Sponge-associated Microbes Research Articles

Marine sponge-derived natural products: trends and opportunities for the decade of 2011-2020

The discovery of natural products derived from marine sources has demonstrated a consistent upward trajectory for the decade of 2011-2020, holding significant promise for the development of novel drugs and many other marine bioproducts. In recent years, the spotlight has shifted away from marine sponges (Porifera) towards marine microorganisms as the primary source of discovery. Despite reports of marine sponges spanning 20 different orders and being the subject of 769 papers between 2011 and 2020, they only contributed to 19.29% of all new compounds discovered, in contrast to 51.94% by marine microorganisms and phytoplankton. 563 new compounds were reported from marine sponge-associated microbes, more than doubling the number for the previous decade (2001-2010). It heralds a positive outlook for a sustainable resource strategy as the extraction of bioactive compounds produced by pure cultures of sponge-associated microbes could overcome supply challenges that arise with isolation from host sponges for the same compound. However, the application of novel marine natural products (MNPs) remains challenging due to the limited yield of compounds from large amounts of sponges. This review covers the literature published between 2011 and 2020, focusing on MNPs isolated from marine sponges. A total of 2603 new compounds are documented, detailing their chemical classification, biological activities, source country or geographic locations, and the taxonomic information of the source organisms, including order, family, genus, and species.

Sponge-associated microbes in the twilight zone of Curaçao

Mesophotic reefs, located in the "Twilight Zone" of the ocean, are coral reefs that exist in relatively deep waters ranging from approximately 30 to 150 m below the surface. These reefs are situated just beyond the reach of conventional SCUBA diving and are typically explored using advanced diving techniques or with the aid of submersibles. In the present study, we used a state-of-the-art submersible to sample 26 sponge specimens belonging to 11 species. High (HMA) or low (LMA) microbial abundance status was assigned to species based on TEM imagery. Prokaryotic communities associated with these sponges were, furthermore, assessed using high-throughput sequencing. Proteobacteria, Chloroflexi, Actinobacteriota, and Acidobacteriota were the most abundant phyla overall. HMA/LMA status proved to be a highly significant predictor of prokaryotic composition. HMA sponges also tended to be more diverse in terms of richness and evenness than LMA sponges. 14 predictor-classes were identified using an exploratory technique based on machine learning including classes within the phyla Chloroflexi (e.g., Dehalococcoidia and JG30-KF-CM66) and Acidobacteriota (Thermoanaerobaculia and Subgroups 11 and 21). Previous studies have demonstrated the prevalence of the HMA/LMA dichotomy in shallow waters and, recently, the deep sea. Our results demonstrate its prevalence in the mesophotic realm.

Induction of antimicrobial, antioxidant metabolites production by co-cultivation of two red-sea-sponge-associated Aspergillus sp. CO2 and Bacillus sp. COBZ21

The growing spread of infectious diseases has become a potential global health threat to human beings. According to WHO reports, in this study, we investigated the impact of co-cultivating the isolated endophytic fungus Aspergillus sp. CO2 and Bacillus sp. COBZ21 as a method to stimulate the production of natural bioactive substances. (GC/MS)-based metabolomics profiling of two sponge-associated microbes, Aspergillus sp. CO2 and Bacillus sp. COBZ21, revealed that the co-culture of these two isolates induced the accumulation of metabolites that were not traced in their axenic cultures. By detection of different activities of extracts of Bacillus sp. COBZ21 and Aspergillus sp. CO2 and coculture between Bacillus sp. COBZ21 and Aspergillus sp. CO2. It was noted that the coculture strategy was the reason for a notable increase in some different activities, such as the antimicrobial activity, which showed potent activity against Escherichia coli ATCC 25,922, Staphylococcus aureus NRRLB-767, and Candida albicans ATCC 10,231. The antibiofilm activity showed significant biofilm inhibitory activity toward Bacillus subtilis ATCC 6633, Pseudomonas aeruginosa ATCC 10,145, and Staph aureus NRRLB-767, with activity up to 53.66, 71.17, and 47.89%, while it showed low activity against E. coli ATCC 25,922, while the antioxidant activity based on the DPPH assay showed maximum activity (75.25%). GC-MS investigations revealed the presence of variable chemical constituents belonging to different chemical categories, which reflected their chemical diversity. The main components are (+-) cis-Deethylburnamine (2.66%), Bis(3,6,9,12-tetraoxapentaethylene) crowno-N,N,N’,N’-tetra methylpphanediamine (2.48%), and 11-phenyl-2,4,6,8-tetra(2-thienyl)-11-aza-5,13-dithiaeteracyclo[7.3.0.1(2,8)0.0(3,7)] trideca-3,6-diene-10,12,13-trione (3.13%), respectively, for Bacillus sp. axenic culture, Aspergillus sp. CO2, Aspergillus sp. CO2, and Bacillus sp. COBZ21 coculture. By studying the ADME-related physicochemical properties of coculture extract, the compound showed log Po/w values above 5 (8.82). The solubility of the substance was moderate. In order to provide a comprehensive definition of medicinal chemistry and leadlikness, it is important to note that the latter did not meet the criteria outlined in the rule of three (RO3). The toxicity prediction of the coculture extract was performed using the ProTox II web server, which showed that the selected compound has no pronounced toxicity.

Current knowledge of the Southern Hemisphere marine microbiome in eukaryotic hosts and the Strait of Magellan surface microbiome project.

Host-microbe interactions are ubiquitous and play important roles in host biology, ecology, and evolution. Yet, host-microbe research has focused on inland species, whereas marine hosts and their associated microbes remain largely unexplored, especially in developing countries in the Southern Hemisphere. Here, we review the current knowledge of marine host microbiomes in the Southern Hemisphere. Our results revealed important biases in marine host species sampling for studies conducted in the Southern Hemisphere, where sponges and marine mammals have received the greatest attention. Sponge-associated microbes vary greatly across geographic regions and species. Nevertheless, besides taxonomic heterogeneity, sponge microbiomes have functional consistency, whereas geography and aging are important drivers of marine mammal microbiomes. Seabird and macroalgal microbiomes in the Southern Hemisphere were also common. Most seabird microbiome has focused on feces, whereas macroalgal microbiome has focused on the epibiotic community. Important drivers of seabird fecal microbiome are aging, sex, and species-specific factors. In contrast, host-derived deterministic factors drive the macroalgal epibiotic microbiome, in a process known as "microbial gardening". In turn, marine invertebrates (especially crustaceans) and fish microbiomes have received less attention in the Southern Hemisphere. In general, the predominant approach to study host marine microbiomes has been the sequencing of the 16S rRNA gene. Interestingly, there are some marine holobiont studies (i.e., studies that simultaneously analyze host (e.g., genomics, transcriptomics) and microbiome (e.g., 16S rRNA gene, metagenome) traits), but only in some marine invertebrates and macroalgae from Africa and Australia. Finally, we introduce an ongoing project on the surface microbiome of key species in the Strait of Magellan. This is an international project that will provide novel microbiome information of several species in the Strait of Magellan. In the short-term, the project will improve our knowledge about microbial diversity in the region, while long-term potential benefits include the use of these data to assess host-microbial responses to the Anthropocene derived climate change.

Cultivation of previously uncultured sponge-associated bacteria using advanced cultivation techniques: A perspective on possible key mechanisms

Most of the microbes from natural habitats cannot be cultivated with standard cultivation in laboratory, and sponge-associated microbes are no exception. We used two advanced methods based on a continuous-flow bioreactor (CF) and in situ cultivation (I-tip) to isolate previously uncultivated marine sponge-associated bacteria. We also characterized the physiological properties of the isolates from each method and attempted to clarify the mechanisms operating in each cultivation method. A greater number of novel bacteria were isolated using CF and in situ cultivation compared to standard direct plating (SDP) cultivation. Most isolates from CF cultivation were poor growers (with lower specific growth rates and saturated cell densities than those of isolates from SDP cultivation), demonstrating that it is effective to carry out pre-enrichment cultivation targeting bacteria that are less competitive on conventional cultivation, especially K-strategists and bacterial types inhibited by their own growth. Isolates from in situ cultivation showed a positive influence on cell recovery stimulated by chemical compounds in the extract of sponge tissue, indicating that some of the bacteria require a “growth initiation factor” that is present in the natural environment. Each advanced cultivation method has its own distinct key mechanisms allowing cultivation of physiologically and phylogenetically different fastidious bacteria for cultivation compared with conventional methods.

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.

Assessing the Diversity and Biomedical Potential of Microbes Associated With the Neptune's Cup Sponge, Cliona patera.

Marine sponges are known to host a complex microbial consortium that is essential to the health and resilience of these benthic invertebrates. These sponge-associated microbes are also an important source of therapeutic agents. The Neptune’s Cup sponge, Cliona patera, once believed to be extinct, was rediscovered off the southern coast of Singapore in 2011. The chance discovery of this sponge presented an opportunity to characterize the prokaryotic community of C. patera. Sponge tissue samples were collected from the inner cup, outer cup and stem of C. patera for 16S rRNA amplicon sequencing. C. patera hosted 5,222 distinct OTUs, spanning 26 bacterial phyla, and 74 bacterial classes. The bacterial phylum Proteobacteria, particularly classes Gammaproteobacteria and Alphaproteobacteria, dominated the sponge microbiome. Interestingly, the prokaryotic community structure differed significantly between the cup and stem of C. patera, suggesting that within C. patera there are distinct microenvironments. Moreover, the cup of C. patera had lower diversity and evenness as compared to the stem. Quorum sensing inhibitory (QSI) activities of selected sponge-associated marine bacteria were evaluated and their organic extracts profiled using the MS-based molecular networking platform. Of the 110 distinct marine bacterial strains isolated from sponge samples using culture-dependent methods, about 30% showed quorum sensing inhibitory activity. Preliminary identification of selected QSI active bacterial strains revealed that they belong mostly to classes Alphaproteobacteria and Bacilli. Annotation of the MS/MS molecular networkings of these QSI active organic extracts revealed diverse classes of natural products, including aromatic polyketides, siderophores, pyrrolidine derivatives, indole alkaloids, diketopiperazines, and pyrone derivatives. Moreover, potential novel compounds were detected in several strains as revealed by unique molecular families present in the molecular networks. Further research is required to determine the temporal stability of the microbiome of the host sponge, as well as mining of associated bacteria for novel QS inhibitors.

Triggering Growth via Growth Initiation Factors in Nature: A Putative Mechanism for in situ Cultivation of Previously Uncultivated Microorganisms.

Most microorganisms resist cultivation under standard laboratory conditions. On the other hand, cultivating microbes in a membrane-bound device incubated in nature (in situ cultivation) can be an effective approach to overcome this limitation. In the present study, we applied in situ cultivation to isolate diverse previously uncultivated marine sponge-associated microbes and comparatively analyzed this method’s efficiencies with those of the conventional method. Then, we attempted to investigate the key and previously unidentified mechanism of growing uncultivated microorganisms by in situ cultivation focusing on growth triggering via growth initiation factor. Significantly more novel and diverse microbial types were isolated via in situ cultivation than by standard direct plating (SDP). We hypothesized that some of environmental microorganisms which resist cultivation are in a non-growing state and require growth initiation factors for the recovery and that these can be provided from the environment (in this study from marine sponge). According to the hypothesis, the effect of the sponge extract on recovery on agar medium was compared between strains derived from in situ and SDP cultivation. Adding small amounts of the sponge extracts to the medium elevated the colony-formation efficiencies of the in situ strains at the starvation recovery step, while it showed no positive effect on that of SDP strains. Conversely, specific growth rates or saturated cell densities of all tested strains were not positively affected. These results indicate that, (1) the sponge extract contains chemical compounds that facilitate recovery of non-growing microbes, (2) these substances worked on the in situ strains, and (3) growth initiation factor in the sponge extract did not continuously promote growth activity but worked as triggers for regrowth (resuscitation from non-growing state).

Nitrification Activity of the Sponge Chondrosia reniformis Under Elevated Concentrations of Ammonium

This study examined the ability of a Mediterranean demosponge Chondrosia reniformis to oxidize exogenous ammonium, simulating N-rich conditions that occur near finfish farms. We hypothesized that as the concentration of ammonium increases in the surrounding seawater, nitrification mediated by microbes associated with C. reniformis will lead to enhancement of ammonium uptake, nitrate excretion and oxygen consumption by the sponge holobiont. To test this hypothesis, we conducted laboratory experiments with C. reniformis explants exposed to ammonium enrichments (300–6667 nM) and to ambient seawater (45–1511 nM ammonium). We analyzed inhaled (IN) and exhaled (EX) water samples for dissolved oxygen, ammonium, nitrates and retention of picoplankton cells. We observed ammonium uptake in nearly half the cases and excretion of nitrate in most experimental outcomes. Yet, the consumption of ammonium and oxygen, as well as the excretion of nitrate by C. reniformis were not related to the concentration of inhaled ammonium, which suggests that the nitrification activity of sponge-associated microbes is not necessarily related to the concentration of ammonium in the surrounding seawater. Further research is required to reveal the sources of nitrate released from sponges and the fate of this nitrate in natural and manipulated ecosystems.

Diversity of a bacterial community associated with Cliona lobata Hancock and Gelliodes pumila (Lendenfeld, 1887) sponges on the South-East coast of India

Marine sponges are sources of various bioactive metabolites, including several anticancer drugs, produced mainly by sponge-associated microbes. Palk Bay, on the south-east coast of India, is an understudied, highly disturbed reef environment exposed to various anthropogenic and climatic stresses. In recent years, Palk Bay suffered from pollution due to the dumping of untreated domestic sewage, effluents from coastal aquaculture, tourism, salt pans, cultivation of exotic seaweeds, and geogenic heavy-metal pollution, especially arsenic, mercury, cadmium, and lead. Low microbial-abundant sponge species, such as Gelliodes pumila and Cliona lobata, were found to be ubiquitously present in this reef environment. Triplicate samples of each of these sponge species were subjected to Illumina MiSeq sequencing using V3–V4 region-specific primers. In both C. lobata and G. pumila, there was an overwhelming dominance (98 and 99%) of phylum Candidatus Saccharibacteria and Proteobacteria, respectively. The overall number of operational taxonomic units (OTUs) was 68 (40 and 13 OTUs unique to G. pumila and C. lobata, respectively; 15 shared OTUs). Alphaproteobacteria was the most abundant class in both the sponge species. Unclassified species of phylum Candidatus Saccharibacteria from C. lobata and Chelotivorans composti from G. pumila were the most abundant bacterial species. The predominance of Alphaproteobacteria also revealed the occurrence of various xenobiotic-degrading, surfactant-producing bacterial genera in both the sponge species, indirectly indicating the possible polluted reef status of Palk Bay. Studies on sponge microbiomes at various understudied geographical locations might be helpful in predicting the status of reef environments.

Dark CO2 fixation into phospholipid-derived fatty acids by the cold-water coral associated sponge Hymedesmia (Stylopus) coriacea (Tisler Reef, NE Skagerrak)

ABSTRACTMany cold-water sponges harbour microorganisms of which the role in the sponge host remains enigmatic. Here, we show a transfer of fixed inorganic carbon by sponge-associated microbes to its host, the cold-water coral encrusting sponge Hymedesmia (Stylopus) coriacea. Sponge were collected at approx. 100 m depth and incubated for 1.5–2.5 days with 13C labelled dissolved inorganic carbon (DIC) as tracer. Total DIC fixation rates ranged from 0.03–0.11 mmol C × mmol Csponge × d−1. 13C-tracer was recovered in bacterial-specific (i.e. short and branched) and sponge-specific (very long-chained) phospholipid-derived fatty acids (PLFA's), but was not incorporated into archaeal lipids. 13C-incorporation in biomarkers such as C16:1w7c and C18:1w7c indicated that nitrifying and/or sulphur-oxidizing bacteria (chemoautotrophs) were likely active in the sponge. Trophic transfer of microbially-fixed carbon to the sponge host was confirmed by recovery of label in very long chain fatty acids (VLCFA's) including C26:2 and C26:3. Tracer accumulation into several VLCFA's continued after removal of 13C-DIC, while tracer in most bacteria-specific PLFA's declined, indicating a transfer and elongation of bacterial-specific PLFA's to sponge-specific PLFA's. This implies that PLFA precursors released from chemo- as well as heterotrophic microbes in sponges contributed to the synthesis of VLCFA's, identifying sponge-associated bacteria as symbionts of the sponge.

Morphological characterization of virus-like particles in coral reef sponges.

Marine sponges host complex microbial consortia that vary in their abundance, diversity and stability amongst host species. While our understanding of sponge-microbe interactions has dramatically increased over the past decade, little is known about how sponges and their microbial symbionts interact with viruses, the most abundant entities in the ocean. In this study, we employed three transmission electron microscopy (TEM) preparation methods to provide the first comprehensive morphological assessment of sponge-associated viruses. The combined approaches revealed 50 different morphologies of viral-like particles (VLPs) represented across the different sponge species. VLPs were visualized within sponge cells, within the sponge extracellular mesohyl matrix, on the sponge ectoderm and within sponge-associated microbes. Non-enveloped, non-tailed icosahedral VLPs were the most commonly observed morphotypes, although tailed bacteriophage, brick-shaped, geminate and filamentous VLPs were also detected. Visualization of sponge-associated viruses using TEM has confirmed that sponges harbor not only diverse communities of microorganisms but also diverse communities of viruses.

Isolation and structure determination of lysiformine from bacteria associated with marine sponge Halichondria okadai

Marine sponge Halichondria okadai is a source of numerous toxic secondary metabolites, which are considered as products of the symbiotic species associated with the sponge. In this study, we tested 720 culture conditions and purified 48 sponge-associated microbes. The ethyl acetate extract from the liquid culture of each species was tested for cytotoxicity, hemolytic activity, and brine shrimp lethal activity. Lysiformine (1) was isolated from Lysinibacillus fusiformis and its structure was determined by HRMS and NMR.

Bioprospecting Sponge-Associated Microbes for Antimicrobial Compounds.

Sponges are the most prolific marine organisms with respect to their arsenal of bioactive compounds including antimicrobials. However, the majority of these substances are probably not produced by the sponge itself, but rather by bacteria or fungi that are associated with their host. This review for the first time provides a comprehensive overview of antimicrobial compounds that are known to be produced by sponge-associated microbes. We discuss the current state-of-the-art by grouping the bioactive compounds produced by sponge-associated microorganisms in four categories: antiviral, antibacterial, antifungal and antiprotozoal compounds. Based on in vitro activity tests, identified targets of potent antimicrobial substances derived from sponge-associated microbes include: human immunodeficiency virus 1 (HIV-1) (2-undecyl-4-quinolone, sorbicillactone A and chartarutine B); influenza A (H1N1) virus (truncateol M); nosocomial Gram positive bacteria (thiopeptide YM-266183, YM-266184, mayamycin and kocurin); Escherichia coli (sydonic acid), Chlamydia trachomatis (naphthacene glycoside SF2446A2); Plasmodium spp. (manzamine A and quinolone 1); Leishmania donovani (manzamine A and valinomycin); Trypanosoma brucei (valinomycin and staurosporine); Candida albicans and dermatophytic fungi (saadamycin, 5,7-dimethoxy-4-p-methoxylphenylcoumarin and YM-202204). Thirty-five bacterial and 12 fungal genera associated with sponges that produce antimicrobials were identified, with Streptomyces, Pseudovibrio, Bacillus, Aspergillus and Penicillium as the prominent producers of antimicrobial compounds. Furthemore culture-independent approaches to more comprehensively exploit the genetic richness of antimicrobial compound-producing pathways from sponge-associated bacteria are addressed.

Pyrosequencing reveals the microbial communities in the Red Sea sponge Carteriospongia foliascens and their impressive shifts in abnormal tissues.

Abnormality and disease in sponges have been widely reported, yet how sponge-associated microbes respond correspondingly remains inconclusive. Here, individuals of the sponge Carteriospongia foliascens under abnormal status were collected from the Rabigh Bay along the Red Sea coast. Microbial communities in both healthy and abnormal sponge tissues and adjacent seawater were compared to check the influences of these abnormalities on sponge-associated microbes. In healthy tissues, we revealed low microbial diversity with less than 100 operational taxonomic units (OTUs) per sample. Cyanobacteria, affiliated mainly with the sponge-specific species "Candidatus Synechococcus spongiarum," were the dominant bacteria, followed by Bacteroidetes and Proteobacteria. Intraspecies dynamics of microbial communities in healthy tissues were observed among sponge individuals, and potential anoxygenic phototrophic bacteria were found. In comparison with healthy tissues and the adjacent seawater, abnormal tissues showed dramatic increase in microbial diversity and decrease in the abundance of sponge-specific microbial clusters. The dominated cyanobacterial species Candidatus Synechococcus spongiarum decreased and shifted to unspecific cyanobacterial clades. OTUs that showed high similarity to sequences derived from diseased corals, such as Leptolyngbya sp., were found to be abundant in abnormal tissues. Heterotrophic Planctomycetes were also specifically enriched in abnormal tissues. Overall, we revealed the microbial communities of the cyanobacteria-rich sponge, C. foliascens, and their impressive shifts under abnormality.

Two Peptides, Cycloaspeptide A and Nazumamide A from a Sponge Associated Marine Actinobacterium Salinispora sp

Marine sponges are a major component of benthic communities and act as a reservoir for microbial species. In terms of biomass, they are the richest source of secondary metabolite production, with the potential to influence both benthic and pelagic systems. In most cases it is the sponge-associated microbes that account for many of the secondary metabolites assigned to the host. Here we report the occurrence of cycloaspeptide A, a fungus-derived cyclic peptide, in a culturable bacterium Salinispora arenicola. We have also identified nazumamide A, a sponge-derived linear tetrapeptide currently used as a thrombin inhibitor, in Salinispora pacifica. Their structures were determined using an integrated approach consisting of: (1) HPLC-UV-Vis-QToF-MS analysis with multimode ionization (ESI and APCI) and fast polarity switching; (2) database searching and matching of monoisotopic masses, retention times, mass spectra of the precursor and product ions of the compounds of interest and the authentic reference standards thereof.

Symbiotic Adaptation Drives Genome Streamlining of the Cyanobacterial Sponge Symbiont “ Candidatus Synechococcus spongiarum”

ABSTRACT“Candidatus Synechococcus spongiarum” is a cyanobacterial symbiont widely distributed in sponges, but its functions at the genome level remain unknown. Here, we obtained the draft genome (1.66 Mbp, 90% estimated genome recovery) of “Ca. Synechococcus spongiarum” strain SH4 inhabiting the Red Sea sponge Carteriospongia foliascens. Phylogenomic analysis revealed a high dissimilarity between SH4 and free-living cyanobacterial strains. Essential functions, such as photosynthesis, the citric acid cycle, and DNA replication, were detected in SH4. Eukaryoticlike domains that play important roles in sponge-symbiont interactions were identified exclusively in the symbiont. However, SH4 could not biosynthesize methionine and polyamines and had lost partial genes encoding low-molecular-weight peptides of the photosynthesis complex, antioxidant enzymes, DNA repair enzymes, and proteins involved in resistance to environmental toxins and in biosynthesis of capsular and extracellular polysaccharides. These genetic modifications imply that “Ca. Synechococcus spongiarum” SH4 represents a low-light-adapted cyanobacterial symbiont and has undergone genome streamlining to adapt to the sponge’s mild intercellular environment.

Evidence of unique and generalist microbes in distantly related sympatric intertidal marine sponges (Porifera: Demospongiae).

The diversity and specificity of microbial communities in marine environments is a key aspect of the ecology and evolution of both the eukaryotic hosts and their associated prokaryotes. Marine sponges harbor phylogenetically diverse and complex microbial lineages. Here, we investigated the sponge bacterial community and distribution patterns of microbes in three sympatric intertidal marine demosponges, Hymeniacidon perlevis, Ophlitaspongia papilla and Polymastia penicillus, from the Atlantic coast of Portugal using classical isolation techniques and 16S rRNA gene clone libraries. Microbial composition assessment, with nearly full-length 16S rRNA gene sequences (ca. 1400 bp) from the isolates (n = 31) and partial sequences (ca. 280 bp) from clone libraries (n = 349), revealed diverse bacterial communities and other sponge-associated microbes. The majority of the bacterial isolates were members of the order Vibrionales and other symbiotic bacteria like Pseudovibrio ascidiaceiocola, Roseobacter sp., Hahellaceae sp. and Cobetia sp. Extended analyses using ecological metrics comprising 142 OTUs supported the clear differentiation of bacterial community profiles among the sponge hosts and their ambient seawater. Phylogenetic analyses were insightful in defining clades representing shared bacterial communities, particularly between H. perlevis and the geographically distantly-related H. heliophila, but also among other sponges. Furthermore, we also observed three distinct and unique bacterial groups, Betaproteobactria (∼81%), Spirochaetes (∼7%) and Chloroflexi (∼3%), which are strictly maintained in low-microbial-abundance host species O. papilla and P. penicillus. Our study revealed the largely generalist nature of microbial associations among these co-occurring intertidal marine sponges.

Phylogenetically Diverse ureC Genes and Their Expression Suggest the Urea Utilization by Bacterial Symbionts in Marine Sponge Xestospongia testudinaria

Urea is one of the dominant organic nitrogenous compounds in the oligotrophic oceans. Compared to the knowledge of nitrogen transformation of nitrogen fixation, ammonia oxidization, nitrate and nitrite reduction mediated by sponge-associated microbes, our knowledge of urea utilization in sponges and the phylogenetic diversity of sponge-associated microbes with urea utilization potential is very limited. In this study, Marinobacter litoralis isolated from the marine sponge Xestospongia testudinaria and the slurry of X. testudinaria were found to have urease activity. Subsequently, phylogenetically diverse bacterial ureC genes were detected in the total genomic DNA and RNA of sponge X. testudinaria, i.e., 19 operative taxonomic units (OTUs) in genomic DNA library and 8 OTUs in cDNA library at 90% stringency. Particularly, 6 OTUs were common to both the genomic DNA library and the cDNA library, which suggested that some ureC genes were expressed in this sponge. BLAST and phylogenetic analysis showed that most of the ureC sequences were similar with the urease alpha subunit of members from Proteobacteria, which were the predominant component in sponge X. testudinaria, and the remaining ureC sequences were related to those from Magnetococcus, Cyanobacteria, and Actinobacteria. This study is the first assessment of the role of sponge bacterial symbionts in the regenerated utilization of urea by the detection of transcriptional activity of ureC gene, as well as the phylogenetic diversity of ureC gene of sponge bacterial symbionts. The results suggested the urea utilization by bacterial symbionts in marine sponge X. testudinaria, extending our understanding of nitrogen cycling mediated by sponge-associated microbiota.

Same, same but different: symbiotic bacterial associations in GBR sponges

Symbioses in marine sponges involve diverse consortia of microorganisms that contribute to the health and ecology of their hosts. The microbial communities of 13 taxonomically diverse Great Barrier Reef (GBR) sponge species were assessed by DGGE and 16S rRNA gene sequencing to determine intra and inter species variation in bacterial symbiont composition. Microbial profiling revealed communities that were largely conserved within different individuals of each species with intra species similarity ranging from 65–100%. 16S rRNA gene sequencing revealed that the communities were dominated by Proteobacteria, Chloroflexi, Acidobacteria, Actinobacteria, Nitrospira, and Cyanobacteria. Sponge-associated microbes were also highly host-specific with no operational taxonomic units (OTUs) common to all species and the most ubiquitous OTU found in only 5 of the 13 sponge species. In total, 91% of the OTUs were restricted to a single sponge species. However, GBR sponge microbes were more closely related to other sponge-derived bacteria than they were to environmental communities with sequences falling within 50 of the 173 previously defined sponge-(or sponge-coral) specific sequence clusters (SC). These SC spanned the Acidobacteria, Actinobacteria, Proteobacteria, Bacteroidetes, Chloroflexi, Cyanobacteria, Gemmatimonadetes, Nitrospira, and the Planctomycetes-Verrucomicrobia-Chlamydiae superphylum. The number of sequences assigned to these sponge-specific clusters across all species ranged from 0 to 92%. No relationship between host phylogeny and symbiont communities were observed across the different sponge orders, although the highest level of similarity was detected in two closely related Xestospongia species. This study identifies the core microbial inhabitants in a range of GBR sponges thereby providing the basis for future studies on sponge symbiotic function and research aiming to predict how sponge holobionts will respond to environmental perturbation.