A comparison of culture-dependent and culture-independent techniques used to characterize bacterial communities on healthy and white plague-diseased corals of the Montastraea annularis species complex

Diseases of hermatypic corals pose a global threat to coral reefs, and investigations of bacterial communities associated with healthy corals and those exhibiting signs of disease are necessary for proper diagnosis. One disease, commonly called white plague (WP), is characterized by acute tissue loss. This investigation compared the bacterial communities associated with healthy coral tissue (N = 15), apparently healthy tissue on WP-diseased colonies (N = 15), and WP-diseased tissues (N = 15) from Montastraea annularis (species complex) colonies inhabiting a Bahamian reef. Aliquots of sediment (N = 15) and water (N = 15) were also obtained from the proximity of each coral colony sampled. Samples for culture-dependent analyses were inoculated onto one-half strength Marine Agar (½ MA) and Thiosulfate Citrate Bile Salts Sucrose Agar to quantify the culturable communities. Length heterogeneity PCR (LH-PCR) of the 16S rRNA gene characterized the bacterial operational taxonomic units (OTU) associated with lesions on corals exhibiting signs of a white plague-like disease as well as apparently healthy tissue from diseased and non-diseased conspecifics. Analysis of Similarity was conducted on the LH-PCR fingerprints, which indicated no significant difference in the composition of bacterial communities associated with apparently healthy and diseased corals. Comparisons of the 16S rRNA gene amplicons from cultured bacterial colonies (½ MA; N = 21) with all amplicons obtained from the whole coral-associated bacterial community indicated ≥39 % of coral-associated bacterial taxa could be cultured. Amplicons from these bacterial cultures matched amplicons from the whole coral-associated bacterial community that, when combined, accounted for >70 % total bacterial abundance. An OTU with the same amplicon length as Aurantimonas coralicida (313.1 bp), the reported etiological agent of WPII, was detected in relatively low abundance (<0.1 %) on all tissue types. These findings suggest a coral disease resembling WP may result from multiple etiologies.     

Potential role of viruses in white plague coral disease

White plague (WP)-like diseases of tropical corals are implicated in reef decline worldwide, although their etiological cause is generally unknown. Studies thus far have focused on bacterial or eukaryotic pathogens as the source of these diseases; no studies have examined the role of viruses. Using a combination of transmission electron microscopy (TEM) and 454 pyrosequencing, we compared 24 viral metagenomes generated from Montastraea annularis corals showing signs of WP-like disease and/or bleaching, control conspecific corals, and adjacent seawater. TEM was used for visual inspection of diseased coral tissue. No bacteria were visually identified within diseased coral tissues, but viral particles and sequence similarities to eukaryotic circular Rep-encoding single-stranded DNA viruses and their associated satellites (SCSDVs) were abundant in WP diseased tissues. In contrast, sequence similarities to SCSDVs were not found in any healthy coral tissues, suggesting SCSDVs might have a role in WP disease. Furthermore, Herpesviridae gene signatures dominated healthy tissues, corroborating reports that herpes-like viruses infect all corals. Nucleocytoplasmic large DNA virus (NCLDV) sequences, similar to those recently identified in cultures of Symbiodinium (the algal symbionts of corals), were most common in bleached corals. This finding further implicates that these NCLDV viruses may have a role in bleaching, as suggested in previous studies. This study determined that a specific group of viruses is associated with diseased Caribbean corals and highlights the potential for viral disease in regional coral reef decline.     

Bacterial communities associated with healthy and Acropora white syndrome-affected corals from American Samoa

Acropora white syndrome (AWS) is characterized by rapid tissue loss revealing the white underlying skeleton and affects corals worldwide; however, reports of causal agents are conflicting. Samples were collected from healthy and diseased corals and seawater around American Samoa and bacteria associated with AWS characterized using both culture-dependent and culture-independent methods, from coral mucus and tissue slurries, respectively. Bacterial 16S rRNA gene clone libraries derived from coral tissue were dominated by the Gammaproteobacteria, and Jaccard's distances calculated between the clone libraries showed that those from diseased corals were more similar to each other than to those from healthy corals. 16S rRNA genes from 78 culturable coral mucus isolates also revealed a distinct partitioning of bacterial genera into healthy and diseased corals. Isolates identified as Vibrionaceae were further characterized by multilocus sequence typing, revealing that whilst several Vibrio spp. were found to be associated with AWS lesions, a recently described species, Vibrio owensii, was prevalent amongst cultured Vibrio isolates. Unaffected tissues from corals with AWS had a different microbiota than normal Acropora as found by others. Determining whether a microbial shift occurs prior to disease outbreaks will be a useful avenue of pursuit and could be helpful in detecting prodromal signs of coral disease prior to manifestation of lesions.     

Bacterial community structure associated with white band disease in the elkhorn coral Acropora palmata determined using culture-independent 16S rRNA techniques

Culture-independent molecular (16S ribosomal RNA) techniques showed distinct differences in bacterial communities associated with white band disease (WBD) Type I and healthy elkhorn coral Acropora palmata. Differences were apparent at all levels, with a greater diversity present in tissues of diseased colonies. The bacterial community associated with remote, non-diseased coral was distinct from the apparently healthy tissues of infected corals several cm from the disease lesion. This demonstrates a whole-organism effect from what appears to be a localised disease lesion, an effect that has also been recently demonstrated in white plague-like disease in star coral Montastraea annularis. The pattern of bacterial community structure changes was similar to that recently demonstrated for white plague-like disease and black band disease. Some of the changes are likely to be explained by the colonisation of dead and degrading tissues by a micro-heterotroph community adapted to the decomposition of coral tissues. However, specific ribosomal types that are absent from healthy tissues appear consistently in all samples of each of the diseases. These ribotypes are closely related members of a group of alpha-proteobacteria that cause disease, notably juvenile oyster disease, in other marine organisms. It is clearly important that members of this group are isolated for challenge experiments to determine their role in the diseases.     

Bacteria associated with the rapid tissue necrosis of stony corals

The rapid tissue necrosis (RTN) is a common disease of both wild and captive stony corals, which causes a fast tissue degradation (peeling) and death of the colony. Here we report the results of an investigation carried out on the stony coral Pocillopora damicornis, affected by an RTN-like disease. Total abundance of prokaryotes in tissue samples, determined by epifluorescence microscopy, was significantly higher in diseased than in healthy corals, as well as bacterial counts on MB2216 agar plates. Further experiments performed by fluorescent in situ hybridization using a 16S rDNA Vibrio-specific probe showed that vibrios were significantly more abundant in diseased than in healthy corals. Accordingly, bacterial counts on TCBS agar plates were higher in diseased than in healthy tissues. 16S rDNA sequencing identified as Vibrio colonies from diseased tissues only. Cultivated vibrios were dominated by a single ribotype, which displayed 99% of similarity with Vibrio harveyi strain LB4. Bacterial ribotype richness, assessed by terminal-restriction fragment length polymorphism analysis of the 16S rDNA, was significantly higher in diseased than in healthy corals. Using an in silico software, we estimated that a single terminal restriction fragment, putatively assigned to a Vibrio sp., accounted for > 15% and < 5% of the total bacterial assemblage, in diseased and healthy corals respectively. These results let us hypothesize that the RTN in stony corals can be an infectious disease associated to the presence of Vibrio harveyi. However, further studies are needed to validate the microbial origin of this pathology.     

Coral Disease Diagnostics: What's between a Plague and a Band?

Recently, reports of coral disease have increased significantly across the world's tropical oceans. Despite increasing efforts to understand the changing incidence of coral disease, very few primary pathogens have been identified, and most studies remain dependent on the external appearance of corals for diagnosis. Given this situation, our current understanding of coral disease and the progression and underlying causes thereof is very limited. In the present study, we use structural and microbial studies to differentiate different forms of black band disease: atypical black band disease and typical black band disease. Atypical black band diseased corals were infected with the black band disease microbial consortium yet did not show any of the typical external signs of black band disease based on macroscopic observations. In previous studies, these examples, here referred to as atypical black band disease, would have not been correctly diagnosed. We also differentiate white syndrome from white diseases on the basis of tissue structure and the presence/absence of microbial associates. White diseases are those with dense bacterial communities associated with lesions of symbiont loss and/or extensive necrosis of tissues, while white syndromes are characteristically bacterium free, with evidence for extensive programmed cell death/apoptosis associated with the lesion and the adjacent tissues. The pathology of coral disease as a whole requires further investigation. This study emphasizes the importance of going beyond the external macroscopic signs of coral disease for accurate disease diagnosis.     

Coral disease diagnostics: what's between a plague and a band?

Recently, reports of coral disease have increased significantly across the world's tropical oceans. Despite increasing efforts to understand the changing incidence of coral disease, very few primary pathogens have been identified, and most studies remain dependent on the external appearance of corals for diagnosis. Given this situation, our current understanding of coral disease and the progression and underlying causes thereof is very limited. In the present study, we use structural and microbial studies to differentiate different forms of black band disease: atypical black band disease and typical black band disease. Atypical black band diseased corals were infected with the black band disease microbial consortium yet did not show any of the typical external signs of black band disease based on macroscopic observations. In previous studies, these examples, here referred to as atypical black band disease, would have not been correctly diagnosed. We also differentiate white syndrome from white diseases on the basis of tissue structure and the presence/absence of microbial associates. White diseases are those with dense bacterial communities associated with lesions of symbiont loss and/or extensive necrosis of tissues, while white syndromes are characteristically bacterium free, with evidence for extensive programmed cell death/apoptosis associated with the lesion and the adjacent tissues. The pathology of coral disease as a whole requires further investigation. This study emphasizes the importance of going beyond the external macroscopic signs of coral disease for accurate disease diagnosis.     

Widespread association of a Rickettsiales-like bacterium with reef-building corals

White band disease type I (WBD I) has been a major cause of the dramatic decline of Acroporid coral populations throughout the Caribbean during the last two decades, yet the aetiological agent of this disease is unknown. In this study, the bacterial communities associated with both healthy and diseased Acropora species were compared by 16S rDNA analyses. The bacterial communities of both healthy and diseased Acropora spp. were dominated by a single ribotype with 90% identity to a bacterium in the order Rickettsiales. Screening by nested PCR specific to the coral-associated Rickettsiales 1 (CAR1) bacterium showed that this microbe was widespread in both healthy and diseased A. cervicornis and A. palmata corals from 'healthy' (i.e. low WBD I incidence) and 'stressed' reefs (i.e. high WBD I incidence). These results indicate that there were no dramatic changes in the composition of the microbial community associated with WBD I. CAR1 was also associated with non-Acroporid corals of the Caribbean, as well as with two Acroporid corals native to the Pacific. CAR1 was not present in the water column. This bacterium was also absent from preserved Caribbean Acroporid samples collected between 1937 and 1980 before the outbreak of WBD I. These results suggest CAR1 is a relatively new bacterial associate of Acroporids and that a non-bacterial pathogen might be the cause of WBD I.     

Does Dark-Spot Syndrome Experimentally Transmit among Caribbean Corals?

Over the last half-century, coral diseases have contributed to the rapid decline of coral populations throughout the Caribbean region. Some coral diseases appear to be potentially infectious, yet little is known about their modes of transmission. This study experimentally tested whether dark-spot syndrome on Siderastrea siderea was directly or indirectly transmissible to neighboring coral colonies. We also tested whether open wounds were necessary to facilitate disease transmission. At the completion of the experiments, we sampled bacterial communities on diseased, exposed, and healthy coral colonies to determine whether bacterial pathogens had transmitted to the susceptible colonies. We saw no evidence of either direct or waterborne transmission of dark-spot syndrome, and corals that received lesions by direct contact with diseased tissue, healed and showed no signs of infection. There were no significant differences among bacterial communities on healthy, exposed, and diseased colonies, although nine individual ribotypes were significantly higher in diseased corals compared with healthy and exposed corals, indicating a lack of transmission. Although our experiments do not fully refute the possibility that dark-spot syndrome is infectious and transmissible, our results suggest that in situ macroscopic signs of dark-spot syndrome are not always contagious.     

The Role of Coral-Associated Bacterial Communities in Australian Subtropical White Syndrome of Turbinaria mesenterina

Australian Subtropical White Syndrome (ASWS) is an infectious, temperature dependent disease of the subtropical coral Turbinaria mesenterina involving a hitherto unknown transmissible causative agent. This report describes significant changes in the coral associated bacterial community as the disease progresses from the apparently healthy tissue of ASWS affected coral colonies, to areas of the colony affected by ASWS lesions, to the dead coral skeleton exposed by ASWS. In an effort to better understand the potential roles of bacteria in the formation of disease lesions, the effect of antibacterials on the rate of lesion progression was tested, and both culture based and culture independent techniques were used to investigate the bacterial communities associated with colonies of T. mesenterina. Culture-independent analysis was performed using the Oligonucleotide Fingerprinting of Ribosomal Genes (OFRG) technique, which allowed a library of 8094 cloned bacterial 16S ribosomal genes to be analysed. Interestingly, the bacterial communities associated with both healthy and disease affected corals were very diverse and ASWS associated communities were not characterized by a single dominant organism. Treatment with antibacterials had a significant effect on the rate of progress of disease lesions (p = 0.006), suggesting that bacteria may play direct roles as the causative agents of ASWS. A number of potential aetiological agents of ASWS were identified in both the culture-based and culture-independent studies. In the culture-independent study an Alphaproteobacterium closely related to Roseovarius crassostreae, the apparent aetiological agent of juvenile oyster disease, was found to be significantly associated with disease lesions. In the culture-based study Vibrio harveyi was consistently associated with ASWS affected coral colonies and was not isolated from any healthy colonies. The differing results of the culture based and culture-independent studies highlight the importance of using both approaches in the investigation of microbial communities.     

<Emphasis Type="Italic">Symbiodinium</Emphasis> associations with diseased and healthy scleractinian corals

Despite recent advances in identifying the causative agents of disease in corals and understanding the impact of epizootics on reef communities, little is known regarding the interactions among diseases, corals, and their dinoflagellate endosymbionts (Symbiodinium spp.). Since the genotypes of both corals and their resident Symbiodinium contribute to colony-level phenotypes, such as thermotolerance, symbiont genotypes might also contribute to the resistance or susceptibility of coral colonies to disease. To explore this, Symbiodinium were identified using the internal transcribed spacer-2 region of ribosomal DNA from diseased and healthy tissues within individual coral colonies infected with black band disease (BB), dark spot syndrome (DSS), white plague disease (WP), or yellow blotch disease (YB) in the Florida Keys (USA) and the US Virgin Islands. Most of the diseased colonies sampled contained B1, B5a, or C1 (depending on host species), while apparently healthy colonies of the same coral species frequently hosted these types and/or additional symbiont diversity. No potentially “parasitic” Symbiodinium types, uniquely associated with diseased coral tissue, were detected. Within most individual colonies, the same dominant Symbiodinium type was detected in diseased and visually healthy tissues. These data indicate that specific Symbiodinium types are not correlated with the infected tissues of diseased colonies and that DSS and WP onset do not trigger symbiont shuffling within infected tissues. However, few diseased colonies contained clade D symbionts suggesting a negative correlation between hosting Symbiodinium clade D and disease incidence in scleractinian corals. Understanding the influence of Symbiodinium diversity on colony phenotypes may play a critical role in predicting disease resistance and susceptibility in scleractinian corals.     

Shifts in bacterial communities of two caribbean reef-building coral species affected by white plague disease

Coral reefs are deteriorating at an alarming rate mainly as a consequence of the emergence of coral diseases. The white plague disease (WPD) is the most prevalent coral disease in the southwestern Caribbean, affecting dozens of coral species. However, the identification of a single causal agent has proved problematic. This suggests more complex etiological scenarios involving alterations in the dynamic interaction between environmental factors, the coral immune system and the symbiotic microbial communities. Here we compare the microbiome of healthy and WPD-affected corals from the two reef-building species Diploria strigosa and Siderastrea siderea collected at the Tayrona National Park in the Caribbean of Colombia. Microbiomes were analyzed by combining culture-dependent methods and pyrosequencing of 16S ribosomal DNA (rDNA) V5-V6 hypervariable regions. A total of 20 410 classifiable 16S rDNA sequences reads were obtained including all samples. No significant differences in operational taxonomic unit diversity were found between healthy and affected tissues; however, a significant increase of Alphaproteobacteria and a concomitant decrease in the Beta- and Gammaproteobacteria was observed in WPD-affected corals of both species. Significant shifts were also observed in the orders Rhizobiales, Caulobacteriales, Burkholderiales, Rhodobacterales, Aleteromonadales and Xanthomonadales, although they were not consistent between the two coral species. These shifts in the microbiome structure of WPD-affected corals suggest a loss of community-mediated growth control mechanisms on bacterial populations specific for each holobiont system.     

Bacterial Communities of Two Ubiquitous Great Barrier Reef Corals Reveals Both Site- and Species-Specificity of Common Bacterial Associates

Background

Coral-associated bacteria are increasingly considered to be important in coral health, and altered bacterial community structures have been linked to both coral disease and bleaching. Despite this, assessments of bacterial communities on corals rarely apply sufficient replication to adequately describe the natural variability. Replicated data such as these are crucial in determining potential roles of bacteria on coral.

Methodology/Principal Findings

Denaturing Gradient Gel Electrophoresis (DGGE) of the V3 region of the 16S ribosomal DNA was used in a highly replicated approach to analyse bacterial communities on both healthy and diseased corals. Although site-specific variations in the bacterial communities of healthy corals were present, host species-specific bacterial associates within a distinct cluster of gamma-proteobacteria could be identified, which are potentially linked to coral health. Corals affected by “White Syndrome” (WS) underwent pronounced changes in their bacterial communities in comparison to healthy colonies. However, the community structure and bacterial ribotypes identified in diseased corals did not support the previously suggested theory of a bacterial pathogen as the causative agent of the syndrome.

Conclusions/Significance

This is the first study to employ large numbers of replicated samples to assess the bacterial communities of healthy and diseased corals, and the first culture-independent assessment of bacterial communities on WS affected Acroporid corals on the GBR. Results indicate that a minimum of 6 replicate samples are required in order to draw inferences on species, spatial or health-related changes in community composition, as a set of clearly distinct bacterial community profiles exist in healthy corals. Coral bacterial communities may be both site and species specific. Furthermore, a cluster of gamma-proteobacterial ribotypes may represent a group of specific common coral and marine invertebrate associates. Finally, the results did not support the contention that a single bacterial pathogen may be the causative agent of WS Acroporids on the GBR.     

Bacterial profiling of White Plague Disease in a comparative coral species framework

Coral reefs are threatened throughout the world. A major factor contributing to their decline is outbreaks and propagation of coral diseases. Due to the complexity of coral-associated microbe communities, little is understood in terms of disease agents, hosts and vectors. It is known that compromised health in corals is correlated with shifts in bacterial assemblages colonizing coral mucus and tissue. However, general disease patterns remain, to a large extent, ambiguous as comparative studies over species, regions, or diseases are scarce. Here, we compare bacterial assemblages of samples from healthy (HH) colonies and such displaying signs of White Plague Disease (WPD) of two different coral species (Pavona duerdeni and Porites lutea) from the same reef in Koh Tao, Thailand, using 16S rRNA gene microarrays. In line with other studies, we found an increase of bacterial diversity in diseased (DD) corals, and a higher abundance of taxa from the families that include known coral pathogens (Alteromonadaceae, Rhodobacteraceae, Vibrionaceae). In our comparative framework analysis, we found differences in microbial assemblages between coral species and coral health states. Notably, patterns of bacterial community structures from HH and DD corals were maintained over species boundaries. Moreover, microbes that differentiated the two coral species did not overlap with microbes that were indicative of HH and DD corals. This suggests that while corals harbor distinct species-specific microbial assemblages, disease-specific bacterial abundance patterns exist that are maintained over coral species boundaries.     

Identification of Candidate Coral Pathogens on White Band Disease-Infected Staghorn Coral

Bacterial diseases affecting scleractinian corals pose an enormous threat to the health of coral reefs, yet we still have a limited understanding of the bacteria associated with coral diseases. White band disease is a bacterial disease that affects the two Caribbean acroporid corals, the staghorn coral Acropora cervicornis and the elkhorn coral A. palmate. Species of Vibrio and Rickettsia have both been identified as putative WBD pathogens. Here we used Illumina 16S rRNA gene sequencing to profile the bacterial communities associated with healthy and diseased A. cervicornis collected from four field sites during two different years. We also exposed corals in tanks to diseased and healthy (control) homogenates to reduce some of the natural variation of field-collected coral bacterial communities. Using a combination of multivariate analyses, we identified community-level changes between diseased and healthy corals in both the field-collected and tank-exposed datasets. We then identified changes in the abundances of individual operational taxonomic units (OTUs) between diseased and healthy corals. By comparing the diseased and healthy-associated bacteria in field-collected and tank-exposed corals, we were able to identify 16 healthy-associated OTUs and 106 consistently disease-associated OTUs, which are good candidates for putative WBD pathogens. A large percentage of these disease-associated OTUs belonged to the order Flavobacteriales. In addition, two of the putative pathogens identified here belong to orders previously suggested as WBD pathogens: Vibronales and Rickettsiales.     

Histopathological methods for the investigation of microbial communities associated with disease lesions in reef corals

AIMS: To determine the spatial structure of microbial communities associated with disease lesions of reef corals (Scleractinia). METHODS AND RESULTS: Agarose pre-embedding preserved the structure of the disease lesion and surrounding tissues prior to demineralization of the carbonate exoskeleton and embedding in resin. Fluorescence in situ hybridization (FISH) was used to localize bacteria in the lesions of various diseases. CONCLUSIONS: The techniques successfully preserved the in situ spatial structure of degenerated coral tissues. In one case (white plague disease), significant bacterial populations were found only in fragmented remnants of degenerated coral tissues at the lesion boundary that would not have been detected using conventional histopathological techniques. SIGNIFICANCE AND IMPACT OF THE STUDY: Determining the composition, spatial structure and dynamics of microbial communities within the disease lesions is necessary to understand the process of disease progression. The methods described may be applicable to a wide range of diseases involving necrotic lesion formation and requiring extensive tissue processing, such as skeleton demineralization.     

Bacterial communities associated with the mucopolysaccharide layers of three coral species affected and unaffected with dark spots disease

Dark spots disease (DSD) is a relatively new coral disease that has become one of the most prevalent afflictions in the Caribbean Sea. To partially characterize bacterial communities associated with DSD, carbon utilization patterns of bacterial strains isolated from the surface mucopolysaccharide layers of healthy and DSD-affected Montastraea annularis, Montastraea faveolata, and Siderastrea siderea were compared with each other and with bacterial strains isolated from the water column by using cluster analysis. Differences between healthy and diseased corals were found and were greatest for M. annularis than for the other species. A metabolic group of bacteria similar to Vibrio carchariae was found to be unique to diseased samples. Inoculation experiments on healthy corals did not result in the development of disease signs. However, our results support the hypothesis that stress (in this case disease) alters the normal microbiota in the coral surface mucopolysaccaride layers. Studies are continuing to determine the pathogenic agent(s) responsible for DSD.     

Microbiological assessment of a disease outbreak on corals from Magnetic Island (Great Barrier Reef, Australia)

Unusual disease lesions were observed in Montipora corals on the fringing reef of Magnetic Island (Great Barrier Reef, Australia) following a period of high water temperature in early January 2002. Tissue death in Montipora spp. appeared as a black layer that spread rapidly across the colony surface, though this appeared as the final phase of disease progression (with three previous disease phases now identified, S. Anthony, unpublished). Culture and molecular-based microbial analysis of this layer did not identify a likely microbial pathogen. Despite this, DNA sequencing of microbial 16S rDNA indicated a shift in the bacterial population associated with affected coral tissue. A clone library of the healthy coral sample predominantly contained sequences within the -Proteobacteria. A disease coral sample representing the margin of the black lesion and healthy coral tissue was dominated by sequences, which demonstrated low sequence identity to a range of -Proteobacteria, -Proteobacteria and cyanobacteria. The microbes identified in the diseased Montipora spp. samples are likely to be opportunistic rather than the causative agent of the observed lesion. Studies are in progress to further characterise the ecology of this disease and describe the potential microbial pathogen(s).     

Experimental antibiotic treatment identifies potential pathogens of white band disease in the endangered Caribbean coral Acropora cervicornis

Coral diseases have been increasingly reported over the past few decades and are a major contributor to coral decline worldwide. The Caribbean, in particular, has been noted as a hotspot for coral disease, and the aptly named white syndromes have caused the decline of the dominant reef building corals throughout their range. White band disease (WBD) has been implicated in the dramatic loss of Acropora cervicornis and Acropora palmata since the 1970s, resulting in both species being listed as critically endangered on the International Union for Conservation of Nature Red list. The causal agent of WBD remains unknown, although recent studies based on challenge experiments with filtrate from infected hosts concluded that the disease is probably caused by bacteria. Here, we report an experiment using four different antibiotic treatments, targeting different members of the disease-associated microbial community. Two antibiotics, ampicillin and paromomycin, arrested the disease completely, and by comparing with community shifts brought about by treatments that did not arrest the disease, we have identified the likely candidate causal agent or agents of WBD. Our interpretation of the experimental treatments is that one or a combination of up to three specific bacterial types, detected consistently in diseased corals but not detectable in healthy corals, are likely causal agents of WBD. In addition, a histophagous ciliate (Philaster lucinda) identical to that found consistently in association with white syndrome in Indo-Pacific acroporas was also consistently detected in all WBD samples and absent in healthy coral. Treatment with metronidazole reduced it to below detection limits, but did not arrest the disease. However, the microscopic disease signs changed, suggesting a secondary role in disease causation for this ciliate. In future studies to identify a causal agent of WBD via tests of Henle–Koch''s postulates, it will be vital to experimentally control for populations of the other potential pathogens identified in this study.     

Caribbean yellow-band syndrome on Montastraea faveolata is not transmitted mechanically under field conditions

Caribbean yellow-band syndrome is a highly prevalent coral disease whose transmission mechanisms are unknown. Affected corals often show multifocal lesions of yellow-colored tissue. We tested the hypothesis that a mechanical vector was responsible for these multifocal lesions. Four presumably non-resistant colonies were experimentally manipulated. Tissue and microbial assemblages were taken from diseased tissue and injected into healthy-looking tissue. Seawater injections were used as controls. The manipulations created a small wound, as would be generated by a coral predator. After 1 mo, all lesions healed and showed no signs of disease. We therefore reject the hypothesis that a mechanical vector, acting in a similar way as our manipulations, was responsible for the multifocal lesions.