Stony coral tissue loss disease intervention with amoxicillin leads to a reversal of disease-modulated gene expression pathways.

Stony coral tissue loss disease (SCTLD) remains an unprecedented disease outbreak due to its high mortality rate and rapid spread throughout Florida's Coral Reef and wider Caribbean. A collaborative effort is underway to evaluate strategies that mitigate the spread of SCTLD across coral colonies and reefs, including restoration of disease-resistant genotypes, genetic rescue, and disease intervention with therapeutics. We conducted an in-situ experiment in Southeast Florida to assess molecular responses among SCTLD-affected Montastraea cavernosa pre- and post-application of the most widely used intervention method, CoreRx Base 2B with amoxicillin. Through Tag-Seq gene expression profiling of apparently healthy, diseased, and treated corals, we identified modulation of metabolomic and immune gene pathways following antibiotic treatment. In a complementary ex-situ disease challenge experiment, we exposed nursery-cultured M. cavernosa and Orbicella faveolata fragments to SCTLD-affected donor corals to compare transcriptomic profiles among clonal individuals from unexposed controls, those exposed and displaying disease signs, and corals exposed and not displaying disease signs. Suppression of metabolic functional groups and activation of stress gene pathways as a result of SCTLD exposure were apparent in both species. Amoxicillin treatment led to a 'reversal' of the majority of gene pathways implicated in disease response, suggesting potential recovery of corals following antibiotic application. In addition to increasing our understanding of molecular responses to SCTLD, we provide resource managers with transcriptomic evidence that disease intervention with antibiotics appears to be successful and may help to modulate coral immune responses to SCTLD. These results contribute to feasibility assessments of intervention efforts following disease outbreaks and improved predictions of coral reef health across the wider Caribbean.

Corals as canaries in the coalmine: Towards the incorporation of marine ecosystems into the 'One Health' concept.

'One World - One Health' is a developing concept which aims to explicitly incorporate linkages between the environment and human society into wildlife and human health care. Past work in the field has concentrated on aspects of disease, particularly emerging zoonoses, and focused on terrestrial systems. Here, we argue that marine environments are crucial components of the 'One World - One Health' framework, and that coral reefs are the epitome of its underlying philosophy. That is, they provide vast contributions to a wide range of ecosystem services with strong and direct links to human well-being. Further, the sensitivity of corals to climate change, and the current emergence of a wide range of diseases, make coral reefs ideal study systems to assess links, impacts, and feedback mechanisms that can affect human and ecosystem health. There are well established protocols for monitoring corals, as well as global networks of coral researchers, but there remain substantial challenges to understanding these complex systems, their health and links to provisioning of ecosystem services. We explore these challenges and conclude with a look at how developing technology offers potential ways of addressing them. We argue that a greater integration of coral reef research into the 'One World - One Health' framework will enrich our understanding of the many links within, and between, ecosystems and human society. This will ultimately support the development of measures for improving the health of both humans and the environment.

Novel metagenomics analysis of stony coral tissue loss disease.

Stony coral tissue loss disease (SCTLD) has devastated coral reefs off the coast of Florida and continues to spread throughout the Caribbean. Although a number of bacterial taxa have consistently been associated with SCTLD, no pathogen has been definitively implicated in the etiology of SCTLD. Previous studies have predominantly focused on the prokaryotic community through 16S rRNA sequencing of healthy and affected tissues. Here, we provide a different analytical approach by applying a bioinformatics pipeline to publicly available metagenomic sequencing samples of SCTLD lesions and healthy tissues from 4 stony coral species. To compensate for the lack of coral reference genomes, we used data from apparently healthy coral samples to approximate a host genome and healthy microbiome reference. These reads were then used as a reference to which we matched and removed reads from diseased lesion tissue samples, and the remaining reads associated only with disease lesions were taxonomically classified at the DNA and protein levels. For DNA classifications, we used a pathogen identification protocol originally designed to identify pathogens in human tissue samples, and for protein classifications, we used a fast protein sequence aligner. To assess the utility of our pipeline, a species-level analysis of a candidate genus, Vibrio, was used to demonstrate the pipeline's effectiveness. Our approach revealed both complementary and unique coral microbiome members compared with a prior metagenome analysis of the same dataset.

Novel metagenomics analysis of stony coral tissue loss disease.

Stony coral tissue loss disease (SCTLD) has devastated coral reefs off the coast of Florida and continues to spread throughout the Caribbean. Although a number of bacterial taxa have consistently been associated with SCTLD, no pathogen has been definitively implicated in the etiology of SCTLD. Previous studies have predominantly focused on the prokaryotic community through 16S rRNA sequencing of healthy and affected tissues. Here, we provide a different analytical approach by applying a bioinformatics pipeline to publicly available metagenomic sequencing samples of SCTLD lesions and healthy tissues from four stony coral species. To compensate for the lack of coral reference genomes, we used data from apparently healthy coral samples to approximate a host genome and healthy microbiome reference. These reads were then used as a reference to which we matched and removed reads from diseased lesion tissue samples, and the remaining reads associated only with disease lesions were taxonomically classified at the DNA and protein levels. For DNA classifications, we used a pathogen identification protocol originally designed to identify pathogens in human tissue samples, and for protein classifications, we used a fast protein sequence aligner. To assess the utility of our pipeline, a species-level analysis of a candidate genus, Vibrio, was used to demonstrate the pipeline's effectiveness. Our approach revealed both complementary and unique coral microbiome members compared to a prior metagenome analysis of the same dataset.

Investigating microbial size classes associated with the transmission of stony coral tissue loss disease (SCTLD).

Effective treatment and prevention of any disease necessitates knowledge of the causative agent, yet the causative agents of most coral diseases remain unknown, in part due to the difficulty of distinguishing the pathogenic microbe(s) among the complex microbial backdrop of coral hosts. Stony coral tissue loss disease (SCTLD) is a particularly destructive disease of unknown etiology, capable of transmitting through the water column and killing entire colonies within a matter of weeks. Here we used a previously described method to (i) isolate diseased and apparently healthy coral colonies within individual mesocosms containing filtered seawater with low microbial background levels; (ii) incubate for several days to enrich the water with coral-shed microbes; (iii) use tangential-flow filtration to concentrate the microbial community in the mesocosm water; and then (iv) filter the resulting concentrate through a sequential series of different pore-sized filters. To investigate the size class of microorganism(s) associated with SCTLD transmission, we used 0.8 µm pore size filters to capture microeukaryotes and expelled zooxanthellae, 0.22 µm pore size filters to capture bacteria and large viruses, and 0.025 µm pore size filters to capture smaller viruses. In an attempt to further refine which size fraction(s) contained the transmissible element of SCTLD, we then applied these filters to healthy "receiver" coral fragments and monitored them for the onset of SCTLD signs over three separate experimental runs. However, several factors outside of our control confounded the transmission results, rendering them inconclusive. As the bulk of prior studies of SCTLD in coral tissues have primarily investigated the associated bacterial community, we chose to characterize the prokaryotic community associated with all mesocosm 0.22 µm pore size filters using Illumina sequencing of the V4 region of the 16S rRNA gene. We identified overlaps with prior SCTLD studies, including the presence of numerous previously identified SCTLD bioindicators within our mesocosms. The identification in our mesocosms of specific bacterial amplicon sequence variants that also appear across prior studies spanning different collection years, geographic regions, source material, and coral species, suggests that bacteria may play some role in the disease.

Coral affected by stony coral tissue loss disease can produce viable offspring.

Stony coral tissue loss disease (SCTLD) has caused high mortality of at least 25 coral species across the Caribbean, with Pseudodiploria strigosa being the second most affected species in the Mexican Caribbean. The resulting decreased abundance and colony density reduces the fertilization potential of SCTLD-susceptible species. Therefore, larval-based restoration could be of great benefit, though precautionary concerns about disease transmission may foster reluctance to implement this approach with SCTLD-susceptible species. We evaluated the performance of offspring obtained by crossing gametes of a healthy P. strigosa colony (100% apparently healthy tissue) with that of a colony affected by SCTLD (>50% tissue loss) and compared these with prior crosses between healthy parents. Fertilization and settlement were as high as prior crosses among healthy parents, and post-settlement survivorship over a year in outdoor tanks was 7.8%. After thirteen months, the diseased-parent recruits were outplanted to a degraded reef. Their survivorship was ∼44% and their growth rate was 0.365 mm ± 1.29 SD per month. This study shows that even diseased parent colonies can be effective in assisted sexual reproduction for the restoration of species affected by SCTLD.

Combining tangential flow filtration and size fractionation of mesocosm water as a method for the investigation of waterborne coral diseases.

The causative agents of most coral diseases today remain unknown, complicating disease response and restoration efforts. Pathogen identifications can be hampered by complex microbial communities naturally associated with corals and seawater, which create complicating "background noise" that can potentially obscure a pathogen's signal. Here, we outline an approach to investigate waterborne coral diseases that use a combination of coral mesocosms, tangential flow filtration, and size fractionation to reduce the impact of this background microbial diversity, compensate for unknown infectious dose, and further narrow the suspect pool of potential pathogens. As proof of concept, we use this method to compare the bacterial communities shed into six Montastraea cavernosa coral mesocosms and demonstrate this method effectively detects differences between diseased and healthy coral colonies. We found several amplicon sequence variants (ASVs) in the diseased mesocosms that represented 100% matches with ASVs identified in prior studies of diseased coral tissue, further illustrating the effectiveness of our approach. Our described method is an effective alternative to using coral tissue or mucus to investigate waterborne coral diseases of unknown etiology and can help more quickly narrow the pool of possible pathogens to better aid in disease response efforts. Additionally, this versatile method can be easily adapted to characterize either the entire microbial community associated with a coral or target-specific microbial groups, making it a beneficial approach regardless of whether a causative agent is suspected or is completely unknown.

Characterization of the Microbiome of Corals with Stony Coral Tissue Loss Disease along Florida's Coral Reef.

Stony coral tissue loss disease (SCTLD) is an emergent and often lethal coral disease that was first reported near Miami, FL (USA) in 2014. Our objective was to determine if coral colonies showing signs of SCTLD possess a specific microbial signature across five susceptible species sampled in Florida's Coral Reef. Three sample types were collected: lesion tissue and apparently unaffected tissue of diseased colonies, and tissue of apparently healthy colonies. Using 16S rRNA high-throughput gene sequencing, our results show that, for every species, the microbial community composition of lesion tissue was significantly different from healthy colony tissue and from the unaffected tissue of diseased colonies. The lesion tissue of all but one species (Siderastrea siderea) had higher relative abundances of the order Rhodobacterales compared with other types of tissue samples, which may partly explain why S. siderea lesions often differed in appearance compared to other species. The order Clostridiales was also present at relatively high abundances in the lesion tissue of three species compared to healthy and unaffected tissues. Stress often leads to the dysbiosis of coral microbiomes and increases the abundance of opportunistic pathogens. The present study suggests that Rhodobacterales and Clostridiales likely play an important role in SCTLD.

Local dynamics of a white syndrome outbreak and changes in the microbial community associated with colonies of the scleractinian brain coral Pseudodiploria strigosa.

Reef corals in the Mexican Reef System have been severely affected by the emergence of a white syndrome that resembles both White Plague II and SCTLD descriptions. Meandroid scleractinian coral species are among the most severely affected. To gain insight into this affliction we conducted a broad study in the brain coral Pseudodiploria strigosa at a rear reef site in the NE Mexican Caribbean. We describe macro and microscopical signals of the disease, characterize the outbreak dynamics, the tissue histopathology, explore immunological responses in the individuals, and compare microbial assemblages associated with the surface mucus layer of healthy and unhealthy colonies. At the study site, the white syndrome outbreak on P. strigosa showed a high incidence rate in summer-fall and a low one in winter, as well as low survival expectation of diseased colonies at the end of the study. After 306 days of observation, out of 96 tracked colonies, eight remained apparently healthy and seven were diseased. No effective resistance to colony disease progression was observed once white syndrome signs developed. Tissue loss rate during the study varied among colonies (mean = 10.8 cm2, s.d. = 7.8 cm2) suggesting a complex relation between causal agents and colony resistance. The deterioration of tissues was evidenced from the basal to the surface body wall of polyps (up to 66% hypertrophy and liquefactive necrosis in unhealthy colonies), implying that microscopic alterations begin before macroscopic signals develop, suggesting this may be a systemic disease. We measured high levels of phenoloxidase (two orders of magnitude higher PO activity than P. strigosa affected by BBD) and antibacterial activity without significant reduction in unhealthy samples from the mucus layer, indicative of an enhanced immunological response. Results showed that opportunistic bacteria dominated damaged colonies, where six genera of the Bacteroidia class were found with significant changes in unhealthy colonies after DeSeq2 analysis. Nevertheless, histological observations did not support infection of the tissues. The opportunistic overload seems to be contained within the mucus layer but may be associated with the mortality of tissues in a yet unclear way. Future research should focus on experimental infections, the tracking of natural infections, and the immunocompetence of corals in the face of environmental pressures due to local, regional, and global impacts. If environmental deterioration is the primary cause of the continuing emergence and re-emergence of lethal coral diseases, as has been proposed by many authors, the only true option to effectively help preserve the coral reef biodiversity and services, is to restore the environmental quality of reef waters at the local scale and reduce greenhouse gases at the global scale.

Rhodobacterales and Rhizobiales Are Associated With Stony Coral Tissue Loss Disease and Its Suspected Sources of Transmission.

In 2014, Stony Coral Tissue Loss Disease (SCTLD) was first detected off the coast of Miami, FL, United States, and continues to persist and spread along the Florida Reef Tractr (FRT) and into the Caribbean. SCTLD can have up to a 61% prevalence in reefs and has affected at least 23 species of scleractinian corals. This has contributed to the regional near-extinction of at least one coral species, Dendrogyra cylindrus. Initial studies of SCTLD indicate microbial community shifts and cessation of lesion progression in response to antibiotics on some colonies. However, the etiology and abiotic sources of SCTLD transmission are unknown. To characterize SCTLD microbial signatures, we collected tissue samples from four affected coral species: Stephanocoenia intersepta, Diploria labyrinthiformis, Dichocoenia stokesii, and Meandrina meandrites. Tissue samples were from apparently healthy (AH) corals, and unaffected tissue (DU) and lesion tissue (DL) on diseased corals. Samples were collected in June 2018 from three zones: (1) vulnerable (ahead of the SCTLD disease boundary in the Lower Florida Keys), (2) endemic (post-outbreak in the Upper Florida Keys), and (3) epidemic (SCTLD was active and prevalent in the Middle Florida Keys). From each zone, sediment and water samples were also collected to identify whether they may serve as potential sources of transmission for SCTLD-associated microbes. We used 16S rRNA gene amplicon high-throughput sequencing methods to characterize the microbiomes of the coral, water, and sediment samples. We identified a relatively higher abundance of the bacteria orders Rhodobacterales and Rhizobiales in DL tissue compared to AH and DU tissue. Also, our results showed relatively higher abundances of Rhodobacterales in water from the endemic and epidemic zones compared to the vulnerable zone. Rhodobacterales and Rhizobiales identified at higher relative abundances in DL samples were also detected in sediment samples, but not in water samples. Our data indicate that Rhodobacterales and Rhizobiales may play a role in SCTLD and that sediment may be a source of transmission for Rhodobacterales and Rhizobiales associated with SCTLD lesions.

A rapid spread of the stony coral tissue loss disease outbreak in the Mexican Caribbean.

Caribbean reef corals have experienced unprecedented declines from climate change, anthropogenic stressors and infectious diseases in recent decades. Since 2014, a highly lethal, new disease, called stony coral tissue loss disease, has impacted many reef-coral species in Florida. During the summer of 2018, we noticed an anomalously high disease prevalence affecting different coral species in the northern portion of the Mexican Caribbean. We assessed the severity of this outbreak in 2018/2019 using the AGRRA coral protocol to survey 82 reef sites across the Mexican Caribbean. Then, using a subset of 14 sites, we detailed information from before the outbreak (2016/2017) to explore the consequences of the disease on the condition and composition of coral communities. Our findings show that the disease outbreak has already spread across the entire region by affecting similar species (with similar disease patterns) to those previously described for Florida. However, we observed a great variability in prevalence and tissue mortality that was not attributable to any geographical gradient. Using long-term data, we determined that there is no evidence of such high coral disease prevalence anywhere in the region before 2018, which suggests that the entire Mexican Caribbean was afflicted by the disease within a few months. The analysis of sites that contained pre-outbreak information showed that this event considerably increased coral mortality and severely changed the structure of coral communities in the region. Given the high prevalence and lethality of this disease, and the high number of susceptible species, we encourage reef researchers, managers and stakeholders across the Western Atlantic to accord it the highest priority for the near future.