Skip to main content
NCBI home page
The ISME Journal logoLink to The ISME Journal
. 2025 Mar 28;19(1):wraf059. doi: 10.1093/ismejo/wraf059

Differential aggregation patterns of Endozoicomonas within tissues of the coral Acropora loripes

Cecilie R Gotze 1,2,✉, Ashley M Dungan 3, Allison M L van de Meene 4,5, Katarina Damjanovic 6, Gayle K Philip 7, Justin Maire 8, Lone Høj 9, Linda L Blackall 10, Madeleine J H van Oppen 11,12
PMCID: PMC12649755  PMID: 40156309

Abstract

Bacteria in the genus Endozoicomonas are well-known coral symbionts commonly found as clusters within tissues of several coral species. Mapping the spatial distribution of these microbial communities is critical to gaining a holistic understanding of the potential role they may play within the coral host. This study focuses on characterizing bacterial aggregates associated with the common reef-building coral, Acropora loripes, from the central Great Barrier Reef, Australia. A conventional cultivation-based method was employed to establish a pure culture collection of 11 undescribed Endozoicomonas strains isolated from A. loripes. Subsequent 16S rRNA gene amplicon sequencing revealed their classification into two distinct phylogenetic clades. To resolve their spatial distribution in hospite, clade-specific fluorescence in situ hybridization probes were designed. Aggregates were consistently observed in the gastrodermal tissue layers surrounding the upper and lower gastrovascular cavity and were predominantly formed by cells from the same phylogenetic clade, with a minor proportion of aggregates formed by Endozoicomonas from both targeted clades. Furthermore, a clear distinction in aggregation pattern was observed; one clade exhibited clusters with regular and contained growth patterns, whereas the other formed clusters lacking clear boundaries and having irregular shapes. Scanning electron microscopy revealed the presence of a membrane of unknown origin associated with bacterial aggregates in two instances, suggesting potential structural or functional differences in these aggregates. These morphological differences highlight the importance of further investigations into the mechanisms governing bacterial aggregate formation in corals.

Keywords: Endozoicomonas, coral symbiosis, microbiome, symbiotic bacteria

Graphical Abstract

Graphical Abstract.

Graphical Abstract

Open in a new tab

Introduction

Coral reefs are known as the rainforests of the sea, as they are among the world’s most biologically diverse ecosystems [1]. Much of the ecological success of reef-building corals is attributed to their endosymbiosis with dinoflagellate algae in the family Symbiodiniaceae [2, 3]. However, corals also form associations with a multitude of other microbes, including protists, fungi, bacteria, archaea, and viruses [4–7]. Together, the coral host and its associated microbial community form a multipartite organism termed a holobiont. Historically, coral microbiology has focused on the coral-Symbiodiniaceae symbiosis, but the last decade has seen increasing recognition of the role bacteria play in holobiont functioning and health [8–10]. Studies aimed at deciphering the roles of bacteria within the holobiont are complicated by the diverse and rich nature of their bacterial communities, with some coral species harboring several thousand different prokaryotic taxa [11]. Additionally, coral-associated bacterial communities have also been demonstrated to differ according to environmental disturbances, seasonality, geographic area, coral genotype, and even within a single coral colony [12–16].

A reductionist approach can be employed by studying coral species that naturally host relatively low-diversity bacterial communities, with some corals naturally associating with as few as 10–20 bacterial species, often dominated by a few highly abundant taxa [17–20]. Among these, the bacterial genus Endozoicomonas has garnered significant interest due to its association with various marine invertebrates, such as cnidarians [21, 22]. The global prevalence and abundance of Endozoicomonas within coral ecosystems suggest a pivotal ecological role, potentially driven by competitive advantages or mutualistic relationships with the coral hosts [23, 24]. Although the functional role of Endozoicomonas in the host microenvironment is not fully understood, they are suggested to be involved in fundamental holobiont processes such as B-vitamin metabolism [25] as well as cycling of nitrogen, sulfur, and phosphorus [26–28].

The generally high relative abundance of Endozoicomonas within corals can be attributed to their capacity to form dense clusters, known as cell-associated microbial aggregates (CAMAs). Although CAMAS have been found in a diversity of coral species throughout the Indo-Pacific [29] and the Caribbean [30, 31], the presence of Endozoicomonas in these aggregates has only been confirmed in a subset of coral species. This includes CAMAs occurring within the epidermal layer of the tentacles of corals from the family Pocilloporidae [28, 32, 33]. Moreover, members of the novel genus, Soroendozoicomonas, within the Endozoicomonadaceae family, were recently identified as forming aggregates in the mesenterial filaments in the coral Pocillopora acuta, which are used for digestion and prey capture [34]. The lack of a consistent pattern in aggregate distribution suggests that the process may be phylotype-specific, host-specific, or influenced by other host physiological factors. However, the microorganisms that reside within these aggregates have not been fully characterized.

Only one recent study has applied species-specific probes to investigate the bacterial composition within CAMAs and found them to be occupied by multiple Endozoicomonas phylotypes, indicating the potential for shared metabolic pathways [28]. Nonetheless, most studies of Endozoicomonas have either described their abundance or genomic repertoire without assessing their spatial distribution and ability to form CAMAs [25, 27, 35, 36]. Consequently, important knowledge on the taxonomic composition of bacteria within CAMAs and how they are distributed throughout different microhabitats of the host is lacking. Such knowledge could help decipher their functional potential, as different locations likely represent distinct metabolic and biochemical niches where members could carry out different functions [37].

This study investigates the bacterial composition and spatial distribution of CAMAs in the reef-building coral Acropora loripes, A. loripes is known to harbor low-diversity bacterial communities, making it an ideal model for targeted analysis [19, 20]. We employed a culture-based approach combined with fluorescence in situ hybridization (FISH) and scanning electron microscopy (SEM) to visualize the spatial distribution, taxonomic composition, and morphology of CAMAs within their native host, obtained from two reef sites on the central Great Barrier Reef (GBR), Australia, i.e. Davies and Backnumbers Reef.

Materials and methods

Coral collection and maintenance

Visually healthy A. loripes colonies at ~25 cm diameter (i.e. only colonies with no signs of tissue necrosis or bleaching) were collected from two sites on the central Great Barrier Reef: Davies Reef (June 2020, February 2021) and Back Numbers Reef (December 2020) (Permit G12/35236.1). Corals were transported to the Australian Institute of Marine Science (AIMS) in aerated seawater and maintained at the National Sea Simulator (SeaSim) under ambient lighting and temperature conditions that mirrored the Davies Reef profile. Corals were supplied with 0.4 μm filtered seawater at a turnover rate of 4.8 times per day and fed daily with Artemia (0.5 nauplii ml −1). Additional collection details and tank parameters are provided in Supplementary Materials.

16S rRNA gene and ITS2 amplicon sequencing

As significant variation in microbial communities both within and among A. loripes colonies, has previously been observed [19], fragments were collected from four distinct branches within each coral colony to account for variations in microbial composition. Following DNA extraction and amplification, samples from each colony were pooled for downstream analysis to ensure a representative assessment of the microbial community. DNA was extracted from 3 to 5 mm fragments using a previously described in-house extraction protocol [38]. The V5–V6 region of the 16S rRNA gene was then amplified using the bacterial primer pair 784F/1061R (Andersson et al., 2008), and the internal transcribed spacer 2 (ITS2) region of Symbiodiniaceae was amplified using forward primer SYM_VAR_5.8S2 and reverse primer SYM_VAR_REV (Hume et al., 2018). Sequencing was performed on a MiSeq platform (Illumina) using v3 (2 × 250 bp) reagents at the Walter and Eliza Hall Institute (Melbourne, Australia). Amplicon data were processed using QIIME2 [39], and ITS2 sequences were analysed with the SymPortal framework [40]. For additional details, including sampling, primer sequences, PCR protocols, and bioinformatics pipelines, see Supplementary Materials.

Bacterial culturing

Coral fragments were processed to isolate tissue-associated bacteria using tissue homogenization, followed by serial dilutions and plating on Marine Agar 2216. Plates were incubated in the dark at 23°C, and bacterial colonies were purified over three passages. DNA from bacterial isolates was screened using PCR with Endozoicomonas-specific primers and sequenced to confirm identity. Further details, including homogenization protocols, culturing conditions, and PCR reagents, are provided in Supplementary Materials.

Phylogenetic analysis

Phylogenetic relationships of cultured Endozoicomonas strains were assessed by constructing a maximum likelihood tree of 16S rRNA gene sequences aligned with near full-length Endozoicomonadaceae sequences from the SILVA database. Sequences were aligned using MAFFT, and the TIM3e + R6 model was selected with ModelFinder. Bootstrap support for tree topology was evaluated with 1000 replicates. Detailed pipeline steps, including software versions and alignment methods, are available in Supplementary Materials.

Design and validation of FISH probes

Phylotype-specific probes for Endozoicomonas Clade-A and Clade-B were designed using the ARB probe design tool with a curated database of 16S rRNA sequences. Probes were tested for specificity using in silico validation and hybridization with pure bacterial isolates. Stringency conditions were optimized using increasing formamide gradients (15%–30%). See Supplementary Materials for probe sequences, specificity testing, and optimization.

Sample fixation, histology, and microscopy

Coral fragments were fixed in 4% paraformaldehyde, decalcified in EDTA, and embedded in paraffin for histology and FISH. Tissue sections were stained with hematoxylin and eosin or hybridized with clade-specific and universal Endozoicomonas probes labeled with Atto550 or Atto647N. Autofluorescence was reduced through methanol quenching, and probe specificity was confirmed via confocal laser scanning microscopy. For SEM, paraformaldehyde-fixed tissue sections where CAMAs were identified were further processed with heavy metal staining (uranyl acetate and lead citrate) to enhance contrast and the CAMA visualized using a scanning electron microscope after correlation with the optical image. Detailed fixation, staining, and imaging protocols are described in supplementary materials.

Results

Microbial community composition within the tissues of the coral A. loripes

To characterize the bacterial community composition associated with A. loripes, 16S rRNA gene amplicon sequence analysis was conducted on coral fragments from 12 of the 14 coral colonies collected from the GBR, Australia. Bulk tissue samples (n = 4 per coral colony) indicated a similar microbial community composition across all sampled colonies, comprising just a few bacterial genera (Fig. 1A). Furthermore, considering all amplicon sequence variants (ASVs), including rare ones, a total of 600 ASVs were identified from 47 samples across 12 A. loripes corals. However, upon excluding low-abundance ASVs (i.e. those with counts representing less than 0.001% relative abundance), a total of 249 ASVs were present across all 47 samples. Endozoicomonas was consistently the dominant genus across almost all replicate samples, with sequences affiliated with Endozoicomonas reaching more than 95% relative abundance (RA) in some samples. Other major taxa included Candidatus Amoebophilus (colony H, ~25% RA), P3OB-42 (family Myxococcaceae, in coral Al06, ~15% RA), and Candidatus Fritschea (family Simkaniaceae, coral Al01, ~10% RA) and an unknown taxon from Gammaproteobacteria (coral Al06 and Al09, ~20% RA).

Figure 1.

Figure 1

Open in a new tab

Relative abundance of A. loripes-associated bacteria. (A) Bar plot visualizing the relative abundance of bacterial genera identified in March 2021 across 12 A. loripes colonies annotated on the vertical axis. Genera with <5% relative abundance were pooled into the “<5% abundance” category. Each coral colony (Al01-Al14) was subsampled across four distinct-colony locations, with replicate samples pooled by genotype for subsequent analysis. Relative abundances are represented as the average across four replicate samples. Three replicate samples of filtered seawater (FSW) from the aquarium where the corals were reared were included in 16S rRNA gene sequencing for comparison. (B) Bar plot showing the relative abundance of the ASVs matching the cultured or uncultured strains across the same 12 A. loripes colonies. Relative abundances are represented as the average across the four replicates and two time points (March and June 2021). The hatched proportion represents ASV’s which did not match any isolate sanger sequences, i.e. these were not obtained in the Endozoicomonas culture collection.

In addition to bacterial community composition analysis, ITS2 amplicon sequencing was performed to assess the dominant Symbiodiniaceae genus across coral genotypes. Analysis of bulk tissue samples (n = 4 per coral colony) revealed that all coral colonies hosted a single Symbiodiniaceae genus, Cladocopium. Although pairwise Adonis comparisons of ITS2-type profiles indicated compositional differences in some coral colonies (P = .005), this had no significant effect on the microbiome composition (Fig. S6).

Culturing the dominant taxa from the microbiome of A. loripes

At the same time as the metabarcoding samples were taken, A. loripes colonies were sampled to establish pure cultures of associated bacteria on Marine Agar. Culturing efforts yielded >500 bacterial pure cultures, among which 87 were identified as belonging to Endozoicomonas through PCR screening with genus-specific primers. Analysis of the 16S rRNA gene sequences from each isolate revealed 11 distinct Endozoicomonas strains. The comparison of isolate-derived near full length 16S rRNA gene sequences with ASV sequences from the metabarcoding data of the coral holobiont demonstrated that the culture collection represented the dominant ASVs associated with A. loripes, with the culturable proportion of Endozoicomonas ASV’s ranging from 28% in coral Al06 to 95% in coral Al05 (Fig. 1B). Among the isolated bacteria, three Endozoicomonas isolates, namely ALB032, ALC066, and ALB115, were the most abundant in A. loripes based on matches to ASVs in the metabarcoding dataset. However, their relative abundance exhibited marked variations across coral colonies, with ALB115 showing low relative abundance (<1%) in colony Al13.

Phylogenetic distinction of newly isolated Endozoicomonas strains

To evaluate the taxonomy and phylogenetic connections among newly isolated Endozoicomonas strains, we constructed a maximum likelihood phylogeny using near-complete 16S rRNA gene sequences (Fig. 2). Alongside our novel isolates, we incorporated 1459 Endozoicomonadaceae 16S rRNA sequences >1200 bp sourced from the SILVA database. The four isolates ALB091, ALE010, ALB115, and ALC013 share >97% sequence identity in their 16S rRNA gene, indicating that they belong to the same species (De Albuquerque and Haag 2023) (Fig. S7), hereafter referred to as Clade-A. Isolates ALB060, ALC020, ALC066, ALD068, ALB122, ALB032, and ALB040 also exhibited >97% sequence identity and together formed a distinct clade, named Clade-B. Pair-wise comparisons between 16S rRNA gene sequences from each clade revealed that Clade-A and Clade-B on average share 94% identity. Moreover, phylogenetic analysis indicated that despite low sequence identity, they both share common recent ancestors with Endozoicomonas sequences obtained from the coral Acropora humilis, sampled from the Red Sea [41, 42].

Figure 2.

Figure 2

Open in a new tab

Phylogenetic placement of isolated Endozoicomonas strains. Placement of 11 A. loripes-associated Endozoicomonas strains within the Endozoicomonadaceae family. Maximum likelihood tree constructed from 1459 bacterial 16S rRNA gene sequences (length of total alignment 2087 bp) obtained from the SILVA database in addition to 16S rRNA gene sequences extracted from 11 novel A. loripes sourced isolates. Bootstrap values >50% based on 1000 replications are shown at the nodes. The tree is rooted at Zoshikella.

Stability and composition of Endozoicomonas after three months in captivity

To assess the stability of the bacterial community composition, coral colonies sampled in March 2021 were re-sampled in June 2021. During this period, corals were kept in an aquarium under conditions mimicking seasonal ambient light and temperature at Davies Reef, GBR. Replicate samples from the same coral colony were pooled for each time point. Metabarcoding analysis revealed that Endozoicomonas remained the dominant taxon across all coral colonies (except Al06, which was excluded early due to tissue sloughing) (Fig. 3). Although some variation in the relative abundance of specific Endozoicomonas ASVs was observed, the overall prevalence of Endozoicomonas persisted over time.

Figure 3.

Figure 3

Open in a new tab

Relative abundance across two timepoints. Relative abundance of the 18 most abundant 16S rRNA ASVs over time in captive A. loripes. Bubble plot showing the relative abundance (%) of the 18 most abundant ASVs across all 12 A. loripes colonies. Each column visualizes ASVs in one representative colony annotated above (Al01-Al14). Relative abundances are represented as the average across four replicate samples. Sampling for 16S rRNA gene metabarcoding analysis was carried out in March and June 2021.

In situ spatial distribution and composition of CAMAs

To examine the spatial distribution of the two major phylotypes (Clade-A and Clade-B) within the host tissue, coral colonies sampled for 16S rRNA gene metabarcoding analysis in June 2021, were concurrently sampled for histological examination. Six replicate fragments (~2 cm branch tips) were sampled from different locations across each coral colony ranging from sun-exposed to shaded areas. A total of 293 CAMAs were identified through H&E staining of the serial-sectioned tissues from distinct colonies.

To validate the presence of Endozoicomonas within the CAMAs, we also conducted FISH with previously published probes targeting either all bacteria [43] or specifically the Endozoicomonas genus [41] (Fig. 4). By employing both probe types on consecutive serial sections, we confirmed that Endozoicomonas constitute the primary and potentially sole, bacterial genus forming CAMAs within A. loripes.

Figure 4.

Figure 4

Open in a new tab

CAMAs found within the tissue of A. loripes. (A) CAMAs displaying hybridization with the 'all-Endozoicomonas mix' probe (red) targeting all Endozoicomonas bacteria are localized within the mesenteries lining the gastrovascular cavity. (B) Whole-mount FISH image showing a CAMA in the actinopharynx hybridized with the 'all-Endozoicomonas mix' probe (red). In both images, red fluorescence corresponds to the 'all-Endozoicomonas mix' probe labeled with Atto647N. Non-EUB probe labeled with Atto550 (negative control) is depicted in white. Cyan indicates DNA stained by DAPI (shown in A only). Host autofluorescence is represented in blue and overlapping signals between the target probe and negative control probe are shown in pink indicating non-specific binding at nematocysts and mucocytes within the Cnidoglandular bands. Sym, Symbiodiniaceae; Nuclei, Host nuclei; Nem, Nematocytes; Tent, tentacle; Cni, Cnidoglandular bands. Scale bars represent 100 μm.

Endozoicomonas CAMAs were extensively distributed across several regions of the coral polyp, predominantly associated with the gastrodermal layer lining the mesenteries, which forms multiple folds that span the upper and lower gastrovascular cavity [44] (Fig. 5A). In total, 78% of CAMAs were detected in the mesenterial gastrodermis, 13% in the gastrodermis lining the actinopharynx, and 7% in the coenenchyme, which acts as gastrovascular tubes between polyps. The remaining 2% could not be confidently assigned to specific tissue regions due to histological artifacts. On average three CAMAs were detected per 2 cm longitudinal coral section. Exceptions were corals Al01 and Al07, in which histological sections appeared not to have been cut deep enough to capture tissues lining the gastrovascular cavity. The average diameter of CAMAs across all corals was 42.8 μm (SD = 17.5, SE = 1.02), with the largest being 97 μm and the smallest 12 μm—a ninefold difference (Fig. 5B-C). This considerable variation likely reflects both biological variation and differences in the plane of sectioning through the 4 μm-thick tissue sections.

Figure 5.

Figure 5

Open in a new tab

CAMA distribution and size proxy across anatomical regions. 2D surface area measurements of 4 μm coral tissue sections showing CAMAs within various tissues of A. loripes. (A) Illustration of a coral polyp with color-coded tissue regions where CAMAs were detected (refer to panels B and C). (B) Pie charts showing the frequency of CAMA occurrence in the various tissue compartments. (B) Dot plots comparing CAMA sizes (surface area in 2D sections), with colors representing the tissue compartment of each CAMA. A total of 293 CAMAs were detected in H&E-stained tissues. Violin plots show the median and interquartile range of CAMA sizes from each coral colony. "NA" denotes CAMAs with indeterminate locations due to histological artifacts. Schematic representation inspired by [45].

Clade-specific aggregation and morphology of CAMAs

To further resolve the composition and spatial distribution of Endozoicomonas within CAMAs, we employed FISH with newly designed clade-specific oligonucleotide probes targeting either Clade-A or Clade-B. Each coral colony underwent an investigation of 30 CAMAs to discern the bacterial composition at the clade level based on observed hybridization patterns. The analysis revealed that the majority of CAMAs consisted of members belonging to Clade-B, comprising 83% of CAMAs observed with phylotype-specific FISH probes. These Clade-B CAMAs were present across all examined coral colonies. In contrast, CAMAs composed of Clade-A members were exclusively identified in corals Al01, Al05, Al06, Al07, and Al11.

Distinct morphological differences were observed between Clade-A and Clade-B CAMAs. Clade-A aggregates displayed a regular and contained aggregation pattern, whereas those formed by Clade-B lacked a clear boundary, exhibiting irregular shapes (Fig. 6). Clade-B CAMA morphologies ranged from highly dispersed growth to somewhat more circular shapes, but they never appeared to be enclosed by a restricted boundary in the outer periphery as consistently observed in Clade-A CAMA morphologies (Fig. S8).

Figure 6.

Figure 6

Open in a new tab

Clade-specific CAMA morphology. (A) CAMA observed in a tissue section, showing hybridization signal from the Clade-A specific probe labeled with Atto647N depicted (depicted in magenta). (B) CAMA in a tissue section hybridized with the Clade-B specific probe labeled with Atto550 depicted in yellow. (C) Three adjacent CAMAs, with two CAMAs hybridized with the Clade-A probe (magenta) and another CAMA hybridized with the Clade-B probe (yellow). Blue indicates host autofluorescence and cyan highlights nuclear structures stained by DAPI. Scale bars represent 50 μm.

Mixed-population CAMAs, containing both Clade-A and Clade-B members, were detected in only six instances across corals Al07, Al08, and Al09. These aggregates exhibited binding of both phylotype-specific probes, with overlapping fluorescent signals or spatial segregation within the same structure (Fig. 7). In some mixed CAMAs, fluorescent signals from the Clade-A specific probe were concentrated at the outer edges, whereas in others, both probes exhibited a homogeneous distribution across the same CAMA. However, no clear patterns were identified regarding the cellular arrangement of Clade-A and Clade-B within mixed-population aggregates. The morphology of all six CAMAs which were comprised of both Endozoicomonas clades closely resembled that of Clade-A (Fig. 6A). Lastly, we found no CAMAs that did not bind phylotype-specific oligonucleotide probes, emphasizing that CAMAs were exclusively formed by members belonging to either Clade-A, Clade-B, or a combination of both.

Figure 7.

Figure 7

Open in a new tab

Detection and co-localization of mixed-CAMA populations. Fluorescent signals from CAMAs binding both phylotype-specific probes. (A) Two adjacent CAMAs in a tissue section hybridized with the 'all-Endozoicomonas mix’ probe (red) and stained with DAPI depicted in cyan. Cyan arrow indicates coral host nuclei at the periphery of a CAMA, suggesting coral cells are overlapping the aggregate. (B) the consecutive section to the one shown in (A) hybridized with Clade-A (magenta) and Clade-B (yellow) specific probes, showing binding of both probes within the same CAMAs. Arrows indicate individual bacterial cells hybridized by the Clade-B probe (top CAMA) and Clade-A probe (bottom CAMA) at the periphery of the CAMAs, whereas individual cells at the center of the CAMA are difficult to observe due to both signals merging in the image (B-C). Coral tissue in (A) is sectioned 8 μm above (B). Blue: Host autofluorescence. Cyan: Nuclear structures stained by DAPI. (C and D) are split channels from the merged image shown in B. White arrows indicating individual cells which can be distinguished in B. Scale bars represent 20 μm.

To further investigate the clade-specific CAMA patterns, we examined sectioned coral tissue using SEM. In coral genotype Al07, this analysis revealed bacterial aggregates enclosed by a distinct membrane of unknown origin (Fig. 8). However, no SEM images of Clade-B CAMAs were obtained, as Clade-B aggregates could not be detected in the examined tissue sections.

Figure 8.

Figure 8

Open in a new tab

Scanning electron micrographs visualizing bacterial aggregates within the tissues of coral genotype Al07 using backscattered electrons. (A) Overall view of a restricted bacterial aggregate within the coral tissue. Host nuclei are marked with black arrows, indicating their spatial relationship adjacent to the aggregate. Scale bar = 20 μm. (B) Close-up view highlighting a membrane-like structure of unknown origin surrounding the bacterial aggregate (indicated by white arrows). Scale bar = 10 μm. C: Detailed close-up of the bacterial aggregate and surrounding structure. Scale bar = 3 μm.

Discussion

Dominant Endozoicomonas species of A. loripes are stable symbionts

Overall, the relative abundance of Endozoicomonas within captive A. loripes colonies persisted over three months, which is consistent with previous findings in A. loripes [19], as well as in other coral families [32, 46]. A consistently high relative abundance of three ASVs was observed, i.e. ALB032, ALC066 from Clade-B and ALB115 from Clade-A, suggesting these isolates could be true symbionts of A. loripes, forming a temporally stable association with their coral host. However, genotype-specific differences in the relative abundance of ALB032, ALC066, and ALB115 were observed, and the distribution of these ASVs varied across individual corals (Fig. 3). This study examined relative abundance only, and fluctuations may also be influenced by the presence of large bacterial aggregates within sampled fragments, causing bacterial abundances to increase [47, 48]. Therefore, to draw more conclusive insights, ASV stability should ideally be assessed using absolute abundance data [20, 49].

Diversity and co-occurrence of Endozoicomonas within A. loripes

Phylogenetic analysis indicated that Endozoicomonas strains isolated from tissues of the coral A. loripes belonged to two distinct species co-occurring within the same host (Fig. 2) [50]. However, not all Endozoicomonas ASVs were detected in all sampled colonies, for instance, ALB112 and ALC020 from Clade-B were only detected in a subset of corals, entirely lacking in seven out of the 12 corals sampled (Fig. 3). A similar pattern has been observed in other Acropora species, where multiple distinct Endozoicomonas phylotypes were obtained from the same colony, although with widely varying abundance among individual corals [35, 51]. This pattern is not only restricted to acroporid corals but has also been observed in pocilloporids [52], suggesting that a subset of strains in this study could be more widespread, possibly originating from differing adaptative strategies despite occurring within the same coral host [10, 25]. These strategies could include variations in resource acquisition mechanisms, metabolic flexibility, or tolerance to specific environmental stressors, allowing distinct Endozoicomonas strains to coexist while occupying different ecological niches of the same coral host [23]. Moreover, local environmental conditions may drive associations with specialized Endozoicomonas phylotypes. For instance, corals in the genus Pocillopora associate with Endozoicomonas phylotypes that are globally distributed, whereas Stylophora exhibits a higher degree of host-microbe specificity where colonies associate with unique or closely related Endozoicomonas strains [33, 36, 41]. Thus, some corals might select specific characteristics or functions that different Endozoicomonas strains and species have and which are required for adaptation to their particular ecological niche or metabolic requirements [53]. Conversely, instead of the host-selection of bacterial symbionts, it is plausible that the bacteria colonize hosts based on their nutritional requirements which may be provided by coral or photosymbiont, as some coral-associated bacteria appear to be correlated with the presence of Symbiodiniaceae [54].

Localization and formation of Endozoicomonas aggregates in A. loripes tissues

In this study, we demonstrated that distinct Endozoicomonas phylotypes were found as aggregates within the gastrodermis of A. loripes. These findings align with several previous studies on other coral species [29, 41, 45]. Most CAMAs observed in this study were located within the mesenteries of the gastrovascular cavity, consistent with the localization of CAMAs in other acroporid corals, such as Acropora hyacinthus [45] and Pocillopora acuta [34]. However, unlike these corals, CAMAs were not observed in the mesenterial filaments, specialized extensions of the mesenteries that can be extruded from the polyp’s mouth during digestion or defense (Fig. 4). Moreover, unlike pocilloporid corals where CAMAs validated as Endozoicomonas are mainly detected in the epidermis of the tentacles [28, 32, 41], no CAMAs were detected within the tentacles of A. loripes. These findings suggest the existence of coral species-specific niches for CAMA formation.

Coral-associated bacteria differ in chemotactic capabilities that allow them to locate optimal niche microhabitats within their host [55, 56]. Therefore, differences in CAMA location could be linked to the chemotactic capabilities of natural populations of coral-associated bacteria towards chemicals released by corals and/or their symbionts or localization near preferred metabolites including carbohydrates, ammonium and dimethyl sulfoniopropionate [57, 58]. Therefore, variations in biochemical and nutritional requirements across different Endozoicomonas strains may impact the selection of their preferred ecological niche within the coral holobiont.

Variations in Symbiodiniaceae composition have been shown to influence coral physiology, including nutrient cycling and thermal tolerance [59, 60], which in turn could have an effect on the coral-associated microbiome [61], e.g. the distribution and abundance of Endozoicomonas. The ITS2 amplicon analysis revealed that all coral genotypes were dominated by the same Symbiodiniaceae genus, Cladocopium, indicating that symbiont composition is less likely to be a confounding factor in the observed differences in Endozoicomonas aggregation and localization.

No CAMAs were found in the epidermis nor the gastrodermis of the tentacles in this study. Instead, all CAMAs co-localized with Symbiodiniaceae in gastrodermal tissues, including the actinopharynx, mesenteries, and coenenchyme. The spatial distribution suggests metabolic interactions between CAMAs and Symbiodiniaceae, where oxygenic photosynthesis generates high oxygen levels during the day. Microhabitats within the mesenteries or gastrovascular cavity may optimize microbial metabolism, with CAMAs positioned in areas with lower oxygen or temporary anoxic conditions during nighttime respiration, supported by localized high nutrient gradients such as ammonium, nitrate, nitrite, and phosphate and vitamin B12 [62–65]. In contrast, CAMAs in the epidermis, as reported in pocilloporid corals [28, 32, 41], may experience lower nutrient availability in surrounding reef waters, particularly during tentacle extension for feeding [63]. These differences suggest that variations in biochemical and nutritional requirements among Endozoicomonas strains influence their tissue localization and preferred ecological niches.

Endozoicomonas composition within aggregates

CAMAs were predominantly formed by cells from the same phylogenetic clade (Clade-A or Clade-B), with a minor proportion (2%) containing the two distantly related Endozoicomonas phylotypes (94% average 16S rRNA sequence identity) coexisting within the same aggregate. Only one other recent study has applied species-specific probes to investigate the bacterial community composition within CAMAs of corals, in this case in Stylophora pistillata [28]. Their findings indicated that CAMAs were predominantly located in the tentacles and contained a single phylotype of Endozoicomonas or a mix of highly similar phylotypes (sequence identity ≥97.7%). Similarly, only closely related Endozoicomonas strains (16S rRNA V5-V6 sequence identity 96 to >99%) were excised from the same CAMA in the coral P. acuta [32].

Different bacterial phylotypes within aggregates can interact antagonistically through secretion systems and compete for nutrients, resulting in growth inhibition of one of the phylotypes [66, 67]. However, microorganisms can also take advantage of the proximity to interacting cells via bidirectional beneficial relationships [68–70]. Such synergistic interactions can lead to the evolution of advantageous traits, such as metabolic complementation [71], which arises when one or multiple species utilize metabolites produced by neighboring species. All these interactions are often facilitated by cross-species communication via quorum sensing [72–74]. Hence, the CAMA development of one Endozoicomonas phylotype could play an important role in governing the sequential recruitment of either conspecific strains or other Endozoicomonas species. Conversely, the dominance of members from Clade-B (Fig. 4), as well as the low proportion of mixed CAMAs in this study compared to S. pistillata [28] could suggest a degree of niche exclusion between phylogenetically distinct Endozoicomonas species. This is corroborated by the finding that mixed CAMAs were only obtained from one geographical region in [28].

Aggregate morphological variation

In this study, differences in CAMA morphology were observed between the two identified clades. Clade-A exhibited CAMAs with regular and contained growth patterns, whereas Clade-B aggregates lacked a clear boundary and exhibited an irregular shape. Although the specific bacterial taxa forming CAMAs remained unknown, a previous study documented cellular-level variations among collective CAMAs within individual A. hyacinthus colonies, including those comprised of rod-shaped, pleomorphic, filamentous-like, or spore-like structures [45]. However, the morphology of bacterial cells within individual CAMAs in A. loripes appeared homogenous. To our knowledge, this is the first study documenting systematic variations in CAMA morphology, highlighting the need for further investigation. SEM confirmed aggregates in genotype Al07 were surrounded by a membrane of unknown origin (Fig. 8). This membrane-like structure formed a clear boundary around the aggregates, potentially indicating compartmentalization within the host tissue. Although SEM and FISH could not be performed on the same CAMAs, the Al07 genotype exhibited a higher relative abundance of Clade-A Endozoicomonas compared to Clade-B Endozoicomonas (Fig. 1). This observation suggests a potential association between Clade-A Endozoicomonas and the membrane-like structures, possibly reflecting an intracellular localization. However, SEM images of Clade-B CAMAs could not be obtained, leaving their ultrastructural characteristics unresolved in this study.

The possible membranes enclosing Clade-A CAMAs imply a controlled process of aggregation, possibly involving specific adhesion molecules, such as fimbriae or targeted secretion systems [75, 76]. In certain cases, secretion systems of both pathogenic and symbiotic bacteria can deliver effector proteins that interact directly with host cell machinery. These effectors can facilitate adherence, promote cell invasion via host immune suppression and support the intracellular survival of bacteria within host cells [77–79]. Conversely, the regular and contained growth pattern of these aggregates within host tissues could also suggest the presence of a membrane. A membrane of unknown origin was recently observed in CAMAs within the tentacles of the coral P. acuta [32] suggesting an intracellular localization. Moreover, CAMAs of unknown composition detected in the coral Porites compressa were found to be contained within a membrane [29]. These observations also extend to the cnidarian model Exaiptasia diaphana, where the presence of a membrane surrounding CAMAs found within the tentacles was dependent on the size of the aggregates, with only aggregates smaller than anemone cells appearing to be intracellular [80]. The absence of a clear margin in CAMAs formed by members of Clade-B CAMAs suggests they may utilize different adhesion molecules or extracellular matrix components, leading to a distinct pattern of aggregation within the coral tissue. Clade-B may have alternative strategies for evading host immune system cues compared with Clade-A, with consequences for subsequent internalization or lack thereof [75, 81].

Recent work has suggested a trade-off between higher Endozoicomonas abundance and increased disease susceptibility, indicating that while Endozoicomonas may promote coral growth, elevated loads could compromise host health [82]. The spatial compartmentalization of Endozoicomonas and the structural differences between Clade-A (restricted, membrane-associated aggregates) and Clade-B (diffuse, irregular aggregates) suggest that aggregation patterns could influence host immune interactions. The membrane-associated aggregates of Clade-A bear similarities to coral symbiosomes, intracellular compartments that regulate nutrient exchange and immune modulation in Symbiodiniaceae-host interactions [83–86]. In contrast, Clade-B’s diffuse growth may indicate a less regulated interaction, potentially eliciting stronger immune responses, aligning with the hypothesis that higher bacterial loads increase disease susceptibility [82]. While the precise function of Clade-A’s membrane-associated aggregates is unknown, they may contribute to a more regulated host–microbe interaction, balancing mutualism and immune activation in coral-associated microbiomes.

Differences in CAMA morphology between Clade-A and Clade-B may also reflect metabolic adaptations. A recent study has demonstrated that bacterial populations of marine Vibrionaceae strains secrete enzymes required for polysaccharide breakdown, which may be reflected in cell aggregation patterns [87]. Specifically, strains with lower levels of lyases tend to aggregate more strongly than those that secrete high levels of enzymes. Furthermore, the study suggested that increased aggregation enhances intercellular synergy within tightly packed aggregates compared to strains that aggregate more diffusely. These findings suggest that metabolic functions can be reflected in the cell aggregation patterns of marine bacteria capable of extracellularly catabolizing polysaccharides.

Clade-B CAMAs exhibited irregular and dispersed formation patterns, possibly indicating a dynamic state of aggregation. These structures may represent bacterial aggregates undergoing dispersal or early stages of colonization within the coral tissues. Corals have been shown to regulate symbiont abundance by digesting excess symbiont cells [88]. A similar regulatory mechanism could be acting on bacterial CAMAs, where the host selectively removes or modulates bacterial aggregates based on environmental or physiological cues.

Conclusion

In summary, this study provides key insights into the role of Endozoicomonas in coral health and functioning. Amplicon sequencing and culturing revealed the dominance of Endozoicomonas in A. loripes colonies, identifying two coexisting clades. FISH experiments showed clade-specific CAMAs in the coral's gastrodermis, with distinct morphologies suggesting differing host–microbe interactions. A membrane of unknown origin was observed enclosing CAMAs dominated by Clade-A, though further validation is required to confirm this structure. Future studies will investigate the genomic potential of these clades and the mechanisms underlying CAMA formation.

Supplementary Material

Gotze_Supplementary_materials_wraf059
gotze_supplementary_materials_wraf059.pdf (10.2MB, pdf)

Acknowledgements

We acknowledge the Wulgurukaba and Bindal peoples as the first scientists and traditional custodians of the lands and sea country where this research was conducted. We extend our sincere gratitude to the SeaSim team for their collaboration and support in coral collection and husbandry. We are also grateful to Chien Lit Cheah for assistance with histology and staining. Additionally, we acknowledge the Biological Optical Microscopy Platform (The University of Melbourne) for confocal microscopy training and support with image analysis, the BioSciences Microscopy Unit and the Ian Holmes Imaging Centre for optical and electron microscopy. C.R.G. also thank AIMS@JCU for facilitating transportation to and from the Australian Institute of Marine Science.

Contributor Information

Cecilie R Gotze, School of BioSciences, The University of Melbourne, Parkville, VIC 3010, Australia; Australian Institute of Marine Science, Townsville, QLD 4810, Australia.

Ashley M Dungan, School of BioSciences, The University of Melbourne, Parkville, VIC 3010, Australia.

Allison M L van de Meene, School of BioSciences, The University of Melbourne, Parkville, VIC 3010, Australia; Ian Holmes Imaging Centre, Bio21, The University of Melbourne, Parkville, VIC 3010, Australia.

Katarina Damjanovic, Australian Institute of Marine Science, Townsville, QLD 4810, Australia.

Gayle K Philip, Melbourne Bioinformatics, The University of Melbourne, Parkville, VIC 3010, Australia.

Justin Maire, School of BioSciences, The University of Melbourne, Parkville, VIC 3010, Australia.

Lone Høj, Australian Institute of Marine Science, Townsville, QLD 4810, Australia.

Linda L Blackall, School of BioSciences, The University of Melbourne, Parkville, VIC 3010, Australia.

Madeleine J H van Oppen, School of BioSciences, The University of Melbourne, Parkville, VIC 3010, Australia; Australian Institute of Marine Science, Townsville, QLD 4810, Australia.

Author contributions

Cecilie R. Gotze (Conceptualization, Formal analysis, Investigation, Methodology, Visualization, Writing—original draft), Ashley M. Dungan (Investigation, Methodology), Allison M.L. van de Meene (Investigation), Katarina Damjanovic (Investigation), Gayle K. Philip (Investigation), Justin Maire (Visualization), Lone Høj (Conceptualization, Methodology, Supervision, Writing—review & editing), Linda L. Blackall (Conceptualization, Funding acquisition, Methodology, Supervision, Writing—review & editing), and Madeleine J.H. van Oppen (Conceptualization, Funding acquisition, Methodology, Supervision, Writing—review & editing)

Conflicts of interest

The authors declare no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding

This research was supported by funding from the Australian Research Council (ARC) FL180100036 (to MJHvO), DP210100630 (to MJHvO and LLB), and the Melbourne Research Scholarship (to CRG). Data analysis was supported by The University of Melbourne's Research Computing Services.

Data availability

The datasets generated during the presented study are available under NCBI BioProject ID PRJNA1198616.

References

  • 1. Fisher  R, O’Leary  RA, Low-Choy  S. et al.  Species richness on coral reefs and the pursuit of convergent global estimates. Curr Biol  2015;25:500–5. 10.1016/j.cub.2014.12.022 [DOI] [PubMed] [Google Scholar]
  • 2. Trench  RK. The cell biology of plant-animal Symbiosis. Annu Rev Plant Physiol  1979;30:485–531. 10.1146/annurev.pp.30.060179.002413 [DOI] [Google Scholar]
  • 3. LaJeunesse  TC, Parkinson  JE, Gabrielson  PW. et al.  Systematic revision of Symbiodiniaceae highlights the antiquity and diversity of coral endosymbionts. Curr Biol  2018;28:2570–2580.e6. 10.1016/j.cub.2018.07.008 [DOI] [PubMed] [Google Scholar]
  • 4. Knowlton  N, Rohwer  F. Multispecies microbial mutualisms on coral reefs: the host as a habitat. Am Nat  2003;162:S51–62. 10.1086/378684 [DOI] [PubMed] [Google Scholar]
  • 5. Bourne  DG, Morrow  KM, Webster  NS. Insights into the coral microbiome: underpinning the health and resilience of reef ecosystems. Ann Rev Microbiol  2016;70:317–40. 10.1146/annurev-micro-102215-095440 [DOI] [PubMed] [Google Scholar]
  • 6. Clerissi  C, Brunet  S, Vidal-Dupiol  J. et al.  Protists within corals: the hidden diversity. Front Microbiol  2018;9:2043. 10.3389/fmicb.2018.02043 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. van Oppen  MJH, Blackall  LL. Coral microbiome dynamics, functions and design in a changing world. Nat Rev Microbiol  2019;17:557–67. 10.1038/s41579-019-0223-4 [DOI] [PubMed] [Google Scholar]
  • 8. Rosenberg  E, Koren  O, Reshef  L. et al.  The role of microorganisms in coral health, disease and evolution. Nat Rev Microbiol  2007;5:355–62. 10.1038/nrmicro1635 [DOI] [PubMed] [Google Scholar]
  • 9. Krediet  CJ, Ritchie  KB, Paul  VJ. et al.  Coral-associated micro-organisms and their roles in promoting coral health and thwarting diseases. Proc Biol Sci  2013;4:20122328. 10.3389/fncom.2010.00160 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Voolstra  CR, Raina  J-B, Dörr  M. et al.  The coral microbiome in sickness, in health and in a changing world. Nat Rev Microbiol  2024;22:460–75. 10.1038/s41579-024-01015-3 [DOI] [PubMed] [Google Scholar]
  • 11. Blackall  LL, Wilson  B, Van Oppen  MJH. Coral-the world’s most diverse symbiotic ecosystem. Mol Ecol  2015;24:5330–47. 10.1111/mec.13400 [DOI] [PubMed] [Google Scholar]
  • 12. Rohwer  F, Seguritan  V, Azam  F. et al.  Diversity and distribution of coral-associated bacteria. Mar Ecol Prog Ser  2002;243:1–10. 10.3354/meps243001 [DOI] [Google Scholar]
  • 13. Wegley  L, Edwards  R, Rodriguez-Brito  B. et al.  Metagenomic analysis of the microbial community associated with the coral Porites astreoides. Environ Microbiol  2007;9:2707–19. 10.1111/j.1462-2920.2007.01383.x [DOI] [PubMed] [Google Scholar]
  • 14. Lee  OO, Yang  J, Bougouffa  S. et al.  Spatial and species variations in bacterial communities associated with corals from the Red Sea as revealed by pyrosequencing. Appl Environ Microbiol  2012;78:7173–84. 10.1128/AEM.01111-12 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15. Li  J, Chen  Q, Zhang  S. et al.  Highly heterogeneous bacterial communities associated with the South China Sea reef corals Porites lutea, Galaxea fascicularis and Acropora millepora. PLoS One  2013;8:e71301. 10.1371/journal.pone.0071301 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Dunphy  CM, Gouhier  TC, Chu  ND. et al.  Structure and stability of the coral microbiome in space and time. Sci Rep  2019;9:6785. 10.1038/s41598-019-43268-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Pollock  FJ, McMinds  R, Smith  S. et al.  Coral-associated bacteria demonstrate phylosymbiosis and cophylogeny. Nat Commun  2018;9:4921. 10.1038/s41467-018-07275-x [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. Gardner  SG, Camp  EF, Smith  DJ. et al.  Coral microbiome diversity reflects mass coral bleaching susceptibility during the 2016 El Niño heat wave. Ecol Evol  2019;9:938–56. 10.1002/ece3.4662 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. Damjanovic  K, Blackall  LL, Peplow  LM. et al.  Assessment of bacterial community composition within and among Acropora loripes colonies in the wild and in captivity. Coral Reefs  2020;39:1245–55. 10.1007/s00338-020-01958-y [DOI] [Google Scholar]
  • 20. Dungan  AM, Geissler  L, Williams  AS. et al.  DNA from non-viable bacteria biases diversity estimates in the corals Acropora loripes and Pocillopora acuta. Environ Microbiome  2023;18:86. 10.1186/s40793-023-00541-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. Neave  MJ, Apprill  A, Ferrier-Pagès  C. et al.  Diversity and function of prevalent symbiotic marine bacteria in the genus Endozoicomonas. Appl Microbiol Biotechnol  2016;100:8315–24. 10.1007/s00253-016-7777-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22. McCauley  M, Goulet  TL, Jackson  CR. et al.  Systematic review of cnidarian microbiomes reveals insights into the structure, specificity, and fidelity of marine associations. Nat Commun  2023;14:4899. 10.1038/s41467-023-39876-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. Pogoreutz  C, Ziegler  M. Frenemies on the reef? Resolving the coral–Endozoicomonas association. Trends Microbiol  2024;32:422–34. 10.1016/j.tim.2023.11.006 [DOI] [PubMed] [Google Scholar]
  • 24. Cardinale  BJ, Srivastava  DS, Duffy  JE. et al.  Effects of biodiversity on the functioning of trophic groups and ecosystems. Nature  2006;443:989–92. 10.1038/nature05202 [DOI] [PubMed] [Google Scholar]
  • 25. Pogoreutz  C, Oakley  CA, Rädecker  N. et al.  Coral holobiont cues prime Endozoicomonas for a symbiotic lifestyle. ISME J  2022;16:1883–95. 10.1038/s41396-022-01226-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Neave  MJ, Michell  CT, Apprill  A. et al.  Endozoicomonas genomes reveal functional adaptation and plasticity in bacterial strains symbiotically associated with diverse marine hosts. Sci Rep  2017;7:1–12. 10.1038/srep40579 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. Tandon  K, Lu  CY, Chiang  PW. et al.  Comparative genomics: dominant coral-bacterium Endozoicomonas acroporae metabolizes dimethylsulfoniopropionate (DMSP). ISME J  2020;14:1290–303. 10.1038/s41396-020-0610-x [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Wada  N, Hsu  M-T, Tandon  K. et al.  High-resolution spatial and genomic characterization of coral-associated microbial aggregates in the coral Stylophora pistillata. Sci Adv  2022;8:eabo2431. 10.1126/sciadv.abo2431 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29. Work  TM, Aeby  GS. Microbial aggregates within tissues infect a diversity of corals throughout the indo-Pacific. Mar Ecol Prog Ser  2014;500:1–9. 10.3354/meps10698 [DOI] [Google Scholar]
  • 30. Peters  EC, Oprandy  JJ, Yevich  PP. Possible causal agent of “white band disease” in caribbean acroporid corals. J Invertebr Pathol  1983;41:394–6. 10.1016/0022-2011(83)90260-4 [DOI] [Google Scholar]
  • 31. Peters  EC. A survey of cellular reactions to environmental stress and disease in Caribbean scleractinian corals. Helgol Mar Res  1984;37:113–37. 10.1007/BF01989298 [DOI] [Google Scholar]
  • 32. Maire  J, Tandon  K, Collingro  A. et al.  Colocalization and potential interactions of Endozoicomonas and chlamydiae in microbial aggregates of the coral Pocillopora acuta. Sci Adv  2023;9:eadg0773. 10.1126/sciadv.adg0773 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33. Neave  MJ, Rachmawati  R, Xun  L. et al.  Differential specificity between closely related corals and abundant Endozoicomonas endosymbionts across global scales. ISME J  2017;11:186–200. 10.1038/ismej.2016.95 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34. Maire  J, Tsang Min Ching  SJ, Damjanovic  K. et al.  Tissue-associated and vertically transmitted bacterial symbiont in the coral Pocillopora acuta. ISME J  2024;18:wrad027. 10.1093/ismejo/wrad027 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Ide  K, Nishikawa  Y, Maruyama  T. et al.  Targeted single-cell genomics reveals novel host adaptation strategies of the symbiotic bacteria Endozoicomonas in Acropora tenuis coral. Microbiome  2022;10:220. 10.1186/s40168-022-01395-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36. Hochart  C, Paoli  L, Ruscheweyh  H-J. et al.  Ecology of Endozoicomonadaceae in three coral genera across the Pacific Ocean. Nat Commun  2023;14:3037. 10.1038/s41467-023-38502-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37. Sweet  MJ, Croquer  A, Bythell  JC. Bacterial assemblages differ between compartments within the coral holobiont. Coral Reefs  2011;30:39–52. 10.1007/s00338-010-0695-1 [DOI] [Google Scholar]
  • 38. Damjanovic  K, Van Oppen  MJH, Menéndez  P. et al.  Experimental inoculation of coral recruits with marine bacteria indicates scope for microbiome manipulation in Acropora tenuis and Platygyra daedalea. Front Microbio  2019;10:1702. 10.3389/fmicb.2019.01702 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39. Bolyen  E, Rideout  JR, Dillon  MR. et al.  Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat Biotechnol  2019;37:852–7. 10.1038/s41587-019-0209-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40. Hume  BCC, Smith  EG, Ziegler  M. et al.  SymPortal: a novel analytical framework and platform for coral algal symbiont next-generation sequencing ITS2 profiling. Mol Ecol Resour  2019;19:1063–80. 10.1111/1755-0998.13004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41. Bayer  T, Neave  MJ, Alsheikh-Hussain  A. et al.  The microbiome of the red sea coral Stylophora pistillata is dominated by tissue-associated Endozoicomonas bacteria. Appl Environ Microbiol  2013;79:4759–62. 10.1128/AEM.00695-13 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42. Stackebrandt  E, Goebel  BM. Taxonomic note: a place for DNA-DNA Reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Evol Microbiol  1994;44:846–9. 10.1099/00207713-44-4-846 [DOI] [Google Scholar]
  • 43. Daims  H, Brühl  A, Amann  R. et al.  The domain-specific probe EUB338 is insufficient for the detection of all bacteria: development and evaluation of a more comprehensive probe set. Syst Appl Microbiol  1999;22:434–44. 10.1016/S0723-2020(99)80053-8 [DOI] [PubMed] [Google Scholar]
  • 44. Peters  EC. Anatomy. In: Woodley  C.M., Downs  C.A., Bruckner  A.W.  et al. (eds.), Diseases of Coral, 1st edn. Hoboken, NJ: Wiley, 2015, 85–107. 10.1002/9781118828502.ch6. [Google Scholar]
  • 45. Wada  N, Ishimochi  M, Matsui  T. et al.  Characterization of coral-associated microbial aggregates (CAMAs) within tissues of the coral Acropora hyacinthus. Sci Rep  2019;9:14662. 10.1038/s41598-019-49651-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46. Ide  K, Nakano  Y, Ito  M. et al.  The effect of co-culture of two coral species on their bacterial composition under captive environments. Mar Biotechnol  2022;24:871–81. 10.1007/s10126-022-10149-5 [DOI] [PubMed] [Google Scholar]
  • 47. Morton  JT, Marotz  C, Washburne  A. et al.  Establishing microbial composition measurement standards with reference frames. Nat Commun  2019;10:2719. 10.1038/s41467-019-10656-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48. Lin  H, Peddada  SD. Analysis of compositions of microbiomes with bias correction. Nat Commun  2020;11:3514. 10.1038/s41467-020-17041-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49. Weinroth  MD, Belk  AD, Dean  C. et al.  Considerations and best practices in animal science 16S ribosomal RNA gene sequencing microbiome studies. J Anim Sci  2022;100:skab346. 10.1093/jas/skab346 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50. De Albuquerque  NRM, Haag  KL. Using average nucleotide identity (ANI) to evaluate microsporidia species boundaries based on their genetic relatedness. J Eukaryot Microbiol  2023;70:e12944. 10.1111/jeu.12944 [DOI] [PubMed] [Google Scholar]
  • 51. Tandon  K, Chiou  Y-J, Yu  S-P. et al.  Microbiome restructuring: dominant coral bacterium Endozoicomonas species respond differentially to environmental changes. mSystems  2022;7:e0035922–2. 10.1128/msystems.00359-22 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52. Buitrago-López  C, Cárdenas  A, Hume  BCC. et al.  Disparate population and holobiont structure of pocilloporid corals across the Red Sea gradient demonstrate species-specific evolutionary trajectories. Mol Ecol  2023;32:2151–73. 10.1111/mec.16871 [DOI] [PubMed] [Google Scholar]
  • 53. Mohamed  AR, Ochsenkühn  MA, Kazlak  AM. et al.  The coral microbiome: towards an understanding of the molecular mechanisms of coral–microbiota interactions. FEMS Microbiol Rev  2023;47:fuad005. 10.1093/femsre/fuad005 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54. Bourne  DG, Dennis  PG, Uthicke  S. et al.  Coral reef invertebrate microbiomes correlate with the presence of photosymbionts. ISME J  2013;7:1452–8. 10.1038/ismej.2012.172 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55. Tout  J, Jeffries  TC, Petrou  K. et al.  Chemotaxis by natural populations of coral reef bacteria. ISME J  2015;9:1764–77. 10.1038/ismej.2014.261 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56. Raina  JB, Lambert  BS, Parks  DH. et al.  Chemotaxis shapes the microscale organization of the ocean’s microbiome. Nature  2022;605:132–8. 10.1038/s41586-022-04614-3 [DOI] [PubMed] [Google Scholar]
  • 57. Raina  JB, Tapiolas  D, Willis  BL. et al.  Coral-associated bacteria and their role in the biogeochemical cycling of sulfur. Appl Environ Microbiol  2009;75:3492–501. 10.1128/AEM.02567-08 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58. Garren  M, Son  K, Raina  J-B. et al.  A bacterial pathogen uses dimethylsulfoniopropionate as a cue to target heat-stressed corals. ISME J  2014;8:999–1007. 10.1038/ismej.2013.210 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59. Baker  AC. Flexibility and specificity in coral-algal Symbiosis: diversity, ecology, and biogeography of Symbiodinium. Annu Rev Ecol Evol Syst  2003;34:661–89. 10.1146/annurev.ecolsys.34.011802.132417 [DOI] [Google Scholar]
  • 60. LaJeunesse  TC. Diversity and community structure of symbiotic dinoflagellates from Caribbean coral reefs. Mar Biol  2002;141:387–400. 10.1007/s00227-002-0829-2 [DOI] [Google Scholar]
  • 61. Quigley  KM, Alvarez Roa  C, Torda  G. et al.  Co-dynamics of Symbiodiniaceae and bacterial populations during the first year of symbiosis with Acropora tenuis juveniles. MicrobiologyOpen  2020;9:e959. 10.1002/mbo3.959 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62. Agostini  S, Suzuki  Y, Casareto  BE. et al.  Coral symbiotic complex: hypothesis through vitamin B12 for a new evaluation. Galaxea, JCRS  2009;11:1–11. 10.3755/galaxea.11.1 [DOI] [Google Scholar]
  • 63. Agostini  S, Suzuki  Y, Higuchi  T. et al.  Biological and chemical characteristics of the coral gastric cavity. Coral Reefs  2012;31:147–56. 10.1007/s00338-011-0831-6 [DOI] [Google Scholar]
  • 64. Bove  CB, Whitehead  RF, Szmant  AM. Responses of coral gastrovascular cavity pH during light and dark incubations to reduced seawater pH suggest species-specific responses to the effects of ocean acidification on calcification. Coral Reefs  2020;39:1675–91. 10.1007/s00338-020-01995-7 [DOI] [Google Scholar]
  • 65. Zhou  Y, Tang  K, Wang  P. et al.  Identification of bacteria-derived urease in the coral gastric cavity. Sci China Earth Sci  2020;63:1553–63. 10.1007/s11430-020-9647-x [DOI] [Google Scholar]
  • 66. Russell  AB, Peterson  SB, Mougous  JD. Type VI secretion system effectors: poisons with a purpose. Nat Rev Microbiol  2014;12:137–48. 10.1038/nrmicro3185 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 67. Serapio-Palacios  A, Woodward  SE, Vogt  SL. et al.  Type VI secretion systems of pathogenic and commensal bacteria mediate niche occupancy in the gut. Cell Rep  2022;39:110731. 10.1016/j.celrep.2022.110731 [DOI] [PubMed] [Google Scholar]
  • 68. Nadell  CD, Xavier  JB, Foster  KR. The sociobiology of biofilms. FEMS Microbiol Rev  2009;33:206–24. 10.1111/j.1574-6976.2008.00150.x [DOI] [PubMed] [Google Scholar]
  • 69. Elias  S, Banin  E. Multi-species biofilms: living with friendly neighbors. FEMS Microbiol Rev  2012;36:990–1004. 10.1111/j.1574-6976.2012.00325.x [DOI] [PubMed] [Google Scholar]
  • 70. Vasudevan  R. Biofilms: microbial cities of scientific significance. JMEN  2014;1:1. 10.15406/jmen.2014.01.00014 [DOI] [Google Scholar]
  • 71. Mori  M, Ponce-de-León  M, Peretó  J. et al.  Metabolic complementation in bacterial communities: necessary conditions and optimality. Front Microbiol  2016;7:1553. 10.3389/fmicb.2016.01553 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 72. Majumdar  S, Pal  S. Cross-species communication in bacterial world. J Cell Commun Signal  2017;11:187–90. 10.1007/s12079-017-0383-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 73. Girard  L. Quorum sensing in vibrio spp.: the complexity of multiple signalling molecules in marine and aquatic environments. Crit Rev Microbiol  2019;45:451–71. 10.1080/1040841X.2019.1624499 [DOI] [PubMed] [Google Scholar]
  • 74. Wellington  S, Greenberg  EP. Quorum sensing signal selectivity and the potential for interspecies cross talk. MBio  2019;10:e00146–19. 10.1128/mBio.00146-19 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 75. Tytgat  HLP, De Vos  WM. Sugar coating the envelope: Glycoconjugates for microbe–host crosstalk. Trends Microbiol  2016;24:853–61. 10.1016/j.tim.2016.06.004 [DOI] [PubMed] [Google Scholar]
  • 76. Rosset  SL, Oakley  CA, Ferrier-Pagès  C. et al.  The molecular language of the cnidarian–dinoflagellate Symbiosis. Trends Microbiol  2021;29:320–33. 10.1016/j.tim.2020.08.005 [DOI] [PubMed] [Google Scholar]
  • 77. Deakin  WJ, Broughton  WJ. Symbiotic use of pathogenic strategies: rhizobial protein secretion systems. Nat Rev Microbiol  2009;7:312–20. 10.1038/nrmicro2091 [DOI] [PubMed] [Google Scholar]
  • 78. Monjarás Feria  J, Valvano  MA. An overview of anti-eukaryotic T6SS effectors. Front Cell Infect Microbiol  2020;10:584751. 10.3389/fcimb.2020.584751 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 79. Hsieh  P-F, Lu  Y-R, Lin  T-L. et al.  Klebsiella pneumoniae type VI secretion system contributes to bacterial competition, cell invasion, Type-1 fimbriae expression, and In vivo colonization. J Infect Dis  2019;219:637–47. 10.1093/infdis/jiy534 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 80. Palincsar  EE, Jones  WR, Palincsar  JS. et al.  Bacterial aggregates within the epidermis of the sea anemone Aiptasia pallida. Biol Bull  1989;177:130–40. 10.2307/1541840 [DOI] [Google Scholar]
  • 81.Chu H, Mazmanian SK. Innate immune recognition of the microbiota promotes host-microbial symbiosis. Nat Immunol 2013;14:668–675. 10.1038/ni.2635 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 82. Epstein  HE, Brown  T, Akinrinade  AO. et al.  Evidence for microbially-mediated tradeoffs between growth and defense throughout coral evolution. Anim microbiome  2025;7:1. 10.1186/s42523-024-00370-z [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 83. Davy  SK, Allemand  D, Weis  VM. Cell biology of cnidarian-dinoflagellate Symbiosis. Microbiol Mol Biol Rev  2012;76:229–61. 10.1128/MMBR.05014-11 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 84. Papina  M, Meziane  T, Van Woesik  R. Symbiotic zooxanthellae provide the host-coral Montipora digitata with polyunsaturated fatty acids. Comp Biochem Physiol B Biochem Mol Biol  2003;135:533–7. 10.1016/S1096-4959(03)00118-0 [DOI] [PubMed] [Google Scholar]
  • 85. Whitehead  LF, Douglas  AE. Metabolite comparisons and the identity of nutrients translocated from symbiotic algae to an animal host. J Exp Biol  2003;206:3149–57. 10.1242/jeb.00539 [DOI] [PubMed] [Google Scholar]
  • 86. Burriesci  MS, Raab  TK, Pringle  JR. Evidence that glucose is the major transferred metabolite in dinoflagellate–cnidarian symbiosis. J Exp Biol  2012;215:3467–77. 10.1242/jeb.070946 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 87. D’Souza  G, Ebrahimi  A, Stubbusch  A. et al.  Cell aggregation is associated with enzyme secretion strategies in marine polysaccharide-degrading bacteria. ISME J  2023;17:703–11. 10.1038/s41396-023-01385-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 88. Wiedenmann  J, D’Angelo  C, Mardones  ML. et al.  Reef-building corals farm and feed on their photosynthetic symbionts. Nature  2023;620:1018–24. 10.1038/s41586-023-06442-5 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Gotze_Supplementary_materials_wraf059
gotze_supplementary_materials_wraf059.pdf (10.2MB, pdf)

Data Availability Statement

The datasets generated during the presented study are available under NCBI BioProject ID PRJNA1198616.

Articles from The ISME Journal are provided here courtesy of Oxford University Press

RESOURCES