Sponge Microbiota Research Articles

Metagenomic and metatranscriptomic exploration of the Egyptian Red Sea sponge Theonella sp. associated microbial community.

Marine sponges associated microorganisms are considered to be prolific source of bioactive natural products. Omics-based techniques such as metagenomics and metatranscriptomics have been used as effective tools to discover natural products. In this study, we used integrated metagenomic and metatranscriptomic analysis of three samples of the Egyptian Red Sea sponge Theonella sp. microbiome to obtain a complete picture of its biosynthetic activity to produce bioactive compounds. Our data revealed high biodiversity of the Egyptian sponge microbiota represented by 38 bacterial phyla with Candidate Phylum Poribacteria as the most abundant phyla with an average of 27.5% of the microbial community. The analysis also revealed high biosynthetic activity of the sponge microbiome through detecting different types of biosynthetic gene clusters (BGCs) with predicted antibacterial, cytotoxic and inhibitory bioactivity and the majority of these clusters were found to be actively transcribed. The complete BGCs of the cytotoxic theonellamide and misakinolide were detected and found to be actively transcribed. The majority of the detected BGCs were predicted to be novel as they did not show any similarity with any known cluster in the MIBiG database.

How does heat stress affect sponge microbiomes? Structure and resilience of microbial communities of marine sponges from different habitats

IntroductionSponges are key components of marine benthic communities, providing many ecosystem functions and establishing close relationships with microorganisms, conforming the holobiont. These symbiotic microbiotas seem to be host species-specific and highly diverse, playing key roles in their sponge host. The effects of elevated seawater temperature on sponges and their microbiota are still poorly known, and whether sponges from polar areas are more sensitive to these impacts respect to temperate and tropical species is totally unknown.MethodsWe analyzed the microbiomes of different sponge species in their natural habitat and after exposure to heat stress in aquaria by 16S rRNA amplicon sequencing to (1) characterize the sponge microbiota covering a latitudinal gradient (polar, temperate and tropical environments), and (2) asses the effects of thermal stress on their microbial communities.ResultsBacterial communities’ structure was different in the different sponge species and also respect the surrounding seawater. The core microbiome is maintained in most sponge species after a heat stress, although whether they would recover to the normal conditions previous to the stress remains yet to be further investigated. We observed increased abundances of transient bacteria from unknown origin in sponge species exposed to heat stress.DiscussionSome of the transient bacteria may be opportunistic bacteria that may benefit from the heat stress-associated dysregulation in the sponge by occupying new niches in the holobiont. According to our results, sponges from Antarctic waters could be more resilient than tropical and temperate sponges. Both the microbiome composition and the changes produced by the heat stress seem to be quite host species-specific, and thus, depend on the sponge species. Under a global change scenario, the microbiomes of the tropical and temperate sponges will probably be those suffering the most the heat stress, and therefore the effects of global change may be dramatic for benthic ecosystems since sponges are a fundamental part of them.

Biosynthetic activity of Marine Sponge Microbiota

Biosynthetic activity of Marine Sponge Microbiota

Microbiome of the freshwater sponge Ephydatia muelleri shares compositional and functional similarities with those of marine sponges.

Sponges are known for hosting diverse communities of microbial symbionts, but despite persistent interest in the sponge microbiome, most research has targeted marine sponges; freshwater sponges have been the focus of less than a dozen studies. Here, we used 16 S rRNA gene amplicon sequencing and shotgun metagenomics to characterize the microbiome of the freshwater sponge Ephydatia muelleri and identify potential indicators of sponge-microbe mutualism. Using samples collected from the Sooke, Nanaimo, and Cowichan Rivers on Vancouver Island, British Columbia, we show that the E. muelleri microbiome is distinct from the ambient water and adjacent biofilms and is dominated by Sediminibacterium, Comamonas, and unclassified Rhodospirillales. We also observed phylotype-level differences in sponge microbiome taxonomic composition among different rivers. These differences were not reflected in the ambient water, suggesting that other environmental or host-specific factors may drive the observed geographic variation. Shotgun metagenomes and metagenome-assembled genomes further revealed that freshwater sponge-associated bacteria share many genomic similarities with marine sponge microbiota, including an abundance of defense-related proteins (CRISPR, restriction-modification systems, and transposases) and genes for vitamin B12 production. Overall, our results provide foundational information on the composition and function of freshwater sponge-associated microbes, which represent an important yet underappreciated component of the global sponge microbiome.

Minority report: small-scale metagenomic analysis of the non-bacterial kitchen sponge microbiota

Kitchen sponges are particularly well known to harbor a high number and diversity of bacteria, including pathogens. Viruses, archaea, and eukaryotes in kitchen sponges, however, have not been examined in detail so far. To increase knowledge on the non-bacterial kitchen sponge microbiota and its potential hygienic relevance, we investigated five used kitchen sponges by means of metagenomic shot-gun sequencing. Viral particles were sought to be enriched by a filter step during DNA extraction from the sponges. Data analysis revealed that ~ 2% of the sequences could be assigned to non-bacterial taxa. Each sponge harbored different virus (phage) species, while the present archaea were predominantly affiliated with halophilic taxa. Among the eukaryotic taxa, besides harmless algae, or amoebas, mainly DNA from food-left-overs was found. The presented work offers new insights into the complex microbiota of used kitchen sponges and contributes to a better understanding of their hygienic relevance.

Distribution of mercury inside the Mediterranean sponge Chondrosia reniformis: A study case from the Tuscan Archipelago National Park (Tyrrhenian Sea)

Trace elements are ubiquitous substances in the marine environment, representing a problematic hazard for organisms since many of them lack any biological function. The main aim of the study was to investigate the variation of the total mercury content (THg) in the Mediterranean sponge Chondrosia reniformis collected in two islands of the Tuscan Archipelago, Italy. The two islands did not show harmful levels of THg (seawater and sediment) and no intraspecific variability was detected in THg values and bioconcentration factors of collected samples. Conversely, THg values recorded in the choanosome of C. reniformis were significantly higher than in the cortex, pointing out a possible involvement of the sponge microbiota in the bioaccumulation process. Our study suggests that C. reniformis could be an attractive bioindicator of toxic elements pollution in coastal waters, highlighting the potential use of these organisms in the monitoring programs of coastal areas, as suggested by various European marine policies.

A Microbial Nitrogen Engine Modulated by Bacteriosyncytia in Hexactinellid Sponges: Ecological Implications for Deep-Sea Communities

Hexactinellid sponges are common in the deep sea, but their functional integration into those ecosystems remains poorly understood. The phylogenetically related species Schaudinnia rosea and Vazella pourtalesii were herein incubated for nitrogen and phosphorous, returning markedly different nutrient fluxes. Transmission electron microscopy (TEM) revealed S. rosea to host a low abundance of extracellular microbes, while Vazella pourtalesii showed higher microbial abundance and hosted most microbes within bacteriosyncytia, a novel feature for Hexactinellida. Amplicon sequences of the microbiome corroborated large between-species differences, also between the sponges and the seawater of their habitats. Metagenome-assembled genome of the V. pourtalesii microbiota revealed genes coding for enzymes operating in nitrification, denitrification, dissimilatory nitrate reduction to ammonium, nitrogen fixation, and ammonia/ammonium assimilation. In the nitrification and denitrification pathways some enzymes were missing, but alternative bridging routes allow the microbiota to close a N cycle in the holobiont. Interconnections between aerobic and anaerobic pathways may facilitate the sponges to withstand the low-oxygen conditions of deep-sea habitats. Importantly, various N pathways coupled to generate ammonium, which, through assimilation, fosters the growth of the sponge microbiota. TEM showed that the farmed microbiota is digested by the sponge cells, becoming an internal food source. This microbial farming demands more ammonium that can be provided internally by the host sponges and some 2.6 million kg of ammonium from the seawater become annually consumed by the aggregations of V. pourtalesii. Such ammonium removal is likely impairing the development of the free-living bacterioplankton and the survival chances of other sponge species that feed on bacterioplankton. Such nutritional competitive exclusion would favor the monospecific character of the V. pourtalesii aggregations. These aggregations also affect the surrounding environment through an annual release of 27.3 million kg of nitrite and, in smaller quantities, of nitrate and phosphate. The complex metabolic integration among the microbiota and the sponge suggests that the holobiont depends critically on the correct functioning of its N-driven microbial engine. The metabolic intertwining is so delicate that it changed after moving the sponges out of their habitat for a few days, a serious warning on the conservation needs of these sponge aggregations.

Author Correction: Vertical transmission of sponge microbiota is inconsistent and unfaithful

Author Correction: Vertical transmission of sponge microbiota is inconsistent and unfaithful

A New Micromonospora Strain with Antibiotic Activity Isolated from the Microbiome of a Mid-Atlantic Deep-Sea Sponge.

To tackle the growing problem of antibiotic resistance, it is essential to identify new bioactive compounds that are effective against resistant microbes and safe to use. Natural products and their derivatives are, and will continue to be, an important source of these molecules. Sea sponges harbour a diverse microbiome that co-exists with the sponge, and these bacterial communities produce a rich array of bioactive metabolites for protection and resource competition. For these reasons, the sponge microbiota constitutes a potential source of clinically relevant natural products. To date, efforts in bioprospecting for these compounds have focused predominantly on sponge specimens isolated from shallow water, with much still to be learned about samples from the deep sea. Here we report the isolation of a new Micromonospora strain, designated 28ISP2-46T, recovered from the microbiome of a mid-Atlantic deep-sea sponge. Whole-genome sequencing reveals the capacity of this bacterium to produce a diverse array of natural products, including kosinostatin and isoquinocycline B, which exhibit both antibiotic and antitumour properties. Both compounds were isolated from 28ISP2-46T fermentation broths and were found to be effective against a plethora of multidrug-resistant clinical isolates. This study suggests that the marine production of isoquinocyclines may be more widespread than previously supposed and demonstrates the value of targeting the deep-sea sponge microbiome as a source of novel microbial life with exploitable biosynthetic potential.

The Bacterial Microbiome of the Long-Term Aquarium Cultured High-Microbial Abundance Sponge Haliclona cnidata – Sustained Bioactivity Despite Community Shifts Under Detrimental Conditions

Marine sponges host highly diverse but specific bacterial communities that provide essential functions for the sponge holobiont, including antimicrobial defense. Here, we characterized the bacterial microbiome of the marine sponge Haliclona cnidata that has been in culture in an artificial marine aquarium system. We tested the hypotheses (1) that the long-term aquarium cultured sponge Haliclona cnidata is tightly associated with typical sponge bacterial microbiota and (2) that the symbiotic Bacteria sustain bioactivity under harmful environmental conditions to facilitate holobiont survival by preventing pathogen invasion. Microscopic and phylogenetic analyses of the bacterial microbiota revealed that H. cnidata represents a high microbial abundance (HMA) sponge with a temporally stable bacterial community that significantly shifts with changing aquaria conditions. A four week incubation experiment was performed in small closed aquaria systems with antibiotic and/or light exclusion treatments to reduce the total bacterial and photosynthetically active sponge-associated microbiota to a treatment specific resilient community. While the holobiont was severely affected by the experimental treatment (i.e. fading of the sponge, reduced bacterial abundance, shifted bacterial community composition), the biological defense and bacterial community interactions (i.e. quorum sensing activity) remained intact. 16S rRNA gene amplicon sequencing revealed a resilient community of 105 bacterial taxa, which remained in the treated sponges. These 105 taxa accounted for a relative abundance of 72-83% of the bacterial sponge microbiota of non-treated sponge fragments that have been cultured under the same conditions. We conclude that a sponge-specific resilient community stays biologically active under harmful environmental conditions, facilitating the resilience of the holobiont. In H. cnidata, bacteria were located in bacteriocytes, which may have contributed to the observed phenomenon.

Vertical transmission of sponge microbiota is inconsistent and unfaithful.

Co-evolutionary theory predicts that if beneficial microbial symbionts improve host fitness, they should be faithfully transmitted to offspring. More recently, the hologenome theory of evolution predicts resemblance between parent and offspring microbiomes and high partner fidelity between host species and their vertically transmitted microbes. Here, we test these ideas in multiple coexisting host species with highly diverse microbiota, leveraging known parent-offspring pairs sampled from eight species of wild marine sponges (Porifera). We found that the processes governing vertical transmission were both neutral and selective. A neutral model was a better fit to larval (R2 = 0.66) than to the adult microbiota (R2 = 0.27), suggesting that the importance of non-neutral processes increases as the sponge host matures. Microbes that are enriched above neutral expectations in adults were disproportionately transferred to offspring. Patterns of vertical transmission were, however, incomplete: larval sponges shared, on average, 44.8% of microbes with their parents, which was not higher than the fraction they shared with nearby non-parental adults. Vertical transmission was also inconsistent across siblings, as larval sponges from the same parent shared only 17% of microbes. Finally, we found no evidence that vertically transmitted microbes are faithful to a single sponge host species. Surprisingly, larvae were as likely to share vertically transmitted microbes with larvae from other sponge species as they were with their own species. Our study demonstrates that common predictions of vertical transmission that stem from species-poor systems are not necessarily true when scaling up to diverse and complex microbiomes.

Disruption of N-acyl-homoserine lactone-specific signalling and virulence in clinical pathogens by marine sponge bacteria.

SummaryIn recent years, the marine environment has been the subject of increasing attention from biotechnological and pharmaceutical industries. A combination of unique physicochemical properties and spatial niche‐specific substrates, in wide‐ranging and extreme habitats, underscores the potential of the marine environment to deliver on functionally novel bioactivities. One such area of ongoing research is the discovery of compounds that interfere with the cell–cell signalling process called quorum sensing (QS). Described as the next generation of antimicrobials, these compounds can target virulence and persistence of clinically relevant pathogens, independent of any growth‐limiting effects. Marine sponges are a rich source of microbial diversity, with dynamic populations in a symbiotic relationship. In this study, we have harnessed the QS inhibition (QSI) potential of marine sponge microbiota and through culture‐based discovery have uncovered small molecule signal mimics that neutralize virulence phenotypes in clinical pathogens. This study describes for the first time a marine sponge Psychrobacter sp. isolate B98C22 that blocks QS signalling, while also reporting dual QS/QSI activity in the Pseudoalteromonas sp. J10 and Paracoccus JM45. Isolation of novel QSI activities has significant potential for future therapeutic development, of particular relevance in the light of the pending perfect storm of antibiotic resistance meeting antibiotic drug discovery decline.

Microbiome analysis and confocal microscopy of used kitchen sponges reveal massive colonization by Acinetobacter, Moraxella and Chryseobacterium species

The built environment (BE) and in particular kitchen environments harbor a remarkable microbial diversity, including pathogens. We analyzed the bacterial microbiome of used kitchen sponges by 454–pyrosequencing of 16S rRNA genes and fluorescence in situ hybridization coupled with confocal laser scanning microscopy (FISH–CLSM). Pyrosequencing showed a relative dominance of Gammaproteobacteria within the sponge microbiota. Five of the ten most abundant OTUs were closely related to risk group 2 (RG2) species, previously detected in the BE and kitchen microbiome. Regular cleaning of sponges, indicated by their users, significantly affected the microbiome structure. Two of the ten dominant OTUs, closely related to the RG2-species Chryseobacterium hominis and Moraxella osloensis, showed significantly greater proportions in regularly sanitized sponges, thereby questioning such sanitation methods in a long term perspective. FISH–CLSM showed an ubiquitous distribution of bacteria within the sponge tissue, concentrating in internal cavities and on sponge surfaces, where biofilm–like structures occurred. Image analysis showed local densities of up to 5.4 * 1010 cells per cm3, and confirmed the dominance of Gammaproteobacteria. Our study stresses and visualizes the role of kitchen sponges as microbiological hot spots in the BE, with the capability to collect and spread bacteria with a probable pathogenic potential.

Sponge Microbiota Are a Reservoir of Functional Antibiotic Resistance Genes.

Wide application of antibiotics has contributed to the evolution of multi-drug resistant human pathogens, resulting in poorer treatment outcomes for infections. In the marine environment, seawater samples have been investigated as a resistance reservoir; however, no studies have methodically examined sponges as a reservoir of antibiotic resistance. Sponges could be important in this respect because they often contain diverse microbial communities that have the capacity to produce bioactive metabolites. Here, we applied functional metagenomics to study the presence and diversity of functional resistance genes in the sponges Aplysina aerophoba, Petrosia ficiformis, and Corticium candelabrum. We obtained 37 insert sequences facilitating resistance to D-cycloserine (n = 6), gentamicin (n = 1), amikacin (n = 7), trimethoprim (n = 17), chloramphenicol (n = 1), rifampicin (n = 2) and ampicillin (n = 3). Fifteen of 37 inserts harbored resistance genes that shared <90% amino acid identity with known gene products, whereas on 13 inserts no resistance gene could be identified with high confidence, in which case we predicted resistance to be mainly mediated by antibiotic efflux. One marine-specific ampicillin-resistance-conferring β-lactamase was identified in the genus Pseudovibrio with 41% global amino acid identity to the closest β-lactamase with demonstrated functionality, and subsequently classified into a new family termed PSV. Taken together, our results show that sponge microbiota host diverse and novel resistance genes that may be harnessed by phylogenetically distinct bacteria.

Metagenomic Analysis of Genes Encoding Nutrient Cycling Pathways in the Microbiota of Deep-Sea and Shallow-Water Sponges.

Sponges host complex symbiotic communities, but to date, the whole picture of the metabolic potential of sponge microbiota remains unclear, particularly the difference between the shallow-water and deep-sea sponge holobionts. In this study, two completely different sponges, shallow-water sponge Theonella swinhoei from the South China Sea and deep-sea sponge Neamphius huxleyi from the Indian Ocean, were selected to compare their whole symbiotic communities and metabolic potential, particularly in element transformation. Phylogenetically diverse bacteria, archaea, fungi, and algae were detected in both shallow-water sponge T. swinhoei and deep-sea sponge N. huxleyi, and different microbial community structures were indicated between these two sponges. Metagenome-based gene abundance analysis indicated that, though the two sponge microbiota have similar core functions, they showed different potential strategies in detailed metabolic processes, e.g., in the transformation and utilization of carbon, nitrogen, phosphorus, and sulfur by corresponding microbial symbionts. This study provides insight into the putative metabolic potentials of the microbiota associated with the shallow-water and deep-sea sponges at the whole community level, extending our knowledge of the sponge microbiota's functions, the association of sponge- microbes, as well as the adaption of sponge microbiota to the marine environment.

Microbial community structure of two freshwater sponges using Illumina MiSeq sequencing revealed high microbial diversity.

Sponges are primitive metazoans that are known to harbour diverse and abundant microbes. All over the world attempts are being made to exploit these microbes for their biotechnological potential to produce, bioactive compounds and antimicrobial peptides. However, the majority of the studies are focussed on the marine sponges and studies on the freshwater sponges have been neglected so far. To increase our understanding of the microbial community structure of freshwater sponges, microbiota of two fresh water sponges namely, Eunapius carteri and Corvospongilla lapidosa is explored for the first time using Next Generation Sequencing (NGS) technology. Overall the microbial composition of these sponges comprises of 14 phyla and on an average, more than 2900 OTUs were obtained from C. lapidosa while E. carteri showed 980 OTUs which is higher than OTUs obtained in the marine sponges. Thus, our study showed that, fresh water sponges also posses highly diverse microbial community than previously thought and it is distinct from the marine sponge microbiota. The present study also revealed that microbial community structure of both the sponges is significantly different from each other and their respective water samples. In the present study, we have detected many bacterial lineages belonging to Firmicutes, Actinobacteria, Proteobacteria, Planctomycetes, etc. that are known to produce compounds of biotechnological importance. Overall, this study gives insight into the microbial composition of the freshwater sponges which is highly diverse and needs to be studied further to exploit their biotechnological capabilities.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0211-2) contains supplementary material, which is available to authorized users.

In four shallow and mesophotic tropical reef sponges from Guam the microbial community largely depends on host identity.

Sponges (phylum Porifera) are important members of almost all aquatic ecosystems, and are renowned for hosting often dense and diverse microbial communities. While the specificity of the sponge microbiota seems to be closely related to host phylogeny, the environmental factors that could shape differences within local sponge-specific communities remain less understood. On tropical coral reefs, sponge habitats can span from shallow areas to deeper, mesophotic sites. These habitats differ in terms of environmental factors such as light, temperature, and food availability, as well as anthropogenic impact. In order to study the host specificity and potential influence of varying habitats on the sponge microbiota within a local area, four tropical reef sponges, Rhabdastrella globostellata, Callyspongia sp., Rhaphoxya sp., and Acanthella cavernosa, were collected from exposed shallow reef slopes and a deep reef drop-off. Based on 16S rRNA gene pyrosequencing profiles, beta diversity analyses revealed that each sponge species possessed a specific microbiota that was significantly different to those of the other species and exhibited attributes that are characteristic of high- and/or low-microbial-abundance sponges. These findings emphasize the influence of host identity on the associated microbiota. Dominant sponge- and seawater-associated bacterial phyla were Chloroflexi, Cyanobacteria, and Proteobacteria. Comparison of individual sponge taxa and seawater samples between shallow and deep reef sites revealed no significant variation in alpha diversity estimates, while differences in microbial beta diversity (variation in community composition) were significant for Callyspongia sp. sponges and seawater samples. Overall, the sponge-associated microbiota is significantly shaped by host identity across all samples, while the effect of habitat differentiation seems to be less predominant in tropical reef sponges.

Temporal dynamics of prokaryotic communities in the marine sponge Sarcotragus spinosulus.

In spite of their putative relevance to host functioning, in-depth knowledge of sponge microbiome stability over time is scarce. This study tackles the temporal maintenance of bacterial and archaeal assemblages in the model host Sarcotragus spinosulus along three successive years. Prokaryotic communities were profiled by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and 454-pyrosequencing of S.spinosulus-derived 16S rRNA gene amplicons. Prevailing bacterial phyla were Actinobacteria, Acidobacteria, Proteobacteria, Poribacteria, PAUC34f, Chloroflexi and Bacteroidetes, with Bacteroidetes, Chloroflexi and Poribacteria showing different abundances over the years. At the approximate species level (operational taxonomic units, OTUs, defined at 97% sequence similarity), no major changes in bacterial richness and composition were found through time. Nearly 50% of all detected bacterial symbionts (96 in 205 OTUs) were recovered from all sampling years, whereas a taxonomically equivalent community of less dominant bacteria characterized the transient sponge microbiota. Despite the evidence for temporal symbiont maintenance, an intriguing cumulative degree of variation between individuals was unravelled, with all the surveyed sponge specimens sharing only 27 bacterial OTUs. Archaeal communities were dominated by one single symbiont of the candidate genus Nitrosopumilus (Thaumarchaeota), known for its ability to aerobically oxidize ammonia to nitrite. Only few bacterial ammonia oxidizers consistently occurred in S.spinosulus across the years as documented by PCR-DGGE fingerprinting. In conclusion, prokaryotic symbionts of S.spinosulus display a state of dynamic stability shaped by the interplay between the maintenance of dominant players and turnover of less prevalent community members, in time and across host individuals, with no apparent consequences to holobiont functioning.

Evidence of a Putative Deep Sea Specific Microbiome in Marine Sponges

The microbiota of four individual deep water sponges, Lissodendoryx diversichela, Poecillastra compressa, Inflatella pellicula, and Stelletta normani, together with surrounding seawater were analysed by pyrosequencing of a region of the 16S rRNA gene common to Bacteria and Archaea. Due to sampling constraints at depths below 700 m duplicate samples were not collected. The microbial communities of L. diversichela, P. compressa and I. pellicula were typical of low microbial abundance (LMA) sponges while S. normani had a community more typical of high microbial abundance (HMA) sponges. Analysis of the deep sea sponge microbiota revealed that the three LMA-like sponges shared a set of abundant OTUs that were distinct from those associated with sponges from shallow waters. Comparison of the pyrosequencing data with that from shallow water sponges revealed that the microbial communities of all sponges analysed have similar archaeal populations but that the bacterial populations of the deep sea sponges were distinct. Further analysis of the common and abundant OTUs from the three LMA-like sponges placed them within the groups of ammonia oxidising Archaea (Thaumarchaeota) and sulphur oxidising γ-Proteobacteria (Chromatiales). Reads from these two groups made up over 70% of all 16S rRNA genes detected from the three LMA-like sponge samples, providing evidence of a putative common microbial assemblage associated with deep sea LMA sponges.

Stability of sponge-associated bacteria over large seasonal shifts in temperature and irradiance.

Complex microbiomes reside in marine sponges and consist of diverse microbial taxa, including functional guilds that may contribute to host metabolism and coastal marine nutrient cycles. Our understanding of these symbiotic systems is based primarily on static accounts of sponge microbiota, while their temporal dynamics across seasonal cycles remain largely unknown. Here, we investigated temporal variation in bacterial symbionts of three sympatric sponges (Ircinia spp.) over 1.5 years in the northwestern (NW) Mediterranean Sea, using replicated terminal restriction fragment length polymorphism (T-RFLP) and clone library analyses of bacterial 16S rRNA gene sequences. Bacterial symbionts in Ircinia spp. exhibited host species-specific structure and remarkable stability throughout the monitoring period, despite large fluctuations in temperature and irradiance. In contrast, seawater bacteria exhibited clear seasonal shifts in community structure, indicating that different ecological constraints act on free-living and on symbiotic marine bacteria. Symbiont profiles were dominated by persistent, sponge-specific bacterial taxa, notably affiliated with phylogenetic lineages capable of photosynthesis, nitrite oxidation, and sulfate reduction. Variability in the sponge microbiota was restricted to rare symbionts and occurred most prominently in warmer seasons, coincident with elevated thermal regimes. Seasonal stability of the sponge microbiota supports the hypothesis of host-specific, stable associations between bacteria and sponges. Further, the core symbiont profiles revealed in this study provide an empirical baseline for diagnosing abnormal shifts in symbiont communities. Considering that these sponges have suffered recent, episodic mass mortalities related to thermal stresses, this study contributes to the development of model sponge-microbe symbioses for assessing the link between symbiont fluctuations and host health.