Cryptic diversity of shallow and mesophotic Stephanocoenia intersepta corals across Florida Keys National Marine Sanctuary.

Population genetic analyses can provide useful data on species' regional connectivity and diversity which can inform conservation and restoration efforts. In this study, we quantified the genetic connectivity and diversity of Stephanocoenia intersepta corals from shallow (<30 m) to mesophotic (30-45 m) depths across Florida Keys National Marine Sanctuary. We generated single nucleotide polymorphism (SNP) markers to identify genetic structuring of shallow and mesophotic S. intersepta corals. We uncovered four distinct, cryptic genetic lineages with varying levels of depth-specificity. Shallow-specific lineages exhibited lower heterozygosity and higher inbreeding relative to depth-generalist lineages found across both shallow and mesophotic reefs. Estimation of recent genetic migration rates demonstrated that mesophotic sites are more prolific sources than shallow sites, particularly in the Lower Keys and Upper Keys. Additionally, we compared endosymbiotic Symbiodiniaceae among sampled S. intersepta using the ITS2 region and SYMPORTAL analysis framework, identifying symbionts from the genera Symbiodinium, Breviolum, and Cladocopium. Symbiodiniaceae varied significantly across depth and location and exhibited significant, but weak correlation with host lineage and genotype. Together, these data demonstrate that despite population genetic structuring across depth, some mesophotic populations may provide refuge for shallow populations moving forward and remain important contributors to the overall genetic diversity of this species throughout the region. This study highlights the importance of including mesophotic as well as shallow corals in population genetic assessments and informs future science-based management, conservation, and restoration efforts within Florida Keys National Marine Sanctuary.

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.

Transcriptomic plasticity of mesophotic corals among natural populations and transplants of Montastraea cavernosa in the Gulf of Mexico and Belize.

While physiological responses to low-light environments have been studied among corals on mesophotic coral ecosystems worldwide (MCEs; 30-150 m), the mechanisms behind acclimatization and adaptation to depth are not well understood for most coral species. Transcriptomic approaches based on RNA sequencing are useful tools for quantifying gene expression plasticity, particularly in slow-growing species such as scleractinian corals, and for identifying potential functional differences among conspecifics. A tag-based RNA-Seq (Tag-Seq) pipeline was applied to quantify transcriptional variation in natural populations of the scleractinian coral Montastraea cavernosa from mesophotic and shallower environments across five sites in Belize and the Gulf of Mexico: Carrie Bow Cay, West and East Flower Garden Banks, Pulley Ridge, and Dry Tortugas. Regional site location was a stronger driver of gene expression patterns than depth. However, mesophotic corals among all sites shared similar regulation of metabolic and cell growth functional pathways that may represent common physiological responses to environmental conditions at depth. Additionally, in a transplant experiment at West and East Flower Garden Banks, colonies transplanted from mesophotic to shallower habitats diverged from the control mesophotic group over time, indicating depth-regulated plasticity of gene expression. When the shallower depth zone experienced a bleaching event, bleaching severity did not differ significantly between transplants and shallow controls, but gene expression patterns indicated variable regulation of stress responses among depth treatments. Coupled observational and experimental studies of gene expression among mesophotic and shallower M. cavernosa provide insights into the ability of this depth-generalist coral species to persist under varying environmental conditions.

Does depth divide? Variable genetic connectivity patterns among shallow and mesophotic Montastraea cavernosa coral populations across the Gulf of Mexico and western Caribbean.

Despite general declines in coral reef ecosystems in the tropical western Atlantic, some reefs, including mesophotic reefs (30-150 m), are hypothesized to function as coral refugia due to their relative isolation from anthropogenic stressors. Understanding the connectivity dynamics among these putative refugia and more degraded reefs is critical to develop effective management strategies that promote coral metapopulation persistence and recovery. This study presents a geographically broad assessment of shallow (<30 m) and mesophotic (>30 m) connectivity dynamics of the depth-generalist coral species Montastraea cavernosa. Over 750 coral genets were collected across the Northwest and Southern Gulf of Mexico, Florida, Cuba, and Belize, and ~5000 SNP loci were generated to quantify high-resolution genetic structure and connectivity among these populations. Generally, shallow and mesophotic populations demonstrated higher connectivity to distant populations within the same depth zone than to adjacent populations across depth zones. However, exceptions to this pattern include the Northwest Gulf of Mexico and the Florida Keys which exhibited relatively high vertical genetic connectivity. Furthermore, estimates of recent gene flow emphasize that mesophotic M. cavernosa populations are not significant sources for their local shallow counterparts, except for the Northwest Gulf of Mexico populations. Location-based differences in vertical connectivity are likely a result of diverse oceanographic and environmental conditions that may drive variation in gene flow and depth-dependent selection. These results highlight the need to evaluate connectivity dynamics and refugia potential of mesophotic coral species on a population-by-population basis and to identify stepping-stone populations that warrant incorporation in future international management approaches.

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.

Assessing the effectiveness of two intervention methods for stony coral tissue loss disease on Montastraea cavernosa.

Stony coral tissue loss disease (SCTLD) was first observed in Florida in 2014 and has since spread to multiple coral reefs across the wider Caribbean. The northern section of Florida's Coral Reef has been heavily impacted by this outbreak, with some reefs experiencing as much as a 60% loss of living coral tissue area. We experimentally assessed the effectiveness of two intervention treatments on SCTLD-affected Montastraea cavernosa colonies in situ. Colonies were tagged and divided into three treatment groups: (1) chlorinated epoxy, (2) amoxicillin combined with CoreRx/Ocean Alchemists Base 2B, and (3) untreated controls. The experimental colonies were monitored periodically over 11 months to assess treatment effectiveness by tracking lesion development and overall disease status. The Base 2B plus amoxicillin treatment had a 95% success rate at healing individual disease lesions but did not necessarily prevent treated colonies from developing new lesions over time. Chlorinated epoxy treatments were not significantly different from untreated control colonies, suggesting that chlorinated epoxy treatments are an ineffective intervention technique for SCTLD. The results of this experiment expand management options during coral disease outbreaks and contribute to overall knowledge regarding coral health and disease.

Quantifying impacts of stony coral tissue loss disease on corals in Southeast Florida through surveys and 3D photogrammetry.

Since 2014, stony coral tissue loss disease (SCTLD) has contributed to substantial declines of reef-building corals in Florida. The emergence of this disease, which impacts over 20 scleractinian coral species, has generated a need for widespread reef monitoring and the implementation of novel survey and disease mitigation strategies. This study paired SCTLD prevalence assessments with colony-level monitoring to help improve understanding of disease dynamics on both individual coral colonies and at reef-wide scales. Benthic surveys were conducted throughout the northern Florida Reef Tract to monitor the presence/absence of disease, disease prevalence, and coral species affected by SCTLD. Observed SCTLD prevalence was lower in Jupiter and Palm Beach than in Lauderdale-by-the-Sea or St. Lucie Reef, but there were no significant changes in prevalence over time. To assess colony-level impacts of the disease, we optimized a low-cost, rapid 3D photogrammetry technique to fate-track infected Montastraea cavernosa coral colonies over four time points spanning nearly four months. Total colony area and healthy tissue area on fate-tracked colonies decreased significantly over time. However disease lesion area did not decrease over time and was not correlated with total colony area. Taken together these results suggest that targeted intervention efforts on larger colonies may maximize preservation of coral cover. Traditional coral surveys combined with 3D photogrammetry can provide greater insights into the spatiotemporal dynamics and impacts of coral diseases on individual colonies and coral communities than surveys or visual estimates of disease progression alone.

Population genetic structure of the great star coral, Montastraea cavernosa, across the Cuban archipelago with comparisons between microsatellite and SNP markers.

Coral reef habitats surrounding Cuba include relatively healthy, well-developed shallow and mesophotic (30-150 m) scleractinian communities at the cross-currents of the Tropical Western Atlantic (TWA). However, Cuba's coral communities are not immune to the declines observed throughout the TWA, and there is limited information available regarding genetic connectivity, diversity, and structure among these populations. This represents an immense gap in our understanding of coral ecology and population dynamics at both local and regional scales. To address this gap, we evaluated the population genetic structure of the coral Montastraea cavernosa across eight reef sites surrounding Cuba. Colonies were genotyped using nine microsatellite markers and > 9,000 single nucleotide polymorphism (SNP) markers generated using the 2bRAD approach to assess fine-scale genetic structure across these sites. Both the microsatellite and SNP analyses identified patterns of genetic differentiation among sample populations. While the microsatellite analyses did not identify significant genetic structure across the seven shallow M. cavernosa sampling sites, the SNP analyses revealed significant pairwise population differentiation, suggesting that differentiation is greater between eastern and western sites. This study provides insight into methodological differences between microsatellite and SNP markers including potential trade-offs between marker-specific biases, sample size, sequencing costs, and the ability to resolve subtle patterns of population genetic structure. Furthermore, this study suggests that locations in western Cuba may play important roles in this species' regional metapopulation dynamics and therefore may merit incorporation into developing international management efforts in addition to the local management the sites receive.

Depth Influences Symbiodiniaceae Associations Among Montastraea cavernosa Corals on the Belize Barrier Reef.

In Belize, shallow populations (10 and 16 m) of the coral species Montastraea cavernosa from the back reef and reef crest are genetically differentiated from deeper populations on the fore reef and reef wall (25 and 35 m). Like many species of scleractinian corals, M. cavernosa has an obligate symbiosis with dinoflagellate microalgae from the family Symbiodiniaceae. Here, we describe the Symbiodiniaceae taxa found within previously sampled and genotyped M. cavernosa populations along a depth gradient on the Belize Barrier Reef by implementing high-throughput sequencing of the ITS2 region of Symbiodiniaceae ribosomal DNA and the SymPortal analysis framework. While Symbiodiniaceae ITS2 type profiles across all sampling depths were almost entirely (99.99%) from the genus Cladocopium (formerly Symbiodinium Clade C), shallow (10 and 16 m) populations had a greater diversity of ITS2 type profiles in comparison to deeper (25 and 35 m) populations. Permutational multivariate analysis of variance (PERMANOVA) confirmed significant differences in ITS2 type profiles between shallow and deep sample populations. Overall Symbiodiniaceae communities changed significantly with depth, following patterns similar to the coral host's population genetic structure. Though physiological differences among species in the cosmopolitan genus Cladocopium are not well-described, our results suggest that although some members of Cladocopium are depth-generalists, shallow M. cavernosa populations in Belize may harbor shallow-specialized Symbiodiniaceae not found in deeper populations.

Montastraea cavernosa corallite structure demonstrates distinct morphotypes across shallow and mesophotic depth zones in the Gulf of Mexico.

This study assessed morphological variation of the depth-generalist coral Montastraea cavernosa across shallow and mesophotic coral ecosystems in the Gulf of Mexico (GOM) using thirteen corallite metrics. While corallite structure differed significantly across sites, we observed that mean corallite diameters were smaller and spacing was greater in mesophotic corals as compared to shallow corals. Additional corallite variation, including greater mean corallite height of mesophotic samples, are hypothesized to be photoadaptive responses to low light environments. Multivariate analyses also revealed two distinct morphotypes identified by significant variation in corallite spacing with >90% accuracy. A 'shallow' morphotype was characterized by larger, more closely-spaced corallites, while a 'depth-generalist' type exhibited smaller, further-spaced corallites. Variable presence of morphotypes within some sites suggests genotypic influence on corallite morphology as there was a slight, but significant, impact of morphotype on genetic structure within shallow zones in the Flower Garden Banks. Patterns of increased algal symbiont (Symbiodiniaceae) density and chlorophyll concentration were retained in the depth-generalist morphotype even in shallow zones, identifying multiple photoadaptive strategies between morphotypes. The results of this study suggest that morphological variation among M. cavernosa represents a combination of genotypic variation and phenotypic plasticity rather than responses to environmental stimuli alone.

Populations of the coral species Montastraea cavernosa on the Belize Barrier Reef lack vertical connectivity.

Larval connectivity among and within coral reefs is important for sustaining coral metapopulations, enhancing ecosystem resilience through species and genetic diversity, and maintaining reef ecosystems' structure and functions. This study characterized genetic structure and assessed horizontal and vertical connectivity among populations of the ubiquitous gonochoric broadcast spawning coral Montastraea cavernosa in Belize. Using nine polymorphic microsatellite loci, we genotyped M. cavernosa colonies from four depth zones at four study sites within Belizean marine management zones. Study sites were selected within South Water Caye Marine Reserve (3 sites) and Glover's Reef Marine Reserve (1 site). Strong contemporary genetic differentiation was observed between relatively shallow M. cavernosa populations (10 m, 16 m) and relatively deep (25 m, 35 m) populations, coinciding with a transition from reef crest to reef slope. These results were consistent across both marine reserves. Vertical and horizontal migration models suggest that all populations were historically panmictic, with little unidirectional migration. The relative local isolation of shallow and mesophotic M. cavernosa populations in Belize, coupled with the importance of Belize's upper mesophotic populations as potential larval sources for other areas in the Tropical Western Atlantic, reinforces the need for management strategies that conserve coral populations across all depth zones.

The presence of the cyanobacterial toxin microcystin in black band disease of corals.

Black band disease (BBD) is a migrating, cyanobacterial dominated, sulfide-rich microbial mat that moves across coral colonies lysing coral tissue. While it is known that BBD sulfate-reducing bacteria contribute to BBD pathogenicity by production of sulfide, additional mechanisms of toxicity may be involved. Using HPLC/MS, the cyanotoxin microcystin was detected in 22 field samples of BBD collected from five coral species on nine reefs of the wider Caribbean (Florida Keys and Bahamas). Two cyanobacterial cultures isolated from BBD, Geitlerinema and Leptolyngbya sp. contained microcystin based on HPLC/MS, with toxic activity confirmed using the protein phosphatase inhibition assay. The gene mcyA from the microcystin synthesis complex was detected in two field samples and from both BBD cyanobacterial cultures. Microcystin was not detected in six BBD samples from a different area of the Caribbean (St Croix, USVI) and the Philippines, suggesting regional specificity for BBD microcystin. This is the first report of the presence of microcystin in a coral disease.

An assessment of the hypervariable domains of the 16S rRNA genes for their value in determining microbial community diversity: the paradox of traditional ecological indices.

Amplicon length heterogeneity PCR (LH-PCR) was investigated for its ability to distinguish between microbial community patterns from the same soil type under different land management practices. Natural sagebrush and irrigated mouldboard-ploughed soils from Idaho were queried as to which hypervariable domains, or combinations of 16S rRNA gene domains, were the best molecular markers. Using standard ecological indices to measure richness, diversity and evenness, the combination of three domains, V1, V3 and V1+V2, or the combined V1 and V3 domains were the markers that could best distinguish the undisturbed natural sagebrush communities from the mouldboard-ploughed microbial communities. Bray-Curtis similarity and multidimensional scaling were found to be better metrics to ordinate and cluster the LH-PCR community profiling data. The use/misuse of traditional ecological indices such as diversity and evenness to study microbial community profiles will remain a major point to consider when performing metagenomic studies.

Coral diseases near Lee Stocking Island, Bahamas: patterns and potential drivers.

The number of coral diseases, coral species they infect, number of reported cases, and range over which these diseases are distributed have all increased dramatically in the past 3 decades, posing a serious threat to coral reef ecosystems worldwide. While some published studies provide data on the distribution of coral diseases at local and regional levels, few studies have addressed the factors that may drive these distributions. We recorded coral disease occurrence, prevalence, and severity along with temperature, sedimentation, and coral population data (species abundance and colony size) over 2 consecutive summers on reefs near Lee Stocking Island (LSI) in the Bahamas' Exuma Chain. In 2002 a total of 11092 coral colonies (all species present) were examined within a survey area of 9420 m2, and 13 973 colonies within 10 362 m2 in 2003. Similar to other reports, relatively large, framework species including Siderastrea siderea, Colpophyllia natans, and Montastraea annularis, along with the smaller Dichocoenia stokesi, were the species most susceptible to coral disease. Recurring infections were observed on individual colonies from 2002 to 2003, and were more likely for black band disease (BBD) than for either white plague (WP) or dark spots syndrome (DS). In 2002, WP and DS demonstrated clumped distributions, while BBD was randomly distributed. However, in 2003 BBD and WP were clumped. This is the first study, to our knowledge, that quantitatively documents coral disease dynamics on reefs surrounding LSI.

Development and field application of a molecular probe for the primary pathogen of the coral disease white plague type II.

One of the current problems in the field of coral disease research is that of tracking coral pathogens in the natural environment. A promising method to do this is by use of pathogen-specific molecular probes. However, this approach has been little used to date. We constructed, and validated in the laboratory, a fluorochrome-labeled molecular probe specific to Aurantimonas coralicida, the bacterial pathogen of the Caribbean coral disease white plague type II (WPIl). We then used the probe to test field samples of diseased coral tissue for the presence of this pathogen. Probe design was based on a unique subset (25 nucleotides) of the complete l6S rRNA gene sequence derived from a pure culture of the pathogen. The pathogen-specific probe was labeled with the fluorochrome GreenStar* FITC (fluorescein isothiocyanate, GeneDetect Ltd, New Zealand). As a control, we used the universal eubacterial probe EUB 338, labeled with a different fluorochrome (TRITC, tetra-methylrhodamine isothiocyanate). Both probes were applied to laboratory samples of pure cultures of bacteria, and field samples collected from the surface of the disease line of corals exhibiting signs of white plague (types I and II), healthy controls, and corals with an uncharacterized disease ("patchy necrosis"). All samples were analyzed using fluorescence in situ hybridization (FISH). We have determined that the probe is specific to our laboratory culture of the coral pathogen, and does not react with other bacterial species (the eubacterial probe does). The WPII pathogen was detected in association with diseased coral samples collected from coral colonies on reefs of the Bahamas (n= 9 samples) exhibiting signs of both WPI and WPII. Diseased (and healthy) tissue samples (n- 4) from corals exhibiting signs of "patchy necrosis" were also assayed. In this case the results were negative, indicating that the same pathogen is not involved in the two diseases. Incorporation and use of pathogen-specific probes can significantly expand our knowledge of the etiology of coral diseases.

Black band disease microbial community variation on corals in three regions of the wider Caribbean.

Black band disease (BBD) is a pathogenic consortium of microorganisms that primarily affects massive framework-building scleractinian corals on reefs worldwide. There has been considerable debate concerning the microbial community composition of BBD. The aim of this study was to utilize microbial profiling to assess overall patterns of variation in the BBD bacterial community with respect to geographic location, host coral species, time, and nutrient regime. Length heterogeneity polymerase chain reaction (LH-PCR) was employed to differentiate BBD communities based on the natural variation in the sequence lengths within hypervariable domains of the 16S rRNA gene. Analysis of LH-PCR profiles of 97 BBD samples using multivariate ordination methods and analysis of similarity revealed significant clustering with respect to geographic region when comparing BBD sampled from reefs near Lee Stocking Island in the Bahamas' Exuma Chain, the Northern Florida Keys (NFK), and St. John in the US Virgin Islands. There was much variability in BBD community composition on a regional basis, between sites in the NFK, and in terms of coral host species. The observed differences among BBD microbial community profiles were driven primarily by variation in relative abundance of 313-316-bp amplicons, which correspond to cyanobacteria and alpha-proteobacteria. The results obtained in this study support previous reports of intrinsic variability and complexity of the BBD microbial community but also suggest that this variability has biogeographic patterns.

Microbial communities in the surface mucopolysaccharide layer and the black band microbial mat of black band-diseased Siderastrea siderea.

Microbial community profiles and species composition associated with two black band-diseased colonies of the coral Siderastrea siderea were studied by 16S rRNA-targeted gene cloning, sequencing, and amplicon-length heterogeneity PCR (LH-PCR). Bacterial communities associated with the surface mucopolysaccharide layer (SML) of apparently healthy tissues of the infected colonies, together with samples of the black band disease (BBD) infections, were analyzed using the same techniques for comparison. Gene sequences, ranging from 424 to 1,537 bp, were retrieved from all positive clones (n = 43 to 48) in each of the four clone libraries generated and used for comparative sequence analysis. In addition to LH-PCR community profiling, all of the clone sequences were aligned with LH-PCR primer sequences, and the theoretical lengths of the amplicons were determined. Results revealed that the community profiles were significantly different between BBD and SML samples. The SML samples were dominated by gamma-proteobacteria (53 to 64%), followed by beta-proteobacteria (18 to 21%) and alpha-proteobacteria (5 to 11%). In contrast, both BBD clone libraries were dominated by alpha-proteobacteria (58 to 87%), followed by verrucomicrobia (2 to 10%) and 0 to 6% each of delta-proteobacteria, bacteroidetes, firmicutes, and cyanobacteria. Alphaproteobacterial sequence types related to the bacteria associated with toxin-producing dinoflagellates were observed in BBD clone libraries but were not found in the SML libraries. Similarly, sequences affiliated with the family Desulfobacteraceae and toxin-producing cyanobacteria, both believed to be involved in BBD pathogenesis, were found only in BBD libraries. These data provide evidence for an association of numerous toxin-producing heterotrophic microorganisms with BBD of corals.

Development and field application of a molecular probe for the primary pathogen of the coral disease white plague type II

Abstract: One of the current problems in the field of coral disease research is that of tracking coral pathogens in the natural environment.A promising method to do this is by use of pathogen-specific molecular probes. However,this approach has been little used to date.We constructed,and validated in the laboratory,a fluoro-chrome-labeled molecular probe specific to Aurantimonas coralicida ,the bacterial pathogen of the Caribbean coral disease white plague type II (WPII).We then used the probe to test field samples of diseased coral tissue for the presence of this pathogen.Probe design was based on a unique subset (25 nucleotides)of the complete16S rRNA gene sequence derived from a pure culture of the pathogen.The pathogen-specific probe was labeled with the fluorochrome GreenStar*™FITC (fluorescein isothiocyanate,GeneDetect Ltd,New Zealand).As a control, we used the universal eubacterial probe EUB 338,labeled with a different fluorochrome (TRITC,tetra-methyl-rhodamine isothiocyanate).Both probes were applied to laboratory samples of pure cultures of bacteria, and field samples collected from the surface of the disease line of corals exhibiting signs of white plague (types I and II),healthy controls,and corals with an uncharacterized disease ("patchy necrosis ").All samples were analyzed using fluorescence in situ hybridization (FISH).We have determined that the probe is specific to our laboratory culture of the coral pathogen,and does not react with other bacterial species (the eubacterial probe does).The WPII pathogen was detected in association with diseased coral samples collected from coral colonies on reefs of the Bahamas (n=9 samples)exhibiting signs of both WPI and WPII.Diseased (and healthy)tissue samples (n=4)from corals exhibiting signs of "patchy necrosis "were also assayed.In this case the results were negative, indicating that the same pathogen is not involved in the two diseases.Incorporation and use of pathogen-specific probes can...