Evaluation of in vitro treatments against the causative agent of Diadema antillarum scuticociliatosis (DaSc).

In the 1980s, a mass die-off of the long-spined sea urchin Diadema antillarum occurred on Florida and Caribbean coral reefs. D. antillarum populations largely did not recover, and in 2022, remaining populations experienced another mass mortality event. A ciliate most similar to Philaster apodigitiformis was identified as the causative agent of the 2022 event, which was named D. antillarum scuticociliatosis (DaSc). Here, we investigated possible treatments for this pathogen. We tested the efficacy of 10 compounds at final concentrations of 100, 50, 25, 12.5, 6.25, and 3.13 µM, or a 10-fold serial dilution series, against ciliates cultured from an infected D. antillarum specimen. Of the tested compounds, 8 induced 100% ciliate mortality at some dose after 24 h. The most effective (defined as those requiring the lowest dose to induce 100% ciliate mortality) were quinacrine and tomatine (both effective at 12.5 µM), followed by furaltadone and plumbagin (25 µM), bithionol sulfoxide and 2'4' dihydroxychalcone (50 µM), and oxyclozanide and carnidazole (100 µM). Toltrazuril and a commercially available anticiliate product containing naphthoquinones were not effective at any dose tested. Shortened (15 min) time trials were performed using ciliate cultures reared in natural seawater to better reflect natural environmental conditions, and revealed that 2 of the compounds (quinacrine and tomatine) induced 100% ciliate mortality at 100 µM, with tomatine also effective at 50 µM. This study identified several treatments effective against the causative agent of DaSc in vitro, but their toxicity and utility in vivo remain unknown.

Transglobal spread of an ecologically relevant sea urchin parasite.

Mass mortality of the dominant coral reef herbivore Diadema antillarum in the Caribbean in the early 1980s contributed to a persistent phase shift from coral- to algal-dominated reefs. In 2022, a scuticociliate most closely related to Philaster apodigitiformis caused further mass mortality of D. antillarum across the Caribbean, leading to >95% mortality at affected sites. Mortality was also reported in the related species Diadema setosum in the Mediterranean in 2022, though the causative agent of the Mediterranean outbreak has not yet been determined. In April 2023, mass mortality of Diadema setosum occurred along the Sultanate of Oman's coastline. Urchins displayed signs compatible with scuticociliatosis including abnormal behavior, drooping and loss of spines, followed by tissue necrosis and death. Here we report the detection of an 18S rRNA gene sequence in abnormal urchins from Muscat, Oman, that is identical to the Philaster strain responsible for D. antillarum mass mortality in the Caribbean. We also show that scuticociliatosis signs can be elicited in Diadema setosum by experimental challenge with the cultivated Philaster strain associated with Caribbean scuticociliatosis. These results demonstrate the Philaster sp. associated with D. antillarum mass mortality has rapidly spread to geographically distant coral reefs, compelling global-scale awareness and monitoring for this devastating condition through field surveys, microscopy, and molecular microbiological approaches, and prompting investigation of long-range transmission mechanisms.

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.

A scuticociliate causes mass mortality of Diadema antillarum in the Caribbean Sea.

Echinoderm mass mortality events shape marine ecosystems by altering the dynamics among major benthic groups. The sea urchin Diadema antillarum, virtually extirpated in the Caribbean in the early 1980s by an unknown cause, recently experienced another mass mortality beginning in January 2022. We investigated the cause of this mass mortality event through combined molecular biological and veterinary pathologic approaches comparing grossly normal and abnormal animals collected from 23 sites, representing locations that were either affected or unaffected at the time of sampling. Here, we report that a scuticociliate most similar to Philaster apodigitiformis was consistently associated with abnormal urchins at affected sites but was absent from unaffected sites. Experimentally challenging naïve urchins with a Philaster culture isolated from an abnormal, field-collected specimen resulted in gross signs consistent with those of the mortality event. The same ciliate was recovered from treated specimens postmortem, thus fulfilling Koch's postulates for this microorganism. We term this condition D. antillarum scuticociliatosis.

A meta-analysis of the stony coral tissue loss disease microbiome finds key bacteria in unaffected and lesion tissue in diseased colonies.

Stony coral tissue loss disease (SCTLD) has been causing significant whole colony mortality on reefs in Florida and the Caribbean. The cause of SCTLD remains unknown, with the limited concurrence of SCTLD-associated bacteria among studies. We conducted a meta-analysis of 16S ribosomal RNA gene datasets generated by 16 field and laboratory SCTLD studies to find consistent bacteria associated with SCTLD across disease zones (vulnerable, endemic, and epidemic), coral species, coral compartments (mucus, tissue, and skeleton), and colony health states (apparently healthy colony tissue (AH), and unaffected (DU) and lesion (DL) tissue from diseased colonies). We also evaluated bacteria in seawater and sediment, which may be sources of SCTLD transmission. Although AH colonies in endemic and epidemic zones harbor bacteria associated with SCTLD lesions, and aquaria and field samples had distinct microbial compositions, there were still clear differences in the microbial composition among AH, DU, and DL in the combined dataset. Alpha-diversity between AH and DL was not different; however, DU showed increased alpha-diversity compared to AH, indicating that, prior to lesion formation, corals may undergo a disturbance to the microbiome. This disturbance may be driven by Flavobacteriales, which were especially enriched in DU. In DL, Rhodobacterales and Peptostreptococcales-Tissierellales were prominent in structuring microbial interactions. We also predict an enrichment of an alpha-toxin in DL samples which is typically found in Clostridia. We provide a consensus of SCTLD-associated bacteria prior to and during lesion formation and identify how these taxa vary across studies, coral species, coral compartments, seawater, and sediment.

Rapid prototyping for quantifying belief weights of competing hypotheses about emergent diseases.

Emerging diseases can have devastating consequences for wildlife and require a rapid response. A critical first step towards developing appropriate management is identifying the etiology of the disease, which can be difficult to determine, particularly early in emergence. Gathering and synthesizing existing information about potential disease causes, by leveraging expert knowledge or relevant existing studies, provides a principled approach to quickly inform decision-making and management efforts. Additionally, updating the current state of knowledge as more information becomes available over time can reduce scientific uncertainty and lead to substantial improvement in the decision-making process and the application of management actions that incorporate and adapt to newly acquired scientific understanding. Here we present a rapid prototyping method for quantifying belief weights for competing hypotheses about the etiology of disease using a combination of formal expert elicitation and Bayesian hierarchical modeling. We illustrate the application of this approach for investigating the etiology of stony coral tissue loss disease (SCTLD) and discuss the opportunities and challenges of this approach for addressing emergent diseases. Lastly, we detail how our work may apply to other pressing management or conservation problems that require quick responses. We found the rapid prototyping methods to be an efficient and rapid means to narrow down the number of potential hypotheses, synthesize current understanding, and help prioritize future studies and experiments. This approach is rapid by providing a snapshot assessment of the current state of knowledge. It can also be updated periodically (e.g., annually) to assess changes in belief weights over time as scientific understanding increases. Synthesis and applications: The rapid prototyping approaches demonstrated here can be used to combine knowledge from multiple experts and/or studies to help with fast decision-making needed for urgent conservation issues including emerging diseases and other management problems that require rapid responses. These approaches can also be used to adjust belief weights over time as studies and expert knowledge accumulate and can be a helpful tool for adapting management decisions.

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.

Identifying mangrove-coral habitats in the Florida Keys.

Coral reefs are degrading due to many synergistic stressors. Recently there have been a number of global reports of corals occupying mangrove habitats that provide a supportive environment or refugium for corals, sheltering them by reducing stressors such as oxidative light stress and low pH. This study used satellite imagery and manual ground-truthing surveys to search for mangrove-coral habitats in the Florida Keys National Marine Sanctuary and then collected basic environmental parameters (temperature, salinity, dissolved oxygen, pHNBS, turbidity) at identified sites using a multi-parameter water quality sonde. Two kinds of mangrove-coral habitats were found in both the Upper and Lower Florida Keys: (1) prop-root corals, where coral colonies were growing directly on (and around) mangrove prop roots, and (2) channel corals, where coral colonies were growing in mangrove channels under the shade of the mangrove canopy, at deeper depths and not in as close proximity to the mangroves. Coral species found growing on and directly adjacent to prop roots included Porites porites (multiple morphs, including P. divaricata and P. furcata), Siderastrea radians, and Favia fragum. Channel coral habitats predominantly hosted S. radians and a few S. siderea, although single colonies of Solenastrea bournoni and Stephanocoenia intersepta were observed. Although clear, low-turbidity water was a consistent feature of these mangrove-coral habitats, the specific combination of environmental factors that determine which mangrove habitats are favorable for coral recruitment remains to be defined. Circumstantial evidence suggests additional coral communities existed on mangrove shorelines of oceanside and backcountry islands until destroyed, likely by Hurricane Irma. These mangrove-coral habitats may be climate refugia for corals and could be included in ecosystem management plans and considered for their applications in coral restoration.

Assessing the water quality impacts of two Category-5 hurricanes on St. Thomas, Virgin Islands.

Managing waterborne and water-related diseases is one of the most critical factors in the aftermath of hurricane-induced natural disasters. The goal of the study was to identify water-quality impairments in order to set the priorities for post-hurricane relief and to guide future decisions on disaster preparation and relief administration. Field investigations were carried out on St. Thomas, U.S. Virgin Islands as soon as the disaster area became accessible after the back-to-back hurricane strikes by Irma and Maria in 2017. Water samples were collected from individual household rain cisterns, the coastal ocean, and street-surface runoffs for microbial concentration. The microbial community structure and the occurrence of potential human pathogens were investigated in samples using next generation sequencing. Loop mediated isothermal amplification was employed to detect fecal indicator bacteria, Enterococcus faecalis. The results showed both fecal indicator bacteria and Legionella genetic markers were prevalent but were low in concentration in the water samples. Among the 22 cistern samples, 86% were positive for Legionella and 82% for Escherichia-Shigella. Enterococcus faecalis was detected in over 68% of the rain cisterns and in 60% of the coastal waters (n = 20). Microbial community composition in coastal water samples was significantly different from cistern water and runoff water. Although identification at bacterial genus level is not direct evidence of human pathogens, our results suggest cistern water quality needs more organized attention for protection of human health, and that preparation and prevention measures should be taken before natural disasters strike.

Stability of temperate coral Astrangia poculata microbiome is reflected across different sequencing methodologies.

The microbiome of the temperate coral Astrangia poculata was first described in 2017 using next-generation Illumina sequencing to examine the coral's bacterial and archaeal associates across seasons and among hosts of differing symbiotic status. To assess the impact of methodology on the detectable diversity of the coral's microbiome, we obtained near full-length Sanger sequences from clone libraries constructed from a subset of the same A. poculata samples. Eight samples were analyzed: two sets of paired symbiotic (brown) and aposymbiotic (white) colonies collected in the fall (September) and two sets collected in the spring (April). Analysis of the Sanger sequences revealed that the microbiome of A. poculata exhibited a high level of richness; 806 OTUs were identified among 1390 bacterial sequences. While the Illumina study revealed that A. poculata's microbial communities did not significantly vary according to symbiotic state, but did vary by season, Sanger sequencing did not expose seasonal or symbiotic differences in the microbiomes. Proteobacteria dominated the microbiome, forming the majority (55% to 80%) of classifiable bacteria in every sample, and the five bacterial classes with the highest mean relative portion (5% to 35%) were the same as those determined by prior Illumina sequencing. Sanger sequencing also captured the same core taxa previously identified by next-generation sequencing. Alignment of all sequences and construction of a phylogenetic tree revealed that both sequencing methods provided similar portrayals of the phylogenetic diversity within A. poculata's bacterial associates. Consistent with previous findings, the results demonstrated that the Astrangia microbiome is stable notwithstanding the choice of sequencing method and the far fewer sequences generated by clone libraries (46 to 326 sequences per sample) compared to next-generation sequencing (3634 to 48481 sequences per sample). Moreover, the near-full length 16S rRNA sequences produced by this study are presented as a resource for the community studying this model system since they provide necessary information for designing primers and probes to further our understanding of this coral's microbiome.

Microbiomes of stony and soft deep-sea corals share rare core bacteria.

BACKGROUND: Numerous studies have shown that bacteria form stable associations with host corals and have focused on identifying conserved "core microbiomes" of bacterial associates inferred to be serving key roles in the coral holobiont. Because studies tend to focus on only stony corals (order Scleractinia) or soft corals (order Alcyonacea), it is currently unknown if there are conserved bacteria that are shared by both. A meta-analysis was done of 16S rRNA amplicon data from multiple studies generated via identical methodology to allow direct comparisons of bacterial associates across seven deep-sea corals, including both stony and soft species: Anthothela grandiflora, Anthothela sp., Lateothela grandiflora, Lophelia pertusa, Paramuricea placomus, Primnoa pacifica, and Primnoa resedaeformis. RESULTS: Twenty-three operational taxonomic units (OTUs) were consistently present in greater than 50% of the coral samples. Seven amplicon sequence variants (ASVs), five of which corresponded to a conserved OTU, were consistently present in greater than 30% of the coral samples including five or greater coral species. A majority of the conserved sequences had close matches with previously identified coral-associated bacteria. While known to dominate tropical and temperate coral microbiomes, Endozoicomonas were extremely rare or absent from these deep-sea corals. An Endozoicomonas OTU associated with Lo. pertusa in this study was most similar to those from shallow-water stony corals, while an OTU associated with Anthothela spp. was most similar to those from shallow-water gorgonians. CONCLUSIONS: Bacterial sequences have been identified that are conserved at the level of class Anthozoa (i.e., found in both stony and soft corals, shallow and deep). These bacterial associates are therefore hypothesized to play important symbiotic roles and are highlighted for targeted future study. These conserved bacterial associates include taxa with the potential for nitrogen and sulfur cycling, detoxification, and hydrocarbon degradation. There is also some overlap with kit contaminants that need to be resolved. Rarely detected Endozoicomonas sequences are partitioned by whether the host is a stony coral or a soft coral, and the finer clustering pattern reflects the hosts' phylogeny.

Comparison of microbiomes of cold-water corals Primnoa pacifica and Primnoa resedaeformis, with possible link between microbiome composition and host genotype.

Cold-water corals provide critical habitats for a multitude of marine species, but are understudied relative to tropical corals. Primnoa pacifica is a cold-water coral prevalent throughout Alaskan waters, while another species in the genus, Primnoa resedaeformis, is widely distributed in the Atlantic Ocean. This study examined the V4-V5 region of the 16S rRNA gene after amplifying and pyrosequencing bacterial DNA from samples of these species. Key differences between the two species' microbiomes included a robust presence of bacteria belonging to the Chlamydiales order in most of the P. pacifica samples, whereas no more than 2% of any microbial community from P. resedaeformis comprised these bacteria. Microbiomes of P. resedaeformis exhibited higher diversity than those of P. pacifica, and the two species largely clustered separately in a principal coordinate analysis. Comparison of P. resedaeformis microbiomes from samples collected in two submarine canyons revealed a significant difference between locations. This finding mirrored significant genetic differences among the P. resedaeformis from the two canyons based upon population genetic analysis of microsatellite loci. This study presents the first report of microbiomes associated with these two coral species.

Biogeographic Comparison of Lophelia-Associated Bacterial Communities in the Western Atlantic Reveals Conserved Core Microbiome.

Over the last decade, publications on deep-sea corals have tripled. Most attention has been paid to Lophelia pertusa, a globally distributed scleractinian coral that creates critical three-dimensional habitat in the deep ocean. The bacterial community associated with L. pertusa has been previously described by a number of studies at sites in the Mediterranean Sea, Norwegian fjords, off Great Britain, and in the Gulf of Mexico (GOM). However, use of different methodologies prevents direct comparisons in most cases. Our objectives were to address intra-regional variation and to identify any conserved bacterial core community. We collected samples from three distinct colonies of L. pertusa at each of four locations within the western Atlantic: three sites within the GOM and one off the east coast of the United States. Amplicon libraries of 16S rRNA genes were generated using primers targeting the V4-V5 hypervariable region and 454 pyrosequencing. The dominant phylum was Proteobacteria (75-96%). At the family level, 80-95% of each sample was comprised of five groups: Pirellulaceae, Pseudonocardiaceae, Rhodobacteraceae, Sphingomonadaceae, and unclassified Oceanospirillales. Principal coordinate analysis based on weighted UniFrac distances showed a clear distinction between the GOM and Atlantic samples. Interestingly, the replicate samples from each location did not always cluster together, indicating there is not a strong site-specific influence. The core bacterial community, conserved in 100% of the samples, was dominated by the operational taxonomic units of genera Novosphingobium and Pseudonocardia, both known degraders of aromatic hydrocarbons. The sequence of another core member, Propionibacterium, was also found in prior studies of L. pertusa from Norway and Great Britain, suggesting a role as a conserved symbiont. By examining more than 40,000 sequences per sample, we found that GOM samples were dominated by the identified conserved core sequences, whereas open Atlantic samples had a much higher proportion of locally consistent bacteria. Further, predictive functional profiling highlights the potential for the L. pertusa microbiome to contribute to chemoautotrophy, nutrient cycling, and antibiotic production.

Bacterial community diversity of the deep-sea octocoral Paramuricea placomus.

Compared to tropical corals, much less is known about deep-sea coral biology and ecology. Although the microbial communities of some deep-sea corals have been described, this is the first study to characterize the bacterial community associated with the deep-sea octocoral, Paramuricea placomus. Samples from five colonies of P. placomus were collected from Baltimore Canyon (379-382 m depth) in the Atlantic Ocean off the east coast of the United States of America. DNA was extracted from the coral samples and 16S rRNA gene amplicons were pyrosequenced using V4-V5 primers. Three samples sequenced deeply (>4,000 sequences each) and were further analyzed. The dominant microbial phylum was Proteobacteria, but other major phyla included Firmicutes and Planctomycetes. A conserved community of bacterial taxa held in common across the three P. placomus colonies was identified, comprising 68-90% of the total bacterial community depending on the coral individual. The bacterial community of P. placomus does not appear to include the genus Endozoicomonas, which has been found previously to be the dominant bacterial associate in several temperate and tropical gorgonians. Inferred functionality suggests the possibility of nitrogen cycling by the core bacterial community.

Coral-Associated Bacterial Diversity Is Conserved across Two Deep-Sea Anthothela Species.

Cold-water corals, similar to tropical corals, contain diverse and complex microbial assemblages. These bacteria provide essential biological functions within coral holobionts, facilitating increased nutrient utilization and production of antimicrobial compounds. To date, few cold-water octocoral species have been analyzed to explore the diversity and abundance of their microbial associates. For this study, 23 samples of the family Anthothelidae were collected from Norfolk (n = 12) and Baltimore Canyons (n = 11) from the western Atlantic in August 2012 and May 2013. Genetic testing found that these samples comprised two Anthothela species (Anthothela grandiflora and Anthothela sp.) and Alcyonium grandiflorum. DNA was extracted and sequenced with primers targeting the V4-V5 variable region of the 16S rRNA gene using 454 pyrosequencing with GS FLX Titanium chemistry. Results demonstrated that the coral host was the primary driver of bacterial community composition. Al. grandiflorum, dominated by Alteromonadales and Pirellulales had much higher species richness, and a distinct bacterial community compared to Anthothela samples. Anthothela species (A. grandiflora and Anthothela sp.) had very similar bacterial communities, dominated by Oceanospirillales and Spirochaetes. Additional analysis of core-conserved bacteria at 90% sample coverage revealed genus level conservation across Anthothela samples. This core included unclassified Oceanospirillales, Kiloniellales, Campylobacterales, and genus Spirochaeta. Members of this core were previously recognized for their functional capabilities in nitrogen cycling and suggest the possibility of a nearly complete nitrogen cycle within Anthothela species. Overall, many of the bacterial associates identified in this study have the potential to contribute to the acquisition and cycling of nutrients within the coral holobiont.

Comparing bacterial community composition of healthy and dark spot-affected Siderastrea siderea in Florida and the Caribbean.

Coral disease is one of the major causes of reef degradation. Dark Spot Syndrome (DSS) was described in the early 1990's as brown or purple amorphous areas of tissue on a coral and has since become one of the most prevalent diseases reported on Caribbean reefs. It has been identified in a number of coral species, but there is debate as to whether it is in fact the same disease in different corals. Further, it is questioned whether these macroscopic signs are in fact diagnostic of an infectious disease at all. The most commonly affected species in the Caribbean is the massive starlet coral Siderastrea siderea. We sampled this species in two locations, Dry Tortugas National Park and Virgin Islands National Park. Tissue biopsies were collected from both healthy colonies and those with dark spot lesions. Microbial-community DNA was extracted from coral samples (mucus, tissue, and skeleton), amplified using bacterial-specific primers, and applied to PhyloChip G3 microarrays to examine the bacterial diversity associated with this coral. Samples were also screened for the presence of a fungal ribotype that has recently been implicated as a causative agent of DSS in another coral species, but the amplifications were unsuccessful. S. siderea samples did not cluster consistently based on health state (i.e., normal versus dark spot). Various bacteria, including Cyanobacteria and Vibrios, were observed to have increased relative abundance in the discolored tissue, but the patterns were not consistent across all DSS samples. Overall, our findings do not support the hypothesis that DSS in S. siderea is linked to a bacterial pathogen or pathogens. This dataset provides the most comprehensive overview to date of the bacterial community associated with the scleractinian coral S. siderea.

Comparing bacterial community composition between healthy and white plague-like disease states in Orbicella annularis using PhyloChip™ G3 microarrays.

Coral disease is a global problem. Diseases are typically named or described based on macroscopic changes, but broad signs of coral distress such as tissue loss or discoloration are unlikely to be specific to a particular pathogen. For example, there appear to be multiple diseases that manifest the rapid tissue loss that characterizes 'white plague.' PhyloChip™ G3 microarrays were used to compare the bacterial community composition of both healthy and white plague-like diseased corals. Samples of lobed star coral (Orbicella annularis, formerly of the genus Montastraea[1]) were collected from two geographically distinct areas, Dry Tortugas National Park and Virgin Islands National Park, to determine if there were biogeographic differences between the diseases. In fact, all diseased samples clustered together, however there was no consistent link to Aurantimonas coralicida, which has been described as the causative agent of white plague type II. The microarrays revealed a large amount of bacterial heterogeneity within the healthy corals and less diversity in the diseased corals. Gram-positive bacterial groups (Actinobacteria, Firmicutes) comprised a greater proportion of the operational taxonomic units (OTUs) unique to healthy samples. Diseased samples were enriched in OTUs from the families Corynebacteriaceae, Lachnospiraceae, Rhodobacteraceae, and Streptococcaceae. Much previous coral disease work has used clone libraries, which seem to be methodologically biased toward recovery of Gram-negative bacterial sequences and may therefore have missed the importance of Gram-positive groups. The PhyloChip™data presented here provide a broader characterization of the bacterial community changes that occur within Orbicella annularis during the shift from a healthy to diseased state.

Comparison of DNA preservation methods for environmental bacterial community samples.

Field collections of environmental samples, for example corals, for molecular microbial analyses present distinct challenges. The lack of laboratory facilities in remote locations is common, and preservation of microbial community DNA for later study is critical. A particular challenge is keeping samples frozen in transit. Five nucleic acid preservation methods that do not require cold storage were compared for effectiveness over time and ease of use. Mixed microbial communities of known composition were created and preserved by DNAgard(™), RNAlater(®), DMSO-EDTA-salt (DESS), FTA(®) cards, and FTA Elute(®) cards. Automated ribosomal intergenic spacer analysis and clone libraries were used to detect specific changes in the faux communities over weeks and months of storage. A previously known bias in FTA(®) cards that results in lower recovery of pure cultures of Gram-positive bacteria was also detected in mixed community samples. There appears to be a uniform bias across all five preservation methods against microorganisms with high G + C DNA. Overall, the liquid-based preservatives (DNAgard(™), RNAlater(®), and DESS) outperformed the card-based methods. No single liquid method clearly outperformed the others, leaving method choice to be based on experimental design, field facilities, shipping constraints, and allowable cost.

PhyloChip™ microarray comparison of sampling methods used for coral microbial ecology.

Interest in coral microbial ecology has been increasing steadily over the last decade, yet standardized methods of sample collection still have not been defined. Two methods were compared for their ability to sample coral-associated microbial communities: tissue punches and foam swabs, the latter being less invasive and preferred by reef managers. Four colonies of star coral, Montastraea annularis, were sampled in the Dry Tortugas National Park (two healthy and two with white plague disease). The PhyloChip™ G3 microarray was used to assess microbial community structure of amplified 16S rRNA gene sequences. Samples clustered based on methodology rather than coral colony. Punch samples from healthy and diseased corals were distinct. All swab samples clustered closely together with the seawater control and did not group according to the health state of the corals. Although more microbial taxa were detected by the swab method, there is a much larger overlap between the water control and swab samples than punch samples, suggesting some of the additional diversity is due to contamination from water absorbed by the swab. While swabs are useful for noninvasive studies of the coral surface mucus layer, these results show that they are not optimal for studies of coral disease.

Characterization of culturable bacteria isolated from the cold-water coral Lophelia pertusa.

Microorganisms associated with corals are hypothesized to contribute to the function of the host animal by cycling nutrients, breaking down carbon sources, fixing nitrogen, and producing antibiotics. This is the first study to culture and characterize bacteria from Lophelia pertusa, a cold-water coral found in the deep sea, in an effort to understand the roles that the microorganisms play in the coral microbial community. Two sites in the northern Gulf of Mexico were sampled over 2 years. Bacteria were cultured from coral tissue, skeleton, and mucus, identified by 16S rRNA genes, and subjected to biochemical testing. Most isolates were members of the Gammaproteobacteria, although there was one isolate each from the Betaproteobacteria and Actinobacteria. Phylogenetic results showed that both sampling sites shared closely related isolates (e.g. Pseudoalteromonas spp.), indicating possible temporally and geographically stable bacterial-coral associations. The Kirby-Bauer antibiotic susceptibility test was used to separate bacteria to the strain level, with the results showing that isolates that were phylogenetically tightly grouped had varying responses to antibiotics. These results support the conclusion that phylogenetic placement cannot predict strain-level differences and further highlight the need for culture-based experiments to supplement culture-independent studies.