Characterization of the microbiome and immune response in corals with chronic Montipora white syndrome.

Coral diseases have increased in frequency and intensity around the tropics worldwide. However, in many cases, little is known about their etiology. Montipora white syndrome (MWS) is a common disease affecting the coral Montipora capitata, a major reef builder in Hawai'i. Chronic Montipora white syndrome (cMWS) is a slow-moving form of the disease that affects M. capitata throughout the year. The effects of this chronic disease on coral immunology and microbiology are currently unknown. In this study, we use prophenoloxidase immune assays and 16S rRNA gene amplicon sequencing to characterize the microbiome and immunological response associated with cMWS. Our results show that immunological and microbiological responses are highly localized. Relative to diseased samples, apparently healthy portions of cMWS corals differed in immune activity and in the relative abundance of microbial taxa. Coral tissues with cMWS showed decreased tyrosinase-type catecholase and tyrosinase-type cresolase activity and increased laccase-type activity. Catecholase and cresolase activity were negatively correlated across all tissue types with microbiome richness. The localized effect of cMWS on coral microbiology and immunology is probably an important reason for the slow progression of the disease. This local confinement may facilitate interventions that focus on localized treatments on tissue types. This study provides an important baseline to understand the interplay between the microbiome and immune system and the mechanisms used by corals to manage chronic microbial perturbations associated with white syndrome.

RNASTAR: an RNA STructural Alignment Repository that provides insight into the evolution of natural and artificial RNAs.

Automated RNA alignment algorithms often fail to recapture the essential conserved sites that are critical for function. To assist in the refinement of these algorithms, we manually curated a set of 148 alignments with a total of 9600 unique sequences, in which each alignment was backed by at least one crystal or NMR structure. These alignments included both naturally and artificially selected molecules. We used principles of isostericity to improve the alignments from an average of 83%-94% isosteric base pairs. We expect that this alignment collection will assist in a wide range of benchmarking efforts and provide new insight into evolutionary principles governing change in RNA structural motifs. The improved alignments have been contributed to the Rfam database.

Chronic nutrient enrichment increases prevalence and severity of coral disease and bleaching.

Nutrient loading is one of the strongest drivers of marine habitat degradation. Yet, the link between nutrients and disease epizootics in marine organisms is often tenuous and supported only by correlative data. Here, we present experimental evidence that chronic nutrient exposure leads to increases in both disease prevalence and severity and coral bleaching in scleractinian corals, the major habitat-forming organisms in tropical reefs. Over 3 years, from June 2009 to June 2012, we continuously exposed areas of a coral reef to elevated levels of nitrogen and phosphorus. At the termination of the enrichment, we surveyed over 1200 scleractinian corals for signs of disease or bleaching. Siderastrea siderea corals within enrichment plots had a twofold increase in both the prevalence and severity of disease compared with corals in unenriched control plots. In addition, elevated nutrient loading increased coral bleaching; Agaricia spp. of corals exposed to nutrients suffered a 3.5-fold increase in bleaching frequency relative to control corals, providing empirical support for a hypothesized link between nutrient loading and bleaching-induced coral declines. However, 1 year later, after nutrient enrichment had been terminated for 10 months, there were no differences in coral disease or coral bleaching prevalence between the previously enriched and control treatments. Given that our experimental enrichments were well within the ranges of ambient nutrient concentrations found on many degraded reefs worldwide, these data provide strong empirical support to the idea that coastal nutrient loading is one of the major factors contributing to the increasing levels of both coral disease and coral bleaching. Yet, these data also suggest that simple improvements to water quality may be an effective way to mitigate some coral disease epizootics and the corresponding loss of coral cover in the future.

Pan-genome of the dominant human gut-associated archaeon, Methanobrevibacter smithii, studied in twins.

The human gut microbiota harbors three main groups of H(2)-consuming microbes: methanogens including the dominant archaeon, Methanobrevibacter smithii, a polyphyletic group of acetogens, and sulfate-reducing bacteria. Defining their roles in the gut is important for understanding how hydrogen metabolism affects the efficiency of fermentation of dietary components. We quantified methanogens in fecal samples from 40 healthy adult female monozygotic (MZ) and 28 dizygotic (DZ) twin pairs, analyzed bacterial 16S rRNA datasets generated from their fecal samples to identify taxa that co-occur with methanogens, sequenced the genomes of 20 M. smithii strains isolated from families of MZ and DZ twins, and performed RNA-Seq of a subset of strains to identify their responses to varied formate concentrations. The concordance rate for methanogen carriage was significantly higher for MZ versus DZ twin pairs. Co-occurrence analysis revealed 22 bacterial species-level taxa positively correlated with methanogens: all but two were members of the Clostridiales, with several being, or related to, known hydrogen-producing and -consuming bacteria. The M. smithii pan-genome contains 987 genes conserved in all strains, and 1,860 variably represented genes. Strains from MZ and DZ twin pairs had a similar degree of shared genes and SNPs, and were significantly more similar than strains isolated from mothers or members of other families. The 101 adhesin-like proteins (ALPs) in the pan-genome (45 ± 6 per strain) exhibit strain-specific differences in expression and responsiveness to formate. We hypothesize that M. smithii strains use their different repertoires of ALPs to create diversity in their metabolic niches, by allowing them to establish syntrophic relationships with bacterial partners with differing metabolic capabilities and patterns of co-occurrence.

Ribosomal RNA diversity predicts genome diversity in gut bacteria and their relatives.

The mammalian gut is an attractive model for exploring the general question of how habitat impacts the evolution of gene content. Therefore, we have characterized the relationship between 16 S rRNA gene sequence similarity and overall levels of gene conservation in four groups of species: gut specialists and cosmopolitans, each of which can be divided into pathogens and non-pathogens. At short phylogenetic distances, specialist or cosmopolitan bacteria found in the gut share fewer genes than is typical for genomes that come from non-gut environments, but at longer phylogenetic distances gut bacteria are more similar to each other than are genomes at equivalent evolutionary distances from non-gut environments, suggesting a pattern of short-term specialization but long-term convergence. Moreover, this pattern is observed in both pathogens and non-pathogens, and can even be seen in the plasmids carried by gut bacteria. This observation is consistent with the finding that, despite considerable interpersonal variation in species content, there is surprising functional convergence in the microbiome of different humans. Finally, we observe that even within bacterial species or genera 16S rRNA divergence provides useful information about average conservation of gene content. The results described here should be useful for guiding strain selection to maximize novel gene discovery in large-scale genome sequencing projects, while the approach could be applied in studies seeking to understand the effects of habitat adaptation on genome evolution across other body habitats or environment types.

One Health Relationships Between Human, Animal, and Environmental Microbiomes: A Mini-Review.

The One Health concept stresses the ecological relationships between human, animal, and environmental health. Much of the One Health literature to date has examined the transfer of pathogens from animals (e.g., emerging zoonoses) and the environment to humans. The recent rapid development of technology to perform high throughput DNA sequencing has expanded this view to include the study of entire microbial communities. Applying the One Health approach to the microbiome allows for consideration of both pathogenic and non-pathogenic microbial transfer between humans, animals, and the environment. We review recent research studies of such transmission, the molecular and statistical methods being used, and the implications of such microbiome relationships for human health. Our review identified evidence that the environmental microbiome as well as the microbiome of animals in close contact can affect both the human microbiome and human health outcomes. Such microbiome transfer can take place in the household as well as the workplace setting. Urbanization of built environments leads to changes in the environmental microbiome which could be a factor in human health. While affected by environmental exposures, the human microbiome also can modulate the response to environmental factors through effects on metabolic and immune function. Better understanding of these microbiome interactions between humans, animals, and the shared environment will require continued development of improved statistical and ecological modeling approaches. Such enhanced understanding could lead to innovative interventions to prevent and manage a variety of human health and disease states.

Coral-associated bacteria demonstrate phylosymbiosis and cophylogeny.

Scleractinian corals' microbial symbionts influence host health, yet how coral microbiomes assembled over evolution is not well understood. We survey bacterial and archaeal communities in phylogenetically diverse Australian corals representing more than 425 million years of diversification. We show that coral microbiomes are anatomically compartmentalized in both modern microbial ecology and evolutionary assembly. Coral mucus, tissue, and skeleton microbiomes differ in microbial community composition, richness, and response to host vs. environmental drivers. We also find evidence of coral-microbe phylosymbiosis, in which coral microbiome composition and richness reflect coral phylogeny. Surprisingly, the coral skeleton represents the most biodiverse coral microbiome, and also shows the strongest evidence of phylosymbiosis. Interactions between bacterial and coral phylogeny significantly influence the abundance of four groups of bacteria-including Endozoicomonas-like bacteria, which divide into host-generalist and host-specific subclades. Together these results trace microbial symbiosis across anatomy during the evolution of a basal animal lineage.

Best practices for analysing microbiomes.

Complex microbial communities shape the dynamics of various environments, ranging from the mammalian gastrointestinal tract to the soil. Advances in DNA sequencing technologies and data analysis have provided drastic improvements in microbiome analyses, for example, in taxonomic resolution, false discovery rate control and other properties, over earlier methods. In this Review, we discuss the best practices for performing a microbiome study, including experimental design, choice of molecular analysis technology, methods for data analysis and the integration of multiple omics data sets. We focus on recent findings that suggest that operational taxonomic unit-based analyses should be replaced with new methods that are based on exact sequence variants, methods for integrating metagenomic and metabolomic data, and issues surrounding compositional data analysis, where advances have been particularly rapid. We note that although some of these approaches are new, it is important to keep sight of the classic issues that arise during experimental design and relate to research reproducibility. We describe how keeping these issues in mind allows researchers to obtain more insight from their microbiome data sets.

Stress and stability: applying the Anna Karenina principle to animal microbiomes.

All animals studied to date are associated with symbiotic communities of microorganisms. These animal microbiotas often play important roles in normal physiological function and susceptibility to disease; predicting their responses to perturbation represents an essential challenge for microbiology. Most studies of microbiome dynamics test for patterns in which perturbation shifts animal microbiomes from a healthy to a dysbiotic stable state. Here, we consider a complementary alternative: that the microbiological changes induced by many perturbations are stochastic, and therefore lead to transitions from stable to unstable community states. The result is an 'Anna Karenina principle' for animal microbiomes, in which dysbiotic individuals vary more in microbial community composition than healthy individuals-paralleling Leo Tolstoy's dictum that "all happy families look alike; each unhappy family is unhappy in its own way". We argue that Anna Karenina effects are a common and important response of animal microbiomes to stressors that reduce the ability of the host or its microbiome to regulate community composition. Patterns consistent with Anna Karenina effects have been found in systems ranging from the surface of threatened corals exposed to above-average temperatures, to the lungs of patients suffering from HIV/AIDs. However, despite their apparent ubiquity, these patterns are easily missed or discarded by some common workflows, and therefore probably underreported. Now that a substantial body of research has established the existence of these patterns in diverse systems, rigorous testing, intensive time-series datasets and improved stochastic modelling will help to explore their importance for topics ranging from personalized medicine to theories of the evolution of host-microorganism symbioses.

Normalization and microbial differential abundance strategies depend upon data characteristics.

BACKGROUND: Data from 16S ribosomal RNA (rRNA) amplicon sequencing present challenges to ecological and statistical interpretation. In particular, library sizes often vary over several ranges of magnitude, and the data contains many zeros. Although we are typically interested in comparing relative abundance of taxa in the ecosystem of two or more groups, we can only measure the taxon relative abundance in specimens obtained from the ecosystems. Because the comparison of taxon relative abundance in the specimen is not equivalent to the comparison of taxon relative abundance in the ecosystems, this presents a special challenge. Second, because the relative abundance of taxa in the specimen (as well as in the ecosystem) sum to 1, these are compositional data. Because the compositional data are constrained by the simplex (sum to 1) and are not unconstrained in the Euclidean space, many standard methods of analysis are not applicable. Here, we evaluate how these challenges impact the performance of existing normalization methods and differential abundance analyses. RESULTS: Effects on normalization: Most normalization methods enable successful clustering of samples according to biological origin when the groups differ substantially in their overall microbial composition. Rarefying more clearly clusters samples according to biological origin than other normalization techniques do for ordination metrics based on presence or absence. Alternate normalization measures are potentially vulnerable to artifacts due to library size. Effects on differential abundance testing: We build on a previous work to evaluate seven proposed statistical methods using rarefied as well as raw data. Our simulation studies suggest that the false discovery rates of many differential abundance-testing methods are not increased by rarefying itself, although of course rarefying results in a loss of sensitivity due to elimination of a portion of available data. For groups with large (~10×) differences in the average library size, rarefying lowers the false discovery rate. DESeq2, without addition of a constant, increased sensitivity on smaller datasets (<20 samples per group) but tends towards a higher false discovery rate with more samples, very uneven (~10×) library sizes, and/or compositional effects. For drawing inferences regarding taxon abundance in the ecosystem, analysis of composition of microbiomes (ANCOM) is not only very sensitive (for >20 samples per group) but also critically the only method tested that has a good control of false discovery rate. CONCLUSIONS: These findings guide which normalization and differential abundance techniques to use based on the data characteristics of a given study.

Alien vs. predator: bacterial challenge alters coral microbiomes unless controlled by Halobacteriovorax predators.

Coral microbiomes are known to play important roles in organismal health, response to environmental stress, and resistance to disease. The coral microbiome contains diverse assemblages of resident bacteria, ranging from defensive and metabolic symbionts to opportunistic bacteria that may turn harmful in compromised hosts. However, little is known about how these bacterial interactions influence the mechanism and controls of overall structure, stability, and function of the microbiome. We sought to test how coral microbiome dynamics were affected by interactions between two bacteria: Vibrio coralliilyticus, a known temperature-dependent pathogen of some corals, and Halobacteriovorax, a unique bacterial predator of Vibrio and other gram-negative bacteria. We challenged reef-building coral with V. coralliilyticus in the presence or absence of Halobacteriovorax predators, and monitored microbial community dynamics with 16S rRNA gene profiling time-series. Vibrio coralliilyticus inoculation increased the mean relative abundance of Vibrios by greater than 35% from the 4 to 8 hour time point, but not in the 24 & 32 hour time points. However, strong secondary effects of the Vibrio challenge were also observed for the rest of the microbiome such as increased richness (observed species), and reduced stability (increased beta-diversity). Moreover, after the transient increase in Vibrios, two lineages of bacteria (Rhodobacterales and Cytophagales) increased in coral tissues, suggesting that V. coralliilyticus challenge opens niche space for these known opportunists. Rhodobacterales increased from 6.99% (±0.05 SEM) to a maximum mean relative abundance of 48.75% (±0.14 SEM) in the final time point and Cytophagales from <0.001% to 3.656%. Halobacteriovorax predators are commonly present at low-abundance on coral surfaces. Based on the keystone role of predators in many ecosystems, we hypothesized that Halobacteriovorax predators might help protect corals by consuming foreign or "alien" gram negative bacteria. Halobacteriovorax inoculation also altered the microbiome but to a lesser degree than V. coralliilyticus, and Halobacteriovorax were never detected after inoculation. Simultaneous challenge with both V. coralliilyticus and predatory Halobacteriovorax eliminated the increase in V. coralliilyticus, ameliorated changes to the rest of the coral microbiome, and prevented the secondary blooms of opportunistic Rhodobacterales and Cytophagales seen in the V. coralliilyticus challenge. These data suggest that, under certain circumstances, host-associated bacterial predators may mitigate the ability of other bacteria to destabilize the microbiome.

Bacterial predation in a marine host-associated microbiome.

In many ecological communities, predation has a key role in regulating community structure or function. Although predation has been extensively explored in animals and microbial eukaryotes, predation by bacteria is less well understood. Here we show that predatory bacteria of the genus Halobacteriovorax are prevalent and active predators on the surface of several genera of reef-building corals. Across a library of 198 16S rRNA samples spanning three coral genera, 79% were positive for carriage of Halobacteriovorax. Cultured Halobacteriovorax from Porites asteroides corals tested positive for predation on the putative coral pathogens Vibrio corallyticus and Vibrio harveyii. Co-occurrence network analysis showed that Halobacteriovorax's interactions with other bacteria are influenced by temperature and inorganic nutrient concentration, and further suggested that this bacterial predator's abundance may be driven by prey availability. Thus, animal microbiomes can harbor active bacterial predators, which may regulate microbiome structure and protect the host by consuming potential pathogens.

Overfishing and nutrient pollution interact with temperature to disrupt coral reefs down to microbial scales.

Losses of corals worldwide emphasize the need to understand what drives reef decline. Stressors such as overfishing and nutrient pollution may reduce resilience of coral reefs by increasing coral-algal competition and reducing coral recruitment, growth and survivorship. Such effects may themselves develop via several mechanisms, including disruption of coral microbiomes. Here we report the results of a 3-year field experiment simulating overfishing and nutrient pollution. These stressors increase turf and macroalgal cover, destabilizing microbiomes, elevating putative pathogen loads, increasing disease more than twofold and increasing mortality up to eightfold. Above-average temperatures exacerbate these effects, further disrupting microbiomes of unhealthy corals and concentrating 80% of mortality in the warmest seasons. Surprisingly, nutrients also increase bacterial opportunism and mortality in corals bitten by parrotfish, turning normal trophic interactions deadly for corals. Thus, overfishing and nutrient pollution impact reefs down to microbial scales, killing corals by sensitizing them to predation, above-average temperatures and bacterial opportunism.

Hidden state prediction: a modification of classic ancestral state reconstruction algorithms helps unravel complex symbioses.

Complex symbioses between animal or plant hosts and their associated microbiotas can involve thousands of species and millions of genes. Because of the number of interacting partners, it is often impractical to study all organisms or genes in these host-microbe symbioses individually. Yet new phylogenetic predictive methods can use the wealth of accumulated data on diverse model organisms to make inferences into the properties of less well-studied species and gene families. Predictive functional profiling methods use evolutionary models based on the properties of studied relatives to put bounds on the likely characteristics of an organism or gene that has not yet been studied in detail. These techniques have been applied to predict diverse features of host-associated microbial communities ranging from the enzymatic function of uncharacterized genes to the gene content of uncultured microorganisms. We consider these phylogenetically informed predictive techniques from disparate fields as examples of a general class of algorithms for Hidden State Prediction (HSP), and argue that HSP methods have broad value in predicting organismal traits in a variety of contexts, including the study of complex host-microbe symbioses.

Combined phylogenetic and genomic approaches for the high-throughput study of microbial habitat adaptation.

High-throughput sequencing technologies provide new opportunities to address longstanding questions about habitat adaptation in microbial organisms. How have microbes managed to adapt to such a wide range of environments, and what genomic features allow for such adaptation? We review recent large-scale studies of habitat adaptation, with emphasis on those that utilize phylogenetic techniques. On the basis of current trends, we summarize methodological challenges faced by investigators, and the tools, techniques and analytical approaches available to overcome them. Phylogenetic approaches and detailed information about each environmental sample will be crucial as the ability to collect genome sequences continues to expand.

Are all horizontal gene transfers created equal? Prospects for mechanism-based studies of HGT patterns.

Detecting patterns of horizontal gene transfer (HGT) in genomic sequences is an important problem, with implications for evolution, ecology, biotechnology and medicine. Extensive genetic, biochemical and genomic studies have provided a good understanding of sequence features that are associated with many (though not all) known mobile elements and mechanisms of gene transfer. This information, however, is not currently incorporated into automated methods for gene transfer detection in genomic data. In this review, we argue that automated annotation of sequence features associated with gene transfer mechanisms could be used both to build more sensitive, mechanism-specific compositional models for the detection of some types of HGT in genomic data, and to ask new questions about the classes of genes most frequently transferred by each mechanism. We then summarize the genes and sequence features associated with different mechanisms of horizontal transfer, emphasizing those that are most useful for distinguishing types of transfer when examining genomic data, and noting those classes of transfers that cannot be distinguished in genomic data using existing techniques. Finally, we describe software, databases and algorithms for identifying particular classes of mobile elements, and outline prospects for better detection of HGT based on specific mechanisms of transfer.