Sequestration of dietary alkaloids by the spongivorous marine mollusc Tylodina perversa

Specimens of the spongivorous Mediterranean opisthobranch Tylodina perversa that had been collected while feeding on Aplysina acerophoba were shown to sequester the brominated isoxazoline alkaloids of their prey. Alkaloids were stored in the hepatopancreas, mantle tissues, and egg masses in an organ-specific manner. Surprisingly, the known sponge alkaloid aerothionin which is found only in A. cavernicola but not in A. aerophoba was also among the metabolites identified in wild caught specimens of T. perversa as well as in opisthobranchs with a documented feeding history on A. aerophoba. Mollusc derived aerothionin is postulated to be derived from a previous feeding encounter with A. cavernicola as T. perversa was found to freely feed on both Aplysina sponges in aquarium bioassays. The possible ecological significance of alkaloid sequestration by T. perversa is still unknown.     

Exploring the links between natural products and bacterial assemblages in the sponge Aplysina aerophoba

The sponge Aplysina aerophoba produces a large diversity of brominated alkaloids (BAs) and hosts a complex microbial assemblage. Although BAs are located within sponge cells, the enzymes that bind halogen elements to organic compounds have been exclusively described in algae, fungi, and bacteria. Bacterial communities within A. aerophoba could therefore be involved in the biosynthesis of these compounds. This study investigates whether changes in both the concentration of BAs and the bacterial assemblages are correlated in A. aerophoba. To do so, we quantified major natural products using high-performance liquid chromatography and analyzed bacterial assemblages using denaturing gradient gel electrophoresis on the 16S rRNA gene. We identified multiple associations between bacteria and natural products, including a strong relationship between a Chloroflexi phylotype and aplysinamisin-1 and between an unidentified bacterium and aerophobin-2 and isofistularin-3. Our results suggest that these bacteria could either be involved in the production of BAs or be directly affected by them. To our knowledge, this is one of the first reports that find a significant correlation between natural products and bacterial populations in any benthic organism. Further investigating these associations will shed light on the organization and functioning of host-endobiont systems such as Aplysina aerophoba.     

Can a sponge feeder be a herbivore? Tylodina perversa (Gastropoda) feeding on Aplysina aerophoba (Demospongiae)

Feeding biology in mollusks has important biological, ecological and evolutionary implications because many of the characteristics we observe in mollusks arise from their co-evolution with diet organisms. We investigated the relationship between the opisthobranch Tylodina perversa and the sponges Aplysina aerophoba and Aplysina cavernicola in order to ascertain the trophic interactions between them. The opisthobranch preferred specimens of Aplysina aerophoba inhabiting shallow overdeep waters, ectosome of Aplysina aerophoba over choanosome, and showed no preference for Aplysina cavernicola . The sponge Aplysina cavernicola lacks the cyanobacteria abundant in the ectosome of Aplysina aerophoba . Our study shows that the opisthobranch Tylodina perversa actively selects for sponges or sponge zones with high concentration of cyanobacteria, i.e. only a fraction of the ingested material is of animal origin. Addition of cyanobacteria to symbiont-free sponge material induced a shift in mollusk preference. Our results cast doubt over the widely recognized qualification of Tylodina perversa as a carnivorous sponge feeder and show evidence that cyanobacteria determine the opisthobranch food selection. Whether this is an isolated example of how symbionts may determine trophic interactions between hosts and predators or it is widespread in benthic organisms remains an open question that requires further investigation.  © 2003 The Linnean Society of London . Biological Journal of the Linnean Society , 2003, 78 , 429–438.     

Organ-specific distribution of dietary alkaloids in the marine opisthobranch Tylodina perversa

Specimens of the opisthobranch Tylodina perversa that were observed while feeding on the sponge Aplysina aerophoba were transferred to seawater tanks along with their prey and kept under controlled conditions. After one week the opisthobranchs were anaesthetized, dissected and studied for sequestered sponge-derived brominated alkaloids. All parts of T. perversa analyzed including feces, mucus and egg masses that had been produced during captivity contained alkaloids derived from A. aerophoba. The highest total alkaloid concentration (24.6 mg g⁻¹ dry wt) was found in mantles of T. perversa (compared to 51.2 mg g⁻¹ dry wt of total alkaloids in A. aerophoba). Hepatopancreas, egg masses and mucus (respective total alkaloid concentrations ranging from 20.4 to 12.5 mg g⁻¹ dry wt) were also rich in alkaloids. Whereas in A. aerophoba the isoxazoline alkaloids aerophobin-2 and isofistularin-3 were present in almost equal concentrations, aerophobin-2 constituted by far the major alkaloid (amounting to approximately 70% of all identified alkaloids) in mantles, mucus and egg masses of T. perversa, indicating selective sequestration by the opisthobranchs. Mantles as well as mucus also contained appreciable concentrations (approximately 20% of all identified compounds) of the brominated alkaloid aerothionin; this is not detected in A. aerophoba. It is possible that aerothionin originates from a previous encounter of T. perversa with the sponge A. cavernicola, the latter being closely related to A. aerophoba. The enrichment of aerophobin-2 (and of aerothionin) in mantles, mucus and egg masses that are vulnerable and exposed (mantles and egg masses) to predators and/or pathogens argues for defensive functions of the respective alkaloids even though this hypothesis still needs to be experimentally corroborated.     

Isolation and phylogenetic analysis of bacteria with antimicrobial activities from the Mediterranean sponges Aplysina aerophoba and Aplysina cavernicola

The aim of this study was to isolate bacteria with antimicrobial activities from the marine sponges Aplysina aerophoba and Aplysina cavernicola. The obtained 27 isolates could be subdivided into eight phylogenetically different clusters based on comparative sequence analysis of their 16S rDNA genes. The sponge isolates were affiliated with the low (Bacillus) and high G+C Gram-positive bacteria (Arthobacter, Micrococcus), as well as the alpha-Proteobacteria (unknown isolate) and gamma-Proteobacteria (Vibrio, Pseudoalteromonas). One novel Bacillus species was identified and two species were closely related to previously uncharacterized strains. Isolates with antimicrobial activity were numerically most abundant in the genera Pseudoalteromonas and the alpha-Proteobacteria. The sponge isolates show antimicrobial activities against Gram-positive and Gram-negative reference strains but not against the fungus Candida albicans. A general pattern was observed in that Gram-positive bacteria inhibited Gram-positive strains while Gram-negative bacteria inhibited Gram-negative isolates. Antimicrobial activities were also found against clinical isolates, i.e. multi-resistant Staphylococcus aureus and Staphylococcus epidermidis strains isolated from hospital patients. The high recovery of strains with antimicrobial activity suggests that marine sponges represent an ecological niche which harbors a hitherto largely uncharacterized microbial diversity and, concomitantly, a yet untapped metabolic potential.     

Physiology, phylogeny and in situ evidence for bacterial and archaeal nitrifiers in the marine sponge Aplysina aerophoba

The potential for nitrification in the Mediterranean sponge Aplysina aerophoba was assessed using a combined physiological and molecular approach. Nitrate excretion rates in whole sponges reached values of up to 344 nmol g(-1) dry weight (wt) h(-1) (unstimulated) and 1325 nmol g(-1) dry wt h(-1) (stimulated). Addition of nitrapyrin, a nitrification-specific inhibitor, effectively inhibited nitrate excretion. Ammonium was taken up by sponges in spring and excreted in fall, the sponges thus serving as either an ammonium sink or ammonium source. Nitrosospira cluster 1 and Crenarchaeota group I.1A 16S rRNA and amoA genes were recovered from A. aerophoba and other sponges from different world's oceans. The archaeal 16S rRNA genes formed a sponge-specific subcluster, indicating that their representatives are members of the stable microbial community of sponges. On the other hand, clustering was not evident for Nitrosospira rRNA genes which is consistent with their presence in sediment and seawater samples. The presence of both Nitrosospira cluster 1 and crenarchaeal group 1 phylotypes in sponge tissue was confirmed using fluorescently labelled 16S rRNA gene probes. This study contributes to an ongoing effort to link microbial diversity with metabolic functions in the phylogenetically diverse, elusive and so far uncultivated microbial communities of marine sponges.     

Morphological,Bacterial, and Secondary Metabolite Changes of <Emphasis Type="Italic">Aplysina aerophoba</Emphasis> upon Long-Term Maintenance Under Artificial Conditions

The aim of this study was to analyze successional changes in the bacterial community over a period of 6 months of cultivation of Aplysina aerophoba sponges under different artificial cultivation conditions by use of denaturing gradient gel electrophoresis (DGGE). The cultivation conditions varied concerning the water temperature (20 ± 2 °C and 25 ± 2 °C) of the aquaria, additional illumination of one aquarium, and feeding of the sponges. Amplicons from DGGE separation of dominant colonizing or variably appearing bacteria were sequenced and aligned for taxonomical identification. In addition, secondary metabolites typically found in A. aerophoba were analyzed to investigate changes in the natural product profile during cultivation. The cultivation of sponges under any given condition did not lead to a depletion of their bacterial community in the course of the experiment. On the contrary, the distinctive set of associated bacteria was maintained in spite of a dramatic loss of biomass and morphological degradation during the cultivation period. Generally, all sequences obtained from the DGGE gels were related to bacteria of five phyla: Actinobacteria, Cyanobacteria, α-Proteobacteria, γ-Proteobacteria, and Chloroflexi. Despite the overall stability of the bacterial community in A. aerophoba, an unambiguous variability was detected for the Cyanobacteria “A. aerophoba clone TK09”. This variability was ascribed to the predominant light conditions. The analysis of the metabolic pattern revealed that the concentration of a class of characteristic-brominated compounds typically found in A. aerophoba, like aeroplysinin-1, aerophobin-1, aerophobin-2, and isofistularin-3, increased over the 6 months of cultivation.     

Shifts in microbial and chemical patterns within the marine sponge Aplysina aerophoba during a disease outbreak

The microbial community composition in affected and unaffected portions of diseased sponges and healthy control sponges of Aplysina aerophoba was assessed to ascertain the role of microbes in the disease process. Sponge secondary metabolites were also examined to assess chemical shifts in response to infection. The microbial profile and aplysinimine levels in unaffected tissue near the lesions closely reflected those of healthy sponge tissue, indicating a highly localized disease process. DGGE detected multiple sequences that were exclusively present in diseased sponges. Most notably, a Deltaproteobacteria sequence with high homology to a coral black band disease strain was detected in all sponge lesions and was absent from all healthy and unaffected regions of diseased sponges. Other potential pathogens identified by DGGE include an environmental Cytophaga strain and a novel Epsilonproteobacteria strain with no known close relatives. The disease process also caused a major shift in prokaryote community structure at a very high taxonomic level. Using 16S rRNA gene sequence analysis, only the diseased sponges were found to contain sequences belonging to the Epsilonproteobacteria and Firmicutes, and there was a much greater number of Bacteroidetes sequences within the diseased sponges. In contrast, only the healthy sponges contained sequences corresponding to the cyanobacteria and 'OP1' candidate division, and the healthy sponges were dominated by Chloroflexi and Gammaproteobacteria sequences. Overall bacterial diversity was found to be considerably higher in diseased sponges than in healthy sponges. These results provide a platform for future cultivation-based experiments to isolate the putative pathogens from A. aerophoba and perform re-infection trials to define the disease aetiology.     

Advances in the production of sponge biomass Aplysina aerophoba--a model sponge for ex situ sponge biomass production

Sponges are a promising source of organic compounds of potential interest regarding industrial and medical applications. For detailed studies on such compounds, large amounts of sponge biomass are required. Obtaining that is at present extremely difficult because most sponges are relatively rare in nature and their mass cultivation in the laboratory has not yet been accomplished. In this study the possibility of culturing Aplysina aerophoba fragments in laboratory was examined. While a substantial biomass increase was not yet observed, we achieved fragmented sponge tissue to develop into a functional sponge as a first success.     

Bioactive natural products from marine sponges and fungal endophytes

This review highlights recent findings of our group on bioactive marine natural products isolated from marine sponges and marine derived fungi. The activated chemical defence of the Mediterranean sponge Aplysina aerophoba is introduced as an example of a dynamic response of marine sponges to wounding. Following tissue disrupture preformed brominated isoxazoline alkaloids are enzymatically cleaved and thereby give rise to aeroplysinin-1 which is believed to protect sponges from invasion of pathogenic bacteria. A preliminary characterization of the membrane bound enzyme(s) involved in the cleavage reaction is presented. Bromotyrosine derived, oxime group bearing peptides, the so called bastadins, obtained from the sponge Ianthella basta and some of their synthetic derivatives were shown to exhibit pronounced antifouling activity against larvae of the barnacle Balanus improvisus. The antifouling activity could be traced to the oxime group as an important pharmacophore that was also found to be present in other sponge derived natural products exhibiting antifouling activity. Marine derived fungi that reside within invertebrates such as sponges or inside Mangrove plants are emerging as a new source of bioactive metabolites as demonstrated for Aspergillus ustus and Alternaria sp. that were isolated from the sponge Suberites domuncula or from the Mangrove plant Sonneratia alba, respectively. The former fungus yielded new moderately cytotoxic sesquiterpenoids of the drimane type whereas the latter was found to produce polyketides such as alternariol that exhibited strong and selective inhibitory activity against several protein kinases, for instance Aurora A and B which are targets for anticancer chemotherapy.     

Reductive dehalogenation of brominated phenolic compounds by microorganisms associated with the marine sponge Aplysina aerophoba

Marine sponges are natural sources of brominated organic compounds, including bromoindoles, bromophenols, and bromopyrroles, that may comprise up to 12% of the sponge dry weight. Aplysina aerophoba sponges harbor large numbers of bacteria that can amount to 40% of the biomass of the animal. We postulated that there might be mechanisms for microbially mediated degradation of these halogenated chemicals within the sponges. The capability of anaerobic microorganisms associated with the marine sponge to transform haloaromatic compounds was tested under different electron-accepting conditions (i.e., denitrifying, sulfidogenic, and methanogenic). We observed dehalogenation activity of sponge-associated microorganisms with various haloaromatics. 2-Bromo-, 3-bromo-, 4-bromo-, 2,6-dibromo-, and 2,4,6-tribromophenol, and 3,5-dibromo-4-hydroxybenzoate were reductively debrominated under methanogenic and sulfidogenic conditions with no activity observed in the presence of nitrate. Monochlorinated phenols were not transformed over a period of 1 year. Debromination of 2,4,6-tribromophenol, and 2,6-dibromophenol to 2-bromophenol was more rapid than the debromination of the monobrominated phenols. Ampicillin and chloramphenicol inhibited activity, suggesting that dehalogenation was mediated by bacteria. Characterization of the debrominating methanogenic consortia by using terminal restriction fragment length polymorphism (TRFLP) and denaturing gradient gel electrophoresis analysis indicated that different 16S ribosomal DNA (rDNA) phylotypes were enriched on the different halogenated substrates. Sponge-associated microorganisms enriched on organobromine compounds had distinct 16S rDNA TRFLP patterns and were most closely related to the delta subgroup of the proteobacteria. The presence of homologous reductive dehalogenase gene motifs in the sponge-associated microorganisms suggested that reductive dehalogenation might be coupled to dehalorespiration.     

Bioactive Brominated Metabolites from the Natural Habitat and Tank‐Maintained Cuttings of the Jamaican Sponge Aplysina fistularis

Cut specimens of the common reef sponge of the Verongid family, Aplysina fistularis, were retained in flow‐through seawater tanks over a six‐week period to assess the metabolite profile of the sponge when subjected to stress, compare the profile with the source material, and assess the preliminary feasibility of the protocol for sponge culture. The living specimens were harvested, extracted with MeOH/CH₂Cl₂ 1 : 1, and subjected to column chromatography to identify metabolites. The brominated isoxazoline compounds, aerothionin ( 1 ) and 11‐oxoaerothionin ( 2 ), along with aeroplysinin 2 ( 3 ) and 2‐(3,5‐dibromo‐4‐hydroxyphenol)acetamide ( 4 ), were detected in the cuttings from the tank‐maintained sponge. An examination of the metabolite profile of the sponge from the natural habitat showed that the compounds 1 and 2 were present. The identities of all the compounds were ascertained by analysis of the mass‐spectral data and NMR spectra (¹H, ¹³C, HMBC, and HSQC) of the compounds, which were compared with reported data. The survival rate was 44% with limited necrosis or exposed skeletal tissue being observed in eight of the 18 cuttings, suggesting that protocol modifications would be required for culturing the sponge.     

Biogeography of sponge chemical ecology: comparisons of tropical and temperate defenses

Examples from both marine and terrestrial systems have supported the hypothesis that predation is higher in tropical than in temperate habitats and that, as a consequence, tropical species have evolved more effective defenses to deter predators. Although this hypothesis was first proposed for marine sponges over 25 years ago, our study provides the first experimental test of latitudinal differences in the effectiveness of sponge chemical defenses. We collected 20 common sponge species belonging to 14 genera from tropical Guam and temperate Northeast Spanish coasts (Indo-Pacific and Mediterranean biogeographic areas) and conducted field-based feeding experiments with large and small fish predators in both geographic areas. We use the term global deterrence to describe the deterrent activity of a sponge extract against all of the predators used in our experiments and to test the hypothesis that sponges from Guam are chemically better defended than their Mediterranean counterparts. Sympatric and allopatric deterrence refer to the average deterrent activity of a sponge against sympatric or allopatric predators. All of the sponges investigated in this study showed deterrent properties against some predators. However, 35% of the sponge species were deterrent in at least one but not in all the experiments, supporting the idea that predators can respond to chemical defenses in a species-specific manner. Tropical and temperate sponges have comparable global, sympatric, and allopatric deterrence, suggesting not only that chemical defenses from tropical and temperate sponges are equally strong but also that they are equally effective against sympatric and allopatric predators. Rather than supporting geographic trends in the production of chemical defenses, our data suggest a recurrent selection for chemical defenses in sponges as a general life-history strategy.     

Distribution of brominated compounds within the sponge<Emphasis Type="Italic"> Aplysina aerophoba</Emphasis>: coupling of X-ray microanalysis with cryofixation techniques

The major secondary metabolites of the sponge Aplysina aerophoba are brominated compounds. X-ray energy dispersive microanalysis was therefore used to locate secondary metabolites via the Br signal in energy emission spectra from sponge sections. To test the reliability of this method in the face of the loss or redistribution of metabolites during processing, we compared the results obtained by conventional aldehyde fixation with those obtained by cryofixation and cryosubstitution with and without cryoembedding. Bromine appeared to be concentrated in two sponge structures, viz. fibres and spherulous cells, when cryofixed material was examined. However, X-ray microanalysis failed to demonstrate the presence of bromine in spherulous cells in chemically fixed samples, showing the need for cryotechniques to avoid the loss of compounds. Cryofixation plus cryosubstitution methods performed best regarding structural preservation and the immobilization of metabolites. The presence of bromine in the spherulous cells suggests that this cell type is the producer of the secondary metabolites, as described for other sponge species. Nevertheless, the presence of bromine in sponge fibres indicates that they can accumulate metabolic substances, although we have been unable to assess whether the chemicals are in their original form or in a modified state within the fibres. A. aerophoba has both bacterial and cyanobacterial symbionts in its mesohyl; the absence of brominated compounds in them contrasts with previous findings in other sponges with prokaryote symbionts.     

Localization of Two Brominated Metabolites,Aerothionin and Homoaerothionin,in Spherulous Cells of the Marine Sponge Aplysina fistularis (=Verongia thiona)

Energy dispersive X-ray microanalysis was used to localize the two brominated natural products (aerothinonin and homoaerothionin) in the tissues of a marine demosponge, Aplysina fistularis. Virtually all of these compounds were localized within the spherules of the spherulous cells in the mesohyl. This is the first localization of any secondary metabolite at the cellular or sub-cellular level in any marine invertebrate. In Aplysina fistularis, as in other species of the same genus studied by Vacelet, the spherulous cells are concentrated just beneath the exopinacoderm and just beneath the endopinacoderm of the excurrent canals. Moreover, there is electron microscopic evidence for degeneration of some spherulous cells throughout the mesohyl. Presumably, this degeneration can release some aerothionin and homoaerothionin, which are known to have antibiotic properties. After release from the spherulous cells, these brominated natural products could function (1) within the mesohyl to exclude some types of bacteria or to aggregate ingested bacteria and/or (2) within the boundary layer of the surrounding seawater for defense or offense, as considered in the discussion section.     

Temporal changes in the sponge holobiont during the course of infection with Aplysina Red Band Syndrome

Coral Reefs - Diseases of marine organisms, including sponges on coral reefs, are being reported with increasing frequency worldwide. Aplysina Red Band Syndrome (ARBS) occurs across the Caribbean...     

Lectins from tropical sponges. Purification and characterization of lectins from genus Aplysina

Only a few animal phyla have been screened for the presence and distribution of lectins. Probably the most intensively studied group is the mollusk. In this investigation, 22 species from 12 families of tropical sponges collected in Los Roques National Park (Venezuela) were screened for the presence of lectins. Nine saline extracts exhibited strong hemagglutinating activity against pronase-treated hamster red blood cells; five of these reacted against rabbit red blood cells, four with trypsin-treated bovine red blood cells, and five with human red blood cells regardless of the blood group type. Extracts from the three species studied from genus Aplysina (archeri, lawnosa, and cauliformis) were highly reactive and panagglutinating against the panel of red blood cells tested. The lectins from A. archeri and A. lawnosa were purified to homogeneity by ammonium sulfate fractionation, affinity chromatography on p-aminobenzyl-beta-1-thiogalactopyranoside-agarose, and gel filtration chromatography. Both lectins exhibited a native molecular mass of 63 kDa and by SDS-polyacrylamide gel electrophoresis under reducing conditions have an apparent molecular mass of 16 kDa, thus suggesting they occur as homotetramers. The purified lectins contain 3-4 mol of divalent cation per molecule, which are essential for their biological activity. Hapten inhibition of hemagglutination was carried out to define the sugar binding specificity of the purified A. archeri lectin. The results indicate a preference of the lectin for nonreducing beta-linked d-Gal residues being the best inhibitors of red blood cells binding methyl-beta-d-Gal and thiodigalactoside (Gal beta 1-4-thiogalactopyranoside). The behavior of several glycans on immobilized lectin affinity chromatography confirmed and extended the specificity data obtained by hapten inhibition.     

Aplysina red band syndrome: a new threat to Caribbean sponges

A substantial and increasing number of reports have documented dramatic changes and continuing declines in Caribbean coral reef communities over the past 2 decades. To date, the majority of disease reports have focused on scleractinian corals, whereas sponge diseases have been less frequently documented. In this study, we describe Aplysina red band syndrome (ARBS) affecting Caribbean rope sponges of the genus Aplysina observed on shallow reefs in the Bahamas. Visible signs of disease presence included 1 or more rust-colored leading edges, with or without a trailing area of necrotic tissue, such that the lesion forms a contiguous band around part or all of the sponge branch. Microscopic examination of the leading edge of the disease margin indicated that a cyanobacterium was consistently responsible for the coloration. Although the presence of this distinctive coloration was used to characterize the diseased state, it is not yet known whether this cyanobacterium is directly responsible for disease causation. The prevalence of ARBS declined significantly from July to October 2004 before increasing above July levels in January 2005. Transmission studies in the laboratory demonstrated that contact with the leading edge of an active lesion was sufficient to spread ARBS to a previously healthy sponge, suggesting that the etiologic agent, currently undescribed, is contagious. Studies to elucidate the etiologic agent of ARBS are ongoing. Sponges are an essential component of coral reef communities and emerging sponge diseases clearly have the potential to impact benthic community structure on coral reefs.     

Molecular evidence for a uniform microbial community in sponges from different oceans

Sponges (class Porifera) are evolutionarily ancient metazoans that populate the tropical oceans in great abundances but also occur in temperate regions and even in freshwater. Sponges contain large numbers of bacteria that are embedded within the animal matrix. The phylogeny of these bacteria and the evolutionary age of the interaction are virtually unknown. In order to provide insights into the species richness of the microbial community of sponges, we performed a comprehensive diversity survey based on 190 sponge-derived 16S ribosomal DNA (rDNA) sequences. The sponges Aplysina aerophoba and Theonella swinhoei were chosen for construction of the bacterial 16S rDNA library because they are taxonomically distantly related and they populate nonoverlapping geographic regions. In both sponges, a uniform microbial community was discovered whose phylogenetic signature is distinctly different from that of marine plankton or marine sediments. Altogether 14 monophyletic, sponge-specific sequence clusters were identified that belong to at least seven different bacterial divisions. By definition, the sequences of each cluster are more closely related to each other than to a sequence from nonsponge sources. These monophyletic clusters comprise 70% of all publicly available sponge-derived 16S rDNA sequences, reflecting the generality of the observed phenomenon. This shared microbial fraction represents the smallest common denominator of the sponges investigated in this study. Bacteria that are exclusively found in certain host species or that occur only transiently would have been missed. A picture emerges where sponges can be viewed as highly concentrated reservoirs of so far uncultured and elusive marine microorganisms.