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The deep ocean hosts a large diversity of azooxanthellate cold-water corals whose associated microbiomes remain to be described. While the bacterial genus Endozoicomonas has been widely identified as a dominant associate of tropical and temperate corals, it has rarely been detected in deep-sea corals. Determining microbial baselines for these cold-water corals is a critical first step to understanding the ecosystem services their microbiomes contribute, while providing a benchmark against which to measure responses to environmental change or anthropogenic effects. Samples of Acanthogorgia aspera, A. spissa, Desmophyllum dianthus, and D. pertusum (Lophelia pertusa) were collected from western Atlantic sites off the US east coast and from the northeastern Gulf of Mexico. Microbiomes were characterized by 16S rRNA gene amplicon surveys. Although D. dianthus and D. pertusum have recently been combined into a single genus due to their genetic similarity, their microbiomes were significantly different. The Acanthogorgia spp. were collected from submarine canyons in different regions, but their microbiomes were extremely similar and dominated by Endozoicomonas. This is the first report of coral microbiomes dominated by Endozoicomonas occurring below 1000 m, at temperatures near 4°C. D. pertusum from 2 Atlantic sites were also dominated by distinct Endozoicomonas, unlike D. pertusum from other sites described in previous studies, including the Gulf of Mexico, the Mediterranean Sea and a Norwegian fjord.

Coral-associated microbiomes play important roles in host health and metabolism. It is therefore critical to understand the factors that shape microbiome taxonomic composition and function. Little is known about the stability of coral microbiomes over hourly fluctuations in environmental conditions. For parameters such as temperature and pH, the magnitude of day–night fluctuations can exceed those observed in mean values over seasons, especially on shallow reefs. Such short-term heterogeneity can affect longer term trends, possibly by influencing the extent to which corals acclimate to stress and increase resilience. This study examined diel dynamics of microbiomes (Bacteria and Archaea) in three species of stony coral (Porites lutea, Porites cylindrica, and Pocillopora damicornis) from a shallow reef in Mo’orea (French Polynesia). We assessed microbiome composition and relative transcriptional activity by analyzing 16S rRNA gene and transcript sequences from six timepoints over 48 h for each species. Both the composition and transcription of microbiome members changed significantly over the diel cycle. Most changes did not exhibit clear diel periodicity but were instead stochastic, a pattern potentially influenced also by microspatial variation within a coral colony. However, a subset of taxa, notably the dinoflagellate-associated bacterium Porticoccus and bacteria of the ubiquitous coral-associated Endozoicomonas genus, exhibited cyclic changes in relative frequency over the 2 d sampling period in the DNA dataset of Porites lutea. These patterns raise the possibility that ecological functions of the microbiome may vary substantially over hourly timescales, notably between day and night. Further studies are needed to compare temporal change in the coral microbiome with that due to other factors, such as spatial distance within and among colonies.

All animals are host to a multitude of microorganisms that are essential to the animal’s health. Host-associated microbes have been shown to defend against potential pathogens, provide essential nutrients, interact with the host’s immune system, and even regulate mood. However, it can be difficult to preserve and obtain nucleic acids from some host-associated microbiomes, making studying their microbial communities challenging. Corals are an example of this, in part due to their potentially remote, underwater locations, their thick surface mucopolysaccharide layer, and various inherent molecular inhibitors. This study examined three different preservatives (RNAlater, DNA/RNA Shield, and liquid nitrogen) and two extraction methods (the Qiagen PowerBiofilm kit and the Promega Maxwell RBC kit with modifications) to determine if there was an optimum combination for examining the coral microbiome. These methods were employed across taxonomically diverse coral species, including deep-sea/shallow, stony/soft, and zooxanthellate/azooxanthellate: Lophelia pertusa, Paragorgia johnsoni, Montastraea cavernosa, Porites astreoides, and Stephanocoenia intersepta. Although significant differences were found between preservative types and extraction methods, these differences were subtle, and varied in nature from coral species to coral species. Significant differences between coral species were far more profound than those detected between preservative or extraction method. We suggest that the preservative types presented here and extraction methods using a bead-beating step provide enough consistency to compare coral microbiomes across various studies, as long as subtle differences in microbial communities are attributed to dissimilar methodologies. Additionally, the inclusion of internal controls such as a mock community and extraction blanks can help provide context regarding data quality, improving downstream analyses.

The microbiome of the temperate coral Astrangia poculata was first described in 2017 using next-generation Illumina sequencing to examine the coral's bacterial and archaeal associates across seasons and among hosts of differing symbiotic status. To assess the impact of methodology on the detectable diversity of the coral's microbiome, we obtained near full-length Sanger sequences from clone libraries constructed from a subset of the same A. poculata samples. Eight samples were analyzed: two sets of paired symbiotic (brown) and aposymbiotic (white) colonies collected in the fall (September) and two sets collected in the spring (April). Analysis of the Sanger sequences revealed that the microbiome of A. poculata exhibited a high level of richness; 806 OTUs were identified among 1390 bacterial sequences. While the Illumina study revealed that A. poculata's microbial communities did not significantly vary according to symbiotic state, but did vary by season, Sanger sequencing did not expose seasonal or symbiotic differences in the microbiomes. Proteobacteria dominated the microbiome, forming the majority (55% to 80%) of classifiable bacteria in every sample, and the five bacterial classes with the highest mean relative portion (5% to 35%) were the same as those determined by prior Illumina sequencing. Sanger sequencing also captured the same core taxa previously identified by next-generation sequencing. Alignment of all sequences and construction of a phylogenetic tree revealed that both sequencing methods provided similar portrayals of the phylogenetic diversity within A. poculata's bacterial associates. Consistent with previous findings, the results demonstrated that the Astrangia microbiome is stable notwithstanding the choice of sequencing method and the far fewer sequences generated by clone libraries (46 to 326 sequences per sample) compared to next-generation sequencing (3634 to 48481 sequences per sample). Moreover, the near-full length 16S rRNA sequences produced by this study are presented as a resource for the community studying this model system since they provide necessary information for designing primers and probes to further our understanding of this coral's microbiome.

Elasmobranchs (sharks, skates and rays) are of broad ecological, economic, and societal value. These globally important fishes are experiencing sharp population declines as a result of human activity in the oceans. Research to understand elasmobranch ecology and conservation is critical and has now begun to explore the role of body-associated microbiomes in shaping elasmobranch health. Here, we review the burgeoning efforts to understand elasmobranch microbiomes, highlighting microbiome variation among gastrointestinal, oral, skin, and blood-associated niches. We identify major bacterial lineages in the microbiome, challenges to the field, key unanswered questions, and avenues for future work. We argue for prioritizing research to determine how microbiomes interact mechanistically with the unique physiology of elasmobranchs, potentially identifying roles in host immunity, disease, nutrition, and waste processing. Understanding elasmobranch–microbiome interactions is critical for predicting how sharks and rays respond to a changing ocean and for managing healthy populations in managed care. Supplementary Information The online version contains supplementary material available at 10.1186/s42523-021-00121-4.

Host-associated microbes play important roles in animal health. Clownfish and anemones form a mutualistic relationship where external surfaces are in constant contact with one another. The effects of this mutualism on the skin mucosal microbiome of clownfish are unknown. We monitored the mucosal microbiome of the clownfish Amphiprion clarkii before, during and after association (hosting) with the anemone Entacmaea quadricolor. Microbiome composition differed significantly between hosting and non-hosting clownfish. Upon separation of clownfish from anemones, the microbiome of post-hosting fish converged to resemble that of non-hosting fish. Bacterial sequence variants that significantly distinguished hosting from non-hosting clownfish were more abundant in the anemone microbiome and that of hosting fish, compared to non-hosting fish, suggesting transfer from or enrichment by the anemone. These results show that direct contact mutualism results in significant but reversible microbiome shifts, raising questions about a potential microbial role in mediating the fish–anemone interaction.

Coral reefs are degrading to algae-dominated reefs worldwide, with alterations of coral microbiomes commonly co-occurring with reef demise. The severe thermal anomaly during the 2016 El Nino event in the South Pacific killed many corals and stressed others. We examined the microbiome of turf algae and of the coral Porites sp. in contact with turf during this thermal event to investigate algal turf effects on the coral microbiome during a period of environmental stress. The microbial composition of turf did not differ between coral-contacted and non-contacted turfs. However, microbiomes of corals in direct contact with turf were similar to those of the turf microbiome, but differed significantly from coral portions 5 cm from the point of turf/coral contact and from portions of the coral that looked most healthy, regardless of location. Although the majority of significant differences occurred in coral samples at the point of contact, a small subset of microbial taxa was enriched in coral tissues taken 5 cm from turf contact compared to all other sample types, including samples from areas of the coral that appeared most healthy. These results suggest that the coral microbiome is susceptible to colonization by microbes from turf, but not vice versa. Results also suggest that algal contact elicits a subtle shift in the coral microbiome just beyond the contact site. The combination of turf microbiome stability and coral microbiome vulnerability at areas of contact may contribute to the continued decline in coral cover and increase in algal cover associated with coral–algae phase shifts.

Background In adults undergoing allogeneic hematopoietic cell transplantation (HCT), higher gut microbiome diversity is associated with reduced bloodstream infections (BSI) and improved overall survival (OS). Rifaximin prophylaxis in adult HCT helps to maintain microbiome diversity. We examine changes in microbiome in a cohort of pediatric patients undergoing HCT. Methods Patients were enrolled in an institutional biorepository (n=82) with a subset enrolled in an ongoing trial using rifaximin (n=21) between 2013–2020. All patients had HCT for a hematologic malignancy, using myeloablative conditioning. Patients in the rifaximin trial received rifaximin starting 7 days before HCT (D-7) through D+28, otherwise, no prophylactic antibiotics were used. Systemic antibiotic timing was categorized as none, early (≤ Day 0, day of HCT), and late (> D0). We performed 16s rRNA sequencing from stool for 73 subjects, at baseline (D-7), and weekly through D+28 (engraftment). Microbiome diversity was assessed by Shannon index. Results Median age was 9 years (range 1–20), 59% male, 41% Caucasian and 29% Black. There were no differences in BSI or mortality by age, sex, or race. Microbiome diversity changed significantly over time (p=0.008). Drop in diversity was most notable in patients who had early antibiotics (Mean=1.4, CI -0.15, 2.94, p=0.077). Higher diversity was seen when patients received none or late versus early antibiotics, but this was not statistically significant (Figure 1, p=0.23). Piperacillin-tazobactam was used empirically in 91% of patients. OS at 1 year was 88.5% (CI 68.4%, 96.1%) for patients with high (≥ median) D+28 diversity compared to 60% (CI 38.4%, 76.1%) for patients with low diversity (Figure 2, p=0.018) Only 1 of 21 (4.8%) in the rifaximin group developed a BSI with a gut bacterium compared to 8 of 61 (13.1%) not on rifaximin within the first 30 days (trial enrollment ongoing). Figure 1. Effect of systemic antibiotic timing on microbiome diversity over time. Figure 2. One-year overall survival of patients with high (>2.77) versus low ( Conclusion We have shown a significant correlation between engraftment microbiome diversity and 1-year OS. Early antibiotic exposure was detrimental to microbiome diversity. Approaches to preserve microbiome diversity and prevent BSI are likely to improve HCT outcomes. Our ongoing trial using rifaximin will provide preliminary data regarding this approach. Disclosures All Authors: No reported disclosures

Coral bleaching and disease are 2 common occurrences that are contributing to global coral cover decline. Understanding the interactions between the coral animal and its microbial associates, and how they may change in the presence of stressors such as warming and acidification, is a crucial component to understanding both susceptibility and resistance to disease and bleaching. The coral Diploria labyrinthiformis has been shown to be more disease resistant than its relative Pseudodiploria strigosa, providing an ideal study system for disease resistance. In this study, we examined the bacterial communities of these 2 coral species on the Florida Reef tract every 6 mo for 18 mo (in situ sampling), and under experimental (laboratory) thermal and pH manipulation. The in situ sampling encompassed wide fluctuations in temperature, including an anomalously warm summer period. The laboratory experiments involved exposure to both increased temperature (31°C) and lowered pH (7.7). The in situ bacterial communities of both coral species were highly similar in the winter, but diverged during summer, with the D. labyrinthiformis bacterial community being more stable than that of P. strigosa. Differences in the bacterial community between the 2 coral species included 29 operational taxonomic units (OTUs) that were specific to D. labyrinthiformis in all seasons, while only 2 OTUs were specific to P. strigosa. The comparative stability of the D. labyrinthiformis microbiome, in addition to harboring a more specific microbiome, may be a key component of the relative disease resistance of this coral.

Denitrification by sulfur-oxidizing bacteria is an effective nitrate removal strategy in engineered aquatic systems. However, the community taxonomic and metabolic diversity of sulfur-driven denitrification (SDN) systems, as well as the relationship between nitrate removal and SDN community structure, remains underexplored. This is particularly true for SDN reactors applied to marine aquaria, despite the increasing use of this technology to supplement filtration. We applied 16S rRNA gene, metagenomic, and metatranscriptomic analyses to explore the microbial basis of SDN reactors operating on Georgia Aquarium9s Ocean Voyager, the largest indoor closed-system seawater exhibit in the United States. The exhibit9s two SDN systems vary in water retention time and nitrate removal efficiency. The systems also support significantly different microbial communities. These communities contain canonical SDN bacteria, including a strain related to Thiobacillus thioparus that dominates the system with the higher water retention time and nitrate removal but is effectively absent from the other system. Both systems contain a wide diversity of other microbes whose metagenome-assembled genomes contain genes of SDN metabolism. These include hundreds of strains of the epsilonproteobacterium Sulfurimonas, as well as gammaproteobacterial sulfur oxidizers of the Thiotrichales and Chromatiales, and a relative of Sedimenticolathiotaurini with complete denitrification potential. The SDN genes are transcribed and the taxonomic richness of the transcript pool varies markedly among the enzymatic steps, with some steps dominated by transcripts from noncanonical SDN taxa. These results indicate complex and variable SDN communities that may involve chemical dependencies among taxa as well as the potential for altering community structure to optimize nitrate removal. IMPORTANCE Engineered aquatic systems such as aquaria and aquaculture facilities have large societal value. Ensuring the health of animals in these systems requires understanding how microorganisms contribute to chemical cycling and waste removal. Focusing on the largest seawater aquarium in the United States, we explore the microbial communities in specialized reactors designed to remove excess nitrogen through the metabolic activity of sulfur-consuming microbes. We show that the diversity of microbes in these reactors is both high and highly variable, with distinct community types associated with significant differences in nitrogen removal rate. We also show that the genes encoding the metabolic steps of nitrogen removal are distributed broadly throughout community members, suggesting that the chemical transformations in this system are likely a result of microbes relying on other microbes. These results provide a framework for future studies exploring the contributions of different community members, both in waste removal and in structuring microbial biodiversity.

Artificial habitats for animals have high commercial and societal value. Microbial communities (microbiomes) in such habitats may play ecological roles similar to those in nature. However, this hypothesis remains largely untested. Georgia Aquarium's Ocean Voyager (OV) exhibit is a closed-system aquatic habitat that mimics the oligotrophic open ocean and houses thousands of large marine animals, including fish, sea turtles, and whale sharks. We present a 14-month time series characterizing the OV water column microbiome. The composition and stability of the microbiome differed from those of natural marine environments with similar chemical features. The composition shifted dramatically over the span of 2 weeks and was characterized by bloom events featuring members of two heterotrophic bacterial lineages with cosmopolitan distributions in the oceans. The relative abundances of these lineages were inversely correlated, suggesting an overlap in ecological niches. Transcript mapping to metagenome-assembled genomes (MAGs) of these taxa identified unique characteristics, including the presence and activity of genes for the synthesis and degradation of cyanophycin, an amino acid polymer linked to environmental stress and found frequently in cyanobacteria but rarely in heterotrophic bacteria. The dominant MAGs also contained and transcribed plasmid-associated sequences, suggesting a role for conjugation in adaptation to the OV environment. These findings indicate a highly dynamic microbiome despite the stability of the physical and chemical parameters of the water column. Characterizing how such fluctuations affect microbial function may inform our understanding of animal health in closed aquaculture systems. IMPORTANCE Public aquariums play important societal roles, for example, by promoting science education and helping conserve biodiversity. The health of aquarium animals depends on interactions with the surrounding microbiome. However, the extent to which aquariums recreate a stable and natural microbial ecosystem is uncertain. This study describes the taxonomic composition of the water column microbiome over 14 months in a large indoor aquatic habitat, the Ocean Voyager exhibit at the Georgia Aquarium. Despite stable water column conditions, the exhibit experienced blooms in which the abundance of a single bacterial strain increased to over 65% of the community. Genome analysis indicated that the OV's dominant strains share unique adaptations, notably genes for storage polymers associated with environmental stress. These results, interpreted alongside data from natural ocean systems and another artificial seawater aquarium, suggest a highly dynamic aquarium microbiome and raise questions of how microbiome stability may affect the ecological health of the habitat.

Teleost fish represent the most diverse of the vertebrate groups and play important roles in food webs, as ecosystem engineers, and as vectors for microorganisms. However, the microbial ecology of fishes remains underexplored for most host taxa and for certain niches on the fish body. This is particularly true for the gills, the key sites of respiration and waste exchange in fishes. Here we provide a comprehensive analysis of the gill microbiome. We focus on ecologically diverse taxa from coral reefs around Moorea, sampling the gills and intestines of adults and juveniles representing 15 families. The gill microbiome composition differed significantly from that of the gut for both adults and juveniles, with fish-associated niches having lower alpha diversity values and higher beta diversity values than those for seawater, sediment, and alga-associated microbiomes. Of ∼45,000 operational taxonomic units (OTUs) detected across all samples, 11% and 13% were detected only in the gill and the intestine, respectively. OTUs most enriched in the gill included members of the gammaproteobacterial genus Shewanella and the family Endozoicimonaceae . In adult fish, both gill and intestinal microbiomes varied significantly among host species grouped by diet category. Gill and intestinal microbiomes from the same individual were more similar to one another than to gill and intestinal microbiomes from different individuals. These results demonstrate that distinct body sites are jointly influenced by host-specific organizing factors operating at the level of the host individual. The results also identify taxonomic signatures unique to the gill and the intestine, confirming fish-associated niches as distinct reservoirs of marine microbial diversity. IMPORTANCE Fish breathe and excrete waste through their gills. The gills are also potential sites of pathogen invasion and colonization by other microbes. However, we know little about the microbial communities that live on the gill and the factors shaping their diversity. Focusing on ecologically distinct types of coral reef fish, we provide a comprehensive analysis of the fish gill microbiome. By comparison to microbiomes of the gut and the surrounding environment, we identify microbes unique to the gill niche. These microbes may be targets for further studies to determine the contribution of the microbiome to waste exchange or host immunity. We also show that despite exhibiting a unique taxonomic signature, the gill microbiome is influenced by factors that also influence the gut microbiome. These factors include the specific identity of the host individual. These results suggest basic principles describing how association with fishes structures the composition of microbial communities.

Background Understanding the associations among corals, their photosynthetic zooxanthella symbionts (Symbiodinium), and coral-associated prokaryotic microbiomes is critical for predicting the fidelity and strength of coral symbioses in the face of growing environmental threats. Most coral-microbiome associations are beneficial, yet the mechanisms that determine the composition of the coral microbiome remain largely unknown. Here, we characterized microbiome diversity in the temperate, facultatively symbiotic coral Astrangia poculata at four seasonal time points near the northernmost limit of the species range. The facultative nature of this system allowed us to test seasonal influence and symbiotic state (Symbiodinium density in the coral) on microbiome community composition. Results Change in season had a strong effect on A. poculata microbiome composition. The seasonal shift was greatest upon the winter to spring transition, during which time A. poculata microbiome composition became more similar among host individuals. Within each of the four seasons, microbiome composition differed significantly from that of surrounding seawater but was surprisingly uniform between symbiotic and aposymbiotic corals, even in summer, when differences in Symbiodinium density between brown and white colonies are the highest, indicating that the observed seasonal shifts are not likely due to fluctuations in Symbiodinium density. Conclusions Our results suggest that symbiotic state may not be a primary driver of coral microbial community organization in A. poculata, which is a surprise given the long-held assumption that excess photosynthate is of importance to coral-associated microbes. Rather, other environmental or host factors, in this case, seasonal changes in host physiology associated with winter quiescence, may drive microbiome diversity. Additional studies of A. poculata and other facultatively symbiotic corals will provide important comparisons to studies of reef-building tropical corals and therefore help to identify basic principles of coral microbiome assembly, as well as functional relationships among holobiont members. Electronic supplementary material The online version of this article (10.1186/s40168-017-0329-8) contains supplementary material, which is available to authorized users.

Coral mechanisms of resilience and resistance to stressors such as increasing sea surface temperature and ocean acidification must first be understood in order to facilitate the survival of coral reefs as we know them. One such mechanism is production of the protective surface mucopolysaccharide layer (SML). In this study, we investigated changes in the thickness of the SML in response to increasing temperature and acidification for the three Caribbean scleractinian coral species of the genus Diploria, which have been shown to exhibit differential resilience to disease and bleaching. Among the three species, Diploria strigosa is known to have a higher susceptibility to disease, Diploria labyrinthiformis is known to bleach more quickly, and Diploria clivosa is relatively unstudied. When temperature was increased from 25 to 31 °C over a 1- or 6-week period, the overall thickness of the SML decreased from 33 to 55 % for all three species. Average SML thickness at 25 °C for all three species ranged from 106 to 156 μm, while average thickness at 31 °C ranged from 64 to 86 μm. SML thickness was significantly different among species at 25 °C, but not at 31 °C. D. labyrinthiformis demonstrated lower fragment mortality due to thermal stress when compared to the other Diploria species. Acidification from pH 8.2 to 7.7 over 5 weeks had no effect on SML thickness for any species. The observed decrease in SML thickness in response to increased temperature might be attributed to a decrease in the production of mucus or an increase in the viscosity of the SML. These findings may help to explain the increased prevalence of coral disease during the warmer months, since increased temperature compromises an important aspect of coral innate immunity, as well as differences in disease and bleaching susceptibilities between Diploria species.

White plague (WP) is a highly destructive coral disease that rapidly kills susceptible coral species by mass tissue lysis. The pathogen and underlying causes of this disease are not known. In this laboratory-based study, we examined a small coral-associated gall crab from the family Cryptochiridae in terms of a possible association with WP-like lesions. A series of experiments was conducted after observations that 2 scleractinian coral species, Diploria labyrinthiformis and Pseudodiploria strigosa, developed signs of WP-like disease within a laboratory holding aquarium and that small gall crabs were physically present in the center of each lesion. Using fragments of D. labyrinthiformis, a crab from one of the lesions was sequentially removed and placed, under controlled conditions, onto apparently healthy coral colonies, resulting in the development of similar lesions. Next-generation sequencing of the 16S rRNA gene was performed to profile the bacterial communities associated with the crab, lesions, and healthy corals. The microbiota of the crab and lesions were highly similar while that of apparently healthy colonies were significantly different. Significant differences were largely due to an increase in Alphaproteobacteria in crab and lesion communities. In particular, the Roseobacter clade had a higher relative abundance in the crab and WP-like lesions. This study suggests that the cryptochirid gall crab may be associated with development of WP-like lesions.

Microorganisms associated with corals are hypothesized to contribute to the function of the host animal by cycling nutrients, breaking down carbon sources, fixing nitrogen, and producing antibiotics. This is the first study to culture and characterize bacteria from Lophelia pertusa, a cold-water coral found in the deep sea, in an effort to understand the roles that the microorganisms play in the coral microbial community. Two sites in the northern Gulf of Mexico were sampled over 2 years. Bacteria were cultured from coral tissue, skeleton, and mucus, identified by 16S rRNA genes, and subjected to biochemical testing. Most isolates were members of the Gammaproteobacteria, although there was one isolate each from the Betaproteobacteria and Actinobacteria. Phylogenetic results showed that both sampling sites shared closely related isolates (e.g. Pseudoalteromonas spp.), indicating possible temporally and geographically stable bacterial–coral associations. The Kirby–Bauer antibiotic susceptibility test was used to separate bacteria to the strain level, with the results showing that isolates that were phylogenetically tightly grouped had varying responses to antibiotics. These results support the conclusion that phylogenetic placement cannot predict strain-level differences and further highlight the need for culture-based experiments to supplement culture-independent studies.

Bacteria associated with the surface mucopolysaccharide layer (SML) of corals have been proposed to be paramount to coral health, and are occasionally studied in aquaria. Using automated ribosomal intergenic spacer analysis (ARISA), this study examined the temporal changes in the SML microbiota of coral fragments (Siderastrea siderea) transferred from the reef to aquaria. In total, 460 amplicon peaks were detected, 155 of which were unique. Extensive microbiota shifts occurred one day after transfer, with stabilization between 14 and 28 days. These results suggest that studies examining coral in laboratory settings should consider the observed temporal dynamics in the SML microbiota.

Both the incidence and prevalence of coral disease are rapidly increasing, and as a consequence, many studies involving coral microbial associates have been conducted. However, very few of these have considered microbial functional genes. This is an underutilized approach for studying coral disease etiology which is capable of revealing the molecular processes of the coral microbial community. This review presents a summary of the known microbial functional genes that have been linked to coral health and disease. Overall functional gene diversity tended to be lower in healthy corals than diseased or bleached corals, and respiration and photosynthesis functional genes appeared to be crucial to coral health. Genes associated with the nitrogen cycle were the most studied, were highly represented within the coral holobiont, and their expression often shifted in diseased or stressed individuals. Carbon metabolism, such as fatty acid and amino acid catabolism, also tended to shift in unhealthy corals. Genes associated with sulfite respiration as well as dimethylsulfoniopropionate degradation have been detected, although they have yet to be directly associated with coral disease. In addition, genes associated with xenobiotic degradation, antibiotic resistance, virulence, and oxidative stress may all be involved in maintaining coral health. However, the links between these functional genes and their roles in interacting with the coral host are not clear. Continuing identification of coral-associated microbial functional genes within the coral holobiont should facilitate advances in the field of coral health and disease.

Field collections of environmental samples, for example corals, for molecular microbial analyses present distinct challenges. The lack of laboratory facilities in remote locations is common, and preservation of microbial community DNA for later study is critical. A particular challenge is keeping samples frozen in transit. Five nucleic acid preservation methods that do not require cold storage were compared for effectiveness over time and ease of use. Mixed microbial communities of known composition were created and preserved by DNAgard(™), RNAlater(®), DMSO-EDTA-salt (DESS), FTA(®) cards, and FTA Elute(®) cards. Automated ribosomal intergenic spacer analysis and clone libraries were used to detect specific changes in the faux communities over weeks and months of storage. A previously known bias in FTA(®) cards that results in lower recovery of pure cultures of Gram-positive bacteria was also detected in mixed community samples. There appears to be a uniform bias across all five preservation methods against microorganisms with high G + C DNA. Overall, the liquid-based preservatives (DNAgard(™), RNAlater(®), and DESS) outperformed the card-based methods. No single liquid method clearly outperformed the others, leaving method choice to be based on experimental design, field facilities, shipping constraints, and allowable cost.

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.