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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.

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.

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.

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.

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.

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

The microbial community cultured from the marine sponge Rhopaloeides odorabile Thompson et al. is dominated by a single bacterium, designated strain NW001. Sequence analysis of 1212 bp of the16S rRNA gene of strain NW001 indicates that it is a member of the α-subgroup of the class Proteobacteria. The association between this bacterium and its host sponge was observed in healthy R. odorabile collected from six different reefs in the Great Barrier Reef representing a geographic distance of 460 km, and in four collections in different seasons in 1997–1998 at Davies Reef (18°49.6′S; 147°34.49′E). The proportion of colonies of strain NW001 in samples from R. odorabile, expressed as a percentage of the total heterotrophic bacterial colony count, showed no significant spatial (range: 81–98%) or temporal differences (range: 81–99%), although colony counts of strain NW001 varied by up to two orders of magnitude between reef sites and sampling periods. The location of strain NW001 within the sponge mesohyl was visualized by in situ hybridization, using fluorescently labeled probes based on the 16S rRNA gene sequence of this strain. Cells of strain NW001 surround the choanocyte chambers, suggesting that these bacteria may play a role in nutrient uptake by the sponge. The absence of strain NW001 from corresponding seawater samples indicates that it has a specific, intimate relationship with R. odorabile and is not being utilized as a food source. A unique cyanobacterium related to the genera Leptolyngbya and Plectonema was also isolated from R. odorabile and characterized by 16S rRNA gene sequencing.

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.

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 alfa) 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 the sponge samples displayed toxicity, when analyzed in vitro using leukemia cells.

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.

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.

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.

Three taxonomically distant sponges Pericharax heteroraphis, Jaspis stellifera and Neofibularia irata contain phenotypically similar bacterial symbionts which differ from bacteria in the ambient water. These symbionts are predominant in the sponges and were detected after computer analysis of 526 heterotrophic bacterial strains tested for 76 characters. These facultative anaerobic symbionts metabolize a wide range of compounds and may be important in removing waste products while the sponges are not circulating water. The bacteria produce sticky-mucoid colonies and thus would contribute to sponge structural rigidity. The fourth sponge Ircinia wistarii contains a mixed aerobic population similar to that in the ambient water. The majority of the bacteria are located around the inhalant canals, facilitating the uptake of dissolved organic matter and oxygen from the incoming water.

Fine structural analysis of living tissue of the sclerosponges Ceratoporella nicholsoni (Hickson) and Stromatospongia norae Hartman, collected near Discovery Bay, Jamaica, between 1984 and 1986, was carried out using transmission and scanning electron microscopy (TEM and SEM). The thick dermal membrane of these sponges is covered by exopinacocytes having a “T” shape in sections perpendicular to the surface. A dense, complex glycocalyx is produced at the surface of these cells. Choanocyte chambers are diplodal and unusually small. The inhalant and exhalant canals of both species are characterized by the presence of valvules, made by transverse lamellipodial processes of the endopinacocytes lining them. An abundant and diversified bacterial community occupies almost 20% of the mesohyl. A single layer of active basopinacocytes lines the mesohyl at the interface between the living tissue and the aragonitic skeleton. Basopinacocytes are presumed to be precursors of the irregular fibrillar organic matrix found in the aragonitic skeleton. Sclerocytes and spongocytes are abundant in the vicinity of the siliceous spicules. Typical lophocytes releasing smooth collagen fibrils are common in the dermal membrane as well as in the choanosome where they can be grouped in bundles. Uniquely, C. nicholsoni elaborates rough intercellular fibrils characterized by periodically spaced thickenings. The endolithic algae Ostreobium sp. is present in the most apical zones of the aragonitic skeleton, but does not seem to interfere with its development. The striking micromorphological resemblances between both species are discussed and compared to demosponges.

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.