Isolation of Oscillatoria spongeliae,the filamentous cyanobacterial symbiont of the marine sponge Dysidea herbacea

The tropical marine sponge Dysidea herbacea (Keller) (Dictyoceratidae: Dysideidae) is always found associated with the filamentous cyanobacterium (blue-green alga) Oscillatoria spongeliae (Schulze) Hauck (Cyanophyceae: Oscillatoriaceae), which occurs abundantly throughout the sponge mesohyl. Intact, metabolically active, trichomes of O. spongeliae were isolated from the sponge by chopping the sponge tissue with a razor blade and squeezing the trichomes into a seawater-based medium containing polyvinylpyrrolidone, bovine serum albumin, dithiothreitol, glycerol, KCl and Na₂CO₃. The isolated cyanobacteria were concentrated by centrifugation and then washed several times in fresh medium. The isolated O. spongeliae have photosynthetic rates which are similar to the intact sponge-alga association for periods of at least 6 h after isolation. Addition of sponge homogenate to the isolated cyanobacteria causes rapid cell lysis.     

A brominated secondary metabolite synthesized by the cyanobacterial symbiont of a marine sponge and accumulation of the crystalline metabolite in the sponge tissue

The dictyoceratid marine sponge Dysidea herbacea (Keller, 1889) is common in shallow waters of the tropical Pacific Ocean. Polybrominated biphenyl ethers such as 2-(2,4-dibromophenyl)-4,6-dibromophenol (1) are characteristic secondary metabolites of some specimens of this sponge and may represent as much as 12% of the dry weight. We have found 1 to be deposited as conspicuous crystals throughout the sponge tissue. The dominant prokaryotic endosymbiont in the mesohyl of the sponge is a filamentous cyanobacterium (Oscillatoria spongeliae), although a vacuole-containing, heterotrophic bacterium is also present. The cyanobacteria were separated from the sponge cells and heterotrophic bacteria by flow cytometry. Coupled gas chromatography—mass spectrometry and proton nuclear magnetic-resonance spectroscopy revealed that the major brominated Compound 1 isolated from the intact symbiotic association is found in the cyanobacteria and not in the sponge cells or heterotrophic bacteria. This suggests that the production of the compound is due to the cyanobacterium, and not to the sponge or symbiotic heterotrophic bacteria, as had been suggested earlier.     

Microbial diversity in the marine sponge Aplysina cavernicola (formerly Verongia cavernicola) analyzed by fluorescence in situ hybridization (FISH)

We have employed electronmicroscopical methods (SEM, TEM) to document the microbial community associated with the marine sponge Aplysina cavernicola (formerly Verongia cavernicola, class Demospongiae). Five dominant bacterial types were identified, three of which resemble the morphotypes originally described by Vacelet (1975). One bacterial type possesses morphological properties that are characteristic of the genus Planctomyces. In addition, morphologically uniform bacteria which reside inside the nuclei of host cells were observed. Using in situ hybridization with fluorescently labelled rRNA probes directed against known bacterial groups, the phylogenetic affiliation of the mesohyl bacteria was assessed. It could be shown that the vast majority of mesohyl bacteria belongs to the domain Bacteria with a low GC content. Among the Bacteria, the delta-Proteobacteria were most abundant, followed by the gamma-Proteobacteria and representatives of the Bacteroides cluster. Clusters of Gram-positive bacteria with a high GC content were also found consistently in low amounts. No hybridization signal was obtained with probes specific to the domain Archaea, to the alpha- and beta-Proteobacteria and to the Cytophaga/Flavobacterium cluster. This study describes for the first time the application of the “top-to-bottom approach” using 16S rRNA probes and in situ hybridization to assess the microbial diversity in Aplysina sponges. Received: 18 December 1998 / Accepted: 12 March 1999     

Evidence for a symbiosis between bacteria of the genus Rhodobacter and the marine sponge Halichondria panicea : harbor also for putatively toxic bacteria?

Halichondria panicea (Pallas) is a marine sponge, abundantly occurring in the Adriatic Sea, North Sea, and Baltic Sea. It was the aim of the present study to investigate if this sponge species harbors bacteria. Cross sections through H. panicea were taken and inspected by electron microscopy. The micrographs showed that this sponge species is colonized by bacteria in its mesohyl compartment. To identify the bacteria, polymerase chain reaction (PCR) analysis of the 16S rRNA gene segment, typical for bacteria, was performed. DNA was isolated from sponge material that had been collected near Rovinj (Adriatic Sea), Helgoland (North Sea), and Kiel (Baltic Sea) and was amplified with bacterial primers by PCR. The data gathered indicate that in all samples bacteria belonging to the genus Rhodobacter (Proteobacteria, subdivision α) are dominant, suggesting that these bacteria live in symbiotic relationship with the sponge. In addition, the results show that the different samples taken contain further bacterial species, some of them belonging to the same genus even though found in sponges from different locations. The possibility of the presence of toxic bacteria was supported by the finding that organic extracts prepared from sponge samples displayed toxicity, when analyzed in vitro using leukemia cells. Received: 7 March 1997 / Accepted: 2 October 1997     

Innate immune defense of the sponge<Emphasis Type="Italic"> Suberites domuncula</Emphasis> against gram-positive bacteria: induction of lysozyme and AdaPTin

Sponges are filter feeders that are exposed to large amounts of bacteria present in their surrounding aqueous milieu. The characteristic cell wall component of gram-positive bacteria, peptidoglycan (PPG), was used as a model molecule to study the responsiveness of cells from the marine demosponge Suberites domuncula toward gram-positive bacteria. The sponge lysozyme, which hydrolyzes PPG, was isolated from the living sponge; in addition its gene was cloned (SDLYS) and expressed in Escherichia coli. Antibodies were raised against the recombinant protein to demonstrate that in the Western blot both molecules give the same signal. In situ hybridization with SDLYS as a probe showed that cells in the mesohyl, the gray cells, strongly react with SDLYS. Subsequent immunofluorescence studies with antibodies raised against lysozyme revealed that only bacteria react with anti-lysozyme and only those that are scattered within the mesohyl of the tissue. An adaptor gene (AdaPTin-1) was isolated from the same sponge species that encodes a putative protein involved in endosome formation. Based on its differential expression we conclude that sponge cells react to PPG with a rapid activation of endocytosis, followed by the release of lysozyme.Communicated by O. Kinne, Oldendorf/LuheThe cDNA sequences from Suberites domuncula have been deposited (EMBL/GenBank): cDNA for the lysozyme (SDLYS) under the accession number AJ699166 and the AdaPTin-1 cDNA (SDAP1) under AJ699167.     

Glycohistochemistry of a marine sponge, Chondrilla nucula (Porifera, Desmospongiae), with remarks on a possibly related antimicrobial defense strategy and a note on exopinacoderm function

Based on carbohydrate histochemistry, including the use of lectins, and TEM, the study describes the distribution of terminal sugars in different structures of the demosponge Chondrilla nucula. The results of the general and specific carbohydrate histochemical approaches confirmed the presence of acidic and neutral glycoconjugates in the cells, and, with declining amounts from the ectosome to the mesohyl, in the extracellular matrix (ECM). AB-PAS staining indicated acidic complex carbohydrates particularly in the exopinacoderm, and more neutral ones in the cells and the ECM of the mesohyl. The PO-lectins applied demonstrated a general spectrum of free saccharide residues (α-d-mannose, α-/β-d-N-acetylglucosamine, α-d-N-acetylgalactosamine, α-d-galactose, β-d-galactose) in both sponge parts; α-l-fucose was only distinct in the ectosome. Sialic acids [siaα(2,3)-galactose, siaα(2,6)-N-acetylgalactosamine] were dominant in the very thin exopinacoderm, indicating O-linked high molecular weight glycoproteins. In this way a glycophysiologically ‘rigid’ outer mucus cover is developed as protection against mechanical hazards. Some of the free sugars (α-d-mannose, N-acetylglucosamine, N-acetylgalactosamine β-d-galactose, α-l-fucose) are known to prevent the adherence of different bacteria and fungi to cellular surfaces. Thus a high concentration of such sugars, may impede massive attacks of micro-inhabitants on mobile sponge cells, pinacocytes, and the exopinacoderm layer.     

Morphological evidence for vertical transmission of symbiotic bacteria in the viviparous sponge<Emphasis Type="Italic"> Halisarca dujardini</Emphasis> Johnston (Porifera,Demospongiae, Halisarcida)

All stages of vertical transmission of symbiotic bacteria, from the penetration into oocytes to the formation of rhagon, were investigated in the White Sea (Arctic) representatives of Halisarca dujardini Johnston (Demospongiae). Small populations of free-living specific symbiotic bacteria inhabit the mesohyl of H. dujardini. They are represented by a single morphotype of small spiral gram-positive bacteria. Vertical transmission of symbiotic bacteria between generations in sponges may occur in different ways. In the case of H. dujardini the bacteria penetrate into growing oocytes by endocytosis. A part of the bacteria plays a trophic role for oocytes and the other part remains undigested in membrane-bound vacuoles within the cytoplasm. In cleaving embryos bacteria are situated between the blastomeres or in the vacuoles. In the blastula all bacteria are disposed in the blastocoel. The symbionts are situated in intercellular spaces in free-swimming larvae and during metamorphosis. Symbiotic bacteria do not play any trophic role in the period of embryonic and postembryonic development of H. dujardini. No signs of destruction and digestion of bacteria were revealed at any stage of development.Communicated by O. Kinne, Oldendorf/Luhe     

Asexual reproduction in homoscleromorph sponges (Porifera; Homoscleromorpha)

Asexual reproduction by external budding in Homoscleromorpha is reported for the first time. Two Mediterranean sponge species were studied, Oscarella lobularis and O. tuberculata. Buds are formed in the marginal basal part of sponge. Budding takes from 1 to 4 days and is defined in three budding stages. First, small irregular protuberances, consisting of external parental tissue, are formed. Second, they elongate and acquire more regular, nipple-like shape. These protuberances are tube like, their internal cavity derived from parental exhalant canal. The wall consists of three layers: (a) external layer is flagellated exopinacoderm, (b) internal one is flagellated endopinacoderm and (c) intermediate one is a thin layer of mesohyl. Third, a spherical bud with a large central cavity is formed. During budding, we did not observe cell proliferation or transdifferentiation either in budding zone or in any special mitotically active region. The bud attached to the substrate is similar to the rhagon developing after larva metamorphosis, it has a syconoid organization. Morphogenetically, budding in Oscarella differes from that in other sponges. Occurring by epithelial morphogenesis, it is similar to morphallaxis during regeneration. The presence in Homoscleromorpha of an epithelial morphogenesis is unique among sponges. This feature is shared by Homoscleromorpha and Eumetazoa.     

Bacterial symbionts as an additional cytological marker for identification of sponges without a skeleton

Symbiotic bacteria from six Oscarella species (adults and embryos) collected in the Mediterranean Sea (O. lobularis, O. tuberculata, O. imperialis, O. microlobata, O. viridis) and the Sea of Japan (O. malakhovi) were investigated by scanning electron microscopy and transmission electron microscopy. In most cases, symbionts are rather numerous. Each sponge species has a definite set of bacterial morphological types. All bacteria are extracellular. Symbionts occupy the mesohyl of adult sponges or intercellular space in embryos and are often in contact with mesohylar filaments or cells. Bacteria of some morphotypes have characteristic blebs. Most symbionts are gram-negative, and two types of bacteria have traits of Archaea and one type of bacteria is similar to Planctomycetes. Data on morphology of bacterial symbionts can be a good additional character for identification of Oscarella species, which have no skeleton.     

New epizooic symbioses between sponges of the genera Plakortis and Xestospongia in cryptic habitats of the Caribbean

Three new cases of sponge symbiosis between species of Plakortis and Xestospongia were found in reef caves and mesophotic reef habitats of the Caribbean. Plakortis sp. 1 from the Bahamas associates exclusively with Xestospongia deweerdtae which was originally described living freely on the deep fore-reef and caves of Jamaica. In addition, we found Plakortis sp. 2 from Puerto Rico which associates with both X. deweerdtae and a different Xestospongia sp. Sponge specimens were identified using cytochrome oxidase subunit 1, 28S rRNA and 18S rRNA gene sequence fragments, spicule analysis, and histological sections with SEM. Unlike previous sponge pairs, Xestospongia spp. not only grew as a thin veneer of tissue over the Plakortis host sponge but through the mesohyl, forming inner channels (0.1–1 cm) that may provide a benefit by facilitating more efficient water transport through the dense Plakortis tissue. Symbioses with both Plakortis spp. were documented from an early recruit stage through adulthood. Spicule measurements conducted on symbiotic versus free-living X. deweerdtae revealed significantly smaller spicule sizes for symbiotic individuals, suggesting a cost in terms of silicon availability, or a benefit in terms of a lower investment in skeleton synthesis for support. This study reveals new specialized symbiotic associations between distantly related sponge genera that likely represent an alternative strategy of adaptation for life in reef caves and mesophotic reefs.     

Transmission,plasticity and the molecular identification of cyanobacterial symbionts in the Red Sea sponge <Emphasis Type="Italic">Diacarnus erythraenus</Emphasis>

Cyanobacterial symbionts in the sponge Diacarnus erythraenus from the Red Sea were identified in both adult sponges and their larvae by 16S rDNA sequencing. A single cyanobacterial type was found in all samples. This cyanobacterial type is closely related to other sponge cyanobacterial symbionts. The cyanobacterial rDNA, together with the morphological analysis by electron and fluorescence microscopy, provided evidence for vertical transmission of the symbionts in this sponge. In addition, we show phenotypic plasticity of the symbionts inside the sponge, probably as a result of variability in light availability inside the sponge tissue. Finally, the reproduction of Diacarnus erythraenus is also described.Matan Oren, Laura Steindler have contributed equally to the work.     

Evidence of nitrification and denitrification in high and low microbial abundance sponges

Aerobic and anaerobic microbial key processes were quantified and compared to microbial numbers and morphological structure in Mediterranean sponges. Direct counts on histological sections stained with DAPI showed that sponges with high microbial abundances (HMA sponges) have a denser morphological structure with a reduced aquiferous system compared to low microbial abundance (LMA) sponges. In Dysidea avara, the LMA sponge, rates of nitrification and denitrification were higher than in the HMA sponge Chondrosia reniformis, while anaerobic ammonium oxidation and sulfate reduction were below detection in both species. This study shows that LMA sponges may host physiologically similar microbes with comparable or even higher metabolic rates than HMA sponges, and that anaerobic processes such as denitrification can be found both in HMA and LMA sponges. A higher concentration of microorganisms in the mesohyl of HMA compared to LMA sponges may indicate a stronger retention of and, hence, a possible benefit from associated microbes.     

Biochemical genetic divergence and systematics in sponges of the genera Corticium and Oscarella (Demospongiae: Homoscleromorpha) in the Mediterranean Sea

The sponge sub-class Homoscleromorpha is generally considered to include just two families, the Oscarellidae (without spicules) and the Plakinidae (with simple spicules). In May 1990, an unusual sponge was found deep inside a submarine cave in the western Mediterranean Sea. On the basis of externally visible characters this sponge appeared indistinguishable from the common plakinid species Corticium candelabrum Schmidt, 1862. However, on closer examination in the laboratory the new sponge proved to be devoid of spicules. Therefore, despite great morphological similarities to C. candelabrum, the new sponge should, by taxonomic convention, have been placed in the Oscarellidae. On the basis of other criteria, the similarities to C. candelabrum were great and the new sponge was at first considered to be conspecific. Thus, the taxonomic position of the new sponge and its relationship to C. candelabrum are highly confusing. It could be an aspiculate morph of C. candelabrum, or a new and undescribed related species or, lacking spicules, it could justifiably be placed in a different family (Oscarellidae). The relationship of the new sponge to C. candelabrum and also to two species of Oscarella (Oscarellidae) was assessed by the use of enzyme electrophoresis to estimate genetic divergence between species. It was found that the new sponge was reproductively isolated from sympatric C. candelabrum, with 6 of 16 loci proving diagnostic. Thus it is clear that the new sponge belongs to a different biological species. Surprisingly it was also found that, although this new species was fairly closely related to C. candelabrum (level of genetic identity, I0.47), the two Oscarella species were similarly closely related to C. candelabrum (I0.31 to 0.41) and rather less closely to the new species (I0.17 to 0.28). Indeed from genetic identity estimates, O. tuberculata is more closely related to C. candelabrum than it is to O. lobularis. It is concluded that all homoscleromorph sponges should be placed in the single family Plakinidae.     

Bacterial communities of the marine sponges Hymeniacidon heliophila and Polymastia janeirensis and their environment in Rio de Janeiro, Brazil

In this study we performed a survey of the bacterial communities associated with the Western Atlantic demosponges Hymeniacidon heliophila and Polymastia janeirensis, based on 16S rRNA sequencing and transmission electron microscopy (TEM). We compared diversity and composition of the sponge-associated bacteria to those of environmental bacteria, represented by free-living bacterioplankton and by bacteria attached to organic particulate matter in superficial sediments. Partial bacterial 16S rRNA sequences from seawater, sediment, and sponges were retrieved by PCR, cloning, and sequencing. Sequences were subjected to rarefaction analyses, phylogenetic tree construction, and LIBSHUFF quantitative statistics to verify coverage and similarity between libraries. Community structure of the free-living bacterioplankton was phylogenetically different from that of the sponge-associated bacterial assemblages. On the other hand, some sediment-attached bacteria were also found in the sponge bacterial community, indicating that sponges may incorporate bacteria together with sediment particles. Rare and few prokaryotic morphotypes were found in TEM analyses of sponge mesohyl matrix of both species. Molecular data indicate that bacterial richness and diversity decreases from bacterioplankton, to particulate organic sediment, and to H. heliophila and P. janeirensis. Sponges from Rio de Janeiro harbor a pool of novel and exclusive sponge-associated bacterial taxa. Sponge-associated bacterial communities are composed of both taxons shared by many sponge groups and by species-specific bacteria.     

Microbial associations in sponges. I. Ecology,physiology and microbial populations of coral reef sponges

Illumination, current strength and physical turbulence influence the distribution of 4 tropical sponges. Three sponges with cyanobacteria in exposed tissues grow only in poen shallow habitats: Pericharax heteroraphis in moderate-current, lowturbulence regions on the reef slope; Jaspis stellifera in low-current, moderate-turbulence regions of the outer reef flat; and Neofibularia irata in moderate-current, high-turbulence areas below the reef crest. Ircinia wistarii contains no cyanobacteria and occurs in deeper, strong-current, high-turbulence regions. N. irata agressively overgrows neighbouring corals and its growth form is influenced by the current strength. The sponges efficiently filter bacteria from the water. The efficiency is related to the aquiferous structure, particularly the size of choanocyte chambers, and is unrelated to the existing bacterial populations in sponge tissue. The numbers of bacteria associated with the sponges are proportional to the sponge mesohyl density, with the dense sponges J. stellifera and I. wistarii containing many bacteria whereas P. heteroraphis is not dense and has few bacteria.     

Predictable annual mass release of gametes by the coral reef sponge Neofibularia nolitangere (Porifera: Demospongiae)

Mass release of gametes of the sponge Neofibularia nolitangere (Duch. & Mich., 1864) occurs simultaneosly along the leeward coast of Curaçao over a period of three subsequent days, without any apparent spatial pattern in the sperad of activity. A population of 99 individual sponges was monitored from August through November 1984 for development and subsequent release of gametes. Release started every day of 3 3-d period (12 to 14 October) at about 1400 hrs and lasted until just after sunset (1830 hrs). Ninety percent of the population showed reproductive activity. Exactly one lunar month later (11 to 12 November), a second release of gametes occurred. In the following year the same sequence of events was observed for the original population (2 to 4 October and 1 to 2 November, 1985). In all instances the first gamete release began on the third day after the full moon. These and earlier observations on this phenomenon show a strong correlation between moon phase and the time of gamete release. Histological and field observations show separate development and release of gametes (sex-ratio males: females 1.5:1) with external fertilization. No sex-reversal occurred in the population during release activity the following year, demonstrating a clear gonochoristic and oviparous type of reproduction for N. nolitangere. First development of oocytes and spermatocytes has been observed at 36 and 7 d, respectively, before the date of first release. In both males and females, a major part of the mesohyl of the total sponge was involved in gamete development. Spermatozoa are released through the osculum as a dense white smoke; eggs consisting of oocyte-nurse cell globules, reinforced with spicules, are separately released en masse with the outgoing water stream of the female sponge. Shortly after release the eggs become sticky and show a negative buoyancy. The reproductive strategy of N. nolitangere appears to be directed at maximizing the number of surviving recruits by maintaining a high reproductive output at a short specific time interval.     

Ecological roles of natural products from the marine sponge Geodia corticostylifera

In the Brazilian coast, high numbers of the small brittle star Ophiactis savignyi usually live associated with the sponge Geodia corticostylifera (Demospongiae, Geodidae), but not with other sympatric sponge species. In order to check whether this association was related only with the physical shelter provided by the sponge body or was chemically mediated, the crude organic extract of G. corticostylifera was added to sponge mimics made of phytagel and spongin skeleton. Control and treated mimics were simultaneously offered to previously sponge-associated O. savignyi in both static seawater and flow-through laboratory experiments. Ophiuroids were allowed to move towards the preferred mimic. The defensive properties of the sponge extract against fish predation and fouling were also evaluated. Chemotaxis assays showed that symbiotic ophiuroids were able to chemically recognize its host sponge, moving significantly more towards mimics containing G. corticostylifera extract. Chemical deterrence assays showed that the natural concentration of the extract of this sponge was also able to inhibit generalist fish predation on field experiments and the attachment of the common mussel Perna perna in laboratory assays. These results indicate that the crude extract of G. corticostylifera plays multiple functions in the marine environment, presumably being responsible for a closer association of this sponge with O. savignyi, providing protection for this ophiuroid and inhibition of epibionts on itself.     

Cortical and endosomal structure of the marine sponge Stelletta grubii

The fine structure of the marine astrophorid sponge Stelletta grubii is described for the first time. The following new data are presented: spongin is present, choanocyte chambers are diplodal, intercellular symbiotic bacteria are numerous and unequally distributed in the cortex and endosome, and collagenous fibril bundles are associated with lophocyte activity and are not elastic fibers. The cortex contains numerous fibril bundles, fewer symbionts, very few cells, and transitional zones with higher archeocyte density near the surface and endosome. Limited phagocytosis of the bacterial symbionts is observed. This species appears to be dioecious and oviparous. These observations suggest that the enigmatic species Chondrosia reniformis is closely related to S. grubii and that it should be placed within or near the astrophorids. The rhizoids of the red alga Phyllophora palmettoides penetrate the sponge tissue without eliciting the development of a structurally specialized contact zone in the sponge matrix or of a limiting epithelium.     

Spatial distribution of bacteria associated with the marine sponge Tethya californiana

Microbial diversity and spatial distribution of the diversity within tissue of the marine sponge Tethya californiana was analyzed based on 16S rRNA gene sequences. One candidate division and nine bacterial phyla were detected, including members of all five subdivisions of Proteobacteria. Moreover, chloroplast-derived Stramenopiles- and Rhodophyta-affiliated 16S rRNA gene sequences were found and Stramenopiles represented the most abundant clones (30%) in the clone library. On the phylum-level, the microbial fingerprint of T. californiana showed a similar pattern as its Mediterranean relative T. aurantium. An interesting difference was that Cyanobacteria that were abundantly present in T. aurantium were not found in T. californiana, but that the latter sponges harbored phototrophic Stramenopiles instead. Surprisingly, the phototrophic microorganisms were evenly distributed over the inner and outer parts of the sponge tissue, which implies that they also reside in regions without direct light exposure. The other phyla were also present in both the outer cortex and the mesohyl of the sponges. These results were confirmed by analysis on the operational taxonomic unit level. This leads to the conclusion that from a qualitative point of view, spatial distribution of microorganisms in T. californiana tissue is quite homogeneous. Thirty-two percent of the operational taxonomic units shared less than 95% similarity with any other known sequence. This indicates that marine sponges are a rich source of previously undetected microbial life.     

Microbial associations in sponges. III. Ultrastructure of the in situ associations in coral reef sponges

Symbiotic cyanobacteria are associated with marine sponges in three ways: the majority are free-living in the mesohyl; large aggregates occur in cyanocytes (specialized, vacuolated archeocytes); and few are present in digestive vacuoles. The cyanobacteria in Jaspis stellifera and Neofibularia irata are morphologically similar to those described in Mediterranean sponges, whereas those in Pericharax heteroraphis are different. The freeliving bacterial populations are morphologically similar, although the number of bacteria varies between the species. The fourth sponge Ircinia wistarii contains a mixed bacterial population unlike those in the other sponges. Sponge digestion of microbial associates is rare and not considered to contribute significant nutrients.