samples; my committee: Dr. Rebecca Vega Thurber, Grace Klinges, and Dr. Andrew

Thurber; and finally, my friends and family for joining me on this journey over the past few years supporting me every step of the way.

INTRODUCTION

Coral Reef Ecosystems

Coral reefs are found across the globe and are known to be hotspots. Despite occurring in waters with very low nutrient content, there are high amounts of productivity that occur in these tropical locations (Vega Thurber et al., 2017). Within these coral reefs there are multiple species of coral, that help create the foundational structures, both by building up calcium carbonate skeletons and trapping sediment. These ecosystems provide homes for many organisms that sustain not only local food webs, but also human populations across the globe, both as food and sources of income (Hughes et al., 2003).

Within shallow tropical reefs one of the most influential groups of these animals are the

“true” corals, or scleractinian (stony) corals. These consist of typical boulder-forming and branching corals, among others (Lewis, 2006; Coni et al., 2012). And while these corals typically are most abundant on reefs, there is another group of organisms, fire corals, that also contains major reef-building species, that, in some cases, replace scleractinian corals as the dominant reef-building organisms (Rojas-Molina et al., 2012; Dubé et al., 2017). Fire corals are also widespread across reefs, found from depths of 1 m up to 40 m below the surface (Lewis,

2006) with different species presenting different morphologies (e.g. thin branches, robust plates) based on their environment that allow them to exist in various turbulent conditions and inhabit different regions of reefs (Rojas-Molina et al., 2012).

Fire corals and scleractinian corals are somewhat related groups of organisms; both belonging to the phylum , but divided into two different classes, the and Anthozoa, respectively. This taxonomic connection at the phylum level indicates that these groups are somewhat distantly related, and indeed, the fossil record shows that by the beginning

of the Cambrian period (~540 million years ago) the separate classes within the phylum had formed (Cartwright et al., 2007). This means that the common ancestor of fire corals and scleractinians existed prior to that time period. Thus despite these two groups being fairly distantly related, they still fill similar niches within the same ecosystem. Further, while the group of fire corals only contains seventeen species (Lewis, 2006). There are ~500 species of stony coral in the Indo-Pacific region alone (Lalli and Parsons, 1997). Yet fire corals still remain major reef-building species, even capable of overgrowing scleractinian corals (Dubé et al., 2016).

Despite these major differences between these two groups, both importantly possess symbiotic relationships with dinoflagellates within the family Symbiodiniaceae, genus

Symbiodinium, (formerly referred to as ) that reside within the coral tissue in specialized vacuoles (Muscatine & Porter, 1977; Morris et al., 2019). These unicellular algal symbionts are integral aspects of the success of corals; their close interactions provide coral cells oxygen, as well as organic matter, in the form of waste products from (Lalli &

Parsons, 1997). This is particularly important for corals since the tropical oceans they are found in are typically nutrient poor (Muscatine & Porter, 1977). As a result, the nutrients that are present are tightly recycled due to these symbioses (Lalli & Parsons, 1997).

Virome of Corals

The study of virology is in its infancy. Research in this relatively young field has mainly been conducted in the past two decades and the majority has been focused on scleractinian corals (Vega Thurber et al., 2017). Viruses play important roles in the marine setting, predominantly as connections between higher trophic levels and other microbes by inducing cell lysing. The process of lysing cells releases nutrients to the water column (referred to as the “viral shunt”) as viruses destroy the cell in an attempt to spread to new hosts (Vega

Thurber et al., 2017). These nutrients support other microbial life, which is even more important on coral reefs given the nutrient-poor conditions where they are found. In addition, viruses are numerous in the water column and on reefs, up to 107 ml-1, aiding in the high frequency of viral infection (Vega Thurber & Correa, 2011). While there are viruses that infect eukaryotes

(eukaryotic viruses), there is another group of viruses that target specifically bacteria, known as bacteriophages, or phages (Vega Thurber et al., 2017). Since all organisms are targeted by viruses, nutrients that would normally only move in one direction in a food chain can thus be recycled. In addition, viruses may also have more direct positive impact on corals by infecting other pathogens. In particular, phages target bacteria that may opportunistically infect corals, thus decreasing coral infections and actually keeping them healthy (Soffer et al., 2015).

While it is important to recognize the benefits that viruses have for connecting food webs in ways that would not normally be possible, in the case of corals, eukaryotic viruses likely have negative impacts by causing infections, and even killing entire colonies, having broader impacts for the greater community (Sharma & Ravindran, 2020). Viral infections act synergistically with other problems facing coral reefs, namely ocean acidification, increased temperatures, and nutrient runoff, which have only increased in recent decades (Vega Thurber & Correa, 2011).

These coral stressors only seem to increase the occurrence of viral infection, helping lead to further coral reef decline (Correa et al., 2016).

Types of Viruses Found in Corals

Viruses are a diverse group of pathogens that have been documented in coral tissue (in total ~50 families infecting coral), encompassing a wide range of sizes and morphologies.

Eukaryotic viruses are found to infect the coral tissue, though they also infect the symbiotic algal cells (Vega Thurber & Correa, 2011). These eukaryotic viruses can range from 20 nm - ~500 nm

in diameter (Vega Thurber & Correa, 2011; Correa et al., 2016). Typical morphologies of these viruses consist of two main features: 1) genetic material (either DNA or RNA) and 2) a capsid, which is a protein-layer surrounding the genetic material (Vega Thurber et al., 2017). A third feature, the envelope, a circular membrane that surrounds the capsid, is also common, but not always found (Vega Thurber et al., 2017). The capsid is usually icosahedral in shape, which appears as a hexagon in cross-section (Correa et al., 2016).

Within eukaryotic viruses there is an important group, the nucleo-cytoplasmic large DNA viruses (NCLDVs). NCLDVs are so named for their genetic material, which is comprised of

DNA, and their replication stages, where their DNA enters the host’s nucleus to replicate, and is then sent out to the cytoplasm for encapsulation in capsids (Vega Thurber et al., 2017). These viruses are important because of their large structural size, but also because of the complex content of their genomes, such as genes for transcription and DNA repair, which blur the line between what is considered alive and not (Colson et al., 2013). This group is ubiquitous throughout corals, and ranges in diameter from 120 nm - 1 μm (Correa et al., 2016). Within

NCLDVs, giant viruses account for the larger of these viruses, such as mimivirus, phycodnavirus, and poxvirus (Abergel et al., 2015).

Bacteriophages possess a distinct structure from eukaryotic viruses, though they still possess genetic material and capsids. However, a unique structure to phage, albeit not ubiquitous, is a tail that extends from the surface of the capsid, allowing the virus to attach to a bacterial cell and inject its genetic material inside the cell (Sweet & Bythell, 2017). Examples of these viruses include myoviruses, podoviruses, and siphoviruses (Suttle, 2005). Since phages target bacterial cells and not eukaryotic cells, there has been some initial research into their use for ‘phage therapy’ on corals, where these viruses are used as a treatment for bacterial diseases

(Vega Thurber et al., 2017). In total, ~50 families of viruses, including eukaryotic viruses and phages, have been identified to be associated with corals (Sweet & Bythell, 2017).

Bacterial Cells in Corals

Bacteria are another major group of coral pathogens implicated as the cause of various coral diseases for decades (Rosenberg et al., 2007; Bourne et al., 2009). One primary example is

White Band Disease, which affects two specific species of branching stony corals in the

Caribbean, cervicornis and A. palmata (Kline & Vollmer, 2011). This disease causes corals to lose their tissue progressively and rapidly, causing an apparent white band of necrotic tissue between sections of bare skeleton and uninfected tissue (Kline & Vollmer, 2011). One suspected culprit is bacteria belonging to Rickettsiales, an order of obligate intracellular bacteria belonging to the class Alphaproteobacteria (Castelli et al., 2016), because of their high abundance in infected corals. Various experiments also show that this bacterial order is present in apparently healthy corals (Casas et al., 2004; Miller et al., 2014) and a particular genus

Aquarickettsia has been identified for Acropora sp. (Klinges et al., 2019). This bacterium still possesses parasitic capabilities, which while it might not lead to disease symptoms, it likely causes these Acropora species to become susceptible to other stressors (Miller et al., 2014;

Klinges et al., 2019). Regardless, some members, though not all as most are non-marine species, of Rickettsiales are frequently a major constituent of the microbiome on corals, and the presence of this order is an important marker of coral health (Klinges et al., 2019).

TEM and SEM Technology

Because viruses and bacteria tend to be smaller than 2 μm, light microscopy methods cannot always be used to describe their structures in great detail (Robson et al., 2018). While this technique can be used quickly, it has a maximum resolution of ~250 nm, which is insufficient to

view VLPs that are often smaller than that (Robson et al., 2018). Electron microscopy, however, can resolve images down to below 1 nm, making this an ideal method for viewing microbes

(Robson et al., 2018). The most common methods of electron microscopy are transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Both of these methods take advantage of electrons moving at wavelengths much faster than visible light, which means the length at which diffraction interferes with imaging is reduced, thus providing a higher resolution (Bibi et al., 2011). In addition, both of these techniques function by placing samples in a vacuum for imaging, since particles in air would disrupt the electrons while they are being used to image (Robson et al., 2018). The main difference between TEM and SEM lies in whether electrons pass through the thin-sectioned (<100 nm) sample (TEM), or across the sample (SEM)

(Robson et al., 2018). The thin sections used in TEM allow for great detail of intracellular structures, while the scans used in SEM allow for clarity of surface level details. These differences result in TEM being better suited for shape and morphology than SEM (Bibi et al.,

2011).

Objectives

Given the importance of both viruses and bacteria on coral health, especially in regards to changing climate and anthropogenic influence, a deep understanding of their diversity and function is necessary to help guide future actions to restore and protect coral reefs. However, while advances have been made in the arena of scleractinians almost nothing is known about viral and bacterial pathogens in fire corals. Since the microbiome and virome of fire corals is not well understood, and because these organisms are also major ecosystem engineers and have a broad impact on the wide biodiversity seen on coral reefs, it is necessary to further study them.

One first step to do this is at the microscopic level. This project aimed to observe, describe, and

quantify viral and bacterial structures present in fire coral tissues and to compare and contrast them to similar morphologies recorded in scleractinian coral tissues.

METHODS

Fire Coral Sampling for TEM Analysis

Fire coral (species ) samples were collected in Papua New Guinea during the TARA Pacific Expedition, a program designed to study chemical and biological gradients across the Pacific Ocean and which sampled coral reefs from multiple islands to explore the reef microbiome (corals, fishes, and water) (Gorsky et al., 2019). Large fragments

(~300 grams) were sampled on SCUBA using hammer and chisel, placed in sterile bags underwater, and transferred at local seawater temperatures back to the ship within 1 hour of collection time. Once on-board Tara, small <2mm subsamples were preserved in 1ml of 2.5% electron grade glutaraldehyde and 10X phosphate buffered saline (PBS) and stored at 4℃ and shipped on ice to Oregon State University.

Acropora Nutrient Dosing Experimental Design

Acropora cervicornis fragments were collected from the Mote Marine Laboratory coral nursery off of Key West by Erich Bartels and staff. Fragments of 2 genotypes of this species approximately 5 cm in length were mounted upright in an underwater epoxy and divided into groups to receive various nutrient treatments: high nitrate (3x ambient concentrations), low nitrate (2x ambient concentrations), high phosphate, low phosphate, high ammonia, low ammonia, high combined nutrients (a mixture of nitrate, phosphate, and ammonia), low combined, and no nutrient amendment (which served as the control) (Klinges et al., in prep).

Genotypes were housed in separate ‘raceways’ (water baths to normalize tank temperature) at the

Mote Marine Laboratory in Summerland Key, FL, USA. The 9 treatments were distributed

across these raceways at random. Within each treatment group, corals were spread across three tanks to control for tank effects on the corals. Over the course of six weeks, the treatments were applied to the corals every 6 hours (4 times every 24 hours). Corals from all treatments were sampled at three timepoints: a T0 timepoint (prior to initiating nutrient treatment), after three weeks of nutrient exposure, and after six weeks of exposure. At each timepoint, one replicate fragment from each treatment was selected for TEM and a single from each fragment was cut off and immediately placed in a 2.5% glutaraldehyde solution for fixation.

Electron Microscopy Sample Preparation and Staining.

Once at OSU, samples were decalcified for 5 weeks using a 10% EDTA (pH 7), with the solution being replaced 3-4 times a week. The solution was replaced frequently due to the formation of white deposits on the fire coral tissue, presumably calcium carbonate that was redepositing after dissolving from the skeleton. After 5 weeks the skeleton was fully dissolved leaving the tissue behind, which was placed in Karvosky fixative (2% paraformaldehyde, 2.5% glutaraldehyde, 0.1 M buffer) overnight.

Once decalcified, samples were embedded in agarose for post-fixation staining performed by Teresa Sawyer at the Oregon State University Electron Microscope Facility. Tissues were first rinsed with 0.1M sodium cacodylate buffer. Post fixation was conducted in 1.5% potassium ferrocyanide and 2% osmium tetroxide in deionized water. Then samples underwent T-O-T-O staining to allow for the fluorescence of nucleic acids, followed by uranylacetate and lead aspartate fixation. Samples were then sequentially dehydrated in the following acetone mixtures:

10%, 30, 50, 70, 90, 95, 100-100% for 10-15 minutes each. Finally, samples were infiltrated with

Araldite resin and ultrathin sectioned. Imaging was conducted on an FEI Helios Nanolab 650 in

STEM mode at the Oregon State University Electron Microscope Facility.

RESULTS

Viruses in Fire Corals

In order to evaluate the viruses present in fire coral tissue, we imaged fire coral samples with TEM. Preliminary findings show high abundances of virus-like particles (VLP) in fire coral tissue (Fig.1). As there was no aspect to this project involving analysis of genetic material, reliable identification of objects as viruses and their taxonomic organization were not possible.

However, an ongoing parallel investigation on the potential genomic identification of the VLPs is underway using metagenomic and meta-transcriptomic methods (Bistolas et al., in prep).

Fig. 1A shows an extremely high density of small viruses (~ 150 nm diameter for the largest pictured VLP and the vast majority 100 nm and smaller in diameter). In this frame, there are over 100 VLPs in a given 2.25 µm2 area, so there are >500 VLPs pictured. While the density of VLPs is not as high in Fig. 1B, the size range is similar to that in Fig. 1A. Fig.

1B shows VLPs more spread out, with ~100 in a 6.25 µm2 area at its least dense, however, there are some sections of this image where the VLPs are very closely situated, even four times as dense than previously stated. In all, Fig. 1B shows nearly 2000 virus- like particles in this one section of this single fire coral sample. These images are representative of multiple instances seen in these samples, though there were not as large congregations of VLPs in areas of the sections where the fire coral tissue was more intact.

It is important to note, however, for both Fig. 1A and 1B and the rest of the TEM images analyzed in this study, we are only able to see a two-dimensional representation of a single part of what is, in reality, a three-dimensional environment. For example, the wide variety of sizes seen in various virus-like particles may be due to different

morphologies, or perhaps because each VLP was not bisected in the center. This results in an apparent increase in the abundance of smaller VLPs since, for example, only small

(~30 - 50 nm diameter) sections of a VLP are shown, while the whole particle may in reality be larger (~70 - ~100 nm diameter).

Figure 1: Virus-like particle density on M. platyphylla, taken from two different coral samples. Both show VLPs that are roughly icosahedral, none seen with envelopes; most VLPs are ~70 - ~100 nm diameter. A) >100 VLPs in a given 2.25 µm2 area, >500 VLPS seen here. B) ~100 in a 6.25 µm2 area (though some areas are 4x as dense), >2000 VLPs present.

VLPs Associated with Fire Coral Tissues

Beyond the high abundance of VLPs in close proximity to one another, TEM results also showed larger VLPs within fire coral tissues, highlighted in Fig. 2. A large VLP (~100 nm) was present in the bottom center of Fig. 2A with a round envelope present around the capsid, though also of note is a slightly smaller VLP (~70 nm) in the top left of this image that appears to have a capsid projection, though no envelope is present. A giant virus-like particle was found (Fig. 2B) and measured to be ~400 nm in diameter from the edge of its envelope to the opposite edge.

Other VLPs reminiscent of that seen in Fig. 2B were observed, both in size and apparent morphology (thick, unstained envelope with an icosahedral capsid). Finally, other large VLPs

(~70 nm - ~100 nm) were observed (Fig. 2C) with visible envelopes, however, they appear to have a generally flattened shape, as opposed to the classic icosahedral morphology.

Figure 2: Large Virus-Like Particles (VLPs) found in fire coral tissue. A) A VLP with a distinct double membrane (~100 nm) [bottom center] and one with a capsid protrusion(~70 nm) [top left], B) a giant VLP with an icosahedral capsid inside an envelope (~400 nm), and C) multiple VLPs with visible double membranes in an enclosed area, some with a flattened appearance, as opposed to round, or icosahedral (~70 ~100 nm).

VLPs Associated with Symbiodiniaceae

These giant virus-like particles were also observed close to significant structures within the fire coral tissue. Fig. 3 shows giant VLPs alongside, and even attached to, the outer membrane of the Symbiodiniaceae cells. These are identifiable as symbiont cells because of the thylakoid structures of the dinoflagellates’ chloroplasts, seen here as the parts of the images with multiple layers (Goodenough & Staehelin, 1971). The three VLPs annotated in Fig. 3 range in

size from 250 - 300 nm. Only Fig. 3C shows a VLP with what appears to be an envelope, however, given this VLP is in contact with the symbiont cell, this may in fact be the formation of a vacuole for phagocytosis.

Figure 3: Giant virus-like particles alongside symbiont (250 - 300 nm), with smaller VLPs alongside (A&B). Arrows show the giant VLPs and T is for thylakoid, indicating the chloroplasts of symbiont cells.

Novel VLP Morphology Identified in Fire Coral Tissues

Along with identifying many VLPs similar in structure to previously described viruses we imagined a group of nine giant VLPs within a presumed host cell. These VLPs are surrounding the same structure. This structure may be the nucleus of the host cell. Interestingly, three of the VLPs are visibly extending stalked projections to the membrane of the structure they surround. The smallest of these objects is ~350 nm in diameter and the largest is ~550 nm at its

longest section from edge to edge. In addition, all of the giant VLPs in this image have clearly defined double membranes, and interestingly, all have a fuzzy halo surrounding the capsids.

Figure 4: 9 Giant VLPs (~350 nm - ~550 nm) found in fire coral tissue. All have a visible envelope around a round capsid. Stalked projections extending from the VLPs to a central structure of the cell, possibly a nucleus or viral factory.

Bacteria in Fire Corals

While initially the main focus of this project was to observe viruses in fire corals, once the samples had been prepared by the electron microscope laboratory and we began viewing the fire coral samples, we noticed many objects that appeared to be bacteria. Many had been cut sagittally and appeared as dark-stained, ovoid structures. Further, a common morphology among most of these potential bacteria was the presence of an unstained (i.e. white) halo encompassing the structure. These bacteria-like objects were quite abundant, averaging between 20 - 40 objects within the panels of Fig. 5. Often, they were in high densities and seemingly extracellular although Fig. 5E shows these potential bacteria spread out amongst VLPs within the fire coral tissue.

Figure 5. Abundant ovoid bacterial cells in coral epithelium. These bacteria-like-objects all have white halos around their cells and are on average 1 - 1.5 μm in length. Frequently seen in large clusters, rarely solitary individuals. F) also shows a giant VLP with a thick envelope (arrow).

Bacteria Alongside Fire Coral Cellular Structures

Despite the constant groupings of these bacteria-like objects, there were instances of these structures in small groupings (of 2-3) or even solitary. Fig. 6A and B (600 nm) show a small grouping of these BLOs and a solitary one, respectively, near mitochondria, as indicated by the ribbed pattern of the matrix within the organelle membrane. Further, Fig. 6C depicts these same bacteria-like objects in the outermost epithelial cells.

Figure 6: bacterial cells (A, B) in close proximity to mitochondria (~600 nm - 1.5 μm), and (C) in epithelium of fire coral tissue (~725 nm - ~1 μm). Also, giant viruses (~400 nm) are shown (arrows) in C.

Bacteria in Acropora

The nutrient dosing experiment in Florida was designed to induce changes in the bacterial population associated with A. cervicornis. TEM imaging of samples taken prior to nutrient exposure and controls (which theoretically would have similar microbial compositions) revealed that there were indeed bacteria-like objects within these scleractinian coral tissues. Fig. 7A shows clustered groups of these BLOs in tight pockets, reminiscent of those seen in the fire corals. These objects (Fig. 7 A, C-E) in the scleractinian coral similarly have halos around them and are in a similar size range to the objects observed in fire corals (~700 nm - 1 μm). In contrast, unique BLOs are seen with an interesting morphology where they have a light area just within the membrane (Fig. 7B). While the majority of images show an ovoid shape to these

structures, there are also instances, such as Fig. 7C, where two BLOs appear roughly as circles, though this is because they have been bisected transversely. These in particular are close to a

Symbiodinium cell, again identifiable by its thylakoid membranes. Although these BLOs are not attached to the dinoflagellate, they are colocated with Symbiodinium in the vacuole created by the coral for the symbiont (called a symbiosome). In the coral epithelial tissue, there were more

BLOs that were among the smallest observed throughout this project (Fig. 7E). They have a length of ~500 nm and are more spread out from each other, not forming tight clusters.

Figure 7: A) bacteria-like-objects (700 nm- 1μm) in a mucocyte, as well as phages (~90 nm), some with tails, in between the BLOs. B) BLOs (~900 nm - 2 μm) also in a mucocyte. These have an interesting morphology where they have a light area just within the membrane C) BLOs (~1.5 μm) in the vacuole space of a symbiont cell (T - thylakoid). D) Bacteria-like-objects (~600 nm) within the tissue layer. E) BLOs (~500 nm) in the epithelium layer of coral tissue.

Viruses in Acropora

Although the main purpose of the nutrient dosing experiment was to track bacterial populations, TEM images revealed a wide array of VLPs present in the coral tissue (Fig. 8).

Some of these were morphologically similar to fire coral VLPs, albeit VLP density did not approach the high density seen in fire corals (Fig. 1). As in the fire coral samples, giant VLPs

(~500 nm) were present in this tissue (Fig. 8C). Here, the icosahedral capsid shape (shown here as hexagonal due to the cross-section) is visible. Interestingly, there appeared to be three VLPs with capsid protrusions attached to them (Fig. 8D), with a similar capsid size (between 50 and

100 nm) and protrusion length to the VLP discussed in Fig. 2A.

Figure 8: Virus-like particles in A. cervicornis tissue. A) VLPs (~150 - ~250 nm) and in a similar layout to that seen in fire corals (Fig. 1b) B) Small (~80 nm) VLPs gathered in three enclosures, possibly viral factories (VF). C) A giant VLP (~500 nm), icosahedral in shape, with a thick envelope. D) VLPs (~50 - 100 nm) with capsid protrusions (arrows).

Unexpected Findings

Throughout this project looking for the microbes in fire coral and scleractinian coral, we focused on identifying putative viruses and bacteria that appeared in the TEM images. However, these were not the only structures observed, as these samples were from coral tissue and not just cultures. As mentioned previously, symbiont cells and mitochondria were observed, though some structures were not easily identifiable (Fig. 9).

Pictured in Fig. 9A is a structure found in scleractinian coral tissue with four membranes present, unlike any other structure seen in the images from these sets of samples. Also interesting is the lack of staining in this structure, except for the membranes. At just under 600 nm at its longest, this structure is large by virus standards, however, it is small when compared to bacteria.

Also different from bacteria are the numerous objects in scleractinian coral tissue (Fig. 9B), which range in size from 500 nm - 850 nm. Unlike BLOs seen in previous images, they are stained less intensely near their center than near their membranes, which is quite the opposite from other images observed (e.g. Fig. 7B). Finally, in contrast to objects being relatively small,

Fig. 9C shows a structure that was repeatedly seen in the fire coral samples which is ~11 μm in length. Key features of this structure are the dark-stained, undulating pattern of the internal area and the slightly dark-stained layer just within the membrane that does not fully circle the structure.

Figure 9: Interesting structures found in TEM images not within the scope of this project. A) an object with 4 membranes (just under 600 nm in length) B) a field of structures less than 1 μm in length (800 nm in length & 500 nm width) C) A massive structure (~ 11μm in length) with a dark-stained interior and non-uniform layer inside the membrane.

DISCUSSION

Over the course of this project, a relatively narrow question and field of view (viruses within fire corals) was expanded as new developments arose, from observing bacteria-like objects in the fire corals to including the scope of scleractinian corals. This has made analysis more complex, however it has also broadened my understanding of this field of knowledge.

Due to the nature of the analysis conducted here (i.e. TEM imaging), it was not possible to positively identify any structures that appeared to be viruses or bacteria. As a result, terminology such as virus-like-particle and bacteria-like-object were used. Identification of microbial genera in these samples will be confirmed using genomic methods including shotgun

metagenomics. TEM imaging nonetheless allows for observations of distinct morphologies, which provide insight to potential identification of these particles. Despite taxonomic differences in hosts, comparisons can be made across the two sets of samples seen in this study.

Viruses

Unsurprisingly, viral-like particles were found in both fire coral and scleractinian coral samples, which is to be expected as viruses are important aspects of coral microbiomes (Vega

Thurber et al., 2017). There were surprising similarities, however, between VLPs observed in the two sets of corals. The vast majority of observed virus-like particles were smaller than 100 nm in diameter, many with what appear to be icosahedral morphologies. In addition, there were a variety of giant viruses present as well, ranging from icosahedral to round in capsid shape.

Potential NCLDVs

The giant VLPs seen in Fig. 2B and 8C have features similar to mimiviruses, including a thick icosahedral capsid is surrounding its core (Colson et al., 2017). Further, the VLPs observed fit into a similar size range, being just over 400 nm (Fig. 2B) and 500 nm (Fig. 8C) in diameter.

While another signature feature of mimiviruses is long fibrils protruding from the entirety of the capsid, these structures are not visible on the particles seen in these figures. This may be due to the resolution of the images that were taken, as the edge of the capsids are not clearly defined. As a result, there is still a possibility that these VLPs do reflect mimiviruses, or a virus closely related to mimivirus. Additionally, it is interesting that these features are seen across host species, and even more so that they were retrieved from separate ocean basins.

Other giant VLPs were seen in close proximity to symbiont cells (Fig. 3 A & B), even attaching to them (Fig. 3C). Symbiont cells were identified by the presence of thylakoids, depicted in TEM as structures with uniform layers within a cell membrane. The similar

morphology seen across these VLPs (round, darkly stained, no envelope) and their proximity to the symbionts suggest that these are viruses that target Symbiodiniaceae, which have been previously described (Correa et al., 2013).

Novel Stalked Viruses

The virus-like particles seen in Fig. 4 show similar structures to each other in that they are all roughly similar in size (350 - 550 nm). In addition, they all have double membranes for their capsids, as well as what appears to be a fuzzy halo around each of the structures, which may in fact be filaments that would be discernable at a higher resolution. However, the truly distinct feature that all of these VLPs share are the protrusions extending from the capsids to the membrane of a large central structure. None of these VLPs have an icosahedral shape to them, they are all ovoid. This makes potential identification interesting, as a large number of giant viruses possess icosahedral capsid morphologies (Abergel et al., 2015). However, many of these characteristics fall in line with those seen in molliviruses. These are described as non-icosahedral giant viruses with “genomes not compacted in electron-dense nucleoids” (Legendre et al., 2015).

Indeed, molliviruses are known to create connecting structures between the capsid and phagosome after penetrating the host cell through phagocytosis. This action then allows them to inject their genetic material into the cytoplasm, initiating the infection of the cell and replication cycle.

Inconsistent with this theory, however, VLPs in Fig. 4 are connected to a singular structure, all of which are within a greater membrane-bound area. This suggests some process other than the predicted cytoplasmic injection. The structure that the VLPs are attached to could be the cell nucleus, though this is unlikely, given common replication cycles of giant viruses where the capsids themselves do not come in contact with the nucleus (Abergel et al., 2015,

Mutsafi et al., 2013). Instead, this structure could be a virus factory, which are frequently used to assemble new viral particles (Colson et al., 2017; Abergel et al., 2015). Alternatively, if this central structure is the cell nucleus, this would be a novel virus as there are no known giant viruses that connect directly to the nucleus, either to inject their DNA or to extract it.

Bacteria

Notable features of fire coral TEM samples included numerous structures with similar morphologies to bacteria, such as the general ovoid shape, the dark-stained area within the membrane, and in general a length of 1 μm. A striking feature, however, repeated throughout the fire coral samples was an electron-lucent area surrounding the bacteria-like object, causing an effect like a halo. This is characteristic of Rickettsiales bacteria, supporting the identification of these objects as Rickettsia-like objects (RLO) (Silverman et al., 1978; Fritsche et al., 1999). This observation has particular ecological relevance, as Rickettsiales species are believed to be involved with White Band Disease, a pervasive disease affecting A. cervicornis (Klinges et al.,

2019) and thus it was influential in using this species as a representative of scleractinian corals from the experiment based in the Florida Keys

Observation of A. cervicornis samples also revealed RLOs with similar features (e.g. ovoid shape, white halo, etc.) to those seen in fire corals. These were found both in tightly packed regions and more spread out, separated from one another. The high abundance of these

RLOs indicates that these structures are important and likely impact the corals’ functions. No bacteria-like objects were found within symbiont cells, though there were instances of them being located nearby symbionts. RLOs were exclusively observed within coral tissue, or in an open space near a mostly enclosed area, possibly a vacuole, though this second scenario is likely

a result of the coral tissue degrading due to sampling, decalcification, and processing for microscopy.

Further evidence of these bacteria-like objects being Rickettsiales is that there were multiple instances of these structures located near mitochondria (Fig. 6A & B). Klinges et al.

(2019) describe the gene possessed by Rickettsiales that allows for the transfer of ATP and ADP across its membrane. This allows the bacteria to acquire ATP from the mitochondria, or intercepting ATP used elsewhere in the cell, while returning ADP, effectively harvesting energy from the host. Close proximity to mitochondria seen in this study indicate that this process may very well be occurring, or alternatively that this RLO is attempting to enter the mitochondria, as has been observed previously (Sassera et al., 2006).

In addition, RLOs were seen clustered in ovoid structures (Fig. 7A), reminiscent of mucocytes (mucus producing cells) (Peters, 2014; Klinges et al., 2019). While Fig. 7B also shows BLOs in a similar grouping inside a mucocyte, these objects possess an interesting morphology in that there is a lighter (unstained) region between the inside of these cells and their membranes. This differs from other RLOs observed as the white halo is typically on the exterior of the cell, not within the membrane. However, the location of these structures needs to be considered, especially given multiple instances of what may be RLOs inside of mucocytes. As a result, there is the possibility that the objects seen in Fig. 7B are also RLOs, just in a different life stage as those seen in Fig. 7A. This may be a result of the increased nutrients that corals were exposed to during the nutrient dosing experiment in Florida. This could induce physical alterations of this bacteria when nutrient levels in the water column change, as well as potentially altering metabolism.

It is also important to note that the tight clusters of RLOs in ovoid structures were not seen in fire coral tissue, though this may be a result of tissue degradation during processing, or the species in fire coral have a different lifestyle and do not grow in host vacuole. There were still multiple examples of large numbers (upwards of 20) of RLOs in fire coral that were also packed densely together (Fig. 5). While this is not the only location that these BLOs were found, the fact that these structures had similar morphologies to the scleractinian corals, they appear to similarly by Rickettsiales-like-objects. RLOs were also found in epithelium cell layers where they were closer to the surface of the coral tissue (Fig. 6C & 7E).

Since Rickettsiales species appear to be abundant throughout both fire coral and scleractinian coral, this further suggests that members of this bacterial order serve functions beyond simply infecting the host and causing White Band Disease, as has been previously thought (Miller et al., 2014; Peters et al., 2014; Klinges et al., 2019). This disease has only been observed in specific scleractinian corals (e.g. A. cervicornis) (Rosenberg et al., 2007; Libro et al., 2013), thus it is reasonable to conclude, as have other studies, that Rickettsiales is not the sole cause of this disease in branching corals (Kline & Vollmer, 2011; Klinges et al., 2019).

Nonetheless, the high abundance of this parasitic bacterial order observed in both fire corals and scleractinian corals likely has deleterious effects on its host. Furthermore, as these bacteria were observed in samples from geographically distinct regions, the ubiquity of these organisms in cnidarians is evident. Not only do the fire coral samples represent multiple islands in the Pacific

Ocean, the stony corals are from the Atlantic Ocean, meaning these similar morphologies, and thus likely similar organisms, are found between ocean basins.

Unexpected Findings

Along the way, while we looked for viruses and bacteria within these coral samples, we also found some structures that were perplexing, for which positive identification was difficult.

For the most part, we were able to identify coral cells, symbiont cells, VLPs and RLOs, however there were many instances of not completely understanding what the TEM images showed. Part of this may be due to the TEM in instances with a lack of clarity, such as the tissue being cut at an odd angle, resulting in structures not being shown fully, or with skewed proportions.

Nevertheless, these objects pose interesting questions, as in some cases it was not a lack of clarity that resulted in confusion, but rather the apparent size, morphology, or abundance of a given structure.

Interestingly, there was an object that had 4 distinct membranes (Fig. 9A). Usually, the maximum amount of membranes that a structure has is two, such as in mitochondria or chloroplasts, which resulted from phagocytosis and then endosymbiosis. If this structure was a result of phagocytosis, there would still be three membranes as a part of its morphology, an unusual characteristic. This object has a length of just under 600 nm at its longest cross section, placing it in the size range of giant viruses, however, since none of this object was stained, it indicates that there is no genetic material within it. While this may be an empty capsid, it does not resolve the issue of the excess membranes.

Also, of note is a clustering of vaguely ovoid objects that was only seen once in the stony coral tissue (Fig. 9B) and not seen at all in the fire coral. These structures range to upwards of

800 nm in length and 500 nm in width. These differ morphologically from the RLOs because the objects seen here have thick (~150 nm), dark-stained membranes, and lightly stained internal areas, different from both morphologies of RLOs discussed previously. This may be another type

of bacteria, though it is interesting that there were no other examples of these structures elsewhere in the tissue. In addition, there are tiny objects interspersed between these larger ovoid objects. While there are some small VLPs (<50 nm in diameter) present in this interstitial space, the structures of interest are thin (~50 nm), long (150-200 nm), dark-stained objects.

Finally, there were enormous structures found multiple times in the fire coral samples that dwarfed the surrounding VLPs and RLOs (Fig. 9C). These objects are almost 11 μm in length and 8 μm in width and one end seems to be slightly narrower than the other, reminiscent of an egg. There seem to be four main parts to these structures, the dark-stained membrane that is on the exterior compared to everything else. Next, a first interior layer that is non uniform as it goes around the structure, it becomes thinner at the narrower end of the structure and thicker in the wider section. The second layer is situated in the reverse order, becoming thinner at the wider end and thicker at the narrow end. This second layer is also very lightly stained, aiding in the identification of separate regions. Finally, there is a core section to this massive object that is darkly stained and has a uniquely wave interface with the second layer. Other TEM images showed these structures where the narrow end had opened, causing the contents of the core to spill out past the layers and membrane surrounding it. Other studies have described similar structures and have identified them as the of fire coral (Rojas-Molina et al., 2012).

This also explains why sometimes the core of these objects were spilling out of the membrane, because of the firing its nematocyst.

CONCLUSION

As has been cited numerous times throughout the literature, viruses and bacteria clearly have a large impact on corals and how they interact with their environment (Kline & Vollmer,

2011; Hester et al., 2015; Sweet & Bythell, 2017; Vega Thurber et al., 2017). From the results of

the samples imaged in this study, viruses and bacteria are more prevalent in the tissues of fire coral than that of scleractinian coral. The abundance of bacteria in fire coral tissue makes it appear as though fire coral are part bacteria themselves. In addition, a large amount of giant virus-like particles was found, hinting at their widespread abundance. Finally, this has profound consequences for understanding the ubiquity of viruses and bacteria, as the samples placed under

TEM here were taken from the Pacific and Atlantic Oceans, meaning that the similarities seen between fire corals and scleractinian corals are present between ocean basins as well.

Future research is needed, such as genomic studies, to evaluate the interactions between these pathogens and their hosts. However, this provides a starting place for evaluating the microbiome and virome of fire corals, since observations allow for the development of hypotheses that can later be explored through experiments, allowing for in depth understanding.

While TEM alone presents challenges to complete identification, when paired with genomic studies (which pose their own issues when used as a standalone technique), compelling evidence can be found (Correa et al., 2016).

REFERENCES

Abergel, C., Legendre, M., & Claverie, J. M. (2015). The rapidly expanding universe of giant

viruses: Mimivirus, Pandoravirus, Pithovirus and Mollivirus. FEMS microbiology

reviews, 39(6), 779-796.

Bibi, S., Kaur, R., Henriksen-Lacey, M., McNeil, S. E., Wilkhu, J., Lattmann, E., Christensen,

D., Mohammed, A. R., & Perrie, Y. (2011). Microscopy imaging of liposomes: from

coverslips to environmental SEM. International journal of pharmaceutics, 417(1-2), 138-

150.

Bourne, D. G., Garren, M., Work, T. M., Rosenberg, E., Smith, G. W., & Harvell, C. D. (2009).

Microbial disease and the coral holobiont. Trends in microbiology, 17(12), 554-562.

Cartwright, P., Halgedahl, S. L., Hendricks, J. R., Jarrard, R. D., Marques, A. C., Collins, A. G.,

& Lieberman, B. S. (2007). Exceptionally preserved from the Middle

Cambrian. PloS one, 2(10).

Casas, V., Kline, D. I., Wegley, L., Yu, Y., Breitbart, M., & Rohwer, F. (2004). Widespread

association of a Rickettsiales‐like bacterium with reef‐building corals. Environmental

microbiology, 6(11), 1137-1148.

Castelli, M., Sassera, D., & Petroni, G. (2016). Biodiversity of “non-model” Rickettsiales and

their association with aquatic organisms. In Rickettsiales (pp. 59-91). Springer, Cham.

Colson, P., De Lamballerie, X., Yutin, N., Asgari, S., Bigot, Y., Bideshi, D. K., Cheng, X.,

Federici, B. A., Van Etten, J. L., Koonin, E. V., La Scola, B., & Raoult, D. (2013).

“Megavirales”, a proposed new order for eukaryotic nucleocytoplasmic large DNA

viruses. Archives of virology, 158(12), 2517-2521.

Colson, P., La Scola, B., Levasseur, A., Caetano-Anolles, G., & Raoult, D. (2017). Mimivirus:

leading the way in the discovery of giant viruses of amoebae. Nature Reviews

Microbiology, 15(4), 243.

Coni, E. O. C., Ferreira, C. M., de Moura, R. L., Meirelles, P. M., Kaufman, L., & Francini-

Filho, R. B. (2013). An evaluation of the use of branching fire-corals (Millepora spp.) as

refuge by reef fish in the Abrolhos Bank, eastern Brazil. Environmental biology of fishes,

96(1), 45-55.

Correa, A. M., Welsh, R. M., & Vega Thurber, R. L. (2013). Unique nucleocytoplasmic dsDNA

and + ssRNA viruses are associated with the dinoflagellate endosymbionts of corals. The

ISME journal, 7(1), 13-27.

Correa, A., Ainsworth, T. D., Rosales, S. M., Thurber, A. R., Butler, C. R., & Vega Thurber, R.

L. (2016). Viral outbreak in corals associated with an in situ bleaching event: atypical

herpes-like viruses and a new megavirus infecting Symbiodinium. Frontiers in

microbiology, 7, 127.

Dubé, C. E., Boissin, E., & Planes, S. (2016). Overgrowth of living scleractinian corals by the

hydrocoral Millepora platyphylla in Moorea, French Polynesia. Marine Biodiversity,

46(2), 329-330.

Dubé, C. E., Boissin, E., Maynard, J. A., & Planes, S. (2017). Fire coral clones demonstrate

phenotypic plasticity among reef habitats. Molecular ecology, 26(15), 3860-3869.

Fritsche, T. R., Horn, M., Seyedirashti, S., Gautom, R. K., Schleifer, K. H., & Wagner, M.

(1999). In situ detection of novel bacterial endosymbionts of Acanthamoeba spp.

phylogenetically related to members of the order Rickettsiales. Appl. Environ.

Microbiol., 65(1), 206-212.

Goodenough, U. W., & Staehelin, L. A. (1971). Structural differentiation of stacked and

unstacked chloroplast membranes: freeze-etch electron microscopy of wild-type and

mutant strains of Chlamydomonas. The Journal of cell biology, 48(3), 594-619.

Gorsky, G., Bourdin, G., Lombard, F., Pedrotti, M. L., Audrain, S., Bin, N., Boss, E., Bowler, C.,

Cassar, N., Caudan, L., Chabot, G., Cohen, N. R., Cron, D., De Vargas, C., Dolan, J. R.,

Douville, E., Elineau, A., Flores, J. M., Ghiglione, J. F., Haëntjens, N., Hertau, M., John,

S. G., Kelly, R. L., Koren, I., Lin, Y., Marie, D., Moulin, C., Moucherie, Y., Pesant, S.,

Picheral, M., Poulain, J., Pujo-Pay, M., Reverdin, G., Romac, S., Sullivan, M. B., Trainic,

M., Tressol, M., Troublé, R., Vardi, A., Voolstra, C. R., Wincker, P., Agostini, S.,

Banaigs, B., Boissin, E., Forcioli, D., Furla, P., Galand, P. E., Gilson, E., Reynaud, S.,

Sunagawa, S., Thomas, O. P., Vega Thurber, R. L. V., Zoccola, D., Planes, S., Allemand,

D., & Karsenti, E. (2019) Expanding Tara Oceans Protocols for Underway, Ecosystemic

Sampling of the Ocean-Atmosphere Interface During Tara Pacific Expedition (2016–

2018). Front. Mar. Sci. 6:750

Hester, E. R., Barott, K. L., Nulton, J., Vermeij, M. J., & Rohwer, F. L. (2016). Stable and

sporadic symbiotic communities of coral and algal holobionts. The ISME Journal, 10(5),

1157-1169.

Hughes, T. P., Baird, A. H., Bellwood, D. R., Card, M., Connolly, S. R., Folke, C., Grosberg, R.,

Hoegh-Guldberg, O., Jackson, J.B.C., Kleypas, J., Lough, J. M., Marshall, P., Nyström,

M., Palumbi, S. R., Pandolfi, J. M., Rosen, B., & Roughgarden, J. (2003). Climate change,

human impacts, and the resilience of coral reefs. science, 301(5635), 929-933.

Kline, D. I., & Vollmer, S. V. (2011). White band disease (type I) of endangered Caribbean

acroporid corals is caused by pathogenic bacteria. Scientific reports, 1, 7.

Klinges JG, Rosales SM, McMinds R, Shaver EC, Shantz AA, Peters EC, Eitel M, Wörheide G,

Sharp KH, Burkepile DE, Silliman BR, Vega Thurber RL (2019) Phylogenetic, genomic,

and biogeographic characterization of a novel and ubiquitous marine invertebrate-

associated Rickettsiales parasite, Candidatus Aquarickettsia rohweri, gen. nov., sp. nov.

ISME J 13:2938–2953.

Lalli, C., & Parsons, T. R. (1997). Biological oceanography: an introduction. Elsevier.

Legendre, M., Lartigue, A., Bertaux, L., Jeudy, S., Bartoli, J., Lescot, M., Alempic, J.M., Ramus,

C., Bruley, C., Labadie, K. & Shmakova, L. (2015). In-depth study of Mollivirus

sibericum, a new 30,000-y-old giant virus infecting Acanthamoeba. Proceedings of the

National Academy of Sciences, 112(38), E5327-E5335.

Lewis, J. B. (2006). Biology and ecology of the hydrocoral Millepora on coral reefs. Advances in

Marine Biology, 50, 1-55.

Libro, S., Kaluziak, S. T., & Vollmer, S. V. (2013). RNA-seq profiles of immune related genes

in the Acropora cervicornis infected with white band disease. PloS one,

8(11).

Miller, M. W., Lohr, K. E., Cameron, C. M., Williams, D. E., & Peters, E. C. (2014). Disease

dynamics and potential mitigation among restored and wild staghorn coral, Acropora

cervicornis. PeerJ, 2, e541.

Morris, L. A., Voolstra, C. R., Quigley, K. M., Bourne, D. G., & Bay, L. K. (2019). Nutrient

availability and metabolism affect the stability of coral–symbiodiniaceae symbioses.

Trends in microbiology.

Muscatine, L., & Porter, J. W. (1977). Reef corals: mutualistic symbioses adapted to nutrient-

poor environments. Bioscience, 27(7), 454-460.

Mutsafi, Y., Shimoni, E., Shimon, A., & Minsky, A. (2013). Membrane assembly during the

infection cycle of the giant Mimivirus. PLoS pathogens, 9(5).

Peters, E. (2014). A Rickettsiales-like (?) bacterium is responsible for the tissue loss diseases of

Caribbean acroporid corals. In Proceedings of the Ocean Science Meeting (pp. 23-28).

Robson, A. L., Dastoor, P. C., Flynn, J., Palmer, W., Martin, A., Smith, D. W., Woldu, A., &

Hua, S. (2018). Advantages and limitations of current imaging techniques for

characterizing liposome morphology. Frontiers in pharmacology, 9, 80.

Rojas-Molina, A., García-Arredondo, A., Ibarra-Alvarado, C., & Bah, M. (2012). Millepora

(“fire corals”) species: toxinological studies until 2011. Advances in environmental

research, 26, 133-48.

Rosenberg, E., Koren, O., Reshef, L., Efrony, R., & Zilber-Rosenberg, I. (2007). The role of

microorganisms in coral health, disease and evolution. Nature Reviews Microbiology,

5(5), 355-362.

Sassera, D., Beninati, T., Bandi, C., Bouman, E. A., Sacchi, L., Fabbi, M., & Lo, N. (2006).

‘Candidatus Midichloria mitochondrii’, an endosymbiont of the tick Ixodes ricinus with a

unique intramitochondrial lifestyle. International journal of systematic and evolutionary

microbiology, 56(11), 2535-2540.

Sharma, D., & Ravindran, C. (2020). Diseases and pathogens of marine invertebrate corals in

Indian reefs. Journal of Invertebrate Pathology, 107373.

Shaver, E. C., Shantz, A. A., McMinds, R., Burkepile, D. E., Vega Thurber, R. L., & Silliman, B.

R. (2017). Effects of predation and nutrient enrichment on the success and microbiome of

a foundational coral. Ecology, 98(3), 830-839.

Silverman, D. J., Wisseman, C. L., Waddell, A. D., & Jones, M. (1978). External layers of

Rickettsia prowazekii and Rickettsia rickettsii: occurrence of a slime layer. Infection and

immunity, 22(1), 233-246.

Soffer, N., Zaneveld, J., & Vega Thurber, R. (2015). Phage–bacteria network analysis and its

implication for the understanding of coral disease. Environmental Microbiology, 17(4),

1203-1218.

Suttle, C. A. (2005). Viruses in the sea. Nature, 437(7057), 356-361.

Sweet, M., & Bythell, J. (2017). The role of viruses in coral health and disease. Journal of

invertebrate pathology, 147, 136-144.

Vega Thurber, R. L., & Correa, A. M. (2011). Viruses of reef-building scleractinian corals.

Journal of Experimental Marine Biology and Ecology, 408(1-2), 102-113.

Vega Thurber, R., Payet, J. P., Thurber, A. R., & Correa, A. M. (2017). Virus–host interactions

and their roles in coral reef health and disease. Nature Reviews Microbiology, 15(4), 205-

216.