Reef Sediments Can Act As a Stony Coral Tissue Loss Disease Vector

Stony coral tissue loss disease (SCTLD) was first observed in 2014 near Virginia Key in Miami-Dade County, Florida. Field sampling, lab experiments, and modeling approaches have suggested that reef sediments may play a role in SCTLD transmission, though a positive link has not been tested experimentally. We conducted an ex situ transmission assay using a statistically-independent disease apparatus to test whether reef sediments can transmit SCTLD in the absence of direct contact between diseased and healthy coral tissue. We evaluated two methods of sediment inoculation: batch inoculation of sediments collected from southeast Florida using whole colonies of diseased Montastraea cavernosa, and individual inoculations of sediments following independent, secondary infections of ∼5 cm2 coral fragments. Healthy fragments of the coral species Orbicella faveolata and M. cavernosa were exposed to these diseased sediment treatments, as well as direct disease contact and healthy sediment controls. SCTLD transmission was observed for both batch and individual diseased sediment inoculation treatments, albeit with lower proportions of infected individuals as compared to disease contact controls. The time to onset of lesions was significantly different between species and among disease treatments, with the most striking infections occurring in the individual diseased sediment treatment in under 24 h. Following infection, tissue samples were confirmed for the presence of SCTLD signs via histological examination, and sediment subsamples were analyzed for microbial community variation between treatments, identifying 16 SCTLD indicator taxa in sediments associated with corals experiencing tissue loss. This study demonstrated that reef sediments can indeed transmit SCTLD through indirect exposure between diseased and healthy corals, and adds credence to the assertion that SCTLD transmission occurs via an infectious agent or agents. This study emphasizes the critical need to understand the roles that sediment microbial communities and coastal development activities may have on the persistence of SCTLD throughout the endemic zone, especially in the context of management and conservation strategies in Florida and the wider Caribbean.

Bacterial Metabolic Potential and Micro-Eukaryotes Enriched in Stony Coral Tissue Loss Disease Lesions

The epizootic disease outbreak known as stony coral tissue loss disease (SCTLD) is arguably the most devastating coral disease in recorded history. SCTLD emerged off the coast of South Florida in 2014 and has since moved into the Caribbean, resulting in coral mortality rates that have changed reef structure and function. Currently, the cause of SCTLD is unknown, but there is evidence from 16S rRNA gene sequencing and bacterial culture studies that the microbial community plays a role in the progression of SCTLD lesions. In this study, we applied shotgun metagenomics to characterize the potential function of bacteria, as well as the composition of the micro-eukaryotic community, associated with SCTLD lesions. We re-examined samples that were previously analyzed using 16S rRNA gene high-throughput sequencing from four coral species: Stephanocoenia intersepta, Diploria labyrinthiformis, Dichocoenia stokesii, and Meandrina meandrites. For each species, tissue from apparently healthy (AH) corals, and unaffected tissue (DU) and lesion tissue (DL) on diseased corals, were collected from sites within the epidemic zone of SCTLD in the Florida Keys. Within the micro-eukaryotic community, the taxa most prominently enriched in DL compared to AH and DU tissue were members of Ciliophora. We also found that DL samples were relatively more abundant in less energy-efficient pathways like the pentose phosphate pathways. While less energy-efficient processes were identified, there were also relatively higher abundances of nucleotide biosynthesis and peptidoglycan maturation pathways in diseased corals compared to AH, which suggests there was more bacteria growth in diseased colonies. In addition, we generated 16 metagenome-assembled genomes (MAGs) belonging to the orders Pseudomonadales, Beggiatoales, Rhodobacterales, Rhizobiales, Rs-D84, Flavobacteriales, and Campylobacterales, and all MAGs were enriched in DL samples compared to AH samples. Across all MAGs there were antibiotic resistance genes that may have implications for the treatment of SCTLD with antibiotics. We also identified genes and pathways linked to virulence, such as nucleotide biosynthesis, succinate dehydrogenase, ureases, nickel/iron transporters, Type-1 secretion system, and metalloproteases. Some of these enzymes/pathways have been previously targeted in the treatment of other bacterial diseases and they may be of interest to mitigate SCTLD lesion progression.

Heat stress reduces the contribution of diazotrophs to coral holobiont nitrogen cycling

Efficient nutrient cycling in the coral-algal symbiosis requires constant but limited nitrogen availability. Coral-associated diazotrophs, i.e., prokaryotes capable of fixing dinitrogen, may thus support productivity in a stable coral-algal symbiosis but could contribute to its breakdown when overstimulated. However, the effects of environmental conditions on diazotroph communities and their interaction with other members of the coral holobiont remain poorly understood. Here we assessed the effects of heat stress on diazotroph diversity and their contribution to holobiont nutrient cycling in the reef-building coral Stylophora pistillata from the central Red Sea. In a stable symbiotic state, we found that nitrogen fixation by coral-associated diazotrophs constitutes a source of nitrogen to the algal symbionts. Heat stress caused an increase in nitrogen fixation concomitant with a change in diazotroph communities. Yet, this additional fixed nitrogen was not assimilated by the coral tissue or the algal symbionts. We conclude that although diazotrophs may support coral holobiont functioning under low nitrogen availability, altered nutrient cycling during heat stress abates the dependence of the coral host and its algal symbionts on diazotroph-derived nitrogen. Consequently, the role of nitrogen fixation in the coral holobiont is strongly dependent on its nutritional status and varies dynamically with environmental conditions.

Effect of species, size, and chimerism on the susceptibility of Caribbean brain coral recruits to stony coral tissue loss disease (SCTLD)

Stony coral tissue loss disease (SCTLD) has devastated coral populations along Florida′s Coral Reef and beyond. Although widespread infection and mortality of adult colonies have been documented, no studies have yet investigated the susceptibility of recruits to this disease. Here, we exposed eight-month-old Diploria labyrinthiformis recruits and four-month-old Colpophyllia natans recruits to two sequential doses of SCTLD in the laboratory to track infection and assess potential resilience. Both species began to develop lesions as early as 48 h after exposure began. During the first dose, 59.0% of C. natans recruits lost all tissue (died) within two to eight days of developing lesions, whereas D. labyrinthiformis recruits experienced significantly slower rates of tissue loss and minimal eventual mortality. In C. natans, larger recruits and those fused into groups of multiple genets (chimeras) exhibited the highest survivorship. In contrast, smaller and/or single (ungrouped) recruits had the lowest survivorship (9.9 - 26.5%). After 20 days, a second SCTLD dose was delivered to further test resistance in remaining recruits, and all recruits of both species succumbed within 6 days. Although no recruits showed absolute resistance to SCTLD following repeated exposures, our results provide evidence that interactions between species, size, and chimerism can impact relative resistance. This study represents the first report of SCTLD in Caribbean coral recruits and carries implications for natural species recovery and reef restoration efforts. Additional research on the susceptibility of coral juveniles to SCTLD is urgently needed, to include different species, locations, parents, and algal symbionts, with the goal of assessing relative susceptibility and identifying potential sources of resilience for this critical life history stage.

Coral Reef Viruses in Kane'ohe Bay, Hawai'i: Abundance, Diversity & Environmental Drivers

<p>Viruses are a ubiquitous component of coral reef ecosystems, with several viral types, from at least seven prokaryotic and 20 eukaryotic virus families currently characterised from the surface mucopolysaccharide layer (SML), coral tissue and the water column. However, little is known about the ecology and function of these viruses. For example, what are the environmental drivers of viral abundance and diversity on coral reefs? In this study, the abundance and distribution of virus-like particles (VLPs) associated with the SML and reef water of the coral Montipora capitata were determined using epifluorescence microscopy, while transmission electron microscopy was employed to determine the morphological diversity of VLPs. Sampling was conducted across the Coconut Island Marine Reserve (CIMR) reef system, Kane’ohe Bay, O’ahu, Hawai’i. Viral abundance was correlated with select environmental drivers and prokaryote abundance, while non-metric multidimensional scaling was used to determine the key environmental drivers of the viral community assemblage. The water column contained high concentrations of VLPs (5.98 × 107 ml-1) and prokaryotes (3.11 × 106 ml-1), consistent with the considerable anthropogenic impacts at this location. In comparison, the SML contained lower concentrations of VLPs (2.61 × 107 ml-1) and prokaryotes (2.08 × 106 ml-1); of note, the densities of viruses and prokaryotes in the SML were strongly coupled while those in the reef water were not. VLP density in the water column varied spatially across the reef, with the most sheltered site and the only one not situated on the reef crest having a greater VLP density than the other sites. Temporal variations in the density of microbes (i.e. viruses and prokaryotes) in the reef water were pronounced, while in the SML microbial densities remained constant. However, no specific environmental drivers of this variability could be identified. In contrast, temperature and water quality were correlated with shifts in the morphological diversity of VLPs across the reef. Small (< 50 nm) polyhedral/spherical VLPs were dominant, and were positively correlated to chlorophyll-a concentration when in the SML. In this same habitat, Fuselloviridae-like VLPs, filamentous VLPs and bead-shaped VLPs were positively correlated to temperature. In the reef water a different pattern was apparent: large (> 100 nm) Podoviridae-like VLPs and elongate Myoviridae-like VLPs, as well as lemon-shaped VLPs of both size classes showed positive associations with turbidity, while large filamentous VLPs, Geminiviridae-like VLPs and rod-shaped VLPs were positively associated with temperature. These results demonstrate that the viral community of Coconut Island’s reef is highly diverse, and subject to spatial and temporal change, especially in the water column. However, while the environmental drivers of viral diversity were partly elucidated, we are still a long way from understanding the drivers of viral abundance. More detailed study, both spatially and temporally, of the CIMR environment is required, as is comprehensive molecular analysis of the viral community of Kane’ohe Bay. Only then can we begin to understand the importance of viruses to the health and function of this, and other reef sites.</p>

Coral Reef Viruses in Kane'ohe Bay, Hawai'i: Abundance, Diversity & Environmental Drivers

<p>Viruses are a ubiquitous component of coral reef ecosystems, with several viral types, from at least seven prokaryotic and 20 eukaryotic virus families currently characterised from the surface mucopolysaccharide layer (SML), coral tissue and the water column. However, little is known about the ecology and function of these viruses. For example, what are the environmental drivers of viral abundance and diversity on coral reefs? In this study, the abundance and distribution of virus-like particles (VLPs) associated with the SML and reef water of the coral Montipora capitata were determined using epifluorescence microscopy, while transmission electron microscopy was employed to determine the morphological diversity of VLPs. Sampling was conducted across the Coconut Island Marine Reserve (CIMR) reef system, Kane’ohe Bay, O’ahu, Hawai’i. Viral abundance was correlated with select environmental drivers and prokaryote abundance, while non-metric multidimensional scaling was used to determine the key environmental drivers of the viral community assemblage. The water column contained high concentrations of VLPs (5.98 × 107 ml-1) and prokaryotes (3.11 × 106 ml-1), consistent with the considerable anthropogenic impacts at this location. In comparison, the SML contained lower concentrations of VLPs (2.61 × 107 ml-1) and prokaryotes (2.08 × 106 ml-1); of note, the densities of viruses and prokaryotes in the SML were strongly coupled while those in the reef water were not. VLP density in the water column varied spatially across the reef, with the most sheltered site and the only one not situated on the reef crest having a greater VLP density than the other sites. Temporal variations in the density of microbes (i.e. viruses and prokaryotes) in the reef water were pronounced, while in the SML microbial densities remained constant. However, no specific environmental drivers of this variability could be identified. In contrast, temperature and water quality were correlated with shifts in the morphological diversity of VLPs across the reef. Small (< 50 nm) polyhedral/spherical VLPs were dominant, and were positively correlated to chlorophyll-a concentration when in the SML. In this same habitat, Fuselloviridae-like VLPs, filamentous VLPs and bead-shaped VLPs were positively correlated to temperature. In the reef water a different pattern was apparent: large (> 100 nm) Podoviridae-like VLPs and elongate Myoviridae-like VLPs, as well as lemon-shaped VLPs of both size classes showed positive associations with turbidity, while large filamentous VLPs, Geminiviridae-like VLPs and rod-shaped VLPs were positively associated with temperature. These results demonstrate that the viral community of Coconut Island’s reef is highly diverse, and subject to spatial and temporal change, especially in the water column. However, while the environmental drivers of viral diversity were partly elucidated, we are still a long way from understanding the drivers of viral abundance. More detailed study, both spatially and temporally, of the CIMR environment is required, as is comprehensive molecular analysis of the viral community of Kane’ohe Bay. Only then can we begin to understand the importance of viruses to the health and function of this, and other reef sites.</p>

Coral Reef Viruses in Kane'ohe Bay, Hawai'i: Abundance, Diversity & Environmental Drivers

<p>Viruses are a ubiquitous component of coral reef ecosystems, with several viral types, from at least seven prokaryotic and 20 eukaryotic virus families currently characterised from the surface mucopolysaccharide layer (SML), coral tissue and the water column. However, little is known about the ecology and function of these viruses. For example, what are the environmental drivers of viral abundance and diversity on coral reefs? In this study, the abundance and distribution of virus-like particles (VLPs) associated with the SML and reef water of the coral Montipora capitata were determined using epifluorescence microscopy, while transmission electron microscopy was employed to determine the morphological diversity of VLPs. Sampling was conducted across the Coconut Island Marine Reserve (CIMR) reef system, Kane’ohe Bay, O’ahu, Hawai’i. Viral abundance was correlated with select environmental drivers and prokaryote abundance, while non-metric multidimensional scaling was used to determine the key environmental drivers of the viral community assemblage. The water column contained high concentrations of VLPs (5.98 × 107 ml-1) and prokaryotes (3.11 × 106 ml-1), consistent with the considerable anthropogenic impacts at this location. In comparison, the SML contained lower concentrations of VLPs (2.61 × 107 ml-1) and prokaryotes (2.08 × 106 ml-1); of note, the densities of viruses and prokaryotes in the SML were strongly coupled while those in the reef water were not. VLP density in the water column varied spatially across the reef, with the most sheltered site and the only one not situated on the reef crest having a greater VLP density than the other sites. Temporal variations in the density of microbes (i.e. viruses and prokaryotes) in the reef water were pronounced, while in the SML microbial densities remained constant. However, no specific environmental drivers of this variability could be identified. In contrast, temperature and water quality were correlated with shifts in the morphological diversity of VLPs across the reef. Small (< 50 nm) polyhedral/spherical VLPs were dominant, and were positively correlated to chlorophyll-a concentration when in the SML. In this same habitat, Fuselloviridae-like VLPs, filamentous VLPs and bead-shaped VLPs were positively correlated to temperature. In the reef water a different pattern was apparent: large (> 100 nm) Podoviridae-like VLPs and elongate Myoviridae-like VLPs, as well as lemon-shaped VLPs of both size classes showed positive associations with turbidity, while large filamentous VLPs, Geminiviridae-like VLPs and rod-shaped VLPs were positively associated with temperature. These results demonstrate that the viral community of Coconut Island’s reef is highly diverse, and subject to spatial and temporal change, especially in the water column. However, while the environmental drivers of viral diversity were partly elucidated, we are still a long way from understanding the drivers of viral abundance. More detailed study, both spatially and temporally, of the CIMR environment is required, as is comprehensive molecular analysis of the viral community of Kane’ohe Bay. Only then can we begin to understand the importance of viruses to the health and function of this, and other reef sites.</p>

Coral Reef Viruses in Kane'ohe Bay, Hawai'i: Abundance, Diversity & Environmental Drivers

<p>Viruses are a ubiquitous component of coral reef ecosystems, with several viral types, from at least seven prokaryotic and 20 eukaryotic virus families currently characterised from the surface mucopolysaccharide layer (SML), coral tissue and the water column. However, little is known about the ecology and function of these viruses. For example, what are the environmental drivers of viral abundance and diversity on coral reefs? In this study, the abundance and distribution of virus-like particles (VLPs) associated with the SML and reef water of the coral Montipora capitata were determined using epifluorescence microscopy, while transmission electron microscopy was employed to determine the morphological diversity of VLPs. Sampling was conducted across the Coconut Island Marine Reserve (CIMR) reef system, Kane’ohe Bay, O’ahu, Hawai’i. Viral abundance was correlated with select environmental drivers and prokaryote abundance, while non-metric multidimensional scaling was used to determine the key environmental drivers of the viral community assemblage. The water column contained high concentrations of VLPs (5.98 × 107 ml-1) and prokaryotes (3.11 × 106 ml-1), consistent with the considerable anthropogenic impacts at this location. In comparison, the SML contained lower concentrations of VLPs (2.61 × 107 ml-1) and prokaryotes (2.08 × 106 ml-1); of note, the densities of viruses and prokaryotes in the SML were strongly coupled while those in the reef water were not. VLP density in the water column varied spatially across the reef, with the most sheltered site and the only one not situated on the reef crest having a greater VLP density than the other sites. Temporal variations in the density of microbes (i.e. viruses and prokaryotes) in the reef water were pronounced, while in the SML microbial densities remained constant. However, no specific environmental drivers of this variability could be identified. In contrast, temperature and water quality were correlated with shifts in the morphological diversity of VLPs across the reef. Small (< 50 nm) polyhedral/spherical VLPs were dominant, and were positively correlated to chlorophyll-a concentration when in the SML. In this same habitat, Fuselloviridae-like VLPs, filamentous VLPs and bead-shaped VLPs were positively correlated to temperature. In the reef water a different pattern was apparent: large (> 100 nm) Podoviridae-like VLPs and elongate Myoviridae-like VLPs, as well as lemon-shaped VLPs of both size classes showed positive associations with turbidity, while large filamentous VLPs, Geminiviridae-like VLPs and rod-shaped VLPs were positively associated with temperature. These results demonstrate that the viral community of Coconut Island’s reef is highly diverse, and subject to spatial and temporal change, especially in the water column. However, while the environmental drivers of viral diversity were partly elucidated, we are still a long way from understanding the drivers of viral abundance. More detailed study, both spatially and temporally, of the CIMR environment is required, as is comprehensive molecular analysis of the viral community of Kane’ohe Bay. Only then can we begin to understand the importance of viruses to the health and function of this, and other reef sites.</p>

Viral-Like Particles Are Associated With Endosymbiont Pathology in Florida Corals Affected by Stony Coral Tissue Loss Disease

Stony coral tissue loss disease (SCTLD) was first documented in 2014 near the Port of Miami, Florida, and has since spread north and south along Florida’s Coral Reef, killing large numbers of more than 20 species of coral and leading to the functional extinction of at least one species, Dendrogyra cylindrus. SCTLD is assumed to be caused by bacteria based on presence of different molecular assemblages of bacteria in lesioned compared to apparently healthy tissues, its apparent spread among colonies, and cessation of spread of lesions in individual colonies treated with antibiotics. However, light microscopic examination of tissues of corals affected with SCTLD has not shown bacteria associated with tissue death. Rather, microscopy shows dead and dying coral cells and symbiotic dinoflagellates (endosymbionts) indicating a breakdown of host cell and endosymbiont symbiosis. It is unclear whether host cells die first leading to death of endosymbionts or vice versa. Based on microscopy, hypotheses as to possible causes of SCTLD include infectious agents not visible at the light microscopy level or toxicosis, perhaps originating from endosymbionts. To clarify this, we examined corals affected with SCTLD and apparently healthy corals using transmission electron microscopy. Endosymbionts in SCTLD-affected and apparently healthy corals consistently had varying degrees of pathology associated with elongated particles compatible in morphology with filamentous positive single-stranded RNA viruses of plants termed anisometric viral-like particles (AVLP). There was apparent progression from early to late replication of AVLP in the cytoplasm of endosymbionts adjacent to or at times within chloroplasts, with morphologic changes in chloroplasts consistent with those seen in plant cells infected by viruses. Coral host cell pathology appeared limited to massive proliferation and lysis of mucus cells. Based on these findings, we hypothesize that SCTLD is a viral disease of endosymbionts leading to coral host death. Efforts to confirm the presence of a virus associated with SCTLD through other means would be appropriate. These include showing the presence of a virus through molecular assays such as deep sequencing, attempts to grow this virus in the laboratory through culture of endosymbionts, localization of virus in tissue sections using immunohistochemistry or in situ hybridization, and experimental infection of known-virus-negative corals to replicate disease at the gross and microscopic level.

Revealing the Systematic Nature of Coral Attachment to Reef Substrates

Reef-building coral colonies propagate by periodic sexual reproduction and continuous asexual fragmentation. The latter depends on successful attachment to the reef substrate through modification of soft tissues and skeletal growth. Despite decades of research examining coral sexual and asexual propagation, the contact response, tissue motion, and cellular reorganisation responsible for attaching to the substrate via a newly formed skeleton have not been documented. Here, we correlated fluorescence and electron microscopy image data with ‘live’ microscopic time-lapse of the coral tissue biomechanics and developed a multiscale imaging approach to establish the first “coral attachment model” (CAM) - identifying three distinct phases that determine the timing and success of attachment during asexual propagation: (i) an initial immune response, followed by (ii) fragment stabilisation through anchoring by the soft tissue and (iii) formation of a “lappet appendage” structure leading to substrate bonding of the tissue for encrustation through the onset of skeletal calcification. In developing CAM, we provide new frameworks and metrics that enable reef researchers, managers and coral restoration practitioners to evaluate attachment effectiveness needed to optimise species-substrate compatibility.