Bacterial Community Associated with Black Band Disease in Corals

Black band disease (BBD) is a virulent polymicrobial disease primarily affecting massive-framework-building species of scleractinian corals. While it has been well established that the BBD bacterial mat is dominated by a cyanobacterium, the quantitative composition of the BBD bacterial mat community has not described previously. Terminal-restriction fragment length polymorphism (T-RFLP) analysis was used to characterize the infectious bacterial community of the bacterial mat causing BBD. These analyses revealed that the bacterial composition of the BBD mat does not vary between different coral species but does vary when different species of cyanobacteria are dominant within the mat. On the basis of the results of a new method developed to identify organisms detected by T-RFLP analysis, our data show that besides the cyanobacterium, five species of the division Firmicutes, two species of the Cytophaga-Flexibacter-Bacteroides (CFB) group, and one species of δ-proteobacteria are also consistently abundant within the infectious mat. Of these dominant taxa, six were consistently detected in healthy corals. However, four of the six were found in much higher numbers in BBD mats than in healthy corals. One species of the CFB group and one species of Firmicutes were not always associated with the bacterial communities present in healthy corals. Of the eight dominant bacteria identified, two species were previously found in clone libraries obtained from BBD samples; however, these were not previously recognized as important. Furthermore, despite having been described as an important component of the pathogenetic mat, a Beggiatoa species was not detected in any of the samples analyzed. These results will permit the dominant BBD bacteria to be targeted for isolation and culturing experiments aimed at deciphering the disease etiology.     

Cyanobacteria Associated with Coral Black Band Disease in Caribbean and Indo-Pacific Reefs

For 30 years it has been assumed that a single species of cyanobacteria, Phormidium corallyticum, is the volumetrically dominant component of all cases of black band disease (BBD) in coral. Cyanobacterium-specific 16S rRNA gene primers and terminal restriction fragment length polymorphism analyses were used to determine the phylogenetic diversity of these BBD cyanobacteria on coral reefs in the Caribbean and Indo-Pacific Seas. These analyses indicate that the cyanobacteria that inhabit BBD bacterial mats collected from the Caribbean and Indo-Pacific Seas belong to at least three different taxa, despite the fact that the corals in each case exhibit similar signs and patterns of BBD mat development.     

The Possible Role of Cyanobacterial Filaments in Coral Black Band Disease Pathology

Black band disease (BBD), characterized by a black mat or line that migrates across a coral colony leaving behind it a bare skeleton, is a persistent disease affecting massive corals worldwide. Previous microscopic and molecular examination of this disease in faviid corals from the Gulf of Eilat revealed a number of possible pathogens with the most prominent being a cyanobacterium identified as Pseudoscillatoria coralii. We examined diseased coral colonies using histopathological and molecular methods in order to further assess the possible role of this cyanobacterium, its mode of entry, and pathological effects on the coral host tissues. Affected areas of colonies with BBD were sampled for examination using both light and transmission electron microscopies. Results showed that this dominant cyanobacterium was found on the coral surface, at the coral–skeletal interface, and invading the polyp tissues and gastrovascular cavity. Although tissues surrounding the invasive cyanobacterial filaments did not show gross morphological alterations, microscopic examination revealed that the coral cells surrounding the lesion were dissociated, necrotic, and highly vacuolated. No amoebocytes were evident in the mesoglea of affected tissues suggesting a possible repression of the coral immune response. Morphological and molecular similarity of the previously isolated BBD-associated cyanobacterium P. coralii to the current samples strengthens the premise that this species is involved in the disease in this coral. These results indicate that the cyanobacteria may play a pivotal role in this disease and that the mode of entry may be via ingestion, penetrating the coral via the gastrodermis, as well as through the skeletal–tissue interface.     

Sporadic Disturbances in Fluctuating Coral Reef Environments: El Nino and Coral Reef Development in the Eastern Pacific

The disastrous effects of the intense 1982–83 El Niño-SouthernOscillation (ENSO) bring new insight into the long-term developmentof eastern Pacific coral reefs. The 1988–83 ENSO sea surfacewarming event caused extensive reef coral bleaching (loss ofsymbiotic zooxanthellae), resulting in up to 70–95% coralmortality on reefs in Costa Rica, Panama, Colombia and Ecuador.In the Galapagos Islands (Ecuador), most coral reefs experienced>95% coral mortality. Also, several coral species experiencedextreme reductions in population size, and local and regionalextinctions. The El Niño event spawned secondary disturbances,such as increased predation and bioerosion, that continue toimpact reef-building corals. The death of Pocillopora colonieswith their crustacean guards eliminated coral barriers now allowingthe corallivore Acanthaster planci access to formerly protectedcoral prey. Sea urchins and other organisms eroded disturbedcorals at rates that exceed carbonate production, potentiallyresulting in the elimination of existing reef buildups. In otherreefbuilding regions following extensive, catastrophic coralmortality, rapid recovery often occurs through the growth ofsurviving corals, recruitment of new corals from nearby sourcepopulations, and survival of consolidated reef surfaces. Inthe eastern Pacific, however, the return of upwelling conditionsand the survival of coral predators and bioeroders hamper coralreef recovery by reducing recruitment success and eroding coralreef substrates. Thus, coral reef growth that occurs betweendisturbance events is not conserved. Repeated El Niñodisturbances, which have occurred throughout the recent geologichistory of the eastern Pacific, prevent coral communities fromincreasing in diversity and limit the development and persistenceof significant reef features. The poor development of easternPacific coral reefs throughout Holocene and perhaps much ofPleistocene time may result from recurrent thermal disturbancesof the intensity of the 1982–83 El Niño event.     

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 α-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.     

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.     

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.Black band disease (BBD) is a polymicrobial disease that affects corals on reefs worldwide. It consists of a migrating microbial mat dominated by cyanobacteria that lyses coral tissue, leading to coral colony death, and is one of the most destructive of coral diseases. Microscopic examination of BBD samples consistently reveals an abundance of nonheterocystous, filamentous cyanobacteria and colorless gliding bacteria with internal elemental sulfur granules characteristic of the genus Beggiatoa (6, 17, 18). It is thought that these are key players in the etiology of BBD. However, with one exception (2), previous molecular studies of BBD consistently detected very low proportions of cyanobacteria (4, 8, 9, 19, 20) and only one study has detected Beggiatoa (19). Instead, all molecular BBD studies indicate a highly variable and diverse composition of heterotrophic bacteria, mostly members of the Alphaproteobacteria.It is unknown why the dominant cyanobacteria and filamentous sulfur-oxidizing bacteria observable microscopically in BBD samples are poorly or not at all detected by molecular methods. It is possible that freezing of the samples in these studies is the cause for low detection of BBD cyanobacteria and sulfur oxidizers. Freezing is the common method of sample processing to extract DNA for microbial community analysis of BBD and has been used in all previous molecular studies. However, this approach may impart a bias to detection of specific BBD bacteria. Suomalainen et al. (22) reported that freezing of samples targeting the fish pathogen Flavobacterium columnare destroyed DNA, suggested to be due to the release of DNase and other enzymes from the cell, leaving most of the F. columnare DNA undetectable by PCR. They noted that DNA from bacteria such as Escherichia coli was not affected (22). Bissett et al. (3) speculated that freezing sediments prior to DNA extraction lysed Beggiatoa filaments and caused their DNA to be lost (3). A recent report showed that algae and cyanobacteria with large cell sizes, including filamentous strains, could not be sufficiently cryopreserved (5). While the above-described studies showed or speculated that freezing of samples affects the detection of some microorganisms in environmental samples, none of these studies included detailed investigation of the mechanism responsible for the effect of freezing or of the effect of freezing on the assessment of microbial community composition.In the present study, we investigated the effect of freezing on molecular analysis of the BBD microbial community by using DNA extracts of frozen and unfrozen BBD samples from two coral hosts (Siderastrea siderea and Diploria clivosa), using both universal and cyanobacterium-specific primers targeting the 16S rRNA gene. BBD samples (i.e., the BBD microbial mat) were collected by suctioning the mat off the coral surface using individual sterile syringes while scuba diving. Samples were transferred to 2-ml cryovials (after decanting seawater) upon return to shore and either immediately frozen and stored at −20°C until DNA extraction or maintained at ambient temperature with DNA extracted within 1 h of collection. Eleven samples were collected from reefs of the Florida Keys (United States), Lee Stocking Island (Bahamas), and St. Croix (United States Virgin Islands).Genomic DNA was extracted by the bead-beating method as previously described (12, 19, 20). Frozen samples were first thawed at room temperature, and 500-μl aliquots were directly transferred into multimix lysing matrix tubes by using trimmed pipette tips, excluding any water. Unfrozen samples were transferred to multimix lysing matrix tubes in the same way. The extracted DNA was verified by gel electrophoresis, and DNA extracts from frozen samples were stored at −20°C, whereas DNA extracts from unfrozen samples were kept at 4°C until used for PCR amplification.DNA extracted from both frozen and unfrozen samples was amplified by PCR using universal bacterial primers 27F and 1492R (14) and cyanobacterium-specific primers CYA359F and CYA781R(B) (15) targeting 16S rRNA genes. The purification of PCR products, cloning, and sequencing of plasmid inserts were done as described previously (20). Primer M13F (11) or CYA359F (15) was used to obtain partial sequences, and an additional primer, 518F (13), M13R (11), or CYA781R(B) (15), was used to obtain full-length sequences. Sequence editing, BLAST (1), and phylogenetic analysis using ARB (10) were done as described previously (19, 20). Sequences that matched at similarity identity values of 97% and above were considered to be of the same operational taxonomic unit. Representative gene sequences that were closely related to cyanobacterial sequences were subjected to maximum-parsimony, neighbor-joining, and maximum-likelihood phylogenetic analyses, and a consensus tree was produced based on maximum-parsimony analysis.The results for universal bacterial primers indicated that all of the frozen BBD samples except one (Fig. ​(Fig.1,1, clone library E) were dominated (44 to 87%) by Alphaproteobacteria (Fig. ​(Fig.1;1; see Tables S1 and S2 in the supplemental material). We previously (19) compared the 16S rRNA gene sequences retrieved from seven of these libraries (Fig. ​(Fig.1,1, libraries A to G), all of which were obtained from frozen BBD samples from the host S. siderea, to investigate the diversity of BBD microorganisms between BBD infections. In the present study, we focus on the differences in results obtained using frozen versus unfrozen BBD samples from S. siderea (Fig. ​(Fig.1,1, libraries G and H) and a second coral host, D. clivosa (Fig. ​(Fig.1,1, libraries I and J). The S. siderea samples (libraries G and H) were taken from different host colonies on the same reef (Butler Bay Reef site), whereas the D. clivosa clone libraries were constructed from subsamples of one BBD sample.Open in a separate windowFIG. 1.Dominant bacterial phylogenetic groups, based on 16S rRNA gene sequence types and universal primers, present in clone libraries produced from frozen and unfrozen BBD samples from the coral hosts Siderastrea siderea and Diploria clivosa. The numbers above the bars represent the numbers of sequences in the respective clone libraries. Libraries A to H, frozen (A to G) and unfrozen (H) BBD from S. siderea. Libraries I and J, frozen (I) and unfrozen (J) BBD from D. clivosa. Sampling locations and sampling dates: libraries A and B, Horseshoe Reef, Lee Stocking Island, Bahamas, 19 July 2004; C, Rainbow Garden Reef, Lee Stocking Island, Bahamas, 16 July 2004; D, Watson''s Reef, Florida Keys, 3 May 2005; E, G, and H, Butler Bay Reef site, St. Croix, U.S. Virgin Islands (USVI), 22 October 2005, 1 June 2005, and 5 June 2006, respectively; F, Frederiksted Reef site, St. Croix, USVI, 1 June 2005; I and J, Frederiksted Reef site, St. Croix, USVI, 7 August 2006. All of the sequences from clone libraries A to G have been previously published by Sekar et al. (19, 20).This approach yielded strikingly different results for the two methods. For example, the clone library produced from one frozen sample (Fig. ​(Fig.1,1, library G) from S. siderea contained only one (of 60) cyanobacterium-related sequence (see {"type":"entrez-nucleotide","attrs":{"text":"EF123584","term_id":"132537907","term_text":"EF123584"}}EF123584 [GenBank sequence accession no.] in Table S1 in the supplemental material), which was phylogenetically related to a sequence from an uncultured planktonic Synechococcus sp. (GenBank sequence accession no. {"type":"entrez-nucleotide","attrs":{"text":"AY172810","term_id":"30525542","term_text":"AY172810"}}AY172810; Fig. ​Fig.2).2). In contrast, the clone library from the corresponding unfrozen sample (Fig. ​(Fig.1,1, library H) was dominated by a cyanobacterial ribotype which represented 37% of the clones. This ribotype was closely related to an Oscillatoria ribotype (GenBank sequence accession no. {"type":"entrez-nucleotide","attrs":{"text":"AY038527","term_id":"21322142","term_text":"AY038527"}}AY038527/{"type":"entrez-nucleotide","attrs":{"text":"AF473936","term_id":"18996878","term_text":"AF473936"}}AF473936) detected in almost all reported BBD molecular studies (2, 4, 7, 23). The sequence was confirmed as the BBD Oscillatoria sequence by phylogenetic analysis using two representative clone sequences (GenBank sequence accession no. {"type":"entrez-nucleotide","attrs":{"text":"EF123639","term_id":"132537962","term_text":"EF123639"}}EF123639 and {"type":"entrez-nucleotide","attrs":{"text":"EF123644","term_id":"132537967","term_text":"EF123644"}}EF123644) (Fig. ​(Fig.2).2). The unfrozen S. siderea clone library additionally produced a dominant epsilonproteobacterial ribotype (14 of 15 clones) (see Table S1 in the supplemental material) that was not detected in the corresponding frozen sample. Phylogenetic analysis of two representative sequences (GenBank sequence accession no. {"type":"entrez-nucleotide","attrs":{"text":"EF123607","term_id":"132537930","term_text":"EF123607"}}EF123607 and {"type":"entrez-nucleotide","attrs":{"text":"EF123613","term_id":"132537936","term_text":"EF123613"}}EF123613, not shown in Fig. ​Fig.2)2) determined that the sequence was related to a sequence from the sulfur-oxidizing bacterium “Candidatus Arcobacter sulfidicus” (GenBank sequence accession no. {"type":"entrez-nucleotide","attrs":{"text":"AY035822","term_id":"18057056","term_text":"AY035822"}}AY035822) (24), a species known to deposit filamentous sulfur (21) and reported previously in BBD (9).Open in a separate windowFIG. 2.Phylogenetic tree derived from the 16S rRNA gene sequences closely related to Synechococcus spp., Xenococcus spp., and Oscillatoria spp. sequences detected in BBD and their neighbors. The tree topology is based on the maximum-parsimony analysis. The bar represents 10% estimated sequence divergence. Boldface type indicates sequences from this study, designated as follows. FRSSBA, UFSSBA, FRSSCY, and UFSSCY indicate sequences retrieved from frozen (FR) and unfrozen (UF) samples of S. siderea (SS) using universal bacterial primers (BA) and cyanobacterium-specific (CY) primers for 16S rRNA gene amplification. FRDCBA, UFDCBA, and UFDCCY indicate sequences retrieved from frozen and unfrozen samples of Diploria clivosa (DC), and the same primer designations are used as for S. siderea sequences. GenBank sequence accession numbers are listed for all sequences. Asterisks designate sequences corresponding to the sequence from the BBD Oscillatoria discussed in the text.Again in clone library I, from the frozen subsample of D. clivosa (see Table S2 in the supplemental material), the Alphaproteobacteria were dominant (44%) and cyanobacteria represented in low percentages (4%). These cyanobacterial sequences were phylogenetically related to sequences of Leptolyngbya spp. (not shown in Fig. ​Fig.2)2) and a planktonic cyanobacterium Xenococcus sp. (GenBank sequence accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF132783","term_id":"5814229","term_text":"AF132783"}}AF132783) (Fig. ​(Fig.2;2; see Table S2 in the supplemental material). The library from the unfrozen BBD subsample of D. clivosa (see Table S2 in the supplemental material) was dominated by Gammaproteobacteria (35%), followed by cyanobacteria (24%) which had the same cyanobacterial sequence type (BBD Oscillatoria) observed in the unfrozen S. siderea sample (see Table S2 in the supplemental material). For corroboration of these results, we constructed an additional clone library, using universal primers, from an unfrozen BBD sample from S. siderea collected during June 2007; in this sample, 47% of the sequences were also related to the sequence from BBD Oscillatoria.The use of cyanobacterium-specific primers produced results similar to the overall pattern we detected using universal primers. Frozen BBD from S. siderea produced 27 sequences, of which 24 were closely related to sequences from planktonic Synechococcus spp. and Xenococcus sp. (see Table S3 in the supplemental material). This was confirmed by phylogenetic analysis (Fig. ​(Fig.2)2) using representative sequences (GenBank sequence accession no. {"type":"entrez-nucleotide","attrs":{"text":"EU019432","term_id":"157704222","term_text":"EU019432"}}EU019432, {"type":"entrez-nucleotide","attrs":{"text":"EU019435","term_id":"157704225","term_text":"EU019435"}}EU019435, {"type":"entrez-nucleotide","attrs":{"text":"EU019439","term_id":"157704229","term_text":"EU019439"}}EU019439, {"type":"entrez-nucleotide","attrs":{"text":"EU019442","term_id":"157704232","term_text":"EU019442"}}EU019442, {"type":"entrez-nucleotide","attrs":{"text":"EU019449","term_id":"157704239","term_text":"EU019449"}}EU019449, and {"type":"entrez-nucleotide","attrs":{"text":"EU019455","term_id":"157704245","term_text":"EU019455"}}EU019455). In contrast, all of the sequences (n = 37) obtained from unfrozen S. siderea samples were closely related to the sequence from the BBD Oscillatoria (see Table S3 in the supplemental material). Representative sequences (GenBank sequence accession no. {"type":"entrez-nucleotide","attrs":{"text":"EU019460","term_id":"157704250","term_text":"EU019460"}}EU019460 and {"type":"entrez-nucleotide","attrs":{"text":"EU019467","term_id":"157704257","term_text":"EU019467"}}EU019467) confirmed this phylogenetic affiliation (Fig. ​(Fig.2).2). Similarly, each of 38 sequences obtained from the unfrozen subsample of D. clivosa with cyanobacterium-specific primers was closely related to the sequence from the BBD Oscillatoria (see Table S3 in the supplemental material), again confirmed by phylogenetic analysis using two representative sequences (GenBank sequence accession no. {"type":"entrez-nucleotide","attrs":{"text":"EU019508","term_id":"157704298","term_text":"EU019508"}}EU019508 and {"type":"entrez-nucleotide","attrs":{"text":"EU019515","term_id":"157704305","term_text":"EU019515"}}EU019515) (Fig. ​(Fig.22).There was very little overlap (6 to 10%) between sequences obtained from frozen versus unfrozen BBD samples collected from both coral hosts when considering all of the BBD bacterial sequences detected (see Tables S1 and S2 in the supplemental material). Only four sequences were common to both frozen and unfrozen clone libraries (6% of 62 sequences detected within 136 clones) for S. siderea and seven sequences (10% of 69 sequences detected within 108 clones) for D. clivosa. Statistical analysis (ANOSIM) showed that the sequence types differed significantly between frozen and unfrozen clone libraries (R = 0.987; P = 0.022). Overall, all frozen libraries (libraries A to G and I) were 69% similar to each other, while the two unfrozen libraries (libraries H and J) were 58% similar.The results of our study are significant for the ongoing investigations into the etiology of BBD. While it is well known that the BBD microbial community consists of photoautotrophs (cyanobacteria), sulfate-reducing bacteria, sulfur-oxidizing bacteria, and heterotrophs (16), we are just beginning to understand the roles of these functional groups in the disease process. A first step in this understanding is the valid and repeatable detection of specific members of the BBD consortium. In summary, we show here that unfrozen samples produce better results for detection of BBD cyanobacteria and sulfur-oxidizing bacteria, while frozen samples are best for detection of heterotrophic proteobacterial sequences. The latter is particularly important because of the consistent finding of Proteobacteria associated with toxic dinoflagellates (19, 20), as well as other marine invertebrate pathogens (4), in BBD. We have not studied the mechanism behind the freezing effect (e.g., release of DNase), which is outside the scope of this study. Though the current study was done with BBD samples, the effect of freezing on other microbial mats or biofilms cannot be ignored. Based on the results of this study, we suggest using multiple sample-processing approaches to characterize the microbial communities associated with BBD and other microbial mats.     

Predictive Modeling of Coral Disease Distribution within a Reef System

Diseases often display complex and distinct associations with their environment due to differences in etiology, modes of transmission between hosts, and the shifting balance between pathogen virulence and host resistance. Statistical modeling has been underutilized in coral disease research to explore the spatial patterns that result from this triad of interactions. We tested the hypotheses that: 1) coral diseases show distinct associations with multiple environmental factors, 2) incorporating interactions (synergistic collinearities) among environmental variables is important when predicting coral disease spatial patterns, and 3) modeling overall coral disease prevalence (the prevalence of multiple diseases as a single proportion value) will increase predictive error relative to modeling the same diseases independently. Four coral diseases: Porites growth anomalies (PorGA), Porites tissue loss (PorTL), Porites trematodiasis (PorTrem), and Montipora white syndrome (MWS), and their interactions with 17 predictor variables were modeled using boosted regression trees (BRT) within a reef system in Hawaii. Each disease showed distinct associations with the predictors. Environmental predictors showing the strongest overall associations with the coral diseases were both biotic and abiotic. PorGA was optimally predicted by a negative association with turbidity, PorTL and MWS by declines in butterflyfish and juvenile parrotfish abundance respectively, and PorTrem by a modal relationship with Porites host cover. Incorporating interactions among predictor variables contributed to the predictive power of our models, particularly for PorTrem. Combining diseases (using overall disease prevalence as the model response), led to an average six-fold increase in cross-validation predictive deviance over modeling the diseases individually. We therefore recommend coral diseases to be modeled separately, unless known to have etiologies that respond in a similar manner to particular environmental conditions. Predictive statistical modeling can help to increase our understanding of coral disease ecology worldwide.     

Restriction Fragment Length Polymorphism Analysis of Large Subunit rDNA of Symbiotic Dinoflagellates from Scleractinian Corals in the Zhubi Coral Reef of the Nansha Islands

Zooxanthellae are very important for the coral reef ecosystem. The diversity of coral hosts Is high in the South China Sea, but the diversity of zooxanthellae has not yet been investigated. We chose the Zhubi Coral Reef of the Nansha Islands as the region to be surveyed in the present study because it represents a typical tropical coral reef of the South China Sea and we investigated zooxanthellae diversity In 10 host scleractinlan coral species using polymerase chain reaction （PCR） of the large subunit rRNA and restriction fragment length polymorphlsm （RFLP） patterns. Poclllopora verrucosa, Acropora pefifera, Acropora mlllepora, Fungla fungltes, Galaxea fasclcularls, and Acropora pruinosa harbor Clade C, Goniastrea aspera harbors Clade D, and Acropora formosa harbors Clades D and C. Therefore, the Clade C is the dominant type in the Zhubi Coral Reef of the Nansha Islands. Furthermore, the results of the present also disprove what has been widely accepted, namely that one coral host harbors only one algal symblont. The coral-algal symbiosis Is flexible, which may be an Important mechanism for surviving coral bleaching. Meanwhile, on the basis of the results of the present study, we think that Symblodlnium Clade D may be more tolerant to stress than Symbiodlnlum Clade C.     

Ecological Physiology of a Coral Pathogen and the Coral Reef Environment

Laboratory studies on the ecological physiology of a coral pathogen were carried out to investigate growth potential in terms of environmental factors that may control coral diseases on reefs. The disease chosen for this study, white plague type II, is considered to be one of the major diseases of Caribbean scleractinian corals, affecting a wide range of coral hosts and causing rapid and widespread tissue loss. It is caused by a single pathogen, the bacterium Aurantimonas coralicida. A series of laboratory experiments using a pure culture of the pathogen was carried out to examine the roles of temperature, pH, and O₂ concentration on growth rate. Results revealed optimal growth between 30 and 35°C, and between pH values of 6 and 8. There was a distinctive synergistic relationship between pH and temperature. Increasing temperature from 25 to 35°C expanded the range of pH tolerance from a minimum of 6.0 down to 5.0. O₂ concentration directly affected growth rate, which increased with increasing O₂. The combined effects of increasing O₂ and increasing temperature resulted in a synergistic effect of more rapid growth. These laboratory results are discussed in terms of the coral host and the range of the environmental factors that occur on coral reefs. We conclude that changing environmental conditions in the reef environment, in particular observed increases in water temperature, may be promoting coral diseases by allowing coral pathogens to expand their ecological niches. In the case of the white plague type II pathogen, elevated temperature would allow A. coralicida to colonize the low pH environment of the coral surface mucopolysaccharide layer as an initial stage of infection. The synergistic effect between temperature and oxygen concentration appeared to be less environmentally relevant for this coral pathogen.     

Effect of Phase Shift from Corals to Zoantharia on Reef Fish Assemblages

Consequences of reef phase shifts on fish communities remain poorly understood. Studies on the causes, effects and consequences of phase shifts on reef fish communities have only been considered for coral-to-macroalgae shifts. Therefore, there is a large information gap regarding the consequences of novel phase shifts and how these kinds of phase shifts impact on fish assemblages. This study aimed to compare the fish assemblages on reefs under normal conditions (relatively high cover of corals) to those which have shifted to a dominance of the zoantharian Palythoa cf. variabilis on coral reefs in Todos os Santos Bay (TSB), Brazilian eastern coast. We examined eight reefs, where we estimated cover of corals and P. cf. variabilis and coral reef fish richness, abundance and body size. Fish richness differed significantly between normal reefs (48 species) and phase-shift reefs (38 species), a 20% reduction in species. However there was no difference in fish abundance between normal and phase shift reefs. One fish species, Chaetodon striatus, was significantly less abundant on normal reefs. The differences in fish assemblages between different reef phases was due to differences in trophic groups of fish; on normal reefs carnivorous fishes were more abundant, while on phase shift reefs mobile invertivores dominated.     

Environmental Records from Great Barrier Reef Corals: Inshore versus Offshore Drivers

The biogenic structures of stationary organisms can be effective recorders of environmental fluctuations. These proxy records of environmental change are preserved as geochemical signals in the carbonate skeletons of scleractinian corals and are useful for reconstructions of temporal and spatial fluctuations in the physical and chemical environments of coral reef ecosystems, including The Great Barrier Reef (GBR). We compared multi-year monitoring of water temperature and dissolved elements with analyses of chemical proxies recorded in Porites coral skeletons to identify the divergent mechanisms driving environmental variation at inshore versus offshore reefs. At inshore reefs, water Ba/Ca increased with the onset of monsoonal rains each year, indicating a dominant control of flooding on inshore ambient chemistry. Inshore multi-decadal records of coral Ba/Ca were also highly periodic in response to flood-driven pulses of terrigenous material. In contrast, an offshore reef at the edge of the continental shelf was subject to annual upwelling of waters that were presumed to be richer in Ba during summer months. Regular pulses of deep cold water were delivered to the reef as indicated by in situ temperature loggers and coral Ba/Ca. Our results indicate that although much of the GBR is subject to periodic environmental fluctuations, the mechanisms driving variation depend on proximity to the coast. Inshore reefs are primarily influenced by variable freshwater delivery and terrigenous erosion of catchments, while offshore reefs are dominated by seasonal and inter-annual variations in oceanographic conditions that influence the propensity for upwelling. The careful choice of sites can help distinguish between the various factors that promote Ba uptake in corals and therefore increase the utility of corals as monitors of spatial and temporal variation in environmental conditions.     

Sex, Symbiosis and Coral Reef Communities

SYNOPSIS. Questions about how today's corals and coral reefswill fare in a future that holds not only increasing directanthropogenic impacts, but also global change, cannot be satisfactorilyanswered if we do not understand the relations of corals andreef systems to today's environmental conditions. This paperdiscusses four aspects of modern reef biology: coral reproduction,coral population biology, the coral-zooxanthella symbiosis,and reef community ecology. Conclusions of this survey of currentknowledge are that complexities of cnidarian reproductive biology,and our rudimentary knowledge of reproductive patterns in reefcnidarians, make forecasting based on current knowledge uncertainat best; new discoveries about the coral algal symbiotic systemsuggest a possible mode of adjustment to environmental changethat warrants a strong research effort; coral communities ofthe future may well be unlike what we are familiar with today;and these new assemblages will be shaped by the interactionof novel environmental conditions and the characteristics ofindividual reef species.     

The Physiological Mechanisms of Acclimatization in Tropical Reef Corals

SYNOPSIS. The ability of scleractinian corals to survive changesthat are predicted in the global environment over the next centurywill lie in their physiological mechanisms of acclimatization.Corals display rapid modifications in behavior, morphology andphysiology enabling them to photoacclimate to changing lightconditions, a scenario that demonstrates considerable biologicalflexibility. Here we argue that the acclimatization mechanismsin corals are fundamentally similar to those exhibited by otherinvertebrate taxa. We discuss protein metabolism as a mechanismunderlying acclimatization responses in reef corals, and explorethe relationship between protein turnover, metabolic rate, growthrate, and acclimatization capacity. Our preliminary analysessuggest that corals with low growth rates (µCa/mgN/h)and high metabolic rates (µO₂/cm²/hr), such as the massivespecies, acclimatize more effectively than those with high growthrates and low metabolic rates, a feature that is characteristicof branching species. We conclude that studies of protein turnover,combined with temporally relevant investigations into the dynamicaspects of coral dinoflagellate symbioses will provide considerableinsight into why corals exhibit such a high level of variationin response to the same environmental challenge. Furthermore,a more detailed understanding of acclimatization mechanismsis essential if we are to predict how a coral assemblage willrespond to present and future environmental challenges.     

Extent and effect of Black Band Disease on a Caribbean reef

The effect of Black Band Disease (BBD) among colonies ofMontastrea annularis, M. cavernosa, Diploria strigosa, D. labryinthiformis, S. siderea andColpophyllia natans was determined at 7 shallow locations in the Virgin Islands. Between September 1988 and November 1988, 0.2% of 9204 colonies of these species were infected with BBD in 6908 m² of reef at 22 randomly chosen areas. Infected colonies were not clumped suggesting that the disease is not highly infectious between colonies. BBD infection rates in areas surveyed 4 times between August 1988 and September 1989 in Greater Lameshur Bay, St. John, USVI, were significantly lower in winter compared to summer. BBDs were found on 5.5% of the colonies ofD. strigosa in Fall 1988, and 7 out of 12 infected colonies lost >75% of their tissue in 6 months. Low level, chronic BBD infections could convert 3.9% of the living cover ofD.strigosa to free space per year, thereby creating substrata for successional processes.     

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.