Tissue-associated and vertically transmitted bacterial symbiont in the coral Pocillopora acuta.

Coral microhabitats are colonized by a myriad of microorganisms, including diverse bacteria which are essential for host functioning and survival. However, the location, transmission, and functions of individual bacterial species living inside the coral tissues remain poorly studied. Here, we show that a previously undescribed bacterial symbiont of the coral Pocillopora acuta forms cell-associated microbial aggregates (CAMAs) within the mesenterial filaments. CAMAs were found in both adults and larval offspring, suggesting vertical transmission. In situ laser capture microdissection of CAMAs followed by 16S rRNA gene amplicon sequencing and shotgun metagenomics produced a near complete metagenome-assembled genome. We subsequently cultured the CAMA bacteria from Pocillopora acuta colonies, and sequenced and assembled their genomes. Phylogenetic analyses showed that the CAMA bacteria belong to an undescribed Endozoicomonadaceae genus and species, which we propose to name Candidatus Sororendozoicomonas aggregata gen. nov sp. nov. Metabolic pathway reconstruction from its genome sequence suggests this species can synthesize most amino acids, several B vitamins, and antioxidants, and participate in carbon cycling and prey digestion, which may be beneficial to its coral hosts. This study provides detailed insights into a new member of the widespread Endozoicomonadaceae family, thereby improving our understanding of coral holobiont functioning. Vertically transmitted, tissue-associated bacteria, such as Sororendozoicomonas aggregata may be key candidates for the development of microbiome manipulation approaches with long-term positive effects on the coral host.

Microbiome ecological memory and responses to repeated marine heatwaves clarify variation in coral bleaching and mortality.

Microbiomes are essential features of holobionts, providing their hosts with key metabolic and functional traits like resistance to environmental disturbances and diseases. In scleractinian corals, questions remain about the microbiome's role in resistance and resilience to factors contributing to the ongoing global coral decline and whether microbes serve as a form of holobiont ecological memory. To test if and how coral microbiomes affect host health outcomes during repeated disturbances, we conducted a large-scale (32 exclosures, 200 colonies, and 3 coral species sampled) and long-term (28 months, 2018-2020) manipulative experiment on the forereef of Mo'orea, French Polynesia. In 2019 and 2020, this reef experienced the two most severe marine heatwaves on record for the site. Our experiment and these events afforded us the opportunity to test microbiome dynamics and roles in the context of coral bleaching and mortality resulting from these successive and severe heatwaves. We report unique microbiome responses to repeated heatwaves in Acropora retusa, Porites lobata, and Pocillopora spp., which included: microbiome acclimatization in A. retusa, and both microbiome resilience to the first marine heatwave and microbiome resistance to the second marine heatwave in Pocillopora spp. Moreover, observed microbiome dynamics significantly correlated with coral species-specific phenotypes. For example, bleaching and mortality in A. retusa both significantly increased with greater microbiome beta dispersion and greater Shannon Diversity, while P. lobata colonies had different microbiomes across mortality prevalence. Compositional microbiome changes, such as changes to proportions of differentially abundant putatively beneficial to putatively detrimental taxa to coral health outcomes during repeated heat stress, also correlated with host mortality, with higher proportions of detrimental taxa yielding higher mortality in A. retusa. This study reveals evidence for coral species-specific microbial responses to repeated heatwaves and, importantly, suggests that host-dependent microbiome dynamics may provide a form of holobiont ecological memory to repeated heat stress.

Revealing General Patterns of Microbiomes That Transcend Systems: Potential and Challenges of Deep Transfer Learning.

A growing body of research has established that the microbiome can mediate the dynamics and functional capacities of diverse biological systems. Yet, we understand little about what governs the response of these microbial communities to host or environmental changes. Most efforts to model microbiomes focus on defining the relationships between the microbiome, host, and environmental features within a specified study system and therefore fail to capture those that may be evident across multiple systems. In parallel with these developments in microbiome research, computer scientists have developed a variety of machine learning tools that can identify subtle, but informative, patterns from complex data. Here, we recommend using deep transfer learning to resolve microbiome patterns that transcend study systems. By leveraging diverse public data sets in an unsupervised way, such models can learn contextual relationships between features and build on those patterns to perform subsequent tasks (e.g., classification) within specific biological contexts.

Best practices for generating and analyzing 16S rRNA amplicon data to track coral microbiome dynamics.

Over the past two decades, researchers have searched for methods to better understand the relationship between coral hosts and their microbiomes. Data on how coral-associated bacteria are involved in their host's responses to stressors that cause bleaching, disease, and other deleterious effects can elucidate how they may mediate, ameliorate, and exacerbate interactions between the coral and the surrounding environment. At the same time tracking coral bacteria dynamics can reveal previously undiscovered mechanisms of coral resilience, acclimatization, and evolutionary adaptation. Although modern techniques have reduced the cost of conducting high-throughput sequencing of coral microbes, to explore the composition, function, and dynamics of coral-associated bacteria, it is necessary that the entire procedure, from collection to sequencing, and subsequent analysis be carried out in an objective and effective way. Corals represent a difficult host with which to work, and unique steps in the process of microbiome assessment are necessary to avoid inaccuracies or unusable data in microbiome libraries, such as off-target amplification of host sequences. Here, we review, compare and contrast, and recommend methods for sample collection, preservation, and processing (e.g., DNA extraction) pipelines to best generate 16S amplicon libraries with the aim of tracking coral microbiome dynamics. We also discuss some basic quality assurance and general bioinformatic methods to analyze the diversity, composition, and taxonomic profiles of the microbiomes. This review aims to be a generalizable guide for researchers interested in starting and modifying the molecular biology aspects of coral microbiome research, highlighting best practices and tricks of the trade.

Inconsistent Patterns of Microbial Diversity and Composition Between Highly Similar Sequencing Protocols: A Case Study With Reef-Building Corals.

16S rRNA gene profiling (amplicon sequencing) is a popular technique for understanding host-associated and environmental microbial communities. Most protocols for sequencing amplicon libraries follow a standardized pipeline that can differ slightly depending on laboratory facility and user. Given that the same variable region of the 16S gene is targeted, it is generally accepted that sequencing output from differing protocols are comparable and this assumption underlies our ability to identify universal patterns in microbial dynamics through meta-analyses. However, discrepant results from a combined 16S rRNA gene dataset prepared by two labs whose protocols differed only in DNA polymerase and sequencing platform led us to scrutinize the outputs and challenge the idea of confidently combining them for standard microbiome analysis. Using technical replicates of reef-building coral samples from two species, Montipora aequituberculata and Porites lobata, we evaluated the consistency of alpha and beta diversity metrics between data resulting from these highly similar protocols. While we found minimal variation in alpha diversity between platform, significant differences were revealed with most beta diversity metrics, dependent on host species. These inconsistencies persisted following removal of low abundance taxa and when comparing across higher taxonomic levels, suggesting that bacterial community differences associated with sequencing protocol are likely to be context dependent and difficult to correct without extensive validation work. The results of this study encourage caution in the statistical comparison and interpretation of studies that combine rRNA gene sequence data from distinct protocols and point to a need for further work identifying mechanistic causes of these observed differences.

Interest in long-acting injectable buprenorphine among syringe services program participants.

OBJECTIVE: To examine syringe services program (SSP) participants' interest in long-acting injectable buprenorphine. DESIGN: SSP participants completed a 136-item questionnaire by phone. Items assessed quantitative ratings of interest in sublingual and injectable buprenorphine, preference for sublingual versus injectable buprenorphine, and reasons for preferences. SETTING: Two large urban SSPs. PARTICIPANTS: SSP participants ≥18 years of age with current or lifetime opioid use disorder (OUD). MAIN OUTCOME MEASURE(S): (1) Interest in sublingual and injectable buprenorphine, respectively, on a scale from 0 to 10 (0 = no interest and 10 = high interest); and (2) preference for sublingual buprenorphine versus injectable buprenorphine. Participants were also asked whether they agreed with statements that presented potential reasons for preferring each formulation. RESULTS: A total of 104 unique participants were interviewed, of which 72 (69 percent) were currently receiving or considering buprenorphine treatment. Among these 72 participants, the median level of interest in starting or continuing sublingual buprenorphine was 8 out of 10 (interquartile range [IQR]: 6-10) and in starting injectable buprenorphine was 5 out of 10 (IQR: 1-9). Thirty-six (50 percent) preferred sublingual, 27 (38 percent) preferred injectable, and 9 (13 percent) preferred neither or declined to answer. Participants who preferred injectable buprenorphine most commonly agreed that the convenience of the monthly injection was the reason for their preference. CONCLUSIONS: Among SSP participants with OUD, we found moderate interest in injectable buprenorphine. Introducing this new form of buprenorphine treatment at SSPs could help meet the needs of individuals who are not well-served by standard OUD treatment models.

Dynamic symbioses reveal pathways to coral survival through prolonged heatwaves.

Prospects for coral persistence through increasingly frequent and extended heatwaves seem bleak. Coral recovery from bleaching is only known to occur after temperatures return to normal, and mitigation of local stressors does not appear to augment coral survival. Capitalizing on a natural experiment in the equatorial Pacific, we track individual coral colonies at sites spanning a gradient of local anthropogenic disturbance through a tropical heatwave of unprecedented duration. Unexpectedly, some corals survived the event by recovering from bleaching while still at elevated temperatures. These corals initially had heat-sensitive algal symbiont communities, endured bleaching, and then recovered through proliferation of heat-tolerant symbionts. This pathway to survival only occurred in the absence of strong local stressors. In contrast, corals in highly disturbed areas were already dominated by heat-tolerant symbionts, and despite initially resisting bleaching, these corals had no survival advantage in one species and 3.3 times lower survival in the other. These unanticipated connections between disturbance, coral symbioses and heat stress resilience reveal multiple pathways to coral survival through future prolonged heatwaves.

Temporal Variation in the Microbiome of Acropora Coral Species Does Not Reflect Seasonality.

The coral microbiome is known to fluctuate in response to environmental variation and has been suggested to vary seasonally. However, most studies to date, particularly studies on bacterial communities, have examined temporal variation over a time frame of less than 1 year, which is insufficient to establish if microbiome variations are indeed seasonal in nature. The present study focused on expanding our understanding of long-term variability in microbial community composition using two common coral species, Acropora hyacinthus, and Acropora spathulata, at two mid-shelf reefs on the Great Barrier Reef. By sampling over a 2-year time period, this study aimed to determine whether temporal variations reflect seasonal cycles. Community composition of both bacteria and Symbiodiniaceae was characterized through 16S rRNA gene and ITS2 rDNA metabarcoding. We observed significant variations in community composition of both bacteria and Symbiodiniaceae among time points for A. hyacinthus and A. spathulata. However, there was no evidence to suggest that temporal variations were cyclical in nature and represented seasonal variation. Clear evidence for differences in the microbial communities found between reefs suggests that reef location and coral species play a larger role than season in driving microbial community composition in corals. In order to identify the basis of temporal patterns in coral microbial community composition, future studies should employ longer time series of sampling at sufficient temporal resolution to identify the environmental correlates of microbiome variation.

Parental and early life stage environments drive establishment of bacterial and dinoflagellate communities in a common coral.

The establishment of coral microbial communities in early developmental stages is fundamental to coral fitness, but its drivers are largely unknown, particularly for bacteria. Using an in situ reciprocal transplant experiment, we examined the influence of parental, planulation and early recruit environments on the microbiome of brooded offspring in the coral Pocillopora damicornis. 16S rRNA and ITS2 rDNA gene metabarcoding showed that bacterial and microalgal endosymbiont communities varied according to parental and planulation environments, but not with early recruit environment. Only a small number of bacterial strains were shared between offspring and their respective parents, revealing bacterial establishment as largely environmentally driven in very early life stages. Conversely, microalgal communities of recruits were highly similar to those of their respective parents, but also contained additional low abundance strains, suggesting both vertical transmission and novel ('horizontal') acquisition. Altogether, recruits harboured more variable microbiomes compared to their parents, indicating winnowing occurs as corals mature.

The molecular basis of differential morphology and bleaching thresholds in two morphs of the coral Pocillopora acuta.

Processes of cnidarian evolution, including hybridization and phenotypic plasticity, have complicated the clear diagnosis of species boundaries within the phylum. Pocillopora acuta, a species of scleractinian coral that was recently split from the widespread Pocillopora damicornis species complex, occurs in at least two distinct morphs on the Great Barrier Reef. Contrasting morphology combined with evidence of differential bleaching thresholds among sympatrically distributed colonies suggest that the taxonomy of this recently described species is not fully resolved and may represent its own species complex. To examine the basis of sympatric differentiation between the two morphs, we combined analyses of micro- and macro-skeletal morphology with genome wide sequencing of the coral host, as well as ITS2 genotyping of the associated Symbiodinium communities. We found consistent differences between morphs on both the macro- and micro-skeletal scale. In addition, we identified 18 candidate functional genes that relate to skeletal formation and morphology that may explain how the two morphs regulate growth to achieve their distinct growth forms. With inconclusive results in endosymbiotic algal community diversity between the two morphs, we propose that colony morphology may be linked to bleaching susceptibility. We conclude that cryptic speciation may be in the early stages within the species P. acuta.

Shifting paradigms in restoration of the world's coral reefs.

Many ecosystems around the world are rapidly deteriorating due to both local and global pressures, and perhaps none so precipitously as coral reefs. Management of coral reefs through maintenance (e.g., marine-protected areas, catchment management to improve water quality), restoration, as well as global and national governmental agreements to reduce greenhouse gas emissions (e.g., the 2015 Paris Agreement) is critical for the persistence of coral reefs. Despite these initiatives, the health and abundance of corals reefs are rapidly declining and other solutions will soon be required. We have recently discussed options for using assisted evolution (i.e., selective breeding, assisted gene flow, conditioning or epigenetic programming, and the manipulation of the coral microbiome) as a means to enhance environmental stress tolerance of corals and the success of coral reef restoration efforts. The 2014-2016 global coral bleaching event has sharpened the focus on such interventionist approaches. We highlight the necessity for consideration of alternative (e.g., hybrid) ecosystem states, discuss traits of resilient corals and coral reef ecosystems, and propose a decision tree for incorporating assisted evolution into restoration initiatives to enhance climate resilience of coral reefs.

Fine-scale detection of pollutants by a benthic marine jellyfish.

Local sources of pollution can vary immensely on small geographic scales and short time frames due to differences in runoff and adjacent land use. This study examined the rate of uptake and retention of trace metals in Cassiopea maremetens, a benthic marine jellyfish, over a short time frame and in the presence of multiple pollutants. This study also validated the ability of C. maremetens to uptake metals in the field. Experimental manipulation demonstrated that metal accumulation in jellyfish tissue began within 24h of exposure to treated water and trended for higher accumulation in the presence of multiple pollutants. C. maremetens was found to uptake trace metals in the field and provide unique signatures among locations. This fine-scale detection and rapid accumulation of metals in jellyfish tissue can have major implications for both biomonitoring and the trophic transfer of pollutants through local ecosystems.