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Coral diseases contribute to the rapid decline in coral reefs worldwide, and yet coral bacterial pathogens have proved difficult to identify because 16S rRNA gene surveys typically identify tens to hundreds of disease-associate bacteria as putative pathogens. An example is white band disease (WBD), which has killed up to 95% of the now-endangered Caribbean Acropora corals since 1979, yet the pathogen is still unknown. The 16S rRNA gene surveys have identified hundreds of WBD-associated bacterial amplicon sequencing variants (ASVs) from at least nine bacterial families with little consensus across studies. We conducted a multi-year, multi-site 16S rRNA gene sequencing comparison of 269 healthy and 143 WBD-infected Acropora cervicornis and used machine learning modelling to accurately predict disease outcomes and identify the top ASVs contributing to disease. Our ensemble ML models accurately predicted disease with greater than 97% accuracy and identified 19 disease-associated ASVs and five healthy-associated ASVs that were consistently differentially abundant across sampling periods. Using a tank-based transmission experiment, we tested whether the 19 disease-associated ASVs met the assumption of a pathogen and identified two pathogenic candidate ASVs-ASV25 Cysteiniphilum litorale and ASV8 Vibrio sp. to target for future isolation, cultivation, and confirmation of Henle-Koch's postulate via transmission assays.

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Coral species in the genus Acropora are key ecological components of coral reefs worldwide and represent the most diverse genus of scleractinian corals. While key species of Indo-Pacific Acropora have annotated genomes, no annotated genome has been published for either of the two species of Caribbean Acropora. Here we present the first fully annotated genome of the endangered Caribbean staghorn coral, Acropora cervicornis. We assembled and annotated this genome using high-fidelity nanopore long-read sequencing with gene annotations validated with mRNA sequencing. The assembled genome size is 318 Mb, with 28,059 validated genes. Comparative genomic analyses with other Acropora revealed unique features in A. cervicornis, including contractions in immune pathways and expansions in signaling pathways. Phylogenetic analysis confirms previous findings showing that A. cervicornis diverged from Indo-Pacific relatives around 41 million years ago, with the closure of the western Tethys Sea, prior to the primary radiation of Indo-Pacific Acropora. This new A. cervicornis genome enriches our understanding of the speciose Acropora and addresses evolutionary inquiries concerning speciation and hybridization in this diverse clade.

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White band disease (WBD) has caused unprecedented declines in the Caribbean Acropora corals, which are now listed as critically endangered species. Highly disease-resistant Acropora cervicornis genotypes exist, but the genetic underpinnings of disease resistance are not understood. Using transmission experiments, a newly assembled genome, and whole-genome resequencing of 76 A. cervicornis genotypes from Florida and Panama, we identified 10 genomic regions and 73 single-nucleotide polymorphisms that are associated with disease resistance and that include functional protein-coding changes in four genes involved in coral immunity and pathogen detection. Polygenic scores calculated from 10 genomic loci indicate that genetic screens can detect disease resistance in wild and nursery stocks of A. cervicornis across the Caribbean.

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Although stability is relatively well understood in macro-organisms, much less is known about its drivers in host-microbial systems where processes operating at multiple levels of biological organisation jointly regulate the microbiome. We conducted an experiment to examine the microbiome stability of three Caribbean corals (Acropora cervicornis, Pseudodiploria strigosa and Porites astreoides) by placing them in aquaria and exposing them to a pulse perturbation consisting of a large dose of broad-spectrum antibiotics before transplanting them into the field. We found that coral hosts harboured persistent, species-specific microbiomes. Stability was generally high but variable across coral species, with A. cervicornis microbiomes displaying the lowest community turnover in both the non-perturbed and the perturbed field transplants. Interestingly, the microbiome of P. astreoides was stable in the non-perturbed field transplants, but unstable in the perturbed field transplants. A mathematical model of host-microbial dynamics helped resolve this paradox by showing that when microbiome regulation is driven by host sanctioning, both resistance and resilience to invasion are low and can lead to instability despite the high direct costs bourne by corals. Conversely, when microbiome regulation is mainly associated with microbial processes, both resistance and resilience to invasion are high and promote stability at no direct cost to corals. We suggest that corals that are mainly regulated by microbial processes can be likened to 'glass cannons' because the high stability they exhibit in the field is due to their microbiome's potent suppression of invasive microbes. However, these corals are susceptible to destabilisation when exposed to perturbations that target the vulnerable members of their microbiomes who are responsible for mounting such powerful attacks against invasive microbes. The differential patterns of stability exhibited by P. astreoides across perturbed and non-perturbed field transplants suggest it is a 'glass cannon' whose microbiome is regulated by microbial processes, whereas A. cervicornis' consistent patterns of stability suggest that its microbiome is mainly regulated by host-level processes. Our results show that understanding how processes that operate at multiple levels of biological organisation interact to regulate microbiomes is critical for predicting the effects of environmental perturbations on host-microbial systems.

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The traditional view of innate immunity in insects is that every exposure to a pathogen triggers an identical and appropriate immune response and that prior exposures to pathogens do not confer any protective (i.e., adaptive) effect against subsequent exposure to the same pathogen. This view has been challenged by experiments demonstrating that encounters with sublethal doses of a pathogen can prime the insect's immune system and, thus, have protective effects against future lethal doses. Immune priming has been reported across several insect species, including the red flour beetle, the honeycomb moth, the bumblebee, and the European honeybee, among others. Immune priming can also be transgenerational where the parent's pathogenic history influences the immune response of its offspring. Phenotypic evidence of transgenerational immune priming (TGIP) exists in the tobacco moth Manduca sexta where first-instar progeny of mothers injected with the bacterium Serratia marcescens exhibited a significant increase of in vivo bacterial clearance. To identify the gene expression changes underlying TGIP in M. sexta, we performed transcriptome-wide, transgenerational differential gene expression analysis on mothers and their offspring after mothers were exposed to S. marcescens. We are the first to perform transcriptome-wide analysis of the gene expression changes associated with TGIP in this ecologically relevant model organism. We show that maternal exposure to both heat-killed and live S. marcescens has strong and significant transgenerational impacts on gene expression patterns in their offspring, including upregulation of peptidoglycan recognition protein, toll-like receptor 9, and the antimicrobial peptide cecropin.

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Anthozoans are a class of Cnidarians that includes scleractinian corals, anemones, and their relatives. Despite a global rise in disease epizootics impacting scleractinian corals, little is known about the immune response of this key group of invertebrates. To better characterize the anthozoan immune response, we used the model anemone Exaiptasia pallida to explore the genetic links between the anthozoan-algal symbioses and immunity in a two-factor RNA-Seq experiment using both symbiotic and aposymbiotic (menthol-bleached) Exaiptasia pallida exposed to the bacterial pathogen Vibrio coralliilyticus. Multivariate and univariate analyses of Exaiptasia gene expression demonstrated that exposure to live Vibrio coralliilyticus had strong and significant impacts on transcriptome-wide gene expression for both symbiotic and aposymbiotic anemones, but we did not observe strong interactions between symbiotic state and Vibrio exposure. There were 4,164 significantly differentially expressed (DE) genes for Vibrio exposure, 1,114 DE genes for aposymbiosis, and 472 DE genes for the additive combinations of Vibrio and aposymbiosis. KEGG enrichment analyses identified 11 pathways-involved in immunity (5), transport and catabolism (4), and cell growth and death (2)-that were enriched due to both Vibrio and/or aposymbiosis. Immune pathways showing strongest differential expression included complement, coagulation, nucleotide-binding, and oligomerization domain (NOD), and Toll for Vibrio exposure and coagulation and apoptosis for aposymbiosis.

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Although it is well established that the microbial communities inhabiting corals perform key functions that promote the health and persistence of their hosts, little is known about their spatial structure and temporal stability. We examined the natural variability of microbial communities associated with six Caribbean coral species from three genera at four reef sites over one year. We identified differences in microbial community composition between coral genera and species that persisted across space and time, suggesting that local host identity likely plays a dominant role in structuring the microbiome. However, we found that microbial community dissimilarity increased with geographical distance, which indicates that regional processes such as dispersal limitation and spatiotemporal environmental heterogeneity also influence microbial community composition. In addition, network analysis revealed that the strength of host identity varied across coral host genera, with species from the genus Acropora having the most influence over their microbial community. Overall, our results demonstrate that despite high levels of microbial diversity, coral species are characterized by signature microbiomes that are stable in both space and time.

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Among the greatest threats to coral reefs are coral epizootics, which are increasing in frequency and severity across many reef ecosystems. In particular, white band disease (WBD) has devastated Caribbean acroporid populations since its initial outbreak in 1979. However, despite its widespread and damaging effects, the aetiology of WBD remains largely unresolved. Here, we examine the role of quorum sensing within bacterial communities associated with WBD-infected Acropora cervicornis. Microbial communities isolated from WBD-infected corals were exposed to quorum sensing inhibitor (QSI) - a N-acyl homoserine lactone autoinducer antagonist - and then dosed onto healthy test corals. WBD-associated bacteria supplemented with QSI lost the ability to establish disease, while healthy corals exposed to uninhibited WBD bacterial communities became infected within two days. Microbial 16S rRNA metagenomic sequencing analyses were then used to identify shifts in bacterial communities due to QSI exposure on WBD-associated bacterial communities. Our results demonstrated that Vibrionaceae and Flavobacteriaceae abundances were strongly inhibited by the addition of QSI to WBD-infected corals, whereas putative coral symbiont Endozoicomonas and Halomonadaceae abundances decrease dramatically in diseased corals.

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Coral diseases are a leading factor contributing to the global decline of coral reefs, and yet mechanisms of disease transmission remain poorly understood. This study tested whether zooplankton can act as a vector for white band disease (WBD) in Acropora cervicornis. Natural zooplankton communities were collected from a coral reef in Bocas del Toro, Panama. Half of the zooplankton were treated with antibiotics for 24 h after which the antibiotic-treated and non-antibiotic-treated zooplankton were incubated with either seawater or tissue homogenates from corals exhibiting WBD-like symptoms. A total of 15 of the 30 asymptomatic A. cervicornis colonies exposed to zooplankton incubated in disease homogenate in tank-based experiments showed signs of WBD, regardless of prior antibiotic incubation. These results indicate that in our experimental conditions zooplankton were a vector for coral disease after exposure to disease-causing pathogens. Given the importance of heterotrophy on zooplankton to coral nutrition, this potential mode of disease transmission warrants further investigation.

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Increased bacterial diversity on diseased corals can obscure disease etiology and complicate our understanding of pathogenesis. To untangle microbes that may cause white band disease signs from microbes responding to disease, we inoculated healthy Acropora cervicornis corals with an infectious dose from visibly diseased corals. We sampled these dosed corals and healthy controls over time for sequencing of the bacterial 16S region. Endozoicomonas were associated with healthy fragments from 4/10 colonies, dominating microbiomes before dosing and decreasing over time only in corals that displayed disease signs, suggesting a role in disease resistance. We grouped disease-associated bacteria by when they increased in abundance (primary vs secondary) and whether they originated in the dose (colonizers) or the previously healthy corals (responders). We found that all primary responders increased in all dosed corals regardless of final disease state and are therefore unlikely to cause disease signs. In contrast, primary colonizers in the families Pasteurellaceae and Francisellaceae increased solely in dosed corals that ultimately displayed disease signs, and may be infectious foreign bacteria involved in the development of disease signs. Moving away from a static comparison of diseased and healthy bacterial communities, we provide a framework to identify key players in other coral diseases.

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The rise of coral diseases has triggered a surge of interest in coral microbial communities. But to fully understand how the coral microbiome may cause or respond to disease, we must first understand structure and variation in the healthy coral microbiome. We used 16S rRNA sequencing to characterize the microbiomes of 100 healthy coral colonies from six Caribbean coral species (Acropora cervicornis, A. palmata, Diploria labyrinthiformis, Diploria strigosa, Porites astreoides and P. furcata) across four reefs and three time points over 1 year. We found host species to be the strongest driver of coral microbiome structure across site and time. Analysis of the core microbiome revealed remarkable similarity in the bacterial taxa represented across coral hosts and many bacterial phylotypes shared across all corals sampled. Some of these widespread bacterial taxa have been identified in Pacific corals, indicating that a core coral microbiome may extend across oceans. Core bacterial phylotypes that were unique to each coral were taxonomically diverse, suggesting that different coral hosts provide persistent, divergent niches for bacteria.

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Coral reefs are declining worldwide due to multiple factors including rising sea surface temperature, ocean acidification, and disease outbreaks. Over the last 30 years, White Band Disease (WBD) alone has killed up to 95% of the Caribbean`s dominant shallow-water corals--the staghorn coral Acropora cervicornis and the elkhorn coral A. palmata. Both corals are now listed on the US Endangered Species Act, and while their recovery has been slow, recent transmission surveys indicate that more than 5% of staghorn corals are disease resistant. Here we compared transcriptome-wide gene expression between resistant and susceptible staghorn corals exposed to WBD using in situ transmission assays. We identified constitutive gene expression differences underlying disease resistance that are independent from the immune response associated with disease exposure. Genes involved in RNA interference-mediated gene silencing, including Argonaute were up-regulated in resistant corals, whereas heat shock proteins (HSPs) were down-regulated. Up-regulation of Argonaute proteins indicates that post-transcriptional gene silencing plays a key, but previously unsuspected role in coral immunity and disease resistance. Constitutive expression of HSPs has been linked to thermal resilience in other Acropora corals, suggesting that the down-regulation of HSPs in disease resistant staghorn corals may confer a dual benefit of thermal resilience.

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BACKGROUND: The American lobster, Homarus americanus, is an important species as an economically valuable fishery, a key member in marine ecosystems, and a well-studied model for central pattern generation, the neural networks that control rhythmic motor patterns. Despite multi-faceted scientific interest in this species, currently our genetic resources for the lobster are limited. In this study, we de novo assemble a transcriptome for Homarus americanus using central nervous system (CNS), muscle, and hybrid neurosecretory tissues and compare gene expression across these tissue types. In particular, we focus our analysis on genes relevant to central pattern generation and the identity of the neurons in a neural network, which is defined by combinations of genes distinguishing the neuronal behavior and phenotype, including ion channels, neurotransmitters, neuromodulators, receptors, transcription factors, and other gene products. RESULTS: Using samples from the central nervous system (brain, abdominal ganglia), abdominal muscle, and heart (cardiac ganglia, pericardial organs, muscle), we used RNA-Seq to characterize gene expression patterns across tissues types. We also compared control tissues with those challenged with the neuropeptide proctolin in vivo. Our transcriptome generated 34,813 transcripts with known protein annotations. Of these, 5,000-10,000 of annotated transcripts were significantly differentially expressed (DE) across tissue types. We found 421 transcripts for ion channels and identified receptors and/or proteins for over 20 different neurotransmitters and neuromodulators. Results indicated tissue-specific expression of select neuromodulator (allostatin, myomodulin, octopamine, nitric oxide) and neurotransmitter (glutamate, acetylcholine) pathways. We also identify differential expression of ion channel families, including kainite family glutamate receptors, inward-rectifying K(+) (IRK) channels, and transient receptor potential (TRP) A family channels, across central pattern generating tissues. CONCLUSIONS: Our transcriptome-wide profiles of the rhythmic pattern generating abdominal and cardiac nervous systems in Homarus americanus reveal candidates for neuronal features that drive the production of motor output in these systems.

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Bacterial diseases affecting scleractinian corals pose an enormous threat to the health of coral reefs, yet we still have a limited understanding of the bacteria associated with coral diseases. White band disease is a bacterial disease that affects the two Caribbean acroporid corals, the staghorn coral Acropora cervicornis and the elkhorn coral A. palmate. Species of Vibrio and Rickettsia have both been identified as putative WBD pathogens. Here we used Illumina 16S rRNA gene sequencing to profile the bacterial communities associated with healthy and diseased A. cervicornis collected from four field sites during two different years. We also exposed corals in tanks to diseased and healthy (control) homogenates to reduce some of the natural variation of field-collected coral bacterial communities. Using a combination of multivariate analyses, we identified community-level changes between diseased and healthy corals in both the field-collected and tank-exposed datasets. We then identified changes in the abundances of individual operational taxonomic units (OTUs) between diseased and healthy corals. By comparing the diseased and healthy-associated bacteria in field-collected and tank-exposed corals, we were able to identify 16 healthy-associated OTUs and 106 consistently disease-associated OTUs, which are good candidates for putative WBD pathogens. A large percentage of these disease-associated OTUs belonged to the order Flavobacteriales. In addition, two of the putative pathogens identified here belong to orders previously suggested as WBD pathogens: Vibronales and Rickettsiales.

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Reef-building corals experience large diel shifts in their environment, both externally due to changes in light intensity, predator activity and prey availability, and internally as a result of diel fluctuations in photosynthesis by their endosymbiotic algae, Symbiodinium. Diel patterns of tentacle behaviour, skeletal growth and gene expression indicate reactions of the coral animal in response to light and through circadian regulation. Some corals, such as the Caribbean Acroporas, have strong within-colony division of labour, including specialized fast-growing apical polyps, accompanied by large gene expression differences. Here we use RNA-seq to evaluate how diel changes in gene expression vary within the branching Caribbean staghorn coral, Acropora cervicornis, between branch tips and branch bases. Multifactor generalized linear model analysis indicated that 6% (3005) of transcripts were differentially expressed between branch tips and bases, while 1% (441) of transcripts were differentially expressed between day and night. The gene expression patterns of 220 transcripts were affected by both time of day and location within the colony. In particular, photoreceptors, putative circadian genes, stress response genes and metabolic genes were differentially expressed between day and night, and some of these, including Amcry1, tef and hebp2, exhibited location-specific regulation within the coral colony as well. These findings indicate that the genetic response of the coral to day and night conditions varies within the colony. Both time of day and location within the colony are factors that should be considered in future coral gene expression experiments.

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Coral reefs have entered a state of global decline party due to an increasing incidence of coral disease. However, the diversity and complexity of coral-associated bacterial communities has made identifying the mechanisms underlying disease transmission and progression extremely difficult. This study explores the effects of coral cell-free culture fluid (CFCF) and autoinducer (a quorum sensing signaling molecule) on coral-associated bacterial growth and on coral tissue loss respectively. All experiments were conducted using the endangered Caribbean coral Acropora cervicornis. Coral-associated microbes were grown on selective media infused with CFCF derived from healthy and white band disease-infected A. cervicornis. Exposure to diseased CFCF increased proliferation of Cytophaga-Flavobacterium spp. while exposure to healthy CFCF inhibited growth of this group. Exposure to either CFCF did not significantly affect Vibrio spp. growth. In order to test whether disease symptoms can be induced in healthy corals, A. cervicornis was exposed to bacterial assemblages supplemented with exogenous, purified autoinducer. Incubation with autoinducer resulted in complete tissue loss in all corals tested in less than one week. These findings indicate that white band disease in A. cervicornis may be caused by opportunistic pathogenesis of resident microbes.

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Discrepancies between morphology-based taxonomy and phylogenetic systematics are common in Scleractinian corals. In Pocillopora corals, nine recently identified genetic lineages disagree fundamentally with the 17 recognized Pocillopora species, including 5 major Indo-Pacific reef-builders. Pocillopora corals hybridize in the Tropical Eastern Pacific, so it is possible that some of the disagreement between the genetics and taxonomy may be due to introgressive hybridization. Here we used 6769 genome-wide SNPs from Restriction-site Associated DNA Sequencing (RAD-Seq) to conduct phylogenomic comparisons among three common, Indo-Pacific Pocillopora species - P. damicornis, P. eydouxi and P. elegans - within and between populations in the Tropical Eastern Pacific (TEP) and the Central Pacific. Genome-wide RAD-Seq comparisons of Central and TEP Pocillopora confirm that the morphospecies P. damicornis, P. eydouxi and P. elegans are not monophyletic, but instead fall into three distinct genetic groups. However, hybrid samples shared fixed alleles with their respective parental species and, even without strict monophyly, P. damicornis share a common set of 33 species-specific alleles across the Pacific. RAD-Seq data confirm the pattern of one-way introgressive hybridization among TEP Pocillopora, suggesting that introgression may play a role in generating shared, polyphyletic lineages among currently recognized Pocillopora species. Levels of population differentiation within genetic lineages indicate significantly higher levels of population differentiation in the Tropical Eastern Pacific than in the Central West Pacific.

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BACKGROUND: Colonial reef-building corals have evolved a broad spectrum of colony morphologies based on coordinated asexual reproduction of polyps on a secreted calcium carbonate skeleton. Though cnidarians have been shown to possess and use similar developmental genes to bilaterians during larval development and polyp formation, little is known about genetic regulation of colony morphology in hard corals. We used RNA-seq to evaluate transcriptomic differences between functionally distinct regions of the coral (apical branch tips and branch bases) in two species of Caribbean Acropora, the staghorn coral, A. cervicornis, and the elkhorn coral, A. palmata. RESULTS: Transcriptome-wide gene profiles differed significantly between different parts of the coral colony as well as between species. Genes showing differential expression between branch tips and bases were involved in developmental signaling pathways, such as Wnt, Notch, and BMP, as well as pH regulation, ion transport, extracellular matrix production and other processes. Differences both within colonies and between species identify a relatively small number of genes that may contribute to the distinct "staghorn" versus "elkhorn" morphologies of these two sister species. CONCLUSIONS: The large number of differentially expressed genes supports a strong division of labor between coral branch tips and branch bases. Genes involved in growth of mature Acropora colonies include the classical signaling pathways associated with development of cnidarian larvae and polyps as well as morphological determination in higher metazoans.

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Thermal stress and predation risk have profound effects on rocky shore organisms, triggering changes in their feeding behaviour, morphology and metabolism. Studies of thermal stress have shown that underpinning such changes in several intertidal species are specific shifts in gene and protein expression (e.g. upregulation of heat-shock proteins). But relatively few studies have examined genetic responses to predation risk. Here, we use next-generation RNA sequencing (RNA-seq) to examine the transcriptomic (mRNA) response of the snail Nucella lapillus to thermal stress and predation risk. We found that like other intertidal species, N. lapillus displays a pronounced genetic response to thermal stress by upregulating many heat-shock proteins and other molecular chaperones. In contrast, the presence of a crab predator (Carcinus maenas) triggered few significant changes in gene expression in our experiment, and this response showed no significant overlap with the snail's response to thermal stress. These different gene expression profiles suggest that thermal stress and predation risk could pose distinct and potentially additive challenges for N. lapillus and that genetic responses to biotic stresses such as predation risk might be more complex and less uniform across species than genetic responses to abiotic stresses such as thermal stress.

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