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Coral reefs are threatened by various local and global stressors, including elevated ocean temperatures due to anthropogenic climate change. Coral cryopreservation could help secure the diversity of threatened corals. Recently, isochoric vitrification was used to demonstrate that coral fragments lived to 24 hr post-thaw; however, in this study, they were stressed post-thaw. The microbial portion of the coral holobiont has been shown to affect host fitness and the impact of cryopreservation treatment on coral microbiomes is unknown. Therefore, we examined the coral-associated bacterial communities pre- and post-cryopreservation treatments, with a view towards informing potential future stress reduction strategies. We characterized the microbiome of the Hawaiian finger coral, Porites compressa in the wild and at seven steps during the isochoric vitrification process. We observed significant changes in microbiome composition, including: 1) the natural wild microbiomes of P. compressa were dominated by Endozoicomonadaceae (76.5 % relative abundance) and consistent between samples, independent of collection location across Kāne'ohe Bay; 2) Endozoicomonadaceae were reduced to <6.9 % in captivity, and further reduced to <0.5 % relative abundance after isochoric vitrification; and 3) Vibrionaceae dominated communities post-thaw (58.5-74.7 % abundance). Thus, the capture and cryopreservation processes, are implicated as possible causal agents of dysbiosis characterized by the loss of putatively beneficial symbionts (Endozoicomonadaceae) and overgrowth of potential pathogens (Vibrionaceae). Offsetting these changes with probiotic restoration treatments may alleviate cryopreservation stress and improve post-thaw husbandry., Competing Interests: Declaration of competing interest On behalf of all authors, the corresponding author states that there is no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Gorgonian octocorals are threatened by global and local stressors that can act synergistically to affect their health. In recent years, mass mortality events triggered by marine heatwaves have caused demographic declines in Mediterranean gorgonian populations that may lead to their collapse. Potential changes in microbiome composition under stressful conditions may further increase the susceptibility of the gorgonian holobiont to disease. Given the low recovery capacity of gorgonians, restoration approaches using transplantation are becoming an increasingly attractive option to counteract their decline. Here, we compared the survival and microbiome diversity of Paramuricea clavata colonies transplanted to sites differing in depth and local environmental conditions. Gorgonians sampled at a greater depth than the transplantation site were more likely to suffer necrosis after 1 year of monitoring. Gorgonian transplantation into environments disturbed by an anthropogenic source of pollution resulted in an imbalance of the microbiome with potential consequences on the success of restoration initiatives., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The gill tissue of bivalve mollusks hosts rich symbiotic microbial communities that may contribute to host health. Spondylus spinosus is an invasive Lessepsian oyster in the Eastern Mediterranean Sea that has become highly abundant while constantly expanding its range northwestward. Using 16S rRNA gene amplicon sequencing, we examined how temperature affects S. spinosus oysters and their gill microbiota in a series of experiments: exposing them to the current annual seawater temperature range, to the colder temperature of the Western Mediterranean Sea, and to the elevated temperature as predicted under global warming scenarios. The bacterial genus Endozoicomonas dominated the communities of the S. spinosus , mainly upon exposure to winter-like (16 °C) temperatures. Exposure to the elevated seawater temperature resulted in a significant change in the bacterial communities, while the oysters maintained normal functioning, suggesting that the oyster may survive a seawater warming scenario. Exposure to 11 °C led to the health deterioration of the oysters, the emergence of opportunistic pathogens, such as Arcobacter , Vibrio , Colwelliaceae , and Pseudoalteromonas , and a decline in the relative abundance of Endozoicomonas , suggesting that S. spinosus might not survive Western Mediterranean Sea winters. Both the host and its gill bacteria are thus greatly affected by temperature, which could consequently restrict the range of expansion of this and other invasive oysters.

Bacteria contribute to many physiological functions of coral holobionts, including responses to bleaching. The bacterial genus, Endozoicomonas , dominates the microbial flora of many coral species and its abundance appears to be correlated with coral bleaching. However, evidences for decoupling of bleaching and Endozoicomonas abundance changes have also been reported. In 2020, a severe bleaching event was recorded at reefs in Taiwan, providing a unique opportunity to re-examine bleaching- Endozoicomonas association using multiple stony corals in natural environments. In this study, we monitored tissue color and microbiome changes in three coral species ( Montipora sp., Porites sp., and Stylophora pistillata ) in Kenting National Park, following the bleaching event. All tagged Montipora sp. and Porites sp. recovered from bleaching within 1 year, while high mortality occurred in S. pistillata . Microbiome analysis found no correlation of Endozoicomonas relative abundance and bleaching severity during the sampling period, but found a stronger correlation when the month in which bleaching occurred was excluded. Moreover, Endozoicomonas abundance increased during recovery months in Montipora sp. and Porites sp., whereas in S. pistillata it was nearly depleted. These results suggest that Endozoicomonas abundance may represent a gauge of coral health and reflect recovery of some corals from stress. Interestingly, even though different Endozoicomonas strains predominated in the three corals, these Endozoicomonas strains were also shared among coral taxa. Meanwhile, several Endozoicomonas strains showed secondary emergence during coral recovery, suggesting possible symbiont switching in Endozoicomonas. These findings indicate that it may be possible to introduce Endozoicomonas to non-native coral hosts as a coral probiotic., Competing Interests: On behalf of all authors, the corresponding author states that there are no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Scleractinian assemblages are threatened by marine heat waves with coral survivorship depending on host genetics and microbiome composition. We documented an extreme marine heat wave in Fiji and the response of corals in two thermally stressed reef flats. Through high-throughput amplicon sequencing of 16S and ITS rDNA phylogenetic markers, we assessed coral microbiomes (Symbiodiniaceae, prokaryotes, fungi, and Apicomplexa) of paired bleached and unbleached colonies of four common coral species representative of dominant genera in the South Pacific. While all coral species exhibited one or more pathways to bleaching resistance, harboring assemblages composed primarily of thermally tolerant photosymbionts did not always result in host bleaching resistance. Montipora and Pocillopora species, which associate with diverse Symbiodiniaceae and vertically transmit their photosymbionts, fared better than Acropora, which acquire their photosymbionts from the environment, and Porites, which associate with a narrow photosymbiont assemblage. Prokaryotic and fungal beta diversity did not differ between bleached and unbleached conspecifics, however, the relative abundance of the fungus Malassezia globosa was significantly greater in unbleached colonies of Montipora digitata. Each coral species harbored distinct assemblages of Symbiodiniaceae, prokaryotes, and Apicomplexa, but not fungi, reiterating the importance of host genetics in structuring components of its microbiome. Terrestrial fungal and prokaryotic taxa were detected at low abundance across coral microbiomes, indicating that allochthonous microbial inputs occur, but that coral microbiomes remain dominated by marine microbial taxa. Our study offers valuable insights into the microbiome assemblages associated with coral tolerance to extreme water temperatures., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Climate change has accelerated the occurrence and severity of heatwaves in the Mediterranean Sea and poses a significant threat to the octocoral species that form the foundation of marine animal forests (MAFs). As coral health intricately relies on the symbiotic relationships established between corals and microbial communities, our goal was to gain a deeper understanding of the role of bacteria in the observed tissue loss of key octocoral species following the unprecedented heatwaves in 2022., Results: Using amplicon sequencing and taxon-specific qPCR analyses, we unexpectedly found that the absolute abundance of the major bacterial symbionts, Spirochaetaceae (C. rubrum) and Endozoicomonas (P. clavata), remained, in most cases, unchanged between colonies with 0% and 90% tissue loss. These results suggest that the impairment of coral health was not due to the loss of the main bacterial symbionts. However, we observed a significant increase in the total abundance of bacterial opportunists, including putative pathogens such as Vibrio, which was not evident when only their relative abundance was considered. In addition, there was no clear relation between bacterial symbiont loss and the intensity of thermal stress, suggesting that factors other than temperature may have influenced the differential response of octocoral microbiomes at different sampling sites., Conclusions: Our results indicate that tissue loss in octocorals is not directly caused by the decline of the main bacterial symbionts but by the proliferation of opportunistic and pathogenic bacteria. Our findings thus underscore the significance of considering both relative and absolute quantification approaches when evaluating the impact of stressors on coral microbiome as the relative quantification does not accurately depict the actual changes in the microbiome. Consequently, this research enhances our comprehension of the intricate interplay between host organisms, their microbiomes, and environmental stressors, while offering valuable insights into the ecological implications of heatwaves on marine animal forests. Video Abstract., (© 2023. The Author(s).)

Environmental perturbations evoke down-regulation of metabolism in some multicellular organisms, leading to dormancy, or torpor. Colonies of the urochordate Botrylloides leachii enter torpor in response to changes in seawater temperature and may survive for months as small vasculature remnants that lack feeding and reproductive organs but possess torpor-specific microbiota. Upon returning to milder conditions, the colonies rapidly restore their original morphology, cytology and functionality while harboring re-occurring microbiota, a phenomenon that has not been described in detail to date. Here we investigated the stability of B. leachii microbiome and its functionality in active and dormant colonies, using microscopy, qPCR, in situ hybridization, genomics and transcriptomics. A novel lineage of Endozoicomonas , proposed here as Candidatus Endozoicomonas endoleachii, was dominant in torpor animals (53-79% read abundance), and potentially occupied specific hemocytes found only in torpid animals. Functional analysis of the metagenome-assembled genome and genome-targeted transcriptomics revealed that Endozoicomonas can use various cellular substrates, like amino acids and sugars, potentially producing biotin and thiamine, but also expressing various features involved in autocatalytic symbiosis. Our study suggests that the microbiome can be linked to the metabolic and physiological states of the host, B. leachii , introducing a model organism for the study of symbioses during drastic physiological changes, such as torpor., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hyams, Rubin-Blum, Rosner, Brodsky, Rinkevich and Rinkevich.)

Bacteria in the coral microbiome play a crucial role in determining coral health and fitness, and the coral host often restructures its microbiome composition in response to external factors. An important but often neglected factor determining this microbiome restructuring is the ability of microbiome members to respond to changes in the environment. To address this issue, we examined how the microbiome structure of Acropora muricata corals changed over 9 months following a reciprocal transplant experiment. Using a combination of metabarcoding, genomics, and comparative genomics approaches, we found that coral colonies separated by a small distance harbored different dominant Endozoicomonas -related phylotypes belonging to two different species, including a novel species, " Candidatus Endozoicomonas penghunesis" 4G, whose chromosome-level (complete) genome was also sequenced in this study. Furthermore, the two dominant Endozoicomonas species had different potentials to scavenge reactive oxygen species, suggesting potential differences in responding to the environment. Differential capabilities of dominant members of the microbiome to respond to environmental change can (i) provide distinct advantages or disadvantages to coral hosts when subjected to changing environmental conditions and (ii) have positive or negative implications for future reefs. IMPORTANCE The coral microbiome has been known to play a crucial role in host health. In recent years, we have known that the coral microbiome changes in response to external stressors and that coral hosts structure their microbiome in a host-specific manner. However, an important internal factor, the ability of microbiome members to respond to change, has been often neglected. In this study, we combine metabarcoding, culturing, and genomics to delineate the differential ability of two dominant Endozoicomonas species, including a novel " Ca. Endozoicomonas penghunesis" 4G, to respond to change in the environment following a reciprocal transplant experiment.

Bacterial members of the coral holobiont play an important role in determining coral fitness. However, most knowledge of the coral microbiome has come from reef-building scleractinian corals, with far less known about the nature and importance of the microbiome of octocorals (subclass Octocorallia), which contribute significantly to reef biodiversity and functional complexity. We examined the diversity and structure of the bacterial component of octocoral microbiomes over summer and winter, with a focus on two temperate (Erythropodium hicksoni, Capnella gaboensis; Sydney Harbour) and two tropical (Sinularia sp., Sarcophyton sp.; Heron Island) species common to reefs in eastern Australia. Bacterial communities associated with these octocorals were also compared to common temperate (Plesiastrea versipora) and tropical (Acropora aspera) hard corals from the same reefs. Using 16S rRNA amplicon sequencing, bacterial diversity was found to be heterogeneous among octocorals, but we observed changes in composition between summer and winter for some species (C. gaboensis and Sinularia sp.), but not for others (E. hicksoni and Sarcophyton sp.). Bacterial community structure differed significantly between all octocoral species within both the temperate and tropical environments. However, on a seasonal basis, those differences were less pronounced. The microbiomes of C. gaboensis and Sinularia sp. were dominated by bacteria belonging to the genus Endozoicomonas, which were a key conserved feature of their core microbiomes. In contrast to previous studies, our analysis revealed that Endozoicomonas phylotypes are shared across different octocoral species, inhabiting different environments. Together, our data demonstrates that octocorals harbour a broad diversity of bacterial partners, some of which comprise 'core microbiomes' that potentially impart important functional roles to their hosts., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Global warming threatens reef-building corals with large-scale bleaching events; therefore, it is important to discover potential adaptive capabilities for increasing their temperature resistance before it is too late. This study presents two coral species ( Platygyra verweyi and Isopora palifera ) surviving on a reef having regular hot water influxes via a nearby nuclear power plant that exhibited completely different bleaching susceptibilities to thermal stress, even though both species shared several so-called "winner" characteristics ( e.g., containing Durusdinium trenchii , thick tissue, etc. ). During acute heating treatment, algal density did not decline in P. verweyi corals within three days of being directly transferred from 25 to 31 °C; however, the same treatment caused I . palifera to lose < 70% of its algal symbionts within 24 h. The most distinctive feature between the two coral species was an overwhelmingly higher constitutive superoxide dismutase (ca. 10-fold) and catalase (ca. 3-fold) in P. verweyi over I. palifera . Moreover, P. verweyi also contained significantly higher saturated and lower mono-unsaturated fatty acids, especially a long-chain saturated fatty acid (C22:0), than I. palifera , and was consistently associated with the symbiotic bacteria Endozoicomonas , which was not found in I. palifera . However, antibiotic treatment and inoculation tests did not support Endozoicomonas having a direct contribution to thermal resistance. This study highlights that, besides its association with a thermally tolerable algal symbiont, a high level of constitutive antioxidant enzymes in the coral host is crucial for coral survivorship in the more fluctuating and higher temperature environments., Competing Interests: The authors declare there are no competing interests., (©2022 Wang et al.)

It has been proposed that an effective approach for predicting whether and how reef-forming corals persist under future climate change is to examine populations thriving in present day extreme environments, such as mangrove lagoons, where water temperatures can exceed those of reef environments by more than 3°C, pH levels are more acidic (pH < 7.9, often below 7.6) and O 2 concentrations are regularly considered hypoxic (<2 mg/L). Defining the physiological features of these "extreme" corals, as well as their relationships with the, often symbiotic, organisms within their microbiome, could increase our understanding of how corals will persist into the future. To better understand coral-microbe relationships that potentially underpin coral persistence within extreme mangrove environments, we therefore conducted a 9-month reciprocal transplant experiment, whereby specimens of the coral Pocillopora acuta were transplanted between adjacent mangrove and reef sites on the northern Great Barrier Reef. Bacterial communities associated with P. acuta specimens native to the reef environment were dominated by Endozoicomonas , while Symbiodiniaceae communities were dominated by members of the Cladocopium genus. In contrast, P. acuta colonies native to the mangrove site exhibited highly diverse bacterial communities with no dominating members, and Symbiodiniaceae communities dominated by Durusdinium. All corals survived for 9 months after being transplanted from reef-to-mangrove, mangrove-to-reef environments (as well as control within environment transplants), and during this time there were significant changes in the bacterial communities, but not in the Symbiodiniaceae communities or their photo-physiological functioning. In reef-to-mangrove transplanted corals, there were varied, but sometimes rapid shifts in the associated bacterial communities, including a loss of "core" bacterial members after 9 months where coral bacterial communities began to resemble those of the native mangrove corals. Bacterial communities associated with mangrove-to-reef P. acuta colonies also changed from their original composition, but remained different to the native reef corals. Our data demonstrates that P. acuta associated bacterial communities are strongly influenced by changes in environmental conditions, whereas Symbiodiniaceae associated communities remain highly stable., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Haydon, Seymour, Raina, Edmondson, Siboni, Matthews, Camp and Suggett.)

Background: The coral microbiome plays a key role in host health by being involved in energy metabolism, nutrient cycling, and immune system formation. Inoculating coral with beneficial bacterial consortia may enhance the ability of this host to cope with complex and changing marine environments. In this study, the coral Pocillopora damicornis was inoculated with a beneficial microorganisms for corals (BMC) consortium to investigate how the coral host and its associated microbial community would respond., Results: High-throughput 16S rRNA gene sequencing revealed no significant differences in bacterial community α-diversity. However, the bacterial community structure differed significantly between the BMC and placebo groups at the end of the experiment. Addition of the BMC consortium significantly increased the relative abundance of potentially beneficial bacteria, including the genera Mameliella and Endozoicomonas. Energy reserves and calcification rates of the coral host were also improved by the addition of the BMC consortium. Co-occurrence network analysis indicated that inoculation of coral with the exogenous BMC consortium improved the physiological status of the host by shifting the coral-associated microbial community structure., Conclusions: Manipulating the coral-associated microbial community may enhance the physiology of coral in normal aquarium conditions (no stress applied), which may hypothetically contribute to resilience and resistance in this host.

Ocean acidification (OA) threatens the growth and function of coral reef ecosystems. A key component to coral health is the microbiome, but little is known about the impact of OA on coral microbiomes. A submarine CO 2 vent at Maug Island in the Northern Mariana Islands provides a natural pH gradient to investigate coral responses to long-term OA conditions. Three coral species ( Pocillopora eydouxi , Porites lobata , and Porites rus ) were sampled from three sites where the mean seawater pH is 8.04, 7.98, and 7.94. We characterized coral bacterial communities (using 16S rRNA gene sequencing) and determined pH of the extracellular calcifying fluid (ECF) (using skeletal boron isotopes) across the seawater pH gradient. Bacterial communities of both Porites species stabilized (decreases in community dispersion) with decreased seawater pH, coupled with large increases in the abundance of Endozoicomonas , an endosymbiont. P. lobata experienced a significant decrease in ECF pH near the vent, whereas P. rus experienced a trending decrease in ECF pH near the vent. In contrast, Pocillopora exhibited bacterial community destabilization (increases in community dispersion), with significant decreases in Endozoicomonas abundance, while its ECF pH remained unchanged across the pH gradient. Our study shows that OA has multiple consequences on Endozoicomonas abundance and suggests that Endozoicomonas abundance may be an indicator of coral response to OA. We reveal an interesting dichotomy between two facets of coral physiology (regulation of bacterial communities and regulation of calcification), highlighting the importance of multidisciplinary approaches to understanding coral health and function in a changing ocean. IMPORTANCE Ocean acidification (OA) is a consequence of anthropogenic CO 2 emissions that is negatively impacting marine ecosystems such as coral reefs. OA affects many aspects of coral physiology, including growth (i.e., calcification) and disrupting associated bacterial communities. Coral-associated bacteria are important for host health, but it remains unclear how coral-associated bacterial communities will respond to future OA conditions. We document changes in coral-associated bacterial communities and changes to calcification physiology with long-term exposure to decreases in seawater pH that are environmentally relevant under midrange IPCC emission scenarios (0.1 pH units). We also find species-specific responses that may reflect different responses to long-term OA. In Pocillopora , calcification physiology was highly regulated despite changing seawater conditions. In Porites spp., changes in bacterial communities do not reflect a breakdown of coral-bacterial symbiosis. Insights into calcification and host-microbe interactions are critical to predicting the health and function of different coral taxa to future OA conditions., (Copyright © 2021 Shore et al.)

Kaimoana (shellfish, seafood) is an important food source and a significant social and cultural component of many New Zealand communities, especially the indigenous Māori. Over the past decade a decline has been detected in shellfish health and an increase in mortality events around New Zealand. Intracellular bacteria termed Rickettsia-like organisms (RLOs) have been observed in New Zealand bivalve molluscs during shellfish mortality events. Affected bivalves include cockles Austrovenus stutchburyi, ringed dosinia Dosinia anus, green-lipped mussels Perna canaliculus, pipi Paphies australis, toheroa Paphies ventricosa, tuatua Paphies subtriangulata, deepwater tuatua Paphies donacina and scallops Pecten novaezelandiae. RLOs are an informal morphology-based classification of intracellular bacteria, with the exact identification often unknown. Using shellfish collected during mortality events from 2014 to 2019 and apparently healthy samples collected in 2018 and 2019, we aimed to identify RLOs in New Zealand shellfish. Bacterial 16S rRNA gene sequences from RLO-infected shellfish showed >95% identity to published Endozoicomonas species. In situ hybridization confirmed the presence of the sequenced gene in the gill epithelium and digestive epithelium of all study species. A genus-specific quantitative PCR, targeting the 16S rRNA gene was developed to detect Endozoicomonas spp. in shellfish tissue. Prevalence of Endozoicomonas spp. in samples from mortality events and healthy shellfish analysed by quantitative PCR was high. Samples collected from mortality events, however, had a significantly higher load of Endozoicomonas spp. than the healthy samples. These results give us a greater understanding of these intracellular bacteria and their presence in populations of New Zealand shellfish.

Intracellular microcolonies of bacteria (IMC), in some cases developing large extracellular cysts (bacterial aggregates), infecting primarily gill and digestive gland, have been historically reported in a wide diversity of economically important mollusk species worldwide, sometimes associated with severe lesions and mass mortality events. As an effort to characterize those organisms, traditionally named as Rickettsia or Chlamydia -like organisms, 1950 specimens comprising 22 mollusk species were collected over 10 countries and after histology examination, a selection of 99 samples involving 20 species were subjected to 16S rRNA gene amplicon sequencing. Phylogenetic analysis showed Endozoicomonadaceae sequences in all the mollusk species analyzed. Geographical differences in the distribution of Operational Taxonomic Units (OTUs) and a particular OTU associated with pathology in king scallop (OTU&#95;2) were observed. The presence of Endozoicomonadaceae sequences in the IMC was visually confirmed by in situ hybridization (ISH) in eight selected samples. Sequencing data also indicated other symbiotic bacteria. Subsequent phylogenetic analysis of those OTUs revealed a novel microbial diversity associated with molluskan IMC infection distributed among different taxa, including the phylum Spirochetes, the families Anaplasmataceae and Simkaniaceae , the genera Mycoplasma and Francisella , and sulfur-oxidizing endosymbionts. Sequences like Francisella halioticida/philomiragia and Candidatus Brownia rhizoecola were also obtained, however, in the absence of ISH studies, the association between those organisms and the IMCs were not confirmed. The sequences identified in this study will allow for further molecular characterization of the microbial community associated with IMC infection in marine mollusks and their correlation with severity of the lesions to clarify their role as endosymbionts, commensals or true pathogens., (Copyright © 2020 Cano, Ryder, Webb, Jones, Brosnahan, Carrasco, Bodinier, Furones, Pretto, Carella, Chollet, Arzul, Cheslett, Collins, Lohrmann, Valdivia, Ward, Carballal, Villalba, Marigómez, Mortensen, Christison, Kevin, Bustos, Christie, Green and Feist.)

The coral-associated Endozoicomonas are dominant bacteria in the coral holobiont. Their relative abundance usually decreases with heat-induced coral bleaching and is proposed to be positively correlated with Symbiodiniaceae abundance. It remains unclear whether this phenomenon of decreased Endozoicomonas abundance is caused by temperature stress or a decreased abundance of Symbiodiniaceae. This study induced bleaching in the coral Euphyllia glabrescens using a dark treatment over 15 weeks. We examined shifts in Endozoicomonas abundance and experimentally reduced Symbiodiniaceae density. 16S rRNA gene amplicon sequencing was used to characterize the changes in bacterial community (incl. Endozoicomonas ) over time, and the 16S rRNA gene copy number of Endozoicomonas was quantified by qPCR. We detected a high abundance of Endozoicomonas in E. glabrescens that underwent dark-induced bleaching. The results reveal that changes in the relative abundance of Endozoicomonas are unrelated to Symbiodiniaceae abundance, indicating that Endozoicomonas can be independent of Symbiodiniaceae in the coral holobiont., (Copyright © 2020 Shiu, Yu, Fong, Ding, Tan, Fan, Lu and Tang.)

The coral holobiont is a complex ecosystem consisting of coral animals and a highly diverse consortium of associated microorganisms including algae, fungi, and bacteria. Several studies have highlighted the importance of coral-associated bacteria and their potential roles in promoting the host fitness and survival. Recently, dynamics of coral-associated microbiomes have been demonstrated to be linked to patterns of coral heat tolerance. Here, we examined the effect of elevated seawater temperature on the structure and diversity of bacterial populations associated with Porites lutea, using full-length 16S rRNA sequences obtained from Pacific Biosciences circular consensus sequencing. We observed a significant increase in alpha diversity indices and a distinct shift in microbiome composition during thermal stress. There was a marked decline in the apparent relative abundance of Gammaproteobacteria family Endozoicomonadaceae after P. lutea had been exposed to elevated seawater temperature. Concomitantly, the bacterial community structure shifted toward the predominance of Alphaproteobacteria family Rhodobacteraceae. Interestingly, we did not observe an increase in relative abundance of Vibrio-related sequences in our heat-stressed samples even though the appearance of Vibrio spp. has often been detected in parallel with the increase in the relative abundance of Rhodobacteraceae during thermal bleaching in other coral species. The ability of full-length 16S rRNA sequences in resolving taxonomic uncertainty of associated bacteria at a species level enabled us to identify 24 robust indicator bacterial species for thermally stressed corals. It is worth noting that the majority of those indicator species were members of the family Rhodobacteraceae. The comparison of bacterial community structure and diversity between corals in ambient water temperature and thermally stressed corals may provide a better understanding on how bacteria symbionts contribute to the resilience of their coral hosts to ocean warming., (© 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

The most recently described bacterial members of the genus Endozoicomonas have been found in association with a wide variety of marine invertebrates. Despite their ubiquity in the host holobiont, limited information is available on the molecular genomic signatures of the symbiotic association of Endozoicomonas with marine sponges. Here, we generated a draft genome of Endozoicomonas sp. OPT23 isolated from the intertidal marine sponge Ophlitaspongia papilla and performed comprehensive comparative genomics analyses. Genome-specific analysis and metabolic pathway comparison of the members of the genus Endozoicomonas revealed the presence of gene clusters encoding for unique metabolic features, such as the utilization of carbon sources through lactate, L-rhamnose metabolism, and a phenylacetic acid degradation pathway in Endozoicomonas sp. OPT23. Moreover, the genome harbors genes encoding for eukaryotic-like proteins, such as ankyrin repeats, tetratricopeptide repeats, and Sel1 repeats, which likely facilitate sponge-bacterium attachment. The genome also encodes major secretion systems and homologs of effector molecules that seem to enable the sponge-associated bacterium to interact with the sponge and deliver the virulence factors for successful colonization. In conclusion, the genome analysis of Endozoicomonas sp. OPT23 revealed the presence of adaptive genomic signatures that might favor their symbiotic lifestyle within the sponge host.

Genotype-specific contributions to the environmental tolerance and disease susceptibility of corals are widely accepted. Yet our understanding of how host genotype influences the composition and stability of the coral microbiome subjected to environmental fluctuations is limited. To gain insight into the community dynamics and environmental stability of microbiomes associated with distinct coral genotypes, we assessed the microbial community associated with Acropora tenuis under single and cumulative pressure experiments. Experimental treatments comprised either a single pulse of reduced salinity (minimum of 28 psu) or exposure to the cumulative pressures of reduced salinity (minimum of 28 psu), elevated seawater temperature (+2 °C), elevated p CO 2 (900 ppm), and the presence of macroalgae. Analysis of 16S rRNA gene amplicon sequence data revealed that A. tenuis microbiomes were highly host-genotype specific and maintained high compositional stability irrespective of experimental treatment. On average, 48% of the A. tenuis microbiome was dominated by Endozoicomonas. Amplicon sequence variants (ASVs) belonging to this genus were significantly different between host individuals. Although no signs of stress were evident in the coral holobiont and the vast majority of ASVs remained stable across treatments, a microbial indicator approach identified 26 ASVs belonging to Vibrionaceae, Rhodobacteraceae, Hahellaceae, Planctomycetes, Phylobacteriaceae, Flavobacteriaceae, and Cryomorphaceae that were significantly enriched in corals exposed to single and cumulative stressors. While several recent studies have highlighted the efficacy of microbial indicators as sensitive markers for environmental disturbance, the high host-genotype specificity of coral microbiomes may limit their utility and we therefore recommend meticulous control of host-genotype effects in coral microbiome research., Competing Interests: The authors declare that they have no competing interests.

Endozoicomonas bacteria are commonly regarded as having a potentially symbiotic relationship with their coral hosts. However, their diversity and phylogeny in samples collected from various sources remain unclear. Therefore, we designed an Endozoicomonas-specific primer paired with a bacterial universal primer to detect the 16S ribosomal RNA (rRNA) genes of this taxon and conducted an in-depth investigation of the Endozoicomonas community structure in reef-building corals. The primer had high specificity in the V3-V4 region (95.6%) and its sensitivity was high, particularly when Endozoicomonas was rare in samples (e.g., in seawater, which had a higher alpha diversity of Endozoicomonas than corals). In coral samples, predominant V3-V4 ribotypes had greater divergence than predominant V1-V2 ribotypes, and were grouped into at least 9 novel clades in a phylogenetic tree, indicating Endozoicomonas had high phylogenetic diversity. Divergence within this genus was potentially higher than that among 7 outgroup genera based on the phylogenetic distances of partial 16S rDNA sequences, suggesting that the taxonomy of this genus needs to be revised. In conclusion, dominant Endozoicomonas populations had variable phylogenies; furthermore, the newly designed primers may be useful molecular tools for the reliable detection of the Endozoicomonas community in marine environments.

Two Gram-stain-negative, facultative anaerobic, motile, rod-shaped strains, S-B4-1U T and JOB-63a, forming small whitish transparent colonies on marine agar, were isolated from a sponge of the genus Haliclona. The strains shared 99.7% 16S rRNA gene sequence identity and a DNA-DNA hybridization value of 100%, but were differentiated by genomic fingerprinting using rep-PCRs. 16S rRNA gene sequence phylogeny placed the strains as a sister branch to the monophyletic genus Endozoicomonas (Oceanospirillales; Gammaproteobacteria) with 92.3-94.3% 16S rRNA gene sequence similarity to Endozoicomonas spp., 91.9 and 92.1% to Candidatus Endonucleobacter bathymodiolin, and 91.9 to 92.1% to the type strains of Kistimonas spp. Core genome based phylogeny of strain S-B4-1U T confirmed the phylogenetic placement. Major fatty acids were summed feature 3 (C 16:1 ω7c/C 16:1 ω6c) and 8 (C 18:1 ω7c/C 18:1 ω6c) followed by C 10:0 3-OH, C 16:0 , and C 18:0 . The G+C content was 50.1-51.4mol%. The peptidoglycan diamino acid of strain S-B4-1U T was meso-diaminopimelic acid, the predominant polyamine spermidine, the major respiratory quinone ubiquinone Q-9; phosphatidylethanolamine, phosphatidylglycerol and phosphatidylserine were major polar lipids. Based on the clear phylogenetic distinction, the genus Parendozoicomonas gen. nov. is proposed, with Parendozoicomonas haliclonae sp. nov. as type species and strain S-B4-1U T (=CCM 8713 T =DSM 103671 T =LMG 29769 T ) as type strain and JOB-63a as a second strain of the species. Based on the 16S rRNA gene sequence phylogeny of the Oceanospirillales within the Gammaproteobacteria, the Endozoicomonaceae fam. nov. is proposed including the genera Endozoicomonas, Parendozoicomonas, and Kistimonas as well as the Candidatus genus Endonucleobacter., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

One of the fastest growing fisheries in the UK is the king scallop ( Pecten maximus L.), also currently rated as the second most valuable fishery. Mass mortality events in scallops have been reported worldwide, often with the causative agent(s) remaining uncharacterized. In May 2013 and 2014, two mass mortality events affecting king scallops were recorded in the Lyme Bay marine protected area (MPA) in Southwest England. Histopathological examination showed gill epithelial tissues infected with intracellular microcolonies (IMCs) of bacteria resembling Rickettsia -like organisms (RLOs), often with bacteria released in vascular spaces. Large colonies were associated with cellular and tissue disruption of the gills. Ultrastructural examination confirmed the intracellular location of these organisms in affected epithelial cells. The 16S rRNA gene sequences of the putative IMCs obtained from infected king scallop gill samples, collected from both mortality events, were identical and had a 99.4% identity to 16S rRNA gene sequences obtained from " Candidatus Endonucleobacter bathymodioli" and 95% with Endozoicomonas species. In situ hybridization assays using 16S rRNA gene probes confirmed the presence of the sequenced IMC gene in the gill tissues. Additional DNA sequences of the bacterium were obtained using high-throughput (Illumina) sequencing, and bioinformatic analysis identified over 1,000 genes with high similarity to protein sequences from Endozoicomonas spp. (ranging from 77 to 87% identity). Specific PCR assays were developed and applied to screen for the presence of IMC 16S rRNA gene sequences in king scallop gill tissues collected at the Lyme Bay MPA during 2015 and 2016. There was 100% prevalence of the IMCs in these gill tissues, and the 16S rRNA gene sequences identified were identical to the sequence found during the previous mortality event. IMPORTANCE Molluscan mass mortalities associated with IMCs have been reported worldwide for many years; however, apart from histological and ultrastructural characterization, characterization of the etiological agents is limited. In the present work, we provide detailed molecular characterization of an Endozoicomonas -like organism (ELO) associated with an important commercial scallop species., (© Crown copyright 2018.)

Populations of key benthic habitat-forming octocoral species have declined significantly in the Mediterranean Sea due to mass mortality events caused by microbial disease outbreaks linked to high summer seawater temperatures. Recently, we showed that the microbial communities of these octocorals are relatively structured; however, our knowledge on the seasonal dynamics of these microbiomes is still limited. To investigate their seasonal stability, we collected four soft gorgonian species (Eunicella singularis, Eunicella cavolini, Eunicella verrucosa and Leptogorgia sarmentosa) and the precious red coral (Corallium rubrum) from two coastal locations with different terrestrial impact levels in the Mediterranean Sea, and used next-generation amplicon sequencing of the 16S rRNA gene. The microbiomes of all soft gorgonian species were dominated by the same 'core microbiome' bacteria belonging to the Endozoicomonas and the Cellvibrionales clade BD1-7, whereas the red coral microbiome was primarily composed of 'core' Spirochaetes, Oceanospirillales ME2 and Parcubacteria. The associations with these bacterial taxa were relatively consistent over time at each location for each octocoral species. However, differences in microbiome composition and seasonal dynamics were observed between locations and could primarily be attributed to locally variant bacteria. Overall, our data provide further evidence of the intricate symbiotic relationships that exist between Mediterranean octocorals and their associated microbes, which are ancient and highly conserved over both space and time, and suggest regulation of the microbiome composition by the host, depending on local conditions.

Gorgonians are key habitat-forming species of Mediterranean benthic communities, but their populations have suffered from mass mortality events linked to high summer seawater temperatures and microbial disease. However, our knowledge on the diversity, dynamics and function of gorgonian-associated microbial communities is limited. Here, we analysed the spatial variability of the microbiomes of five sympatric gorgonian species (Eunicella singularis, Eunicella cavolini, Eunicella verrucosa, Leptogorgia sarmentosa and Paramuricea clavata), collected from the Mediterranean Sea over a scale of ∼1100 km, using next-generation amplicon sequencing of the 16S rRNA gene. The microbiomes of all gorgonian species were generally dominated by members of the genus Endozoicomonas, which were at very low abundance in the surrounding seawater. Although the composition of the core microbiome (operational taxonomic units consistently present in a species) was found to be unique for each host species, significant overlap was observed. These spatially consistent associations between gorgonians and their core bacteria suggest intricate symbiotic relationships and regulation of the microbiome composition by the host. At the same time, local variations in microbiome composition were observed. Functional predictive profiling indicated that these differences could be attributed to seawater pollution. Taken together, our data indicate that gorgonian-associated microbiomes are composed of spatially conserved bacteria (core microbiome members) and locally variant members, and that local pollution may influence these local associations, potentially impacting gorgonian health.

Endozoicomonas bacteria are emerging as extremely diverse and flexible symbionts of numerous marine hosts inhabiting oceans worldwide. Their hosts range from simple invertebrate species, such as sponges and corals, to complex vertebrates, such as fish. Although widely distributed, the functional role of Endozoicomonas within their host microenvironment is not well understood. In this review, we provide a summary of the currently recognized hosts of Endozoicomonas and their global distribution. Next, the potential functional roles of Endozoicomonas, particularly in light of recent microscopic, genomic, and genetic analyses, are discussed. These analyses suggest that Endozoicomonas typically reside in aggregates within host tissues, have a free-living stage due to their large genome sizes, show signs of host and local adaptation, participate in host-associated protein and carbohydrate transport and cycling, and harbour a high degree of genomic plasticity due to the large proportion of transposable elements residing in their genomes. This review will finish with a discussion on the methodological tools currently employed to study Endozoicomonas and host interactions and review future avenues for studying complex host-microbial symbioses., Competing Interests: Compliance with ethical standards Funding We would like to acknowledge the following sources for funding: this work was supported by a KAUST-WHOI Post-doctoral Partnership Award to MJN and a KAUST-WHOI Special Academic Partnership Funding Reserve Award to CRV and AA. Research in this study was further supported by baseline research funds to CRV by KAUST and NSF award OCE-1233612 to AA. Conflict of interest The authors declare that they have no conflict of interest. Ethical approval This article does not contain any studies with animals performed by any of the authors. Referenced studies adhere to applicable international, national and/or institutional guidelines for the care and use of animals.

The octocoral Erythropodium caribaeorum is an important species in the Caribbean coral reef community and a source of the cytotoxic natural product desmethyleleutherobin. We utilized 16S small subunit rRNA gene amplicon pyrosequencing to characterize the microbiome of E. caribaeorum collected from Florida, USA and San Salvador, The Bahamas at multiple time points. This coral was found to have a very high microbial richness with an average Chao1 estimated richness of 1464 ± 707 operational taxonomic units and average Shannon diversity index of 4.26 ± 1.65. The taxonomic class Gammaproteobacteria was a dominant member in all samples and the genus Endozoicomonas accounted for an average of 37.7% ± 30.0% of the total sequence reads. One Endozoicomonas sp. was found to be a stable member of all E. caribaeorum sequence libraries regardless of location or time of collection and accounted for 30.1% of all sequence reads. This is the first report characterizing the microbiome associated with the encrusting octocoral E. caribaeorum., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The Pomacentridae (damselfish) and Apogonidae (cardinalfish) are among the most common fish families on coral reefs and in the aquarium trade. Members of both families undergo a pelagic larvae phase prior to settlement on the reef, where adults play key roles in benthic habitat structuring and trophic interactions. Fish-associated microbial communities (microbiomes) significantly influence fish health and ecology, yet little is known of how microbiomes change with life stage. We quantified the taxonomic (16S rRNA gene) composition of whole gut microbiomes from ten species of damselfish and two species of cardinalfish from Lizard Island, Australia, focusing specifically on comparisons between pelagic larvae prior to settlement on the reef versus post-settlement juvenile and adult individuals. On average, microbiome phylogenetic diversity increased from pre- to post-settlement, and was unrelated to the microbial composition in the surrounding water column. However, this trend varied among species, suggesting stochasticity in fish microbiome assembly. Pre-settlement fish were enriched with bacteria of the Endozoicomonaceae, Shewanellaceae, and Fusobacteriaceae, whereas settled fish harbored higher abundances of Vibrionaceae and Pasteurellaceae. Several individual operational taxonomic units, including ones related to Vibrio harveyi, Shewanella sp., and uncultured Endozoicomonas bacteria, were shared between both pre and post-settlement stages and may be of central importance in the intestinal niche across development. Richness of the core microbiome shared among pre-settlement fish was comparable to that of settled individuals, suggesting that changes in diversity with adulthood are due to the acquisition or loss of host-specific microbes. These results identify a key transition in microbiome structure across host life stage, suggesting changes in the functional contribution of microbiomes over development in two ecologically dominant reef fish families., Competing Interests: The authors declare there are no competing interests.

Ascidians are marine filter feeders and harbor diverse microbiota that can exhibit a high degree of host-specificity. Pharyngeal samples of Scandinavian and Mediterranean ascidians were screened for consistently associated bacteria by culture-dependent and -independent approaches. Representatives of the Endozoicomonas (Gammaproteobacteria, Hahellaceae) clade were detected in the ascidian species Ascidiella aspersa, Ascidiella scabra, Botryllus schlosseri, Ciona intestinalis, Styela clava, and multiple Ascidia/Ascidiella spp. In total, Endozoicomonas was detected in more than half of all specimens screened, and in 25-100% of the specimens for each species. The retrieved Endozoicomonas 16S rRNA gene sequences formed an ascidian-specific subclade, whose members were detected by fluorescence in situ hybridization (FISH) as extracellular microcolonies in the pharynx. Two strains of the ascidian-specific Endozoicomonas subclade were isolated in pure culture and characterized. Both strains are chemoorganoheterotrophs and grow on mucin (a mucus glycoprotein). The strains tested negative for cytotoxic or antibacterial activity. Based on these observations, we propose ascidian-associated Endozoicomonas to be commensals, living off the mucus continuously secreted into the pharynx. Members of the ascidian-specific Endozoicomonas subclade were also detected in seawater from the Scandinavian sampling site, which suggests acquisition of the symbionts by horizontal transmission. The combined results indicate a host-specific, yet facultative symbiosis between ascidians and Endozoicomonas.

Two gram-negative, facultative anaerobic, chemoorganoheterotrophic, motile and rod-shaped bacteria, strains AVMART05(T) and KASP37, were isolated from ascidians (Tunicata, Ascidiaceae) of the genus Ascidiella collected at Gullmarsfjord, Sweden. The strains are the first cultured representatives of an ascidian-specific lineage within the genus Endozoicomonas (Gammaproteobacteria, Oceanospirillales, Hahellaceae). Both strains feature three distinct 16S rRNA gene paralogs, with identities of 98.9-99.1% (AVMART05(T)) and 97.7-98.8% (KASP37) between paralogs. The strains are closely related to Endozoicomonas atrinae and Endozoicomonas elysicola, with which they share 97.3-98.0% 16S rRNA gene sequence identity. Digital DNA-DNA hybridization, average nucleotide identity, and tetra-nucleotide correlation analysis indicate that both strains belong to a single species distinct from their closest relatives. Both strains feature similar DNA G+C contents of 46.70mol% (AVMART05(T)) and 44.64mol% (KASP37). The fatty acid patterns of AVMART05(T) and KASP37 are most similar to those of Endozoicomonas euniceicola and Endozoicomonas gorgoniicola. Based on the polyphasic approach, we propose the species Endozoicomonas ascidiicola sp. nov. to accommodate the newly isolated strains. E. ascidiicola sp. nov. is represented by the type strain AVMART05(T) (=DSM 100913(T)=LMG 29095(T)) and strain KASP37 (=DSM 100914=LMG 29096)., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

The bacterial genus Endozoicomonas was commonly detected in healthy corals in many coral-associated bacteria studies in the past decade. Although, it is likely to be a core member of coral microbiota, little is known about its ecological roles. To decipher potential interactions between bacteria and their coral hosts, we sequenced and investigated the first culturable endozoicomonal bacterium from coral, the E. montiporae CL-33(T). Its genome had potential sign of ongoing genome erosion and gene exchange with its host. Testosterone degradation and type III secretion system are commonly present in Endozoicomonas and may have roles to recognize and deliver effectors to their hosts. Moreover, genes of eukaryotic ephrin ligand B2 are present in its genome; presumably, this bacterium could move into coral cells via endocytosis after binding to coral's Eph receptors. In addition, 7,8-dihydro-8-oxoguanine triphosphatase and isocitrate lyase are possible type III secretion effectors that might help coral to prevent mitochondrial dysfunction and promote gluconeogenesis, especially under stress conditions. Based on all these findings, we inferred that E. montiporae was a facultative endosymbiont that can recognize, translocate, communicate and modulate its coral host.