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As coral reef ecosystems experience unprecedented change, effective monitoring of reef features supports management, conservation, and intervention efforts. Omic techniques show promise in quantifying key components of reef ecosystems including dissolved metabolites and microorganisms that may serve as invisible sensors for reef ecosystem dynamics. Dissolved metabolites are released by reef organisms and transferred among microorganisms, acting as chemical currencies and contributing to nutrient cycling and signaling on reefs. Here, we applied four omic techniques (taxonomic microbiome via amplicon sequencing, functional microbiome via shotgun metagenomics, targeted metabolomics, and untargeted metabolomics) to waters overlying Floridas Coral Reef, as well as microbiome profiling on individual coral colonies from these reefs to understand how microbes and dissolved metabolites reflect biogeographical, benthic, and nutrient properties of this 500-km barrier reef. We show that the microbial and metabolite omic approaches each differentiated reef habitats based on geographic zone. Further, seawater microbiome profiling and targeted metabolomics were significantly related to more reef habitat characteristics, such as amount of hard and soft coral, compared to metagenomic sequencing and untargeted metabolomics. Across five coral species, microbiomes were also significantly related to reef zone, followed by species and disease status, suggesting that the geographic water circulation patterns in Florida also impact the microbiomes of reef builders. A combination of differential abundance and indicator species analyses revealed metabolite and microbial signatures of specific reef zones, which demonstrates the utility of these techniques to provide new insights into reef microbial and metabolite features that reflect broader ecosystem processes.

Stony coral tissue loss disease (SCTLD), one of the most pervasive and virulent coral diseases on record, affects over 22 species of reef-building coral and is decimating reefs throughout the Caribbean. To understand how different coral species and their algal symbionts (family Symbiodiniaceae) respond to this disease, we examine the gene expression profiles of colonies of five species of coral from a SCTLD transmission experiment. The included species vary in their purported susceptibilities to SCTLD, and we use this to inform gene expression analyses of both the coral animal and their Symbiodiniaceae. We identify orthologous coral genes exhibiting lineage-specific differences in expression that correlate to disease susceptibility, as well as genes that are differentially expressed in all coral species in response to SCTLD infection. We find that SCTLD infection induces increased expression of rab7, an established marker of in situ degradation of dysfunctional Symbiodiniaceae, in all coral species accompanied by genus-level shifts in Symbiodiniaceae photosystem and metabolism gene expression. Overall, our results indicate that SCTLD infection induces symbiophagy across coral species and that the severity of disease is influenced by Symbiodiniaceae identity.

Quiescence, or dormancy, is a response to stressful conditions in which an organism slows or halts physiological functioning. Although most species that undergo dormancy maintain complex microbiomes, there is little known about how dormancy influences and is influenced by the host's microbiome, including in the temperate coral Astrangia poculata. Northern populations of A. poculata undergo winter quiescence. Here, we characterized wild A. poculata microbiomes in a high-resolution sampling time series before, during, and after quiescence using 16S rRNA gene sequencing on active (RNA) and present (DNA) microbiomes. We observed a restructuring of the coral microbiome during quiescence that persisted after reemergence. Upon entering quiescence, corals shed copiotrophic microbes, including putative pathogens, suggesting a removal of these taxa as corals cease normal functioning. During and after quiescence, bacteria and archaea associated with nitrification were enriched, suggesting that the quiescent microbiome may replace essential functions through supplying nitrate to corals and/or microbes. Overall, this study demonstrates that key microbial groups related to quiescence in A. poculata may play a role in the onset or emergence from dormancy and long-term regulation of the microbiome composition. The predictability of dormancy in A. poculata provides an ideal natural manipulation system to further identify factors that regulate host-microbial associations. IMPORTANCE Using a high-resolution sampling time series, this study is the first to demonstrate a persistent microbial community shift with quiescence (dormancy) in a marine organism, the temperate coral Astrangia poculata. Furthermore, during this period of community turnover, there is a shedding of putative pathogens and copiotrophs and an enhancement of the ammonia-oxidizing bacteria (Nitrosococcales) and archaea ("Candidatus Nitrosopumilus"). Our results suggest that quiescence represents an important period during which the coral microbiome can reset, shedding opportunistic microbes and enriching for the reestablishment of beneficial associates, including those that may contribute nitrate while the coral animal is not actively feeding. We suggest that this work provides foundational understanding of the interplay of microbes and the host's dormancy response in marine organisms.

Marine multicellular organisms host a diverse collection of bacteria, archaea, microbial eukaryotes, and viruses that form their microbiome. Such host-associated microbes can significantly influence the host's physiological capacities; however, the identity and functional role(s) of key members of the microbiome ("core microbiome") in most marine hosts coexisting in natural settings remain obscure. Also unclear is how dynamic interactions between hosts and the immense standing pool of microbial genetic variation will affect marine ecosystems' capacity to adjust to environmental changes. Here, we argue that significantly advancing our understanding of how host-associated microbes shape marine hosts' plastic and adaptive responses to environmental change requires (i) recognizing that individual host-microbe systems do not exist in an ecological or evolutionary vacuum and (ii) expanding the field toward long-term, multidisciplinary research on entire communities of hosts and microbes. Natural experiments, such as time-calibrated geological events associated with well-characterized environmental gradients, provide unique ecological and evolutionary contexts to address this challenge. We focus here particularly on mutualistic interactions between hosts and microbes, but note that many of the same lessons and approaches would apply to other types of interactions.

Coral reef health depends on an intricate relationship among the coral animal, photosynthetic algae, and a complex microbial community. The holobiont can impact the nutrient balance of their hosts amid an otherwise oligotrophic environment, including by cycling physiologically important nitrogen compounds. Here we use 15N-tracer experiments to produce the first simultaneous measurements of ammonium oxidation, nitrate reduction, and nitrous oxide (N2O) production among five iconic species of reef-building corals (Acropora palmata, Diploria labyrinthiformis, Orbicella faveolata, Porites astreoides, and Porites porites) in the highly protected Jardines de la Reina reefs of Cuba. Nitrate reduction is present in most species, but ammonium oxidation is low potentially due to photoinhibition and assimilatory competition. Coral-associated rates of N2O production indicate a widespread potential for denitrification, especially among D. labyrinthiformis, at rates of ~1 nmol cm-2 d-1. In contrast, A. palmata displays minimal active nitrogen metabolism. Enhanced rates of nitrate reduction and N2O production are observed coincident with dark net respiration periods. Genomes of bacterial cultures isolated from multiple coral species confirm that microorganisms with the ability to respire nitrate anaerobically to either dinitrogen gas or ammonium exist within the holobiont. This confirmation of anaerobic nitrogen metabolisms by coral-associated microorganisms sheds new light on coral and reef productivity.

Biofilm-forming bacteria have the potential to contribute to the health, physiology, behavior and ecology of the host and serve as its first line of defense against adverse conditions in the environment. While metabarcoding and metagenomic information furthers our understanding of microbiome composition, fewer studies use cultured samples to study the diverse interactions among the host and its microbiome, as cultured representatives are often lacking. This study examines the surface microbiomes cultured from three shallow-water coral species and two whale species. These unique marine animals place strong selective pressures on their microbial symbionts and contain members under similar environmental and anthropogenic stress. We developed an intense cultivation procedure, utilizing a suite of culture conditions targeting a rich assortment of biofilm-forming microorganisms. We identified 592 microbial isolates contained within 15 bacterial orders representing 50 bacterial genera, and two fungal species. Culturable bacteria from coral and whale samples paralleled taxonomic groups identified in culture-independent surveys, including 29% of all bacterial genera identified in the Megaptera novaeangliae skin microbiome through culture-independent methods. This microbial repository provides raw material and biological input for more nuanced studies which can explore how members of the microbiome both shape their micro-niche and impact host fitness.

There are a few baseline reef-systems available for understanding the microbiology of healthy coral reefs and their surrounding seawater. Here, we examined the seawater microbial ecology of 25 Northern Caribbean reefs varying in human impact and protection in Cuba and the Florida Keys, USA, by measuring nutrient concentrations, microbial abundances, and respiration rates as well as sequencing bacterial and archaeal amplicons and community functional genes. Overall, seawater microbial composition and biogeochemistry were influenced by reef location and hydrogeography. Seawater from the highly protected crown jewel offshore reefs in Jardines de la Reina, Cuba had low concentrations of nutrients and organic carbon, abundant Prochlorococcus, and high microbial community alpha diversity. Seawater from the less protected system of Los Canarreos, Cuba had elevated microbial community beta-diversity whereas waters from the most impacted nearshore reefs in the Florida Keys contained high organic carbon and nitrogen concentrations and potential microbial functions characteristic of microbialized reefs. Each reef system had distinct microbial signatures and within this context, we propose that the protection and offshore nature of Jardines de la Reina may preserve the oligotrophic paradigm and the metabolic dependence of the community on primary production by picocyanobacteria.

The skin is the first line of defense between an animal and its environment, and disruptions in skin-associated microorganisms can be linked to an animals health and nutritional state. To better understand the skin microbiome of large whales, high-throughput sequencing of partial small subunit rRNA genes was used to study the skin-associated bacteria of 89 seemingly healthy humpback whales (Megaptera novaeangliae) sampled along the Western Antarctic Peninsula (WAP) during early (2010) and late (2013) austral summers. Six core groups of bacteria were present in 93% or more of all humpback skin samples. A shift was observed in the average relative abundances of these core bacteria over time, with the emergence of four additional core groups of bacteria that corresponded to a decrease in water temperature, possibly caused by season- or foraging-related changes in skin biochemistry that influenced microbial growth, or other temporal factors. The skin microbiome differed between whales sampled at several regional locations along the WAP, suggesting that environmental factors or population may also influence the whale skin microbiome. Overall, the skin microbiome of humpback whales appears to provide insight into animal- and environment-related factors and may serve as a useful indicator for animal health or ecosystem alterations.IMPORTANCE The microbiomes of wild animals are currently understudied but may provide information about animal health and/or animal-environment interactions. In the largest sampling of any marine mammal microbiome, this study demonstrates conservation in the skin microbiome of 89 seemingly healthy humpback whales sampled in the Western Antarctic Peninsula, with shifts in the bacterial community composition related to temporal and regional variability. This study is important because it suggests that the skin microbiome of humpback whales could provide insight into animal nutritional or seasonal/environment-related factors, which are becoming increasingly important to recognize due to unprecedented rates of climate change and anthropogenic impact on ocean ecosystems.

Acoustic cues of healthy reefs are known to support critical settlement behaviors for one reef-building coral, but acoustic responses have not been demonstrated in additional species. Settlement of Favia fragum larvae in response to replayed coral reef soundscapes were observed by exposing larvae in aquaria and reef settings to playback sound treatments for 24–72 h. Settlement increased under 24 h sound treatments in both experiments. The results add to growing knowledge that acoustically mediated settlement may be widespread among stony corals with species-specific attributes, suggesting sound could be one tool employed to rehabilitate and build resilience within imperiled reef communities. [ABSTRACT FROM AUTHOR]

The recent rise of 'omics and other molecular research technologies alongside improved techniques for tissue preservation have broadened the scope of marine mammal research. Collecting biological samples from wild marine mammals is both logistically challenging and expensive. To enhance the power of marine mammal research, great effort has been made in both the field and the laboratory to ensure the scientific integrity of samples from collection through processing, supporting the long‐term use of precious samples across a broad range of studies. However, identifying the best methods of sample preservation can be challenging, especially as this technological toolkit continues to evolve and expand. Standardizing best practices could maximize the scientific value of biological samples, foster multi‐institutional collaborative efforts across fields, and improve the quality of individual studies by removing potential sources of error from the collection, handling, and preservation processes. With these aims in mind, we summarize relevant literature, share current expert knowledge, and suggest best practices for sample collection and preservation. This manuscript is intended as a reference resource for scientists interested in exploring collaborative studies and preserving samples in a suitable manner for a broad spectrum of analyses, emphasizing support for 'omics technologies. [ABSTRACT FROM AUTHOR]

Globally, coral reef ecosystems are undergoing significant change related to climate change and anthropogenic activities. Yet, the Cuban archipelago of Jardines de la Reina (JR) has experienced fewer stressors due to its geographical remoteness and high level of conservation. This study examines the surface and benthic reef water microbial communities associated with 32 reef sites along the JR archipelago and explores the relationship between the community composition of reef microorganisms examined using bacterial and archaeal small subunit ribosomal RNA gene (16S rRNA gene) sequencing compared to geographic, conservation/protection level, environmental, physicochemical, and reef benthic and pelagic community features. Reef nutrient concentrations were low and microbial communities dominated by picocyanobacteria and SAR11 and SAR86 clade bacteria, characteristic of an oligotrophic system. Reef water microbial community alpha and beta diversity both varied throughout the archipelago and were strongly related to geography. Three sites in the western archipelago showed unique microbial communities, which may be related to the hydrogeography and influences of the channels linking the Ana Maria gulf with the Caribbean Sea. Overall, this work provides the first extensive description of the reef microbial ecology of the Caribbean's 'Crown Jewel' reef system and a framework to evaluate the influence of ongoing stressors on the reef microorganisms. [ABSTRACT FROM AUTHOR]

Coral reefs are biodiverse marine ecosystems that are undergoing rapid changes, making monitoring vital as we seek to manage and mitigate stressors. Healthy reef soundscapes are rich with sounds, enabling passive acoustic recording and soundscape analyses to emerge as cost-effective, long-term methods for monitoring reef communities. Yet most biological reef sounds have not been identified or described, limiting the effectiveness of acoustic monitoring for diversity assessments. Machine learning offers a solution to scale such analyses but has yet to be successfully applied to characterize the diversity of reef fish sounds. Here we sought to characterize and categorize coral reef fish sounds using unsupervised machine learning methods. Pulsed fish and invertebrate sounds from 480 min of data sampled across 10 days over a 2-month period on a US Virgin Islands reef were manually identified and extracted, then grouped into acoustically similar clusters using unsupervised clustering based on acoustic features. The defining characteristics of these clusters were described and compared to determine the extent of acoustic diversity detected on these reefs. Approximately 55 distinct calls were identified, ranging in centroid frequency from 50 Hz to 1,300 Hz. Within this range, two main sub-bands containing multiple signal types were identified from 100 Hz to 400 Hz and 300 Hz-700 Hz, with a variety of signals outside these two main bands. These methods may be used to seek out acoustic diversity across additional marine habitats. The signals described here, though taken from a limited dataset, speak to the diversity of sounds produced on coral reefs and suggest that there might be more acoustic niche differentiation within soniferous fish communities than has been previously recognized. [ABSTRACT FROM AUTHOR]

Designing primers for PCR-based taxonomic surveys that amplify a broad range of phylotypes in varied community samples is a difficult challenge, and the comparability of data sets amplified with varied primers requires attention. Here, we examined the performance of modified 16S rRNA gene and internal transcribed spacer (ITS) primers for archaea/bacteria and fungi, respectively, with nonaquatic samples. We moved primer bar codes to the 5' end, allowing for a range of different 3' primer pairings, such as the 515f/926r primer pair, which amplifies variable regions 4 and 5 of the 16S rRNA gene. We additionally demonstrated that modifications to the 515f/806r (variable region 4) 16S primer pair, which improves detection of Thaumarchaeota and clade SAR11 in marine samples, do not degrade performance on taxa already amplified effectively by the original primer set. Alterations to the fungal ITS primers did result in differential but overall improved performance compared to the original primers. In both cases, the improved primers should be widely adopted for amplicon studies. IMPORTANCE We continue to uncover a wealth of information connecting microbes in important ways to human and environmental ecology. As our scientific knowledge and technical abilities improve, the tools used for microbiome surveys can be modified to improve the accuracy of our techniques, ensuring that we can continue to identify groundbreaking connections between microbes and the ecosystems they populate, from ice caps to the human body. It is important to confirm that modifications to these tools do not cause new, detrimental biases that would inhibit the field rather than continue to move it forward. We therefore demonstrated that two recently modified primer pairs that target taxonomically discriminatory regions of bacterial and fungal genomic DNA do not introduce new biases when used on a variety of sample types, from soil to human skin. This confirms the utility of these primers for maintaining currently recommended microbiome research techniques as the state of the art.

The diverse prokaryotic communities associated with reef-building corals may provide important ecological advantages to their threatened hosts. The consistency of relationships between corals and specific prokaryotes, however, is debated, and the locations where microbially mediated processes occur in the host are not resolved. Here, we examined how the prokaryotic associates of five common Caribbean corals with different evolutionary and ecological traits differ across mucus and tissue habitats. We used physical and chemical separation of coral mucus and tissue and sequencing of partial small-subunit rRNA genes of bacteria and archaea from these samples to demonstrate that coral tissue and mucus harbor unique reservoirs of prokaryotes, with 23 to 49% and 31 to 56% of sequences exclusive to the tissue and mucus habitats, respectively. Across all coral species, we found that 46 tissue- and 22 mucus-specific microbial members consistently associated with the different habitats. Sequences classifying as Candidatus Amoebophilus, Bacteroidetes-affiliated intracellular symbionts of amoebae, emerged as previously unrecognized tissue associates of three coral species. This study demonstrates how coral habitat differentiation enables highly resolved examination of ecological interactions between corals and their associated microorganisms and identifies previously unrecognized tissue and mucus associates of Caribbean corals for future targeted study. IMPORTANCE This study demonstrates that coral tissue or mucus habitats structure the microbiome of corals and that separation of these habitats facilitates identification of consistent microbial associates. Using this approach, we demonstrated that sequences related to Candidatus Amoebophilus, recognized intracellular symbionts of amoebae, were highly associated with the tissues of Caribbean corals and possibly endosymbionts of a protistan host within corals, adding a further degree of intricacy to coral holobiont symbioses. Examining specific habitats within complex hosts such as corals is useful for targeting important microbial associations that may otherwise be masked by the sheer microbial diversity associated with all host habitats.

An understanding of the mechanistic drivers of animal‐microbial symbiosis and associations generally requires experiments that manipulate specific symbionts or microbial communities. As part of an ongoing study of the mechanisms that drive microbial communities in coral reef fishes, and specifically the role of cleanerfish in microbial transmission, we tested the effects of the commonly used antibiotics Enrofloxacin and Nitrofurazone on the skin microbiome of three ornamental reef fish species: the four‐eyed butterflyfish, Chaetodon capistratus (Chaetodontidae), the cleanerfish neon goby Elacatinus oceanops (Gobiidae) and the beaugregory damselfish Stegastes leucostictus (Pomacentridae). Our main aim was to characterize dysbiosis prompted by the delivery of the two antibiotics and understand whether both could be used to establish good starting points for microbial transmission experiments. We bathed the fish in antibiotic (or no exposure in the controls), sampled the skin microbiota via swabbing at zero (before treatment) and 3 and 7 days during the treatments, and examined the microbial community using a 16S rRNA gene sequencing approach. Nitrofurazone reduced skin‐associated microbial diversity in all species, whereas the same effect for Enrofloxacin was only seen in S. leucostictus. Although each antibiotic had its own, unique impact in microbial community, all treatments showed positive and negative shifts in the most abundant microbial taxa over time. Moreover, soon after the delivery of both antibiotics, increases in the abundance of opportunistic bacteria or potential pathogens, such as Alteromonas and Vibrio, were observed. Although both antibiotics are effective, Nitrofurazone more successfully reduces microbial diversity and therefore may be more ideal for experiments seeking to disrupt fish microbiomes. [ABSTRACT FROM AUTHOR]

Coral reef ecosystems are now commonly affected by major climate and disease disturbances. Disturbance impacts are typically recorded using reef benthic cover, but this may be less reflective of other ecosystem processes. To explore the potential for reef water‐based disturbance indicators, we conducted a 7‐year time series on US Virgin Island reefs where we examined benthic cover and reef water nutrients and microorganisms from 2016 to 2022, which included two major disturbances: hurricanes Irma and Maria in 2017 and the stony coral tissue loss disease outbreak starting in 2020. The disease outbreak coincided with the largest changes in the benthic habitat, with increases in the percent cover of turf algae and Ramicrusta, an invasive alga. While sampling timepoint contributed most to changes in reef water nutrient composition and microbial community beta diversity, both disturbances led to increases in ammonium concentration, a mechanism likely contributing to observed microbial community shifts. We identified 10 microbial taxa that were sensitive and predictive of increasing ammonium concentration. This included the decline of the oligotrophic and photoautotrophic Prochlorococcus and the enrichment of heterotrophic taxa. As disturbances impact reefs, the changing nutrient and microbial regimes may foster a type of microbialization, a process that hastens reef degradation. [ABSTRACT FROM AUTHOR]