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 [¹⁴C] NaHCO₃ 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 μM 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.     

Nutrient enrichment enhances black band disease progression in corals

Infectious diseases are recognized as significant contributors to the dramatic loss of corals observed worldwide. However, the causes of increased coral disease prevalence and severity are not well understood. One potential factor is elevated nutrient concentration related to localized anthropogenic activities such as inadequate waste water treatment or terrestrial runoff. In this study the effect of nutrient enrichment on the progression of black band disease (BBD) was investigated using both in situ and laboratory experiments. Experimental increases in localized nutrient availability using commercial time release fertilizer in situ resulted in doubling of BBD progression and coral tissue loss in the common reef framework coral Siderastrea siderea. Laboratory experiments in which artificially infected S. siderea colonies were exposed to increased nitrate concentrations (up to 3 μM) demonstrated similar increases in BBD progression. These findings provide evidence that the impacts of this disease on coral populations are exacerbated by nutrient enrichment and that management to curtail excess nutrient loading may be important for reducing coral cover loss due to BBD.     

Changes in sulfate-reducing bacterial populations during the onset of black band disease

Factors that facilitate the onset of black band disease (BBD) of corals remain elusive, though anoxic conditions under the complex microbial mat and production of sulfide are implicated in necrosis of underlying coral tissues. This study investigated the diversity and quantitative shifts of sulfate-reducing bacterial (SRB) populations during the onset of BBD using real-time PCR (RT-PCR) and cloning approaches targeting the dissimilatory (bi)sulfite reductase (dsrA) gene. A quantitativePCR (qPCR) assay targeting the 16S rRNA gene also provided an estimate of total bacteria, and allowed the relative percentage of SRB within the lesions to be determined. Three Montipora sp. coral colonies identified with lesions previously termed cyanobacterial patches (CPs) (comprising microbial communities unlike those of BBD lesions), were tagged and followed through time as CP developed into BBD. The dsrA-targeted qPCR detected few copies of the gene in the CP samples (<65 per ng DNA), though copy numbers increased in BBD lesions (>2500 per ng DNA). SRB in CP samples were less than 1% of the bacterial population, though represented up to 7.5% of the BBD population. Clone libraries also demonstrated a shift in the dominant dsrA sequences as lesions shifted from CP into BBD. Results from this study confirm that SRB increase during the onset of BBD, likely increasing sulfide concentrations at the base of the microbial mat and facilitating the pathogenesis of BBD.     

Dynamics of seasonal outbreaks of black band disease in an assemblage of Montipora species at Pelorus Island (Great Barrier Reef,Australia)

Recurring summer outbreaks of black band disease (BBD) on an inshore reef in the central Great Barrier Reef (GBR) constitute the first recorded BBD epizootic in the region. In a 2.7 year study of 485 colonies of Montipora species, BBD affected up to 10 per cent of colonies in the assemblage. Mean maximum abundance of BBD reached 16±6 colonies per 100 m² (n=3 quadrats, each 100 m²) in summer, and decreased to 0–1 colony per 100 m² in winter. On average, BBD lesions caused 40 per cent tissue loss and 5 per cent of infections led to whole colony mortality. BBD reappearance on previously infected colonies and continuous tissue loss after the BBD signs had disappeared suggest that the disease impacts are of longer duration than indicated by the presence of characteristic signs. Rates of new infections and linear progression of lesions were both positively correlated with seasonal fluctuations in sea water temperatures and light, suggesting that seasonal increases in these environmental parameters promote virulence of the disease. Overall, the impacts of BBD are greater than previously reported on the GBR and likely to escalate with ocean warming.     

Integrating microbiological, microsensor, molecular, and physiologic techniques in the study of coral disease pathogenesis

The study of coral diseases requires an integrated approach that includes a combination of field and laboratory methods. By combining and building upon information available from multiple disciplines, within both field and laboratory applications, we have been successful in characterizing a number of coral diseases. To illustrate the utility of the integrative approach two very different coral diseases, black band disease and plague, are discussed in detail. Comparison of our ongoing characterization of each disease demonstrates that, within the integrative approach, different combinations of microbiological, microsensor, molecular, and physiologic techniques are required. The pathobiology of black band disease, which consists of a complicated, synergistic microbial consortium functioning around a dynamic sulfur cycle, is slowly being unraveled using a combination of methods. Our study of plague, on the other hand, has progressed in a very different manner that is controlled by the fact that this disease has, to date, emerged in three forms on reefs of the Florida Keys. The study of plague types I, II, and III will be detailed to illustrate the difficulty of characterizing a disease that rapidly evolves in the natural environment of the reef. Our ongoing study of additional (also very different) coral diseases will be summarized from the perspective of combined methodologies to illustrate the range and magnitude of questions that must be addressed and answered in order to understand coral disease pathogenesis and thus coral disease etiology.     

Oxygen and sulfide dynamics in a horizontally migrating cyanobacterial mat: Black band disease of corals

Abstract: Black band disease is caused by a horizontally migrating microbial consortium which overgrows and kills reef-building corals in many areas of the world. The cyanobacterium Phormidium corallyticum , the sulfide-oxidizing bacterium Beggiatoa sp., fungi, and sulfate-reducing bacteria dominate the consortium, which is generally several mm to 1 cm in width and ca. 1 mm in thickness. Microelectrode measurements revealed photosynthetically produced O₂-supersaturation in upper layers during day, although conditions at the band-coral interface were consistently anoxic and, at night, sulfide-rich. Diel distributions of oxygen and sulfide resembled those from cyanobacterial mats in sulfur springs, intertidal mats and hypersaline lagoons.     

The roles of temperature and light in black band disease (BBD) progression on corals of the genus Diploria in Bermuda

On Bermuda reefs the brain coral Diploria labyrinthiformis is rarely documented with black band disease (BBD), while BBD-affected colonies of Diploria strigosa are common. D. labyrinthiformis on these reefs may be more resistant to BBD or less affected by prevailing environmental conditions that potentially diminish host defenses. To determine whether light and/or temperature influence BBD differently on these two species, infection experiments were conducted under the following experimental treatments: (1) 26 °C, ambient light; (2) 30 °C, ambient light; (3) 30 °C, low light; and (4) 30 °C, high light. A digital photograph of the affected area of each coral was taken each day for 7 days and analyzed with ImageJ image processing software. The final affected area was not significantly different between species in any of the four treatments. BBD lesions were smaller on both species infected under ambient light at 26 °C versus 30 °C. Low light at 30 °C significantly reduced the lesion size on both species when compared to colonies infected at the same temperature under ambient light. Under high light at 30 °C, BBD lesions were larger on colonies of D. strigosa and smaller on colonies of D. labyrinthiformis when compared to colonies infected under ambient light at the same temperature. The responses of both species suggests that BBD progression on both D. strigosa and D. labyrinthiformis is similarly influenced by a combination of light and temperature and that other factors present before infections become established likely contribute to the difference in BBD prevalence in Bermuda.     

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.     

Farming behaviour of reef fishes increases the prevalence of coral disease associated microbes and black band disease

Microbial community structure on coral reefs is strongly influenced by coral–algae interactions; however, the extent to which this influence is mediated by fishes is unknown. By excluding fleshy macroalgae, cultivating palatable filamentous algae and engaging in frequent aggression to protect resources, territorial damselfish (f. Pomacentridae), such as Stegastes, mediate macro-benthic dynamics on coral reefs and may significantly influence microbial communities. To elucidate how Stegastes apicalis and Stegastes nigricans may alter benthic microbial assemblages and coral health, we determined the benthic community composition (epilithic algal matrix and prokaryotes) and coral disease prevalence inside and outside of damselfish territories in the Great Barrier Reef, Australia. 16S rDNA sequencing revealed distinct bacterial communities associated with turf algae and a two to three times greater relative abundance of phylotypes with high sequence similarity to potential coral pathogens inside Stegastes''s territories. These potentially pathogenic phylotypes (totalling 30.04% of the community) were found to have high sequence similarity to those amplified from black band disease (BBD) and disease affected corals worldwide. Disease surveys further revealed a significantly higher occurrence of BBD inside S. nigricans''s territories. These findings demonstrate the first link between fish behaviour, reservoirs of potential coral disease pathogens and the prevalence of coral disease.     

Biogeochemical conditions determine virulence of black band disease in corals

The microenvironmental dynamics of the microbial mat of black band disease (BBD) and its less virulent precursor, cyanobacterial patch (CP), were extensively profiled using microsensors under different light intensities with respect to O₂, pH and H₂S. BBD mats exhibited vertical stratification into an upper phototrophic and lower anoxic and sulphidic zone. At the progression front of BBD lesions, high sulphide levels up to 4977 μM were measured in darkness along with lower than ambient levels of pH (7.43±0.20). At the base of the coral–BBD microbial mat, conditions were hypoxic or anoxic depending on light intensity exposure. In contrast, CP mats did not exhibit strong microchemical stratification with mostly supersaturated oxygen conditions throughout the mats at all light intensities and with levels of pH generally higher than in BBD. Two of three replicate CP mats were devoid of sulphide, while the third replicate showed only low levels of sulphide (up to 42 μM) present in darkness and at intermediate light levels. The level of oxygenation and sulphide correlated well with lesion migration rates, that is virulence of the mats, which were greater in BBD than in CP. The results suggest that biogeochemical microgradients of BBD shaped by the complex microbial community, rather than a defined pathogen, are the major trigger for high virulence and the associated derived coral mortality of this disease.     

Characterization of black band disease in Red Sea stony corals

Microbial communities associated with black band disease (BBD) in massive stony corals from the Northern Red Sea (Eilat) were examined for the first time using molecular tools and microscopy. A high microbial diversity was revealed in the affected tissue in comparison with the healthy area of the same colony. Microscopy revealed the penetration of cyanobacteria into the coral mesoglea and adjacent tissues. Cyanobacterial sequences from Red Sea BBD-affected corals formed a cluster with sequences previously identified from black band and red band diseased corals from the Indo-Pacific and Caribbean. In addition, 11 sequences belonging to the genus Vibrio were retrieved. This group was previously documented as pathogenic to corals. Sulfate-reducing bacteria, a group known to be associated with BBD and produce toxic sulfide, were studied using specific primers for the amplification of the dissimilatory sulfite reductase gene (dsrA). This technique facilitated and improved the resolution of the study of diversity of this group. All the sequences obtained were closely related to sequences of the genus Desulfovibrio and 46% showed high homology to Desulfovibrio desulfuricans. The complex nature of BBD and the lack of success in isolating a single causative agent suggest that BBD may be considered a polymicrobial disease.     

Crystal structures of protein phosphatase-1 bound to motuporin and dihydromicrocystin-LA: elucidation of the mechanism of enzyme inhibition by cyanobacterial toxins

The microcystins and nodularins are tumour promoting hepatotoxins that are responsible for global adverse human health effects and wildlife fatalities in countries where drinking water supplies contain cyanobacteria. The toxins function by inhibiting broad specificity Ser/Thr protein phosphatases in the host cells, thereby disrupting signal transduction pathways. A previous crystal structure of a microcystin bound to the catalytic subunit of protein phosphatase-1 (PP-1c) showed distinct changes in the active site region when compared with protein phosphatase-1 structures bound to other toxins. We have elucidated the crystal structures of the cyanotoxins, motuporin (nodularin-V) and dihydromicrocystin-LA bound to human protein phosphatase-1c (gamma isoform). The atomic structures of these complexes reveal the structural basis for inhibition of protein phosphatases by these toxins. Comparisons of the structures of the cyanobacterial toxin:phosphatase complexes explain the biochemical mechanism by which microcystins but not nodularins permanently modify their protein phosphatase targets by covalent addition to an active site cysteine residue.     

Recruitment of scleractinians onto the skeletons of corals killed by black band disease

To determine what happens to scleractinian corals that have been killed by black band disease (BBD), massive corals with BBD were monitored for 11 years on a shallow reef (<10 m depth) in St. John, US Virgin Islands. Small quadrats (0.039 m²) were used to compare the rates of scleractinian recruitment to the skeletons of corals killed by either BBD or physical disturbance (Hurricane Hugo 1989). Coral recruitment was also quantified on the adjacent fringing reef using larger quadrats (0.25 m²) to detect possible biases associated with using small, permanent quadrats to assess recruitment to BBD-killed corals. Of 28 tagged colonies with BBD in 1988, 43% were lost to Hurricane Hugo in 1989, 7% were lost to unknown causes between 1991 and 1992, and 14 were monitored annually for 11 years; of these, 71% were dead and still in their original growth position in 1998. Between 1988 and 1997, corals recruited to the BBD-killed surfaces at a rate of 1.1 ± 0.3 recruits · 0.039 m⁻² · decade⁻¹ (mean ± SE, n = 14), although mortality reduced the density to 0.3 ± 0.2 recruits · 0.039 m⁻² by 1997. The rate of recruitment and the taxonomic composition of the coral recruits to BBD-killed corals were indistinguishable statistically from those to corals killed by Hurricane Hugo. This demonstrates that BBD creates space that is functionally the same as other dead coral surfaces in providing a substratum for coral recruitment. However, because coral recruits are dispersed widely, clumped in distribution and temporally variable in density on the fringing reef as a whole, it is unlikely that they will be found on monitored coral colonies that have been killed by BBD. While this hypothesis is consistent with the higher density of recruits on the fringing reef compared with BBD-killed corals, further studies are required to investigate alternative explanations such as the role of substratum age in favoring recruitment to surfaces other than those killed recently by BBD. Accepted: 26 August 1999     

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.     

Chemistry and structure of skeletal growth rings in the black coral Antipathes fiordensis (Cnidaria,Antipatharia)

The skeleton of the black coral Antipathes fiordensis is a laminar structure displaying major and minor growth rings. The skeleton is composed primarily of protein and chitin organized into successive microlayers. Each microlayer is tightly bound to the next by an organic cement that is periodically opaque in section, and occurs as clusters throughout the skeleton. In contrast, the skeletal microlayers themselves are never opaque. The apposition of multiple, opaque cement lines between skeletal layers is primarily responsible for the growth ring pattern.     

Isolation and characterization of microsatellite loci in Aspergillus sydowii, a pathogen of Caribbean sea fan corals

Here we report on nine microsatellite loci designed for Aspergillus sydowii, a widely distributed soil saprobe that is also the pathogenic agent of aspergillosis in Caribbean sea fan corals. Primers were tested on 20 A. sydowii isolates from the Caribbean, 17 from diseased sea fans and three from environmental sources. All loci were polymorphic and exhibited varying degrees of allelic diversity (three to nine alleles). Gene diversity (expected heterozygosity) ranged from 0.353 to 0.821. These primers will enable future research into the epidemiology of A. sydowii as an emergent infectious disease.