PCR Primers with Enhanced Specificity for Nematode-Trapping Fungi (<Emphasis Type="Italic">Orbiliales</Emphasis>)

In search for microbiological indicators of coral health and coral diseases, community profiles of coral-associated epizoic prokaryotes were investigated because of their dual potential as a source of coral pathogens and their antagonists. In pairwise samples of visually healthy and diseased coral specimens from Bolinao Bay (Pangasinan, Philippines), mixed biofilm communities of ectoderm- and mucus-colonizing epizoic prokaryotes were compared using fluorescent in situ hybridization (FISH). Oligonucleotide probes targeted 13 phylotypes representing the main taxonomic groups of marine prokaryotes. Coral taxa tended to show specific community profiles. An attempt to separate the profiles of healthy and diseased specimens by applying principal component analysis (PCA) to a (nonselective) collection of corals (affected by various diseases) proved unsuccessful. On the other hand, separate PCA clusters were obtained from healthy and diseased corals belonging to a single species (Pocillopora damicornis) only. This cluster formation was dominated by principal component 1 with the genus Vibrio accounting for 18%. At the same time, reef-site-specific clusters were formed as well. At a reef site exposed to pollution from intensive fish cage (Chanos chanos) farming, healthy P. damicornis were mainly (93%) colonized by unicellular cyanobacteria. The formal calculation of diversity parameters suggested that evenness in particular was driven by both health status and reef site location. Despite the low resolution of taxonomic levels achieved with FISH probes targeting only large phylotype groups, significant differences between healthy and diseased corals and also between polluted and nonpolluted reef sites were observed.     

Differences in microbial communities between healthy and bleached coral Acropora solitaryensis from Xisha Islands,South China Sea

Serious bleaching events have been observed in Acropora solitaryensis, one of the main species of reef-building coral in the area of the Xisha islands in the South China Sea, during 2008–2011. The microbial communities of healthy and bleached coral samples were compared to explore the difference in the bacterial composition using 16S rRNA gene sequencing and denaturing gradient gel electrophoresis (DGGE). Analysis of 16S rRNA gene sequences showed that the diversity of bacteria from the corals was different between the healthy and the bleached. Albeit both the healthy coral and bleached coral displayed similar dominant bacterial species (α- and γ-proteobacteria), the ratio of Vibrio spp. increased sharply in the latter. As the bleaching developed, the diversity of the microbial community was dramatically decreased and the dominant species were replaced by γ-proteobacteria where Vibrio spp. and Escherichia spp. overwhelmed other genera. Similar results were gained by the DGGE technique though the abundance was lower. Furthermore, in the bleached tissues, Vibrio coralliilyticus was identified using both methods. These results indicated that pathogenic Vibrio spp. might be one of the factors causing A. solitaryensis bleaching in the coral reef of the Xisha islands.     

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.     

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.     

Responses of coral‐associated bacterial communities to heat stress differ with Symbiodinium type on the same coral host

This study compared the effect of heat stress on coral‐associated bacterial communities among juveniles of the coral, Acropora tenuis, hosting different Symbiodinium types. In comparison to a control temperature treatment (28 °C), we documented dramatic changes in bacterial associates on juvenile corals harbouring ITS 1 type D Symbiodinium when placed in a high (32 °C) temperature treatment. In particular, there was a marked increase in the number of retrieved Vibrio affiliated sequences, which coincided with a 44% decline in the photochemical efficiency of the D‐juveniles. Interestingly, these Vibrio sequences affiliated most closely with the coral pathogen, Vibrio coralliilyticus, which has been implicated in some coral disease outbreaks. In contrast, A. tenuis hosting ITS 1 type C1 Symbiodinium did not exhibit major bacterial shifts in the elevated temperature treatment, indicating a more stable bacterial community during thermal stress; concomitantly a decline (10%) in photochemical efficiency was minimal for this group. D juveniles that had been exposed to moderately elevated sea temperatures (30 °C) in the field before being placed in the control temperature treatment displayed a decrease in the number of Vibrio affiliated sequences and bacterial profiles shifted to become more similar to profiles of corals harbouring type C1 Symbiodinium. In combination, these results demonstrate that thermal stress can result in shifts in coral‐associated bacterial communities, which may lead to deteriorating coral health. The lower resilience of A. tenuis to thermal stress when harbouring Symbiodinium D highlights the importance of inter‐kingdom interactions among the coral host, dinoflagellate endosymbiont and bacterial associates for coral health and resilience.     

Bacterial profiling of White Plague Disease across corals and oceans indicates a conserved and distinct disease microbiome

Coral diseases are characterized by microbial community shifts in coral mucus and tissue, but causes and consequences of these changes are vaguely understood due to the complexity and dynamics of coral‐associated bacteria. We used 16S rRNA gene microarrays to assay differences in bacterial assemblages of healthy and diseased colonies displaying White Plague Disease (WPD) signs from two closely related Caribbean coral species, Orbicella faveolata and Orbicella franksi. Analysis of differentially abundant operational taxonomic units (OTUs) revealed strong differences between healthy and diseased specimens, but not between coral species. A subsequent comparison to data from two Indo‐Pacific coral species (Pavona duerdeni and Porites lutea) revealed distinct microbial community patterns associated with ocean basin, coral species and health state. Coral species were clearly separated by site, but also, the relatedness of the underlying bacterial community structures resembled the phylogenetic relationship of the coral hosts. In diseased samples, bacterial richness increased and putatively opportunistic bacteria were consistently more abundant highlighting the role of opportunistic conditions in structuring microbial community patterns during disease. Our comparative analysis shows that it is possible to derive conserved bacterial footprints of diseased coral holobionts that might help in identifying key bacterial species related to the underlying etiopathology. Furthermore, our data demonstrate that similar‐appearing disease phenotypes produce microbial community patterns that are consistent over coral species and oceans, irrespective of the putative underlying pathogen. Consequently, profiling coral diseases by microbial community structure over multiple coral species might allow the development of a comparative disease framework that can inform on cause and relatedness of coral diseases.     

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).     

PhyloChip™ microarray comparison of sampling methods used for coral microbial ecology

Interest in coral microbial ecology has been increasing steadily over the last decade, yet standardized methods of sample collection still have not been defined. Two methods were compared for their ability to sample coral-associated microbial communities: tissue punches and foam swabs, the latter being less invasive and preferred by reef managers. Four colonies of star coral, Montastraea annularis, were sampled in the Dry Tortugas National Park (two healthy and two with white plague disease). The PhyloChip™ G3 microarray was used to assess microbial community structure of amplified 16S rRNA gene sequences. Samples clustered based on methodology rather than coral colony. Punch samples from healthy and diseased corals were distinct. All swab samples clustered closely together with the seawater control and did not group according to the health state of the corals. Although more microbial taxa were detected by the swab method, there is a much larger overlap between the water control and swab samples than punch samples, suggesting some of the additional diversity is due to contamination from water absorbed by the swab. While swabs are useful for noninvasive studies of the coral surface mucus layer, these results show that they are not optimal for studies of coral disease.     

Comparing Bacterial Community Composition between Healthy and White Plague-Like Disease States in Orbicella annularis Using PhyloChip? G3 Microarrays

Coral disease is a global problem. Diseases are typically named or described based on macroscopic changes, but broad signs of coral distress such as tissue loss or discoloration are unlikely to be specific to a particular pathogen. For example, there appear to be multiple diseases that manifest the rapid tissue loss that characterizes ‘white plague.’ PhyloChip™ G3 microarrays were used to compare the bacterial community composition of both healthy and white plague-like diseased corals. Samples of lobed star coral (Orbicella annularis, formerly of the genus Montastraea [1]) were collected from two geographically distinct areas, Dry Tortugas National Park and Virgin Islands National Park, to determine if there were biogeographic differences between the diseases. In fact, all diseased samples clustered together, however there was no consistent link to Aurantimonas coralicida, which has been described as the causative agent of white plague type II. The microarrays revealed a large amount of bacterial heterogeneity within the healthy corals and less diversity in the diseased corals. Gram-positive bacterial groups (Actinobacteria, Firmicutes) comprised a greater proportion of the operational taxonomic units (OTUs) unique to healthy samples. Diseased samples were enriched in OTUs from the families Corynebacteriaceae, Lachnospiraceae, Rhodobacteraceae, and Streptococcaceae. Much previous coral disease work has used clone libraries, which seem to be methodologically biased toward recovery of Gram-negative bacterial sequences and may therefore have missed the importance of Gram-positive groups. The PhyloChip™data presented here provide a broader characterization of the bacterial community changes that occur within Orbicella annularis during the shift from a healthy to diseased state.     

Vibrio Zinc-Metalloprotease Causes Photoinactivation of Coral Endosymbionts and Coral Tissue Lesions

Background

Coral diseases are emerging as a serious threat to coral reefs worldwide. Of nine coral infectious diseases, whose pathogens have been characterized, six are caused by agents from the family Vibrionacae, raising questions as to their origin and role in coral disease aetiology.

Methodology/Principal Findings

Here we report on a Vibrio zinc-metalloprotease causing rapid photoinactivation of susceptible Symbiodinium endosymbionts followed by lesions in coral tissue. Symbiodinium photosystem II inactivation was diagnosed by an imaging pulse amplitude modulation fluorometer in two bioassays, performed by exposing Symbiodinium cells and coral juveniles to non-inhibited and EDTA-inhibited supernatants derived from coral white syndrome pathogens.

Conclusion/Significance

These findings demonstrate a common virulence factor from four phylogenetically related coral pathogens, suggesting that zinc-metalloproteases may play an important role in Vibrio pathogenicity in scleractinian corals.     

<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.     

Climate change affects key nitrogen-fixing bacterial populations on coral reefs

Coral reefs are at serious risk due to events associated with global climate change. Elevated ocean temperatures have unpredictable consequences for the ocean''s biogeochemical cycles. The nitrogen cycle is driven by complex microbial transformations, including nitrogen fixation. This study investigated the effects of increased seawater temperature on bacteria able to fix nitrogen (diazotrophs) that live in association with the mussid coral Mussismilia harttii. Consistent increases in diazotroph abundances and diversities were found at increased temperatures. Moreover, gradual shifts in the dominance of particular diazotroph populations occurred as temperature increased, indicating a potential future scenario of climate change. The temperature-sensitive diazotrophs may provide useful bioindicators of the effects of thermal stress on coral reef health, allowing the impact of thermal anomalies to be monitored. In addition, our findings support the development of research on different strategies to improve the fitness of corals during events of thermal stress, such as augmentation with specific diazotrophs.     

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.     

Variability in Microbial Community Composition and Function Between Different Niches Within a Coral Reef

To explore how microbial community composition and function varies within a coral reef ecosystem, we performed metagenomic sequencing of seawater from four niches across Heron Island Reef, within the Great Barrier Reef. Metagenomes were sequenced from seawater samples associated with (1) the surface of the coral species Acropora palifera, (2) the surface of the coral species Acropora aspera, (3) the sandy substrate within the reef lagoon and (4) open water, outside of the reef crest. Microbial composition and metabolic function differed substantially between the four niches. The taxonomic profile showed a clear shift from an oligotroph-dominated community (e.g. SAR11, Prochlorococcus, Synechococcus) in the open water and sandy substrate niches, to a community characterised by an increased frequency of copiotrophic bacteria (e.g. Vibrio, Pseudoalteromonas, Alteromonas) in the coral seawater niches. The metabolic potential of the four microbial assemblages also displayed significant differences, with the open water and sandy substrate niches dominated by genes associated with core house-keeping processes such as amino acid, carbohydrate and protein metabolism as well as DNA and RNA synthesis and metabolism. In contrast, the coral surface seawater metagenomes had an enhanced frequency of genes associated with dynamic processes including motility and chemotaxis, regulation and cell signalling. These findings demonstrate that the composition and function of microbial communities are highly variable between niches within coral reef ecosystems and that coral reefs host heterogeneous microbial communities that are likely shaped by habitat structure, presence of animal hosts and local biogeochemical conditions.     

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.     

Phage therapy of the white plague-like disease of Favia favus in the Red Sea

Coral disease is a major factor in the global decline of coral reefs. At present, there are no known procedures for preventing or treating infectious diseases of corals. Immunization is not possible because corals have a restricted adaptive immune system and antibiotics are neither ecologically safe nor practical in an open system. Thus, we tested phage therapy as an alternative therapeutic method for treating diseased corals. Phage BA3, specific to the coral pathogen Thalassomonas loyana, inhibited the progression of the white plague-like disease and transmission to healthy corals in the Gulf of Aqaba, Red Sea. Only one out of 19 (5?%) of the healthy corals became infected when placed near phage-treated diseased corals, whereas 11 out of 18 (61?%) healthy corals were infected in the no-phage control. This is the first successful treatment for a coral disease in the sea. We posit that phage therapy of certain coral diseases is achievable in situ.     

Metagenomic Analysis of Healthy and White Plague-Affected Mussismilia braziliensis Corals

Coral health is under threat throughout the world due to regional and global stressors. White plague disease (WP) is one of the most important threats affecting the major reef builder of the Abrolhos Bank in Brazil, the endemic coral Mussismilia braziliensis. We performed a metagenomic analysis of healthy and WP-affected M. braziliensis in order to determine the types of microbes associated with this coral species. We also optimized a protocol for DNA extraction from coral tissues. Our taxonomic analysis revealed Proteobacteria, Bacteroidetes, Firmicutes, Cyanobacteria, and Actinomycetes as the main groups in all healthy and WP-affected corals. Vibrionales, members of the Cytophaga–Flavobacterium–Bacteroides complex, Rickettsiales, and Neisseriales were more abundant in the WP-affected corals. Diseased corals also had more eukaryotic metagenomic sequences identified as Alveolata and Apicomplexa. Our results suggest that WP disease in M. braziliensis is caused by a polymicrobial consortium.     

A bacterial pathogen uses dimethylsulfoniopropionate as a cue to target heat-stressed corals

Diseases are an emerging threat to ocean ecosystems. Coral reefs, in particular, are experiencing a worldwide decline because of disease and bleaching, which have been exacerbated by rising seawater temperatures. Yet, the ecological mechanisms behind most coral diseases remain unidentified. Here, we demonstrate that a coral pathogen, Vibrio coralliilyticus, uses chemotaxis and chemokinesis to target the mucus of its coral host, Pocillopora damicornis. A primary driver of this response is the host metabolite dimethylsulfoniopropionate (DMSP), a key element in the global sulfur cycle and a potent foraging cue throughout the marine food web. Coral mucus is rich in DMSP, and we found that DMSP alone elicits chemotactic responses of comparable intensity to whole mucus. Furthermore, in heat-stressed coral fragments, DMSP concentrations increased fivefold and the pathogen''s chemotactic response was correspondingly enhanced. Intriguingly, despite being a rich source of carbon and sulfur, DMSP is not metabolized by the pathogen, suggesting that it is used purely as an infochemical for host location. These results reveal a new role for DMSP in coral disease, demonstrate the importance of chemical signaling and swimming behavior in the recruitment of pathogens to corals and highlight the impact of increased seawater temperatures on disease pathways.     

The bacterial ecology of a plague-like disease affecting the Caribbean coral Montastrea annularis

The bacterial communities associated with the Caribbean coral Montastrea annularis showing tissue lesions indicative of a White Plague (WP)-like disease were investigated. Two molecular screening techniques using bacterial 16S rDNA genes were used and demonstrated distinct differences between the bacterial communities of diseased and non-diseased coral tissues, and also in relation to the proximity of tissue lesions on diseased corals. Differences between non-diseased corals and the apparently healthy tissues remote from the tissue lesion in affected corals indicates a 'whole coral' response to a relatively small area of infection with a perturbation in the normal microbial flora occurring prior to the onset of visible signs of disease. These whole organism changes in the microbial flora may serve as a bioindicator of environmental stress and disease. There were striking similarities between the 16S rDNA sequence composition associated with the WP-like disease studied here and that previously reported in association with black band disease (BBD) in coral. Similarities included the presence of a potential pathogen, an alpha-proteobacterium identified as the causal agent of juvenile oyster disease (JOD). The WP-like disease studied here is apparently different to WP Type ii because the bacterial species previously identified as the causal agent of WP Type ii was not detected, although the symptoms of the two diseases are similar.