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.

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.

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.

Transmission of growth anomalies between Indo-Pacific Porites corals.

In this study we provide experimental evidence of transmission of growth anomalies (GAs) between corals. Twenty-four aquaria (16 experimental, 8 containing only apparently healthy corals) were set-up on Negros Island, Philippines, to test for direct-contact and waterborne transmission of GAs. Within seven weeks, two of 16 apparently healthy colonies placed in direct contact with colonies having GAs developed multiple GA lesions whose size and number increased over time. One of 16 apparently healthy colonies in experimental aquaria not touching any diseased colony also developed a GA, exhibiting a single lesion that did not increase in size. Apparently healthy colonies (n=24) in aquaria without a diseased colony remained unchanged.

Coral disease dynamics in the central Philippines.

Limited quantitative research has been conducted on coral disease in the Philippines and baseline data are much needed. Field surveys for prevalence and distribution patterns were conducted from November 2002 to August 2003. Sites included the islands of Negros, Cebu, Siquijor, Panglao, Olango, Sumilon, Bantayan, Pescador, Balicassag and Palawan. In 154 belt transects, 10 026 Porites colonies were examined at 28 sites covering 3080 m2. Two syndromes, Porites ulcerative white spot (PUWS) and coral tumors, occurred at high prevalence. Tumors as high as 39.1% occurred among massive Porites, and PUWS was as high as 53.7% among massive and branching Porites. In 8 mo, 116 tagged colonies showed slow progression and low mortality. Along a 41 km human impact gradient centered on Dumaguete City (Negros), 15 sites were examined. Correlation analyses linked higher disease prevalence to anthropogenic influence (Spearman's rank correlation coefficient [r(s)] = -0.54, p = 0.04 for tumors and r(s) = -0.69, p = 0.005 for PUWS). In most sites disease prevalence was lower than in the sites near Dumaguete. High PUWS prevalence near uninhabited Sumilon Island appeared to be linked to the highly diseased reefs near Dumaguete City due to transmission of disease along a cross-shelf front formed between the Tañon Strait and Bohol Sea. Other observations included 12 potential new host species for PUWS (4 new genera and 1 octocorallia) and 5 likely new hosts for black band disease (BBD) in the Philippines, and a relatively high prevalence (7.8%) of BBD in 1 site in western Palawan.

Coral diseases on Philippine reefs: genus Porites is a dominant host.

While it is generally assumed that Indo-Pacific reefs are not widely affected by diseases, limited data suggest a number of diseases and syndromes that appear to differ from those currently under study in the Caribbean. This report presents the results of a baseline survey of coral diseases in 2 regions in the Philippines: the Central Visayas and the Lingayen Gulf. Mean prevalence for all diseases observed was 8.3 +/- 1.2% (mean +/- SE; n = 8 reefs), with Central Visayas reefs showing higher disease prevalence (11.6 +/- 2.8%; n = 4 reefs) than those of Lingayen Gulf (5.1 +/- 1.4%; n = 4 reefs). Five diseases and syndromes were described; 3 of these-Porites ulcerative white spot disease (PUWS) (prevalence = 8.96 +/- 2.2%), tumors (prevalence = 1.0 +/- 0.5%) and pigmentation response (prevalence = 0.5 +/- 0.2%)--occurred frequently in both regions and targeted the genus Porites. Correlation between disease prevalence and number of Porites colonies was fairly strong (r2 = 43.4), though not significant, and no correlation was seen between prevalence and either the amount or diversity of hard coral. Porites is a major reef-builder in the Indo-Pacific comprising 30% of hard coral colonies on our surveyed reefs, and is generally thought to be a hardy, long-lived genus. Diseases targeting this robust group present an as yet unquantified risk to Philippine reefs and could result in major changes in reef structure.