Microbiome dynamics of two differentially resilient corals.

Coral bleaching and disease are 2 common occurrences that are contributing to global coral cover decline. Understanding the interactions between the coral animal and its microbial associates, and how they may change in the presence of stressors such as warming and acidification, is a crucial component to understanding both susceptibility and resistance to disease and bleaching. The coral Diploria labyrinthiformis has been shown to be more disease resistant than its relative Pseudodiploria strigosa, providing an ideal study system for disease resistance. In this study, we examined the bacterial communities of these 2 coral species on the Florida Reef tract every 6 mo for 18 mo (in situ sampling), and under experimental (laboratory) thermal and pH manipulation. The in situ sampling encompassed wide fluctuations in temperature, including an anomalously warm summer period. The laboratory experiments involved exposure to both increased temperature (31°C) and lowered pH (7.7). The in situ bacterial communities of both coral species were highly similar in the winter, but diverged during summer, with the D. labyrinthiformis bacterial community being more stable than that of P. strigosa. Differences in the bacterial community between the 2 coral species included 29 operational taxonomic units (OTUs) that were specific to D. labyrinthiformis in all seasons, while only 2 OTUs were specific to P. strigosa. The comparative stability of the D. labyrinthiformis microbiome, in addition to harboring a more specific microbiome, may be a key component of the relative disease resistance of this coral.

Elevated temperature enhances short- to medium-chain acyl homoserine lactone production by black band disease-associated vibrios.

Black band disease (BBD) of corals is a horizontally migrating, pathogenic, polymicrobial mat community which is active above a temperature threshold of 27.5°C on the reef. Bacterial isolates from BBD, the surface mucopolysaccharide layer (SML) of healthy corals and SML of healthy areas of BBD-infected corals were tested for production of short- to medium-chain acyl homoserine lactones (AHLs) using the Chromobacterium violaceum CV026 reporter strain. Of 110 bacterial isolates tested, 19 produced AHLs and 15 of these were from BBD. Eight AHLs were identified using LC-MS/MS, with 3OHC4 the most commonly produced, followed by C6. AHL-producing isolates exposed to three temperatures (24°C, 27°C, 30°C) revealed that production of three AHLs (3OHC4, 3OHC5 and 3OHC6) significantly increased at 30°C when compared to 24°C. 16S rRNA gene sequencing revealed that all of the AHL-producing BBD isolates were vibrios. Metagenomic data of BBD communities showed the presence of AHL (and autoinducer-2) genes, many of which are known to be associated with vibrios. These findings suggest that quorum sensing may be involved in BBD pathobiology and community structure due to enhanced production of quorum-sensing signal molecules (AHLs) above the temperature threshold of this globally distributed coral disease.

Possible links between white plague-like disease, scleractinian corals, and a cryptochirid gall crab.

White plague (WP) is a highly destructive coral disease that rapidly kills susceptible coral species by mass tissue lysis. The pathogen and underlying causes of this disease are not known. In this laboratory-based study, we examined a small coral-associated gall crab from the family Cryptochiridae in terms of a possible association with WP-like lesions. A series of experiments was conducted after observations that 2 scleractinian coral species, Diploria labyrinthiformis and Pseudodiploria strigosa, developed signs of WP-like disease within a laboratory holding aquarium and that small gall crabs were physically present in the center of each lesion. Using fragments of D. labyrinthiformis, a crab from one of the lesions was sequentially removed and placed, under controlled conditions, onto apparently healthy coral colonies, resulting in the development of similar lesions. Next-generation sequencing of the 16S rRNA gene was performed to profile the bacterial communities associated with the crab, lesions, and healthy corals. The microbiota of the crab and lesions were highly similar while that of apparently healthy colonies were significantly different. Significant differences were largely due to an increase in Alphaproteobacteria in crab and lesion communities. In particular, the Roseobacter clade had a higher relative abundance in the crab and WP-like lesions. This study suggests that the cryptochirid gall crab may be associated with development of WP-like lesions.

Unraveling the Physiological Roles of the Cyanobacterium Geitlerinema sp. BBD and Other Black Band Disease Community Members through Genomic Analysis of a Mixed Culture.

Black band disease (BBD) is a cyanobacterial-dominated polymicrobial mat that propagates on and migrates across coral surfaces, necrotizing coral tissue. Culture-based laboratory studies have investigated cyanobacteria and heterotrophic bacteria isolated from BBD, but the metabolic potential of various BBD microbial community members and interactions between them remain poorly understood. Here we report genomic insights into the physiological and metabolic potential of the BBD-associated cyanobacterium Geitlerinema sp. BBD 1991 and six associated bacteria that were also present in the non-axenic culture. The essentially complete genome of Geitlerinema sp. BBD 1991 contains a sulfide quinone oxidoreductase gene for oxidation of sulfide, suggesting a mechanism for tolerating the sulfidic conditions of BBD mats. Although the operon for biosynthesis of the cyanotoxin microcystin was surprisingly absent, potential relics were identified. Genomic evidence for mixed-acid fermentation indicates a strategy for energy metabolism under the anaerobic conditions present in BBD during darkness. Fermentation products may supply carbon to BBD heterotrophic bacteria. Among the six associated bacteria in the culture, two are closely related to organisms found in culture-independent studies of diseased corals. Their metabolic pathways for carbon and sulfur cycling, energy metabolism, and mechanisms for resisting coral defenses suggest adaptations to the coral surface environment and biogeochemical roles within the BBD mat. Polysulfide reductases were identified in a Flammeovirgaceae genome (Bacteroidetes) and the sox pathway for sulfur oxidation was found in the genome of a Rhodospirillales bacterium (Alphaproteobacteria), revealing mechanisms for sulfur cycling, which influences virulence of BBD. Each genomic bin possessed a pathway for conserving energy from glycerol degradation, reflecting adaptations to the glycerol-rich coral environment. The presence of genes for detoxification of reactive oxygen species and resistance to antibiotics suggest mechanisms for combating coral defense strategies. This study builds upon previous research on BBD and provides new insights into BBD disease etiology.

Microbiota shifts in the surface mucopolysaccharide layer of corals transferred from natural to aquaria settings.

Bacteria associated with the surface mucopolysaccharide layer (SML) of corals have been proposed to be paramount to coral health, and are occasionally studied in aquaria. Using automated ribosomal intergenic spacer analysis (ARISA), this study examined the temporal changes in the SML microbiota of coral fragments (Siderastrea siderea) transferred from the reef to aquaria. In total, 460 amplicon peaks were detected, 155 of which were unique. Extensive microbiota shifts occurred one day after transfer, with stabilization between 14 and 28days. These results suggest that studies examining coral in laboratory settings should consider the observed temporal dynamics in the SML microbiota.

Ecology and Physiology of the Pathogenic Cyanobacterium Roseofilum reptotaenium.

Roseofilum reptotaenium is a gliding, filamentous, phycoerythrin-rich cyanobacterium that has been found only in the horizontally migrating, pathogenic microbial mat, black band disease (BBD) on Caribbean corals. R. reptotaenium dominates the BBD mat in terms of biomass and motility, and the filaments form the mat fabric. This cyanobacterium produces the cyanotoxin microcystin, predominately MC-LR, and can tolerate high levels of sulfide produced by sulfate reducing bacteria (SRB) that are also associated with BBD. Laboratory cultures of R. reptotaenium infect coral fragments, suggesting that the cyanobacterium is the primary pathogen of BBD, but since this species cannot grow axenically and Koch's Postulates cannot be fulfilled, it cannot be proposed as a primary pathogen. However, R. reptotaenium does play several major pathogenic roles in this polymicrobial disease. Here, we provide an overview of the ecology of this coral pathogen and present new information on R. reptotaenium ecophysiology, including roles in the infection process, chemotactic and other motility responses, and the effect of pH on growth and motility. Additionally, we show, using metabolomics, that exposure of the BBD microbial community to the cyanotoxin MC-LR affects community metabolite profiles, in particular those associated with nucleic acid biosynthesis.

Quorum sensing signal production and microbial interactions in a polymicrobial disease of corals and the coral surface mucopolysaccharide layer.

Black band disease (BBD) of corals is a complex polymicrobial disease considered to be a threat to coral reef health, as it can lead to mortality of massive reef-building corals. The BBD community is dominated by gliding, filamentous cyanobacteria with a highly diverse population of heterotrophic bacteria. Microbial interactions such as quorum sensing (QS) and antimicrobial production may be involved in BBD disease pathogenesis. In this study, BBD (whole community) samples, as well as 199 bacterial isolates from BBD, the surface mucopolysaccharide layer (SML) of apparently healthy corals, and SML of apparently healthy areas of BBD-infected corals were screened for the production of acyl homoserine lactones (AHLs) and for autoinducer-2 (AI-2) activity using three bacterial reporter strains. AHLs were detected in all BBD (intact community) samples tested and in cultures of 5.5% of BBD bacterial isolates. Over half of a subset (153) of the isolates were positive for AI-2 activity. AHL-producing isolates were further analyzed using LC-MS/MS to determine AHL chemical structure and the concentration of (S)-4,5-dihydroxy-2,3-pentanedione (DPD), the biosynthetic precursor of AI-2. C6-HSL was the most common AHL variant detected, followed by 3OC4-HSL. In addition to QS assays, 342 growth challenges were conducted among a subset of the isolates, with 27% of isolates eliciting growth inhibition and 2% growth stimulation. 24% of BBD isolates elicited growth inhibition as compared to 26% and 32% of the bacteria from the two SML sources. With one exception, only isolates that exhibited AI-2 activity or produced DPD inhibited growth of test strains. These findings demonstrate for the first time that AHLs are present in an active coral disease. It is possible that AI-2 production among BBD and coral SML bacteria may structure the microbial communities of both a polymicrobial infection and the healthy coral microbiome.

Sulfate reducing bacteria as secondary and necessary pathogens in black band disease of corals / Bacterias reductoras de sulfato como patógenos secundarios y necesarios en la enfermedad de la banda negra de coral

Black band disease (BBD) is a complex, polymicrobial disease that consists of cyanobacteria, sulfide-oxidizing and sulfate-reducing bacteria (SRB), and heterotrophic bacteria. The cyanobacterium Roseofilum reptotaenium has been implicated as the primary pathogen of BBD, but other consortium members may be secondary pathogens that are necessary to the development of the disease. It is known that populations of the sulfate-reducing bacterium Desulfovibrio are present in BBD and that these populations generate sulfide within the band as a byproduct of dissimilatory sulfate reduction. It is also known that exposure of healthy corals to sulfide leads to cell lysis and coral tissue death. Previous work showed that when freshly collected BBD, which easily infects healthy corals, is exposed to sodium molybdate, a specific inhibitor of sulfate reduction, infection does not occur. In this study we examined the effect of sodium molybdate on infection of corals by a unialgal culture of R. reptotaenium. Coral fragments of Montastraea cavernosa and Siderastrea siderea were transferred into two experimental aquaria, one a control with only artificial seawater (ASW) and the second containing ASW and 2mM sodium molybdate. Small mats of cultured R. reptotaenium were inoculated onto the surface of experimental coral fragments. Both M. cavernosa (n = 6) and S. siderea (n=4) became infected and developed BBD-like infections in the control tank, while there were temporary attachments to, but no successful infection of M. cavernosa (n=3) or S. siderea (n=2) in the experimental tank containing sodium molybdate. The results of this study reveal that a secondary pathogen is essential to the infection process and development of BBD in scleractinian corals. Specifically, SRB such as Desulfovibrio are required for the development of BBD on the coral host. This is the first step in understanding the roles of secondary pathogens in a complex, polymicrobial coral disease.

La enfermedad de la Banda Negra (BBD) es una enfermedad polimicrobiana compleja, que consiste en las cianobacterias, sulfuro oxidante y bacterias reductoras de sulfato (SRB) y bacterias heterotróficas. La cianobacteria Roseofilum reptotaenium se considera como el principal patógeno de BBD, pero otros miembros del consorcio pueden ser patógenos secundarios que son necesarios para el desarrollo de la enfermedad. Se conoce que las poblaciones de la bacteria reductora de sulfato Desulfovibrio están presentes en BBD y que estas poblaciones generan sulfuro dentro de la banda como un subproducto de la reducción del sulfato. También se conoce que la exposición de los corales sanos a sulfuro conduce a la lisis celular y la muerte del tejido de coral. Trabajos previos muestran que cuando recién colectado el tejido con BBD, que infecta fácilmente corales sanos, se expone al molibdato de sodio, un inhibidor específico de la reducción del sulfato, la infección no se produce. En este estudio analizamos el efecto del molibdato de sodio sobre la infección de los corales en un cultivo unialgal de R. reptotaenium. Fragmentos de coral de Montastraea cavernosa y Siderastrea siderea se transfirieron a dos acuarios experimentales, uno control con solo agua de mar artificial (ASW) y el segundo que contenía ASW y molibdato de sodio 2mM. Pequeñas esteras de cultivo R. reptotaenium se inocularon en la superficie de fragmentos experimentales de coral. Tanto M. cavernosa (n=6) y S. siderea (n=4) se infectaron y desarrollaron infecciones BBD- como en el tanque de control, mientras que había adjuntos temporales, sin infección exitosa en M. cavernosa (n=3) o S. siderea (n=2) en el tanque experimental con molibdato de sodio. Los resultados de este estudio revelan que un patógeno secundario es esencial para el proceso de infección y el desarrollo de BBD en corales escleractíneos. Específicamente, SRB como Desulfovibrio son necesarios para el desarrollo de BBD en el coral hospedero. Este es el primer paso en la comprensión de las funciones de los patógenos secundarios en una enfermedad polimicrobiana compleja de coral.

Insights into Migration and Development of Coral Black Band Disease Based on Fine Structure Analysis

In many diverse ecosystems, ranging from natural surfaces in aquatic ecosystems to the mammalian gut and medical implants, bacterial populations and communities exist as biofilms. While the process of biofilm development has been well-studied for those produced by unicellular bacteria such Pseudomonas aeruginosa, little is known about biofilm development associated with filamentous microorganisms. Black band disease (BBD) of corals is characterized as a polymicrobial biofilm (mat) community, visually-dominated by filamentous cyanobacteria. The mat migrates across a living coral host, completely lysing coral tissue and leaving behind exposed coral skeleton. It is the only known cyanobacterial biofilm that migrates across a substratum, thus eliciting questions about the mechanisms and unique characteristics of this system. Fragments of the coral Montastraea annularis, five artificially infected with BBD and two collected from a naturally BBD-infected colony, were used to address these questions by detailed examination using scanning and transmission electron microscopy (SEM and TEM). In areas close to the interface of coral tissue and the mature disease band two types of clusters of cyanobacteria were observed, one with random orientation and one with parallel orientation of filaments. The latter exhibited active secretion of extracellular polysaccharide (EPS) while the randomly oriented clusters did not. Within the well developed band cyanobacterial filaments were observed to be embedded in EPS and were present as layers of filaments in parallel orientation. These observations suggest that BBD cyanobacteria orient themselves and produce EPS in a sequential process during migration to form the complex BBD matrix.

En muchos ecosistemas diversos, que van desde ecosistemas acuáticos hasta los intestinos de mamíferos e implantes médicos, las poblaciones y comunidades de bacterias existen como biopelículas (biofilms). El proceso de desarrollo de las biopelículas ha sido bien estudiado para aquellos producidos por bacterias unicelulares como Pseudomonas aeruginosa, pero se conoce muy poco acerca del desarrollo de biopelículas asociadas con microorganismos filamentosos. La Enfermedad de Banda Negra (EBN) de coral es caracterizada como una comunidad polimicrobiana que forma una biopelícula (lecho), visualmente-dominada por una cianobacteria filamentosa. El lecho migra a través de un huésped de coral vivo, rompiendo completamente el tejido del coral y dejando atrás el esqueleto de coral expuesto. Es la única biopelícula cianobacteriana que migra a través de un sustrato, por lo tanto esto genera preguntas acerca de los mecanismos y las características únicas de este sistema. Fragmentos del coral Montastraea annularis, cinco artificialmente infectados con EBN y dos colectados de una colonia EBN-infectada, fueron usados para abordar estas preguntas mediante exámenes detallados con microscopía electrónica de barrido y de transmisión (MEB y MET). En zonas cercanas a la interfaz de tejido del coral y la banda de la enfermedad madura, se han observado dos tipos de grupos de cianobacterias, uno con orientación aleatoria y otro con una orientación paralela de los filamentos. Este último exhibe la secreción activa de polisacáridos extra-celulares (PEC), mientras que los grupos orientados al azar no lo hicieron. Dentro de la banda de filamentos cianobacterianas bien desarrollados se observó que estaban integradas en PEC y que se presentaban como capas de cianobacteria con orientación paralela. Estas observaciones sugieren que la cianobacteria de EBN se orienta a sí misma y produce PEC en un proceso secuencial durante la migración para formar la matriz complejo de EBN.

Arnfried Antonius, coral diseases, and the AMLC / Arnfried Antonius, las enfermedades de los corales y la AMLC

The study of coral diseases, coral pathogens, and the effects of diseases on tropical and subtropical coral reefs are all current, high-profile research areas. This interest has grown steadily since the first report of a coral disease in 1973. The author of this report was Arnfried Antonius and the publication was an abstract in the proceedings of a scientific meeting of the Association of Marine Laboratories of the Caribbean, or AMLC (then known as the Association of Island Marine Laboratories of the Caribbean). Since Antonius’ pioneering communication he continued working on coral diseases on reefs throughout the world, often documenting the first observation of a novel pathology in a novel location. Each of the coral diseases Antonius first described, in particular black band disease, is the subject of current and ongoing investigations addressing pathogens, etiology, and their effects on coral reefs. Many of the points and observations he made in his early papers are highly relevant to research today. This paper reviews aspects of Antonius’ early work, highlighting contributions he made that include the first in situ experimental studies aimed at discerning coral epizootiology and the first quantitative assessments of the role of environmental factors in coral disease. Antonius’ early findings are discussed in terms of relevant current controversies in this research area.

El estudio de las enfermedades de los corales, los patogenos de los corales y los efectos de estas enfermedades sobre los arrecifes tropicales y subtropicales son actualmente areas importantes de investigacion. El interés en este tema ha crecido continuamente desde el primer informe sobre una enfermedad de coral que se publico en 1973. El autor de este informe fue Arnfried Antonius y la publicacion fue un resumen en el Libro de Programa y Resumenes de la Decima Reunion de la Asociacion de Laboratorios Marinos Islenos del Caribe (conocida ahora como la Asociacion de Laboratorios Marinos del Caribe). Desde esta comunicacion pionera, Antonius siguio trabajando sobre las enfermedades de los corales en arrecifes alrededor del mundo, a menudo documentando la primera observacion de una nueva enfermedad en un nuevo lugar. Cada enfermedad de coral descrita por primera vez por Antonius es actualmente el objeto de investigaciones actuales en lo que se refiere a patogenos, ecologia de las enfermedades y los efectos sobre los arrecifes de coral. Muchas de las observaciones en sus trabajos tempranos siguen siendo relevantes en la investigacion actual. Este trabajo examinara ciertos aspectos de los estudios tempranos de Antonius sobre las enfermedades de los corales, poniendo de relieve sus contribuciones novedosas que incluyen los primeros experimentos in situ que tenian como objetivo el estudio de la etiologia de las enfermedades de los corales y los primeros analisis cuantitativos de la incidencia de las enfermedades de corales y de los patrones de distribución en funcion de los factores ambientales. Las contribuciones iniciales de Antonius se discuten en terminos de las controversias actuales sobre el tema.

Fine structure analysis of black band disease (BBD) infected coral and coral exposed to the BBD toxins microcystin and sulfide.

Black band disease (BBD) of corals is a complex pathogenic polymicrobial mat community that lyses coral tissue as it migrates over an infected colony. Two known toxins are produced by BBD microorganisms - sulfide, produced by sulfate-reducing bacteria, and microcystin, produced by cyanobacteria. Experiments were carried out to determine the effects of exposing healthy coral fragments to variable concentrations of purified microcystin, sulfide at a concentration known to exist in BBD, and a combination of the two. Healthy fragments of the coral Montastraea annularis were placed into experimental chambers with known toxin/s for 18-22.5 h. Fine structural analysis using scanning electron microscopy (SEM) showed that toxin exposure resulted in thinning or removal of the coral epidermal layer coupled with degradation of the gastrodermis. These effects were exacerbated when both toxins were used in combination. Exposure to sulfide and the highest concentration of microcystin caused zooxanthellae to dissociate from the coral tissue and to form clusters on the coral surface. Examination of coral fragments infected with BBD was carried out for comparison. It was determined that the effects of exposure to sulfide and microcystin on coral fine structure were consistent, both quantitatively and qualitatively, with the effects of artificially induced and naturally occurring BBD on M. annularis.

Antibacterial activity of marine and black band disease cyanobacteria against coral-associated bacteria.

Black band disease (BBD) of corals is a cyanobacteria-dominated polymicrobial disease that contains diverse populations of heterotrophic bacteria. It is one of the most destructive of coral diseases and is found globally on tropical and sub-tropical reefs. We assessed ten strains of BBD cyanobacteria, and ten strains of cyanobacteria isolated from other marine sources, for their antibacterial effect on growth of heterotrophic bacteria isolated from BBD, from the surface mucopolysaccharide layer (SML) of healthy corals, and three known bacterial coral pathogens. Assays were conducted using two methods: co-cultivation of cyanobacterial and bacterial isolates, and exposure of test bacteria to (hydrophilic and lipophilic) cyanobacterial cell extracts. During co-cultivation, 15 of the 20 cyanobacterial strains tested had antibacterial activity against at least one of the test bacterial strains. Inhibition was significantly higher for BBD cyanobacteria when compared to other marine cyanobacteria. Lipophilic extracts were more active than co-cultivation (extracts of 18 of the 20 strains were active) while hydrophilic extracts had very limited activity. In some cases co-cultivation resulted in stimulation of BBD and SML bacterial growth. Our results suggest that BBD cyanobacteria are involved in structuring the complex polymicrobial BBD microbial community by production of antimicrobial compounds.

Fine-structural analysis of black band disease-infected coral reveals boring cyanobacteria and novel bacteria.

Examination of coral fragments infected with black band disease (BBD) at the fine- and ultrastructural levels using scanning (SEM) and transmission electron microscopy (TEM) revealed novel features of the disease. SEM images of the skeleton from the host coral investigated (Montastraea annularis species complex) revealed extensive boring underneath the BBD mat, with cyanobacterial filaments present within some of the bore holes. Cyanobacteria were observed to penetrate into the overlying coral tissue from within the skeleton and were present throughout the mesoglea between tissue layers (coral epidermis and gastrodermis). A population of novel, as yet unidentified, small filamentous bacteria was found at the leading edge of the migrating band. This population increased in number within the band and was present within degrading coral epithelium, suggesting a role in disease etiology. In coral tissue in front of the leading edge of the band, cyanobacterial filaments were observed to be emerging from bundles of sloughed-off epidermal tissue. Degraded gastrodermis that contained actively dividing zooxanthellae was observed using both TEM and SEM. The BBD mat contained cyanobacterial filaments that were twisted, characteristic of negative-tactic responses. Some evidence of boring was found in apparently healthy control coral fragments; however, unlike in BBD-infected fragments, there were no associated cyanobacteria. These results suggest the coral skeleton as a possible source of pathogenic BBD cyanobacteria. Additionally, SEM revealed the presence of a potentially important group of small, filamentous BBD-associated bacteria yet to be identified.

Microcystin production and ecological physiology of Caribbean black band disease cyanobacteria.

Molecular studies of black band disease (BBD), a coral disease found on tropical and subtropical reefs worldwide, have shown that one 16S rRNA gene sequence is ubiquitous. This sequence has been reported to be a member of the cyanobacterial genus Oscillatoria. In this study, extracts of two cultured laboratory strains of BBD Oscillatoria, and for comparison two strains of BBD Geitlerinema, all isolated from reefs of the wider Caribbean, were analysed using Ultra-Performance Liquid Chromatography-Tandem Quad Mass Spectrometry (UPLC-MS/MS). The cyanotoxin microcystin-LR (MC-LR) was found in all strains, and one Geitlerinema strain additionally produced MC-YR. Growth experiments that monitored toxin production using enzyme-linked immunosorbent assay (ELISA) showed that BBD Oscillatoria produced yields of MC-LR equivalent (0.02-0.04 mg g(-1)) independent of biomass and culture conditions (varying temperature, pH, light and organic carbon). This pattern is different from BBD Geitlerinema, which increased production of MC-LR equivalent in the presence of organic carbon in the light and dark and at a relatively lower temperature. These results indicate that different species and strains of BBD cyanobacteria, which can occur in the same BBD infection, may contribute to BBD pathobiology by producing different toxins and different amounts of toxin at different stages in the disease process. This is the first detailed study of laboratory cultures of the ubiquitous BBD cyanobacterium Oscillatoria sp. isolated from Caribbean reefs.

A meta-analysis of 16S rRNA gene clone libraries from the polymicrobial black band disease of corals.

Black band disease (BBD) is a polymicrobial disease affecting corals on reefs worldwide. Since 2002, researchers have constructed clone libraries from the BBD consortium using 16S rRNA gene primers targeting a variety of phyla. In the present study, a meta-analysis was conducted of published libraries from 2002 through the present that contain bacterial sequence data associated with individual clones and BBD samples. The libraries analyzed were from 87 BBD samples, collected from 16 species of scleractinian corals in 10 different geographic locations, and included 327 unique operational taxonomic units (OTUs). One OTU (cyanobacterial) was present in 62 (71%) samples, and three (one Cytophaga-Flavobacter-Bacteriodetes and two alphaproteobacterial) were present in 11 (13%) of the samples. The remaining 323 unique OTUs were present in <10% of the samples. The Alphaproteobacteria was the most diversely represented group. Analysis of clone libraries using nonmetric multidimensional scaling indicated strong regional specificity of BBD microbial populations, but limited host coral specificity. The results of this analysis support the hypotheses that: (1) a specific cyanobacterium may be the primary pathogen of BBD; (2) additional functional groups, required for BBD pathobiology, are represented by variable opportunistic species; and (3) opportunistic BBD microorganisms are primarily derived from the environment.

Cyanotoxins from black band disease of corals and from other coral reef environments.

Many cyanobacteria produce cyanotoxins, which has been well documented from freshwater environments but not investigated to the same extent in marine environments. Cyanobacteria are an obligate component of the polymicrobial disease of corals known as black band disease (BBD). Cyanotoxins were previously shown to be present in field samples of BBD and in a limited number of BBD cyanobacterial cultures. These toxins were suggested as one of the mechanisms contributing to BBD-associated coral tissue lysis and death. In this work, we tested nine cyanobacterial isolates from BBD and additionally nine isolated from non-BBD marine sources for their ability to produce toxins. The presence of toxins was determined using cell extracts of laboratory grown cyanobacterial cultures using ELISA and the PP2A assay. Based on these tests, it was shown that cyanobacterial toxins belonging to the microcystin/nodularin group were produced by cyanobacteria originating from both BBD and non-BBD sources. Several environmental factors that can be encountered in the highly dynamic microenvironment of BBD were tested for their effect on both cyanobacterial growth yield and rate of toxin production using two of the BBD isolates of the genera Leptolyngbya and Geitlerinema. While toxin production was the highest under mixotrophic conditions (light and glucose) for the Leptolyngbya isolate, it was highest under photoautotrophic conditions for the Geitlerinema isolate. Our results show that toxin production among marine cyanobacteria is more widespread than previously documented, and we present data showing three marine cyanobacterial genera (Phormidium, Pseudanabaena, and Spirulina) are newly identified as cyanotoxin producers. We also show that cyanotoxin production by BBD cyanobacteria can be affected by environmental factors that are present in the microenvironment associated with this coral disease.

Effect of freezing on PCR amplification of 16S rRNA genes from microbes associated with black band disease of corals.

Molecular analysis of black band disease of corals revealed that samples frozen immediately after collection yielded more proteobacterial 16S rRNA sequences, while unfrozen samples produced more cyanobacterial and sulfur-oxidizing bacterial sequences. These results suggest the need to use multiple approaches for preparation of samples to characterize this complex polymicrobial disease.

Sulfide, microcystin, and the etiology of black band disease.

Black band disease (BBD) consists of a cyanobacterial-dominated, sulfide-rich microbial mat that migrates across coral colonies, degrading coral tissue. The mat contains diverse bacteria that include photoautotrophs (cyanobacteria), sulfate-reducers, sulfide-oxidizers, and organoheterotrophs. BBD sulfate-reducers contribute to BBD pathobiology by production of sulfide, which causes coral tissue lysis and death, and the cyanotoxin microcystin is produced by BBD cyanobacteria. Here we used a model system of coral fragments to investigate the roles of sulfide and microcystin in BBD by exposure to the metabolic inhibitors sodium molybdate and 3-(3', 4'-dichlorophenyl)-1, 1-dimethylurea (DCMU), which inhibit sulfate reduction and oxygenic photosynthesis, respectively. Exposure of BBD inocula to sodium molybdate prior to inoculation prevented infection of healthy fragments but did not prevent continued band migration and coral tissue lysis by active BBD infections. Exposure to DCMU did not inhibit either the initiation of BBD or continued migration of active BBD. Exposure of healthy coral fragments to sulfide, purified microcystin, and a combination of both revealed that both microcystin and sulfide are toxic to coral and act synergistically. Measurement of growth of bacteria isolated from BBD and the healthy coral surface mucopolysaccharide layer (SML) during exposure to microcystin revealed that growth of relatively more BBD than SML isolates was stimulated, although effects were not uniform and the majority exhibited no effect. Our results indicate that sulfide is required for initiation of BBD, both microcystin and sulfide are involved in BBD pathobiology, and microcystin may structure the BBD bacterial community.

Adaptation of cyanobacteria to the sulfide-rich microenvironment of black band disease of coral.

Black band disease (BBD) is a cyanobacteria-dominated microbial mat that migrates across living coral colonies lysing coral tissue and leaving behind exposed coral skeleton. The mat is sulfide-rich due to the presence of sulfate-reducing bacteria, integral members of the BBD microbial community, and the sulfide they produce is lethal to corals. The effect of sulfide, normally toxic to cyanobacteria, on the photosynthetic capabilities of five BBD cyanobacterial isolates of the genera Geitlerinema (3), Leptolyngbya (1), and Oscillatoria (1) and six non-BBD cyanobacteria of the genera Leptolyngbya (3), Pseudanabaena (2), and Phormidium (1) was examined. Photosynthetic experiments were performed by measuring the photoincorporation of [(14)C] NaHCO(3) under the following conditions: (1) aerobic (no sulfide), (2) anaerobic with 0.5 mM sulfide, and (3) anaerobic with 0.5 mM sulfide and 10 microM 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU). All five BBD cyanobacterial isolates tolerated sulfide by conducting sulfide-resistant oxygenic photosynthesis. Five of the non-BBD cyanobacterial isolates did not tolerate sulfide, although one Pseudanabaena isolate continued to photosynthesize in the presence of sulfide at a considerably reduced rate. None of the isolates conducted anoxygenic photosynthesis with sulfide as an electron donor. This is the first report on the physiology of a culture of Oscillatoria sp. found globally in BBD.

Molecular detection and ecological significance of the cyanobacterial genera Geitlerinema and Leptolyngbya in black band disease of corals.

Black band disease (BBD) is a pathogenic, sulfide-rich microbial mat dominated by filamentous cyanobacteria that infect corals worldwide. We isolated cyanobacteria from BBD into culture, confirmed their presence in the BBD community by using denaturing gradient gel electrophoresis (DGGE), and demonstrated their ecological significance in terms of physiological sulfide tolerance and photosynthesis-versus-irradiance values. Twenty-nine BBD samples were collected from nine host coral species, four of which have not previously been investigated, from reefs of the Florida Keys, the Bahamas, St. Croix, and the Philippines. From these samples, seven cyanobacteria were isolated into culture. Cloning and sequencing of the 16S rRNA gene using universal primers indicated that four isolates were related to the genus Geitlerinema and three to the genus Leptolyngbya. DGGE results, obtained using Cyanobacteria-specific 16S rRNA primers, revealed that the most common BBD cyanobacterial sequence, detected in 26 BBD field samples, was related to that of an Oscillatoria sp. The next most common sequence, 99% similar to that of the Geitlerinema BBD isolate, was present in three samples. One Leptolyngbya- and one Phormidium-related sequence were also found. Laboratory experiments using isolates of BBD Geitlerinema and Leptolyngbya revealed that they could carry out sulfide-resistant oxygenic photosynthesis, a relatively rare characteristic among cyanobacteria, and that they are adapted to the sulfide-rich, low-light BBD environment. The presence of the cyanotoxin microcystin in these cultures and in BBD suggests a role in BBD pathogenicity. Our results confirm the presence of Geitlerinema in the BBD microbial community and its ecological significance, which have been challenged, and provide evidence of a second ecologically significant BBD cyanobacterium, Leptolyngbya.