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.     

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.     

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.     

Algae as Reservoirs for Coral Pathogens

Benthic algae are associated with coral death in the form of stress and disease. It''s been proposed that they release exudates, which facilitate invasion of potentially pathogenic microbes at the coral-algal interface, resulting in coral disease. However, the original source of these pathogens remains unknown. This study examined the ability of benthic algae to act as reservoirs of coral pathogens by characterizing surface associated microbes associated with major Caribbean and Indo-Pacific algal species/types and by comparing them to potential pathogens of two dominant coral diseases: White Syndrome (WS) in the Indo-Pacific and Yellow Band Disease (YBD) in the Caribbean. Coral and algal sampling was conducted simultaneously at the same sites to avoid spatial effects. Potential pathogens were defined as those absent or rare in healthy corals, increasing in abundance in healthy tissues adjacent to a disease lesion, and dominant in disease lesions. Potentially pathogenic bacteria were detected in both WS and YBD and were also present within the majority of algal species/types (54 and 100% for WS and YBD respectively). Pathogenic ciliates were associated only with WS and not YBD lesions and these were also present in 36% of the Indo-Pacific algal species. Although potential pathogens were associated with many algal species, their presence was inconsistent among replicate algal samples and detection rates were relatively low, suggestive of low density and occurrence. At the community level, coral-associated microbes irrespective of the health of their host differed from algal-associated microbes, supporting that algae and corals have distinctive microbial communities associated with their tissue. We conclude that benthic algae are common reservoirs for a variety of different potential coral pathogens. However, algal-associated microbes alone are unlikely to cause coral death. Initial damage or stress to the coral via other competitive mechanisms is most likely a prerequisite to potential transmission of these pathogens.     

Ciliate and bacterial communities associated with White Syndrome and Brown Band Disease in reef-building corals

White Syndrome (WS) and Brown Band Disease (BrB) are important causes of reef coral mortality for which causal agents have not been definitively identified. Here we use culture-independent molecular techniques (DGGE and clone libraries) to characterize ciliate and bacterial communities in these diseases. Bacterial (16S rRNA gene) and ciliate (18S rRNA gene) communities were highly similar between the two diseases. Four bacterial and nine ciliate ribotypes were observed in both diseases, but absent in non-diseased specimens. Only one of the bacteria, Arcobacter sp. (JF831360) increased substantially in relative 16S rRNA gene abundance and was consistently represented in all diseased samples. Four of the eleven ciliate morphotypes detected contained coral algal symbionts, indicative of the ingestion of coral tissues. In both WS and BrB, there were two ciliate morphotypes consistently represented in all disease lesion samples. Morph1 (JN626268) was observed to burrow into and underneath the coral tissues at the lesion boundary. Morph2 (JN626269), previously identified in BrB, appears to play a secondary, less invasive role in pathogenesis, but has a higher population density in BrB, giving rise to the visible brown band. The strong similarity in bacterial and ciliate community composition of these diseases suggests that they are actually the same syndrome.     

Bacterial Communities Associated with Porites White Patch Syndrome (PWPS) on Three Western Indian Ocean (WIO) Coral Reefs

The scleractinian coral Porites lutea, an important reef-building coral on western Indian Ocean reefs (WIO), is affected by a newly-reported white syndrome (WS) the Porites white patch syndrome (PWPS). Histopathology and culture-independent molecular techniques were used to characterise the microbial communities associated with this emerging disease. Microscopy showed extensive tissue fragmentation generally associated with ovoid basophilic bodies resembling bacterial aggregates. Results of 16S rRNA sequence analysis revealed a high variability between bacterial communities associated with PWPS-infected and healthy tissues in P. lutea, a pattern previously reported in other coral diseases such as black band disease (BBD), white band disease (WBD) and white plague diseases (WPD). Furthermore, substantial variations in bacterial communities were observed at the different sampling locations, suggesting that there is no strong bacterial association in Porites lutea on WIO reefs. Several sequences affiliated with potential pathogens belonging to the Vibrionaceae and Rhodobacteraceae were identified, mainly in PWPS-infected coral tissues. Among them, only two ribotypes affiliated to Shimia marina (NR043300.1) and Vibrio hepatarius (NR025575.1) were consistently found in diseased tissues from the three geographically distant sampling localities. The role of these bacterial species in PWPS needs to be tested experimentally.     

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.     

Characterisation of the Bacterial and Fungal Communities Associated with Different Lesion Sizes of Dark Spot Syndrome Occurring in the Coral Stephanocoenia intersepta

The number and prevalence of coral diseases/syndromes are increasing worldwide. Dark Spot Syndrome (DSS) afflicts numerous coral species and is widespread throughout the Caribbean, yet there are no known causal agents. In this study we aimed to characterise the microbial communities (bacteria and fungi) associated with DSS lesions affecting the coral Stephanocoenia intersepta using nonculture molecular techniques. Bacterial diversity of healthy tissues (H), those in advance of the lesion interface (apparently healthy AH), and three sizes of disease lesions (small, medium, and large) varied significantly (ANOSIM R  = 0.052 p<0.001), apart from the medium and large lesions, which were similar in their community profile. Four bacteria fitted into the pattern expected from potential pathogens; namely absent from H, increasing in abundance within AH, and dominant in the lesions themselves. These included ribotypes related to Corynebacterium ({"type":"entrez-nucleotide","attrs":{"text":"KC190237","term_id":"441431659","term_text":"KC190237"}}KC190237), Acinetobacter ({"type":"entrez-nucleotide","attrs":{"text":"KC190251","term_id":"441431673","term_text":"KC190251"}}KC190251), Parvularculaceae (KC19027), and Oscillatoria ({"type":"entrez-nucleotide","attrs":{"text":"KC190271","term_id":"441431693","term_text":"KC190271"}}KC190271). Furthermore, two Vibrio species, a genus including many proposed coral pathogens, dominated the disease lesion and were absent from H and AH tissues, making them candidates as potential pathogens for DSS. In contrast, other members of bacteria from the same genus, such as V. harveyii were present throughout all sample types, supporting previous studies where potential coral pathogens exist in healthy tissues. Fungal diversity varied significantly as well, however the main difference between diseased and healthy tissues was the dominance of one ribotype, closely related to the plant pathogen, Rhytisma acerinum, a known causal agent of tar spot on tree leaves. As the corals’ symbiotic algae have been shown to turn to a darker pigmented state in DSS (giving rise to the syndromes name), the two most likely pathogens are R. acerinum and the bacterium Oscillatoria, which has been identified as the causal agent of the colouration in Black Band Disease, another widespread coral disease.     

Fluorescence In Situ Hybridization and Spectral Imaging of Coral-Associated Bacterial Communities

Microbial communities play important roles in the functioning of coral reef communities. However, extensive autofluorescence of coral tissues and endosymbionts limits the application of standard fluorescence in situ hybridization (FISH) techniques for the identification of the coral-associated bacterial communities. This study overcomes these limitations by combining FISH and spectral imaging.     

Coral microbial communities, zooxanthellae and mucus along gradients of seawater depth and coastal pollution

The high incidence of coral disease in shallow coastal marine environments suggests seawater depth and coastal pollution have an impact on the microbial communities inhabiting healthy coral tissues. A study was undertaken to determine how bacterial communities inhabiting tissues of the coral Montastraea annularis change at 5 m, 10 m and 20 m water depth in varying proximity to the urban centre and seaport of Willemstad, Cura?ao, Netherlands Antilles. Analyses of terminal restriction fragment length polymorphisms (TRFLP) of 16S rRNA gene sequences show significant differences in bacterial communities of polluted and control localities only at the shallowest seawater depth. Furthermore, distinct differences in bacterial communities were found with increasing water depth. Comparisons of TRFLP peaks with sequenced clone libraries indicate the black band disease cyanobacterium clone CD1C11 is common and most abundant on healthy corals in less than 10 m water depth. Similarly, sequences belonging to a previously unrecognized group of likely phototrophic bacteria, herein referred to as CAB-I, were also more common in shallow water. To assess the influence of environmental and physiologic factors on bacterial community structure, canonical correspondence analysis was performed using explanatory variables associated with: (i) light availability; (ii) seawater pollution; (iii) coral mucus composition; (iv) the community structure of symbiotic algae; and (v) the photosynthetic activity of symbiotic algae. Eleven per cent of the variation in bacterial communities was accounted for by covariation with these variables; the most important being photosynthetically active radiation (sunlight) and the coral uptake of sewage-derived compounds as recorded by the delta(15)N of coral tissue.     

Fluorescence in situ hybridization and spectral imaging of coral-associated bacterial communities

Microbial communities play important roles in the functioning of coral reef communities. However, extensive autofluorescence of coral tissues and endosymbionts limits the application of standard fluorescence in situ hybridization (FISH) techniques for the identification of the coral-associated bacterial communities. This study overcomes these limitations by combining FISH and spectral imaging.     

The coral immune response facilitates protection against microbes during tissue regeneration

Increasing physical damage on coral reefs from predation, storms and anthropogenic disturbances highlights the need to understand the impact of injury on the coral immune system. In this study, we examined the regulation of the coral immune response over 10 days following physical trauma artificially inflicted on in situ colonies of the coral Acropora aspera, simultaneously with bacterial colonization of the lesions. Corals responded to injury by increasing the expression of immune system‐related genes involved in the Toll‐like and NOD‐like receptor signalling pathways and the lectin–complement system in three phases (<2, 4 and 10 days post‐injury). Phenoloxidase activity was also significantly upregulated in two phases (<3 and 10 days post‐injury), as were levels of non‐fluorescent chromoprotein. In addition, green fluorescent protein expression was upregulated in response to injury from 4 days post‐injury, while cyan fluorescent protein expression was reduced. No shifts in the composition of coral‐associated bacterial communities were evident following injury based on 16S rRNA gene amplicon pyrosequencing. Bacteria‐specific fluorescence in situ hybridization also showed no evidence of bacterial colonization of the wound or regenerating tissues. Coral tissues showed near‐complete regeneration of lesions within 10 days. This study demonstrates that corals exhibit immune responses that support rapid recovery following physical injury, maintain coral microbial homeostasis and prevent bacterial infestation that may compromise coral fitness.     

珊瑚病原微生物鉴定及其分子诊断技术进展

珊瑚礁生态系统是热带海洋最突出、最具代表性的生态系统,具有极高的生态价值和经济价值,然而由珊瑚疾病引起的珊瑚礁退化已经成为珊瑚礁生态系统的主要威胁之一。许多微生物(主要包括细菌、真菌、病毒)被认为与珊瑚疾病发生密切相关,确定珊瑚疾病的病原并建立其快速诊断的方法是开展珊瑚疾病流行病学调查和制定防控措施的必由之路。本文主要综述珊瑚疾病的病原微生物及其分子诊断技术的研究进展。     

Selective feeding by coral reef fishes on coral lesions associated with brown band and black band disease

Recent studies have suggested that corallivorous fishes may be vectors for coral disease, but the extent to which fishes actually feed on and thereby potentially transmit coral pathogens is largely unknown. For this study, in situ video observations were used to assess the level to which fishes fed on diseased coral tissues at Lizard Island, northern Great Barrier Reef. Surveys conducted at multiple locations around Lizard Island revealed that coral disease prevalence, especially of brown band disease (BrB), was higher in lagoon and backreef locations than in exposed reef crests. Accordingly, video cameras were deployed in lagoon and backreef habitats to record feeding by fishes during 1-h periods on diseased sections of each of 44 different coral colonies. Twenty-five species from five fish families (Blennidae, Chaetodontidae, Gobiidae, Labridae and Pomacentridae) were observed to feed on infected coral tissues of staghorn species of Acropora that were naturally infected with black band disease (BBD) or brown band disease (BrB). Collectively, these fishes took an average of 18.6 (±5.6 SE) and 14.3 (±6.1 SE) bites per hour from BBD and BrB lesions, respectively. More than 40% (408/948 bites) and nearly 25% (314/1319 bites) of bites were observed on lesions associated with BBD and BrB, respectively, despite these bands each representing only about 1% of the substratum available. Moreover, many corallivorous fishes (Labrichthys unilineatus, Chaetodon aureofasciatus, C. baronessa, C. lunulatus, C. trifascialis, Cheiloprion labiatus) selectively targeted disease lesions over adjacent healthy coral tissues. These findings highlight the important role that reef fishes may play in the dynamics of coral diseases, either as vectors for the spread of coral disease or in reducing coral disease progression through intensive and selective consumption of diseased coral tissues.     

Regulation of Bacterial Communities Through Antimicrobial Activity by the Coral Holobiont

Interactions between corals and associated bacteria and amongst these bacterial groups are likely to play a key role in coral health. However, the complexity of these interactions is poorly understood. We investigated the functional role of specific coral-associated bacteria in maintaining microbial communities on the coral Acropora millepora (Ehrenberg 1834) and the ability of coral mucus to support or inhibit bacterial growth. Culture-independent techniques were used to assess bacterial community structures whilst bacterial culture was employed to assess intra- and inter-specific antimicrobial activities of bacteria. Members of Pseudoalteromonas and ribotypes closely related to Vibrio coralliilyticus displayed potent antimicrobial activity against a range of other cultured isolates and grew readily on detached coral mucus. Although such bacterial ribotypes would be expected to have a competitive advantage, they were rare or absent on intact and healthy coral colonies growing in situ (analysed using denaturing gradient gel electrophoresis and 16S rRNA gene sequencing). The most abundant bacterial ribotypes found on healthy corals were Gammaproteobacteria, previously defined as type A coral associates. Our results indicate that this group of bacteria and specific members of the Alphaproteobacteria described here as ‘type B associates’ may be important functional groups for coral health. We suggest that bacterial communities on coral are kept in check by a combination of host-derived and microbial interactions and that the type A associates in particular may play a key role in maintaining stability of microbial communities on healthy coral colonies.     

Assessing the accuracy of acoustic seabed classification for mapping coral reef environments in South Florida (Broward County, USA)

The Atlantic coast of Broward County, Florida (USA) is paralleled by a series of progressively deeper, shore-parallel coral reef communities. Two of these reef systems are drowned early Holocene coral reefs of 5 ky and 7 ky uncorrected radiocarbon age. Despite the case of access to these reefs, and their major contribution to the local economy, accurate benthic habitat maps of the area are not available. Ecological studies have shown that different benthic communities (i.e. communities composed of different biological taxa) exist along several spatial gradients on all reefs. Since these studies are limited by time and spatial extent, acoustic surveys with the QTCView V bottom classification system based on a 50 kHz transducer were used as an alternative method of producing habitat maps. From the acoustic data of a 3.1 km(2) survey area, spatial prediction maps were created for the area. These were compared with habitat maps interpreted from in situ data and Laser Airborne Depth Sounder (LADS) bathymetry, in order to ground-truth the remotely sensed data. An error matrix was used to quantitatively determine the accuracy of the acoustically derived spatial prediction model against the maps derived from the in situ and LADS data sets. Confusion analysis of 100 random points showed that the system was able to distinguish areas of reef from areas of rubble and sand with an overall accuracy of 61%. When asked to detect more subtle spatial differences, for example, those between distinct reef communities, the classification was only about 40% accurate. We discuss to what degree a synthesis of acoustic and in situ techniques can provide accurate habitat maps in coral reef environments, and conclude that acoustic methods were able to reflect the spatial extent and composition of at least three different biological communities.