Symbiodiniaceae diversity varies by host and environment across thermally distinct reefs.

Endosymbiotic dinoflagellates (Symbiodiniaceae) influence coral thermal tolerance at both local and regional scales. In isolation, the effects of host genetics, environment, and thermal disturbances on symbiont communities are well understood, yet their combined effects remain poorly resolved. Here, we investigate Symbiodiniaceae across 1300 km in Australia's Coral Sea Marine Park to disentangle these interactive effects. We identified Symbiodiniaceae to species-level resolution for three coral species (Acropora cf humilis, Pocillopora verrucosa, and Pocillopora meandrina) by sequencing two genetic markers of the symbiont (ITS2 and psbAncr), paired with genotype-by-sequencing of the coral host (DArT-seq). Our samples predominantly returned sequences from the genus Cladocopium, where Acropora cf humilis affiliated with C3k, Pocillopora verrucosa with C. pacificum, and Pocillopora meandrina with C. latusorum. Multivariate analyses revealed that Acropora symbionts were driven strongly by local environment and thermal disturbances. In contrast, Pocillopora symbiont communities were both partitioned 2.5-fold more by host genetic structure than by environmental structure. Among the two Pocillopora species, the effects of environment and host genetics explained four times more variation in symbionts for P. meandrina than P. verrucosa. The concurrent bleaching event in 2020 had variable impacts on symbiont communities, consistent with patterns in P. verrucosa and A. cf humilis, but not P. meandrina. Our findings demonstrate how symbiont macroscale community structure responses to environmental gradients depend on host species and their respective population structure. Integrating host, symbiont, and environmental data will help forecast the adaptive potential of corals and their symbionts amidst a rapidly changing environment.

Validation of key sponge symbiont pathways using genome-centric metatranscriptomics.

The sponge microbiome underpins host function through provision and recycling of essential nutrients in a nutrient poor environment. Genomic data suggest that carbohydrate degradation, carbon fixation, nitrogen metabolism, sulphur metabolism and supplementation of B-vitamins are central microbial functions. However, validation beyond the genomic potential of sponge symbiont pathways is rarely explored. To evaluate metagenomic predictions, we sequenced the metagenomes and metatranscriptomes of three common coral reef sponges: Ircinia ramosa, Ircinia microconulosa and Phyllospongia foliascens. Multiple carbohydrate active enzymes were expressed by Poribacteria, Bacteroidota and Cyanobacteria symbionts, suggesting these lineages have a central role in assimilating dissolved organic matter. Expression of entire pathways for carbon fixation and multiple sulphur compound transformations were observed in all sponges. Gene expression for anaerobic nitrogen metabolism (denitrification and nitrate reduction) were more common than aerobic metabolism (nitrification), where only the I. ramosa microbiome expressed the nitrification pathway. Finally, while expression of the biosynthetic pathways for B-vitamins was common, the expression of additional transporter genes was far more limited. Overall, we highlight consistencies and disparities between metagenomic and metatranscriptomic results when inferring microbial activity, while uncovering new microbial taxa that contribute to the health of their sponge host via nutrient exchange.

Life-stage specificity and cross-generational climate effects on the microbiome of a tropical sea urchin (Echinodermata: Echinoidea).

Microbes play a critical role in the development and health of marine invertebrates, though microbial dynamics across life stages and host generations remain poorly understood in most reef species, especially in the context of climate change. Here, we use a 4-year multigenerational experiment to explore microbe-host interactions under the Intergovernmental Panel on Climate Change (IPCC)-forecast climate scenarios in the rock-boring tropical urchin Echinometra sp. A. Adult urchins (F0 ) were exposed for 18 months to increased temperature and pCO2 levels predicted for years 2050 and 2100 under RCP 8.5, a period which encompassed spawning. After rearing F1 offspring for a further 2 years, spawning was induced, and F2 larvae were raised under current day and 2100 conditions. Cross-generational climate effects were also explored in the microbiome of F1 offspring through a transplant experiment. Using 16S rRNA gene sequence analysis, we determined that each life stage and generation was associated with a distinct microbiome, with higher microbial diversity observed in juveniles compared to larval stages. Although life-stage specificity was conserved under climate conditions projected for 2050 and 2100, we observed changes in the urchin microbial community structure within life stages. Furthermore, we detected a climate-mediated parental effect when juveniles were transplanted among climate treatments, with the parental climate treatment influencing the offspring microbiome. Our findings reveal a potential for cross-generational impacts of climate change on the microbiome of a tropical invertebrate species.

The effects of marine heatwaves on acute heat tolerance in corals.

Scleractinian coral populations are increasingly exposed to conditions above their upper thermal limits due to marine heatwaves, contributing to global declines of coral reef ecosystem health. However, historic mass bleaching events indicate there is considerable inter- and intra-specific variation in thermal tolerance whereby species, individual coral colonies and populations show differential susceptibility to exposure to elevated temperatures. Despite this, we lack a clear understanding of how heat tolerance varies across large contemporary and historical environmental gradients, or the selective pressures that underpin this variation. Here we conducted standardised acute heat stress experiments to identify variation in heat tolerance among species and isolated reefs spanning a large environmental gradient across the Coral Sea Marine Park. We quantified the photochemical yield (Fv /Fm ) of coral samples in three coral species, Acropora cf humilis, Pocillopora meandrina, and Pocillopora verrucosa, following exposure to four temperature treatments (local ambient temperatures, and + 3°C, +6°C and + 9°C above local maximum monthly mean). We quantified the temperature at which Fv /Fm decreased by 50% (termed ED50) and used derived values to directly compare acute heat tolerance across reefs and species. The ED50 for Acropora was 0.4-0.7°C lower than either Pocillopora species, with a 0.3°C difference between the two Pocillopora species. We also recorded 0.9°C to 1.9°C phenotypic variation in heat tolerance among reefs within species, indicating spatial heterogeneity in heat tolerance across broad environmental gradients. Acute heat tolerance had a strong positive relationship to mild heatwave exposure over the past 35 years (since 1986) but was negatively related to recent severe heatwaves (2016-2020). Phenotypic variation associated with mild thermal history in local environments provides supportive evidence that marine heatwaves are selecting for tolerant individuals and populations; however, this adaptive potential may be compromised by the exposure to recent severe heatwaves.

Testing cophylogeny between coral reef invertebrates and their bacterial and archaeal symbionts.

Marine invertebrates harbour a complex suite of bacterial and archaeal symbionts, a subset of which are probably linked to host health and homeostasis. Within a complex microbiome it can be difficult to tease apart beneficial or parasitic symbionts from nonessential commensal or transient microorganisms; however, one approach is to detect strong cophylogenetic patterns between microbial lineages and their respective hosts. We employed the Procrustean approach to cophylogeny (PACo) on 16S rRNA gene derived microbial community profiles paired with COI, 18S rRNA and ITS1 host phylogenies. Second, we undertook a network analysis to identify groups of microbes that were co-occurring within our host species. Across 12 coral, 10 octocoral and five sponge species, each host group and their core microbiota (50% prevalence within host species replicates) had a significant fit to the cophylogenetic model. Independent assessment of each microbial genus and family found that bacteria and archaea affiliated to Endozoicomonadaceae, Spirochaetaceae and Nitrosopumilaceae have the strongest cophylogenetic signals. Further, local Moran's I measure of spatial autocorrelation identified 14 ASVs, including Endozoicomonadaceae and Spirochaetaceae, whose distributions were significantly clustered by host phylogeny. Four co-occurring subnetworks were identified, each of which was dominant in a different host group. Endozoicomonadaceae and Spirochaetaceae ASVs were abundant among the subnetworks, particularly one subnetwork that was exclusively comprised of these two bacterial families and dominated the octocoral microbiota. Our results disentangle key microbial interactions that occur within complex microbiomes and reveal long-standing, essential microbial symbioses in coral reef invertebrates.

Insights into the Coral Microbiome: Underpinning the Health and Resilience of Reef Ecosystems.

Corals are fundamental ecosystem engineers, creating large, intricate reefs that support diverse and abundant marine life. At the core of a healthy coral animal is a dynamic relationship with microorganisms, including a mutually beneficial symbiosis with photosynthetic dinoflagellates (Symbiodinium spp.) and enduring partnerships with an array of bacterial, archaeal, fungal, protistan, and viral associates, collectively termed the coral holobiont. The combined genomes of this coral holobiont form a coral hologenome, and genomic interactions within the hologenome ultimately define the coral phenotype. Here we integrate contemporary scientific knowledge regarding the ecological, host-specific, and environmental forces shaping the diversity, specificity, and distribution of microbial symbionts within the coral holobiont, explore physiological pathways that contribute to holobiont fitness, and describe potential mechanisms for holobiont homeostasis. Understanding the role of the microbiome in coral resilience, acclimation, and environmental adaptation is a new frontier in reef science that will require large-scale collaborative research efforts.

Efficient COI barcoding using high throughput single-end 400 bp sequencing.

BACKGROUND: Over the last decade, the rapid development of high-throughput sequencing platforms has accelerated species description and assisted morphological classification through DNA barcoding. However, the current high-throughput DNA barcoding methods cannot obtain full-length barcode sequences due to read length limitations (e.g. a maximum read length of 300 bp for the Illumina's MiSeq system), or are hindered by a relatively high cost or low sequencing output (e.g. a maximum number of eight million reads per cell for the PacBio's SEQUEL II system). RESULTS: Pooled cytochrome c oxidase subunit I (COI) barcodes from individual specimens were sequenced on the MGISEQ-2000 platform using the single-end 400 bp (SE400) module. We present a bioinformatic pipeline, HIFI-SE, that takes reads generated from the 5' and 3' ends of the COI barcode region and assembles them into full-length barcodes. HIFI-SE is written in Python and includes four function modules of filter, assign, assembly and taxonomy. We applied the HIFI-SE to a set of 845 samples (30 marine invertebrates, 815 insects) and delivered a total of 747 fully assembled COI barcodes as well as 70 Wolbachia and fungi symbionts. Compared to their corresponding Sanger sequences (72 sequences available), nearly all samples (71/72) were correctly and accurately assembled, including 46 samples that had a similarity score of 100% and 25 of ca. 99%. CONCLUSIONS: The HIFI-SE pipeline represents an efficient way to produce standard full-length barcodes, while the reasonable cost and high sensitivity of our method can contribute considerably more DNA barcodes under the same budget. Our method thereby advances DNA-based species identification from diverse ecosystems and increases the number of relevant applications.

Dual RNA-sequencing analyses of a coral and its native symbiont during the establishment of symbiosis.

Despite the ecological significance of the mutualistic relationship between Symbiodiniaceae and reef-building corals, the molecular interactions during establishment of this relationship are not well understood. This is particularly true of the transcriptional changes that occur in the symbiont. In the current study, a dual RNA-sequencing approach was used to better understand transcriptional changes on both sides of the coral-symbiont interaction during the colonization of Acropora tenuis by a compatible Symbiodiniaceae strain (Cladocopium goreaui; ITS2 type C1). Comparison of transcript levels of the in hospite symbiont 3, 12, 48 and 72 hr after exposure to those of the same strain in culture revealed that extensive and generalized down-regulation of symbiont gene expression occurred during the infection process. Included in this "symbiosis-derived transcriptional repression" were a range of stress response and immune-related genes. In contrast, a suite of symbiont genes implicated in metabolism was upregulated in the symbiotic state. The coral data support the hypothesis that immune-suppression and arrest of phagosome maturation play important roles during the establishment of compatible symbioses, and additionally imply the involvement of some SCRiP family members in the colonization process. Consistent with previous ecological studies, the transcriptomic data suggest that active translocation of metabolites to the host may begin early in the colonization process, and thus that the mutualistic relationship can be established at the larval stage. This dual RNA-sequencing study provides insights into the transcriptomic remodelling that occurs in C. goreaui during transition to a symbiotic lifestyle and the novel coral genes implicated in symbiosis.

Transcriptomic analysis reveals protein homeostasis breakdown in the coral Acropora millepora during hypo-saline stress.

BACKGROUND: Coral reefs can experience salinity fluctuations due to rainfall and runoff; these events can have major impacts on the corals and lead to bleaching and mortality. On the Great Barrier Reef (GBR), low salinity events, which occur during summer seasons and can involve salinity dropping ~ 10 PSU correlate with declines in coral cover, and these events are predicted to increase in frequency and severity under future climate change scenarios. In other marine invertebrates, exposure to low salinity causes increased expression of genes involved in proteolysis, responses to oxidative stress, and membrane transport, but the effects that changes in salinity have on corals have so far received only limited attention. To better understand the coral response to hypo-osmotic stress, here we investigated the transcriptomic response of the coral Acropora millepora in both adult and juvenile life stages to acute (1 h) and more prolonged (24 h) exposure to low salinity. RESULTS: Differential gene expression analysis revealed the involvement of both common and specific response mechanisms in Acropora. The general response to environmental stressors included up-regulation of genes involved in the mitigation of macromolecular and oxidative damage, while up-regulation of genes involved in amino acid metabolism and transport represent specific responses to salinity stress. CONCLUSIONS: This study is the first comprehensive transcriptomic analysis of the coral response to low salinity stress and provides important insights into the likely consequences of heavy rainfall and runoff events on coral reefs.

DMSP biosynthesis by an animal and its role in coral thermal stress response.

Globally, reef-building corals are the most prolific producers of dimethylsulphoniopropionate (DMSP), a central molecule in the marine sulphur cycle and precursor of the climate-active gas dimethylsulphide. At present, DMSP production by corals is attributed entirely to their algal endosymbiont, Symbiodinium. Combining chemical, genomic and molecular approaches, we show that coral juveniles produce DMSP in the absence of algal symbionts. DMSP levels increased up to 54% over time in newly settled coral juveniles lacking algal endosymbionts, and further increases, up to 76%, were recorded when juveniles were subjected to thermal stress. We uncovered coral orthologues of two algal genes recently identified in DMSP biosynthesis, strongly indicating that corals possess the enzymatic machinery necessary for DMSP production. Our results overturn the paradigm that photosynthetic organisms are the sole biological source of DMSP, and highlight the double jeopardy represented by worldwide declining coral cover, as the potential to alleviate thermal stress through coral-produced DMSP declines correspondingly.

Disentangling causation: complex roles of coral-associated microorganisms in disease.

Rapidly changing climate regimes combined with other anthropogenic pressures are implicated in increased disease epizootics among reef building corals, resulting in changing habitat structure. These accumulated stressors directly contribute to disease outbreaks by compromising the coral host immune system, modulating virulence of microbial pathogens and/or disrupting the balance within the microbiome of the holobiont. Disentangling coral disease causation has been challenging, and while progress has been made for certain diseases in terms of the roles the associated microorganisms play, it is evident that like in other marine or terrestrial systems, compromised host health cannot always be attributed to a single causative agent. Here, we summarize the current state in knowledge of microbial induced coral diseases, and discuss challenges and strategies to further disentangle disease causation. With the major environmental pressures coral reefs face over the next century, understanding interactions between host, environmental and microbial causative agent(s) that lead to disease, is still a priority to enable development of effective strategies for building resilience into coral populations.

Crown-of-Thorns Sea Star Acanthaster cf. solaris Has Tissue-Characteristic Microbiomes with Potential Roles in Health and Reproduction.

Outbreaks of coral-eating crown-of-thorns sea stars (CoTS; Acanthaster species complex) cause substantial coral loss; hence, there is considerable interest in developing prevention and control strategies. We characterized the microbiome of captive CoTS and assessed whether dysbiosis was evident in sea stars during a disease event. Most tissue types had a distinct microbiome. The exception was female gonads, in which the microbiomes were highly variable among individuals. Male gonads were dominated (>97% of reads) by a single Mollicutes-related operational taxonomic unit (OTU). Detailed phylogenetic and microscopy analysis demonstrated the presence of a novel Spiroplasma-related bacterium in the spermatogenic layer. Body wall samples had high relative abundance (43 to 64% of reads) of spirochetes, likely corresponding to subcuticular symbionts reported from many echinoderms. Tube feet were characterized by Hyphomonadaceae (24 to 55% of reads). Pyloric cecal microbiomes had high alpha diversity, comprising many taxa commonly found in gastrointestinal systems. The order Oceanospirillales (genera Endozoicomonas and Kistimonas) was detected in all tissues. A microbiome shift occurred in diseased individuals although differences between tissue types were retained. The relative abundance of spirochetes was significantly reduced in diseased individuals. Kistimonas was present in all diseased individuals and significantly associated with diseased tube feet, but its role in disease causation is unknown. While Arcobacter was significantly associated with diseased tissues and Vibrionaceae increased in diversity, no single OTU was detected in all diseased individuals, suggesting opportunistic proliferation of these taxa in this case. This study shows that CoTS have tissue-characteristic bacterial communities and identifies taxa that could play a role in reproduction and host health.IMPORTANCE Coral-eating crown-of-thorns sea stars (CoTS; Acanthaster species complex) are native to the Indo-Pacific, but during periodic population outbreaks they can reach extreme densities (>1,000 starfish per hectare) and function as a pest species. On the Great Barrier Reef, Australia, CoTS have long been considered one of the major contributors to coral loss. There has been significant investment in a targeted control program using lethal injection, and there is interest in developing additional and complementary technologies that can increase culling efficiencies. The biology of CoTS has been studied extensively, but little is known about their associated microbiome. This cultivation-independent analysis of the CoTS microbiome provides a baseline for future analyses targeting the functional role of symbionts, the identification of pathogens, or the development of reproduction manipulators.

Antimicrobial and stress responses to increased temperature and bacterial pathogen challenge in the holobiont of a reef-building coral.

Global increases in coral disease prevalence have been linked to ocean warming through changes in coral-associated bacterial communities, pathogen virulence and immune system function. However, the interactive effects of temperature and pathogens on the coral holobiont are poorly understood. Here, we assessed three compartments of the holobiont (host, Symbiodinium and bacterial community) of the coral Montipora aequituberculata challenged with the pathogen Vibrio coralliilyticus and the commensal bacterium Oceanospirillales sp. under ambient (27°C) and elevated (29.5 and 32°C) seawater temperatures. Few visual signs of bleaching and disease development were apparent in any of the treatments, but responses were detected in the holobiont compartments. V. coralliilyticus acted synergistically and negatively impacted the photochemical efficiency of Symbiodinium at 32°C, while Oceanospirillales had no significant effect on photosynthetic efficiency. The coral, however, exhibited a minor response to the bacterial challenges, with the response towards V. coralliilyticus being significantly more pronounced, and involving the prophenoloxidase-activating system and multiple immune system-related genes. Elevated seawater temperatures did not induce shifts in the coral-associated bacterial community, but caused significant gene expression modulation in both Symbiodinium and the coral host. While Symbiodinium exhibited an antiviral response and upregulated stress response genes, M. aequituberculata showed regulation of genes involved in stress and innate immune response processes, including immune and cytokine receptor signalling, the complement system, immune cell activation and phagocytosis, as well as molecular chaperones. These observations show that M. aequituberculata is capable of maintaining a stable bacterial community under elevated seawater temperatures and thereby contributes to preventing disease development.

Transcriptomic analysis of the response of Acropora millepora to hypo-osmotic stress provides insights into DMSP biosynthesis by corals.

BACKGROUND: Dimethylsulfoniopropionate (DMSP) is a small sulphur compound which is produced in prodigious amounts in the oceans and plays a pivotal role in the marine sulfur cycle. Until recently, DMSP was believed to be synthesized exclusively by photosynthetic organisms; however we now know that corals and specific bacteria can also produce this compound. Corals are major sources of DMSP, but the molecular basis for its biosynthesis is unknown in these organisms. RESULTS: Here we used salinity stress, which is known to trigger DMSP production in other organisms, in conjunction with transcriptomics to identify coral genes likely to be involved in DMSP biosynthesis. We focused specifically on both adults and juveniles of the coral Acropora millepora: after 24 h of exposure to hyposaline conditions, DMSP concentrations increased significantly by 2.6 fold in adult corals and 1.2 fold in juveniles. Concomitantly, candidate genes enabling each of the necessary steps leading to DMSP production were up-regulated. CONCLUSIONS: The data presented strongly suggest that corals use an algal-like pathway to generate DMSP from methionine, and are able to rapidly change expression of the corresponding genes in response to environmental stress. However, our data also indicate that DMSP is unlikely to function primarily as an osmolyte in corals, instead potentially serving as a scavenger of ROS and as a molecular sink for excess methionine produced as a consequence of proteolysis and osmolyte catabolism in corals under hypo-osmotic conditions.

Allelochemicals Produced by Brown Macroalgae of the Lobophora Genus Are Active against Coral Larvae and Associated Bacteria, Supporting Pathogenic Shifts to Vibrio Dominance.

Pervasive environmental stressors on coral reefs are attributed with shifting the competitive balance in favor of alternative dominants, such as macroalgae. Previous studies have demonstrated that macroalgae compete with corals via a number of mechanisms, including the production of potent primary and secondary metabolites that can influence coral-associated microbial communities. The present study investigates the effects of the Pacific brown macroalga Lobophora sp. (due to the shifting nature of the Lobophora species complex, it will be referred to here as Lobophora sp.) on coral bacterial isolates, coral larvae, and the microbiome associated with the coral Porites cylindrica. Crude aqueous and organic macroalgal extracts were found to inhibit the growth of coral-associated bacteria. Extracts and fractions were also shown to inhibit coral larval settlement and cause mortality at concentrations lower (<0.3 mg · ml-1) than calculated natural concentrations (4.4 mg · ml-1). Microbial communities associated with coral tissues exposed to aqueous (e.g., hydrophilic) crude extracts demonstrated a significant shift to Vibrio dominance and a loss of sequences related to the putative coral bacterial symbiont, Endozoicomonas sp., based on 16S rRNA amplicon sequencing. This study contributes to growing evidence that macroalgal allelochemicals, dissolved organic material, and native macroalgal microbial assemblages all play a role in shifting the microbial equilibrium of the coral holobiont away from a beneficial state, contributing to a decline in coral fitness and a shift in ecosystem structure. IMPORTANCE: Diverse microbial communities associate with coral tissues and mucus, providing important protective and nutritional services, but once disturbed, the microbial equilibrium may shift from a beneficial state to one that is detrimental or pathogenic. Macroalgae (e.g., seaweeds) can physically and chemically interact with corals, causing abrasion, bleaching, and overall stress. This study contributes to a growing body of evidence suggesting that macroalgae play a critical role in shifting the coral holobiont equilibrium, which may promote the invasion of opportunistic pathogens and cause coral mortality, facilitating additional macroalgal growth and invasion in the reef. Thus, macroalgae not only contribute to a decline in coral fitness but also influence coral reef ecosystem structure.

White Syndrome-Affected Corals Have a Distinct Microbiome at Disease Lesion Fronts.

Coral tissue loss diseases, collectively known as white syndromes (WSs), induce significant mortality on reefs throughout the Indo-Pacific, yet definitive confirmation of WS etiologies remains elusive. In this study, we integrated ecological disease monitoring, bacterial community profiling, in situ visualization of microbe-host interactions, and cellular responses of the host coral through an 18-month repeated-sampling regime. We assert that the observed pathogenesis of WS lesions on acroporid corals at Lizard Island (Great Barrier Reef) is not the result of apoptosis or infection by Vibrio bacteria, ciliates, fungi, cyanobacteria, or helminths. Histological analyses detected helminths, ciliates, fungi, and cyanobacteria in fewer than 25% of WS samples, and helminths and fungi were also observed in 12% of visually healthy samples. The abundances of Vibrio-affiliated sequences (assessed using 16S rRNA amplicon sequencing) did not differ significantly between health states and never exceeded 3.3% of reads in any individual sample. In situ visualization detected Vibrio bacteria only in summer WS lesion samples and revealed no signs of these bacteria in winter disease samples (or any healthy tissue samples), despite continued disease progression year round. However, a 4-fold increase in Rhodobacteraceae-affiliated bacterial sequences at WS lesion fronts suggests that this group of bacteria could play a role in WS pathogenesis and/or serve as a diagnostic criterion for disease differentiation. While the causative agent(s) underlying WSs remains elusive, the microbial and cellular processes identified in this study will help to identify and differentiate visually similar but potentially distinct WS etiologies. IMPORTANCE: Over the past decade, a virulent group of coral diseases known as white syndromes have impacted coral reefs throughout the Indian and Pacific Oceans. This article provides a detailed case study of white syndromes to combine disease ecology, high-throughput microbial community profiling, and cellular-scale host-microbe visualization over seasonal time scales. We provide novel insights into the etiology of this devastating disease and reveal new diagnostic criteria that could be used to differentiate visually similar but etiologically distinct forms of white syndrome.

Diversity and stability of coral endolithic microbial communities at a naturally high pCO2 reef.

The health and functioning of reef-building corals is dependent on a balanced association with prokaryotic and eukaryotic microbes. The coral skeleton harbours numerous endolithic microbes, but their diversity, ecological roles and responses to environmental stress, including ocean acidification (OA), are not well characterized. This study tests whether pH affects the diversity and structure of prokaryotic and eukaryotic algal communities associated with skeletons of Porites spp. using targeted amplicon (16S rRNA gene, UPA and tufA) sequencing. We found that the composition of endolithic communities in the massive coral Porites spp. inhabiting a naturally high pCO2 reef (avg. pCO2 811 µatm) is not significantly different from corals inhabiting reference sites (avg. pCO2 357 µatm), suggesting that these microbiomes are less disturbed by OA than previously thought. Possible explanations may be that the endolithic microhabitat is highly homeostatic or that the endolithic micro-organisms are well adapted to a wide pH range. Some of the microbial taxa identified include nitrogen-fixing bacteria (Rhizobiales and cyanobacteria), algicidal bacteria in the phylum Bacteroidetes, symbiotic bacteria in the family Endozoicomoniaceae, and endolithic green algae, considered the major microbial agent of reef bioerosion. Additionally, we test whether host species has an effect on the endolithic community structure. We show that the endolithic community of massive Porites spp. is substantially different and more diverse than that found in skeletons of the branching species Seriatopora hystrix and Pocillopora damicornis. This study reveals highly diverse and structured microbial communities in Porites spp. skeletons that are possibly resilient to OA.

Integrated approach to understanding the onset and pathogenesis of black band disease in corals.

Emerging infectious diseases are contributing to global declines in coral reef ecosystems, highlighting a growing need for aetiological knowledge to develop effective management strategies. In this review, we focus on black band disease (BBD), one of the most virulent diseases and the only polymicrobial disease so far known to affect corals. A multipartite microbial consortium dominated by Cyanobacteria, but also including sulfur-cycling bacteria, other bacterial groups and members of the Archaea and Eukarya, forms a sulfide-rich anaerobic mat that migrates across the surface of coral colonies, killing the underlying tissues. The polymicrobial nature of the disease challenges classic aetiological approaches to unravelling disease causation. Here, we synthesize current knowledge on the range of pathogens forming the microbial consortium with recent studies on the transmission, biogeochemistry and environmental drivers of BBD to develop a conceptual model of BBD pathogenesis. The model illustrates how the development of BBD virulence factors is linked to a cascade of microbial community shifts and associated functional roles that progressively develop the microbial consortium from comparatively benign cyanobacterial patches to virulent BBD lesions. This review showcases how an approach that integrates multiple key aspects of the disease provides insights essential to elucidating the aetiology of BBD.

The coral immune response facilitates protection against microbes during tissue regeneration.

Increasing physical damage on coral reefs from predation, storms and anthropogenic disturbances highlights the need to understand the impact of injury on the coral immune system. In this study, we examined the regulation of the coral immune response over 10 days following physical trauma artificially inflicted on in situ colonies of the coral Acropora aspera, simultaneously with bacterial colonization of the lesions. Corals responded to injury by increasing the expression of immune system-related genes involved in the Toll-like and NOD-like receptor signalling pathways and the lectin-complement system in three phases (<2, 4 and 10 days post-injury). Phenoloxidase activity was also significantly upregulated in two phases (<3 and 10 days post-injury), as were levels of non-fluorescent chromoprotein. In addition, green fluorescent protein expression was upregulated in response to injury from 4 days post-injury, while cyan fluorescent protein expression was reduced. No shifts in the composition of coral-associated bacterial communities were evident following injury based on 16S rRNA gene amplicon pyrosequencing. Bacteria-specific fluorescence in situ hybridization also showed no evidence of bacterial colonization of the wound or regenerating tissues. Coral tissues showed near-complete regeneration of lesions within 10 days. This study demonstrates that corals exhibit immune responses that support rapid recovery following physical injury, maintain coral microbial homeostasis and prevent bacterial infestation that may compromise coral fitness.

Amplicon pyrosequencing reveals spatial and temporal consistency in diazotroph assemblages of the Acropora millepora microbiome.

Diazotrophic bacteria potentially play an important functional role in supplying fixed nitrogen to the coral holobiont, but the value of such a partnership depends on the stability of the association. Here we evaluate the composition of diazotroph assemblages associated with the coral Acropora millepora throughout four seasons and at two reefs, an inshore and an offshore (mid-shelf) reef on the Great Barrier Reef, Australia. Amplicon pyrosequencing of the nifH gene revealed that diazotrophs are ubiquitous members of the bacterial community associated with A. millepora. Rhizobia (65% of the overall nifH sequences retrieved) and particularly Bradyrhizobia sp.-affiliated sequences (> 50% of rhizobia sequences) dominated diazotrophic assemblages across all coral samples from the two sites throughout the year. In contrast to this consistency in the spatial and temporal patterns of occurrence of diazotroph assemblages, the overall coral-associated bacterial community, assessed through amplicon sequencing of the general bacterial 16S ribosomal RNA gene, differed between inshore and mid-shelf reef locations. Sequences associated with the Oceanospirillales family, particularly with Endozoicomonas sp., dominated bacterial communities associated with inshore corals. Although rhizobia represented a variable and generally small fraction of the overall bacterial community associated with A. millepora, consistency in the structure of these diazotrophic assemblages suggests that they have a functional role in the coral holobiont.