Localization of granulatimide alkaloids in the tissues of the ascidian <Emphasis Type="Italic">Didemnum</Emphasis><Emphasis Type="Italic">granulatum</Emphasis>

Ascidians are a notable source of nitrogen-bearing secondary metabolites with a wide range of biological activities. Although many biologically active compounds have been isolated from ascidians, it is often unclear whether the animal or associated microbial symbionts such as bacteria or fungi are the true biosynthetic source of the metabolites. We have addressed the question of the biosynthetic source of the alkaloids granulatimide and isogranulatimides by localizing these compounds within the ascidian. In this work, we demonstrate that granulatimide is stored in Didemnum granulatum tunic bladder cells. Analysis by confocal fluorescence microscopy at the granulatimide emission range indicated the presence of fluorescent cells as highly vacuolated cells found dispersed in the ascidian tunic. Since this is the most exposed ascidian tissue, it is possible that these alkaloids may have a protective role, either as sunscreens and/or as feeding deterrents.     

Microbial associations in sponges. II. Numerical analysis of sponge and water bacterial populations

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.     

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.     

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.     

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.     

Host specificity of the symbiotic cyanobacterium<Emphasis Type="Italic"> Oscillatoria spongeliae</Emphasis> in marine sponges,<Emphasis Type="Italic"> Dysidea</Emphasis> spp.

Marine sponges can host a variety of cyanobacterial and bacterial symbionts, but it is often unclear whether these symbionts are generalists that occur in many host species or specialists that occur only in certain species or populations of sponges. The filamentous cyanobacterium Oscillatoria spongeliae is found in the sponges Dysidea n. sp. aff. herbacea 1A and 1B, and similar cyanobacteria are found in D. n. sp. aff. granulosa. We amplified and sequenced sponge nuclear ribosomal DNA (rDNA) and cyanobacterial 16S rDNA from specimens of these three sponges. We then used these sequences to construct phylogenies for host sponges and their symbiotic cyanobacteria. Each of these three sponge species hosts a distinct cyanobacterial clade, suggesting a high degree of host specificity and potential coevolution between symbiotic cyanobacteria and their host sponges.     

The cellular localization of dercitamide in the Palauan sponge Oceanapia sagittaria

The isolation of a class of bioactive aromatic alkaloid compounds known as pyridoacridines from members of four phyla (Porifera, Chordata-Subphylum Tunicata, Mollusca and Cnidaria) caused some to speculate that they were produced by associated symbionts. We tested this hypothesis by localizing specific metabolites in cells using a combination of visualization methods, including laser-scanning confocal, epifluorescence, and transmission electron microscopy, as well as cell-separation techniques, and chemical analysis. This study demonstrates that large quantities of the pyridoacridine alkaloid dercitamide (=Kuanoniamine C) are localized exclusively in bacteria-free sponge cells in the marine sponge Oceanapia sagittaria (Sollas), and are probably not produced by intracellular symbiotic organisms. We hypothesize that it is unlikely that the pyridoacridines are produced by extracellular bacteria and then transferred to specific sponge cells. The localization of dercitamide in significant concentrations in specific cells throughout the sponge suggests important biological and ecological functions, such as chemical defense against predators and possibly microbial pathogens. If pyridoacridines are produced by the host organism in other phyla, this may be a case of convergent evolution of an efficient and useful biosynthetic pathway.     

Rapid tissue reduction and recovery in the sponge <Emphasis Type="Italic">Aplysinella</Emphasis> sp.

We observed a pronounced, yet reversible tissue reduction in the tropical sponge Aplysinella sp. under non-experimental conditions in its natural habitat, after transfer into seawater tanks, as well as after transplantation from deep to shallow water in the field. Tissue reduction resulted in the formation of small “reduction bodies” tightly attached to the sponge skeleton. Although volume loss and gain were substantial, both tissue reduction and regeneration were often remarkably rapid, occurring within few hours. Microscopic analysis of the reduction bodies revealed morphological similarities to previously described sponge primmorphs, with densely packed archaeocytes and spherulous cells enclosed by a thin layer of epithelial-like cells. Denaturing gradient gel electrophoresis (DGGE) revealed pronounced changes in the sponge-associated microbial community upon tissue reduction during laboratory and field experiments and following changes in ambient conditions after transplantation in the field. Generally, the microbial community associated with this sponge proved less stable, less abundant, and less diverse than those of other, previously investigated Verongid sponges. However, one single phylotype was consistently present in DGGE profiles of Aplysinella sp. This phylotype clustered with γ-proteobacterial sequences found previously in other sponge species of different taxonomic affiliations and geographic provenances, as well as in sponge larvae. No apparent changes in the total secondary metabolite content (per dry weight) occurred in Aplysinella sp. upon tissue reduction; however, comparative analysis of intact and reduced tissue suggested changes in the concentrations of two minor compounds. Besides being ecologically interesting, the tissue reduction phenomenon in Aplysinella sp. provides an experimentally manipulable system for studies on sponge/microbe symbioses. Moreover, it may prove useful as a model system to investigate molecular mechanisms of basic Metazoan traits in vivo, complementing the in vitro sponge primmorph system currently used in this context.     

Identification of the antifungal peptide-containing symbiont of the marine sponge Theonella swinhoei as a novel δ-proteobacterium, “Candidatus Entotheonella palauensis”

 The Palauan sponge Theonella swinhoei (class Demospongiae, order Lithistida, family Theonellidae) harbors filamentous bacterial symbionts that contain theopalauamide, an antifungal, bicyclic glycopeptide. In this study, the filamentous symbionts were shown to be novel bacteria belonging to the δ-subdivision of proteobacteria. The 16S rRNA gene sequence was determined using a combination of denaturing gradient-gel electrophoresis (DGGE) and specific polymerase chain-reaction (PCR) primers, and its source was confirmed by in situ hybridization. In a series of culture experiments, the filamentous bacteria were propagated in a mixed culture on agar plates. Related 16S rRNA gene sequences were isolated from related sponges with slightly different chemistry. The taxonomic status “Candidatus Entotheonella palauensis” is proposed for the theopalauamide-containing filamentous bacteria from T. swinhoei. Received: 12 June 1999 / Accepted: 22 January 2000     

Cytoenzymatic investigation of intracellular digestion in the symbiont-bearing hydrothermal bivalve <Emphasis Type="Italic">Bathymodiolus azoricus</Emphasis>

Invertebrates harbouring endosymbiotic chemoautotrophic bacteria are widely distributed in a variety of reducing marine habitats, including deep-sea hydrothermal vents. Bathymodiolids are dominants of the biomass at geochemically distinct vent sites of the Mid Atlantic Ridge (MAR) and thus are good candidates to study biological processes in response to site-specific conditions. To satisfy their nutritional requirements, these organisms depend to varying extent on two types of chemoautotrophic symbionts and on filterfeeding. The quantitative relationships of the nutritional modes are poorly understood. Using enzyme cytochemistry, electron microscopy and X-ray microanalysis, the structural and functional aspects of the cellular equipment necessary for lysosomal digestion was studied. We provide evidence for the following: (1) the basis of intracellular digestion of symbionts in Bathymodiolus azoricus from two geochemically distinct vent sites was not mainly in the large lysosomal bodies as previously thought (based on the membranous content resembling bacteria); (2) senescent bacteria are autolysed, possibly by bacterial acid phosphatase, that is more likely a cell cycling of the symbionts rather than an active lysosomal digestion by the host; (3) the consistent absence of hydrolases may indicate the improper use of the name “lysosome” for large vesicles at the base of the gill bacteriocytes (4) nutrient transfer in B. azoricus, therefore, may more likely be accomplished through leaking of metabolites from the symbiont to the host, not excluding lysosomal resorption of dead bacteria as an auxiliary strategy for organic molecule transfer; (5) evidence is provided for microvillar transfer of substances from the seawater that may indicate filter-feeding, in non-symbiotic ciliated gill cells of mussels from Lucky Strike; (6) two types of lysosomal vesicles can be distinguished in digestive cells based on their enzymatic content and their elemental composition.     

Metabolic integration between symbiotic cyanobacteria and sponges: a possible mechanism

Metabolic relationships between symbiotic cyanobacteria and host sponge have been investigated in the marine species Chondrilla nucula and Petrosia ficiformis (collected in the Ligurian Sea in 1992). DNA, RNA, total protein, cytosolic protein, total sugar, cytosolic sugar, total lipid, nonprotein sulfhydryl groups, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were assayed in cortex-free sponge tissue, where cyanobacteria are all but absent. For both species, biochemical parameters were determined in specimens living in illuminated habitats and in dark caves, where sponges are virtually aposymbiotic for cyanobacteria. As C. nucula is unable to colonize dark sites, specimens of this species were artificially transferred to a cave and maintained in dark conditions for 6 mo. Results showed that in the absence of light (i.e., in the absence of cyanobacteria) C. nucula undergo metabolic collapse and thiol depletion. In contrast, P. ficiformis activates heterotrophic metabolism and mechanisms which balance the loss of cell reducing power. This suggests that cyanobacteria effectively participate in controlling the redox potential of the host cells by the transfer of reducing equivalents. Only P. ficiformis is capable of counteracting, by means of heterotrophic metabolism, the loss of the contribution from symbionts which is caused by dark conditions. This explains the differences in the ecological requirements of the two species. Because cyanobacterial symbionts release fixed carbon in the form of glycerol and other small organic phosphate (Wilkinson 1979), a model based on the glycerol 3-phosphate shuttle (typically occurring in chloroplasts and mitochondria) is suggested. The mechanism proposed appears to be an ancient biochemical adaptation which arose among ancestral symbiotic systems, and further developed in the relationships between endosymbiotic organelles and cytoplasm.     

Susceptibility to oxidative stress of the Mediterranean demosponge Petrosia ficiformis : role of endosymbionts and solar irradiance

 The effects of elevated pO₂ and irradiance as inducers of prooxidant conditions have been investigated in the Mediterranean demosponge Petrosia ficiformis (Poiret, 1789). This species lives symbiotically with the autotrophic cyanobacterium Aphanocapsa feldmanni, the abundance of which is controlled by the intensity of light irradiance. In the presence of symbionts, tissues of P. ficiformis were characterized by a general enhancement of antioxidant defenses as compared to aposymbiotic specimens. The main differences included higher activities of several antioxidant enzymes and a greater capability to neutralize various forms of oxyradicals, as indicated by the total oxyradical scavenging capacity (TOSC) assay. Elevated pO₂, more than light, appeared to be the primary factor inducing prooxidant pressure in the Mediterranean sponge; in fact, irrespective of the solar irradiance experienced by the sponge, symbiotic specimens showed comparable activities of antioxidant enzymes and a similar scavenging capacity towards various reactive oxygen species. However, the potential toxicity of photodynamic production of reactive oxygen species was demonstrated in organisms from more irradiated sites, as the levels of antioxidant defenses were lowered in the outer layer of the sponge. The role of enhanced antioxidant defenses in protecting symbiotic specimens, also from oxyradical-mediated toxicity of light exposure, was supported by translocation experiments; aposymbiotic sponges did not survive when moved to conditions of elevated solar irradiance, while no effects were observed in symbiotic specimens if translocated and/or deprived of symbionts. Received: 23 November 1999 / Accepted: 13 June 2000     

Sponge-specific bacterial symbionts in the Caribbean sponge, Chondrilla nucula (Demospongiae, Chondrosida)

Marine sponges harbor dense and highly diverse bacterial communities, and some percentage of the microflora appears to be specialized for the sponge habitat. Bacterial diversity was examined in Chondrilla nucula Schmidt to test the hypothesis that some subset of sponge symbiont communities is highly similar regardless of the species of host or habitat requirements of the host. C. nucula was collected from a mangrove channel on Lower Matcumbe Key in the Florida Keys (25°53′N; 80°42′W) in August 1999. Domain-specific universal bacterial primers were used to amplify the 16S rDNA gene from genomic DNA that had been extracted from sponges and the surrounding water. An RFLP technique was used to assess diversity of sponge-associated and environmental bacterial communities. The clone library from C. nucula contained 21 operational taxonomic units (OTUs). None of the 53 OTUs from adjacent water samples were found in the C. nucula library indicating that a distinct community was present in the sponge. Sequence analysis indicated that C. nucula harbors a microbial community as diverse as the microbes from other sponges in different habitats around the world. Phylogenetic analysis placed several C. nucula clones in clades dominated by bacteria that appear to be sponge specialists (e.g., Acidobacteria, Bacteroidetes, and Cyanobacteria). Proportional representation of major bacterial taxonomic groups represented in symbiont communities was compared as a function of geographic location of sponge hosts. This study supports the hypothesis that sponges from different oceans existing in dissimilar habitats harbor closely related bacteria that are distinct from other bacterial lineages and appear specialized for residing within sponges.     

Sulfide assimilation by ectosymbionts of the sessile ciliate, <Emphasis Type="Italic">Zoothamnium niveum</Emphasis>

We investigated the constraints on sulfide uptake by bacterial ectosymbionts on the marine peritrich ciliate Zoothamnium niveum by a combination of experimental and numerical methods. Protists with symbionts were collected on large blocks of mangrove-peat. The blocks were placed in a flow cell with flow adjusted to in situ velocity. The water motion around the colonies was then characterized by particle tracking velocimetry. This shows that the feather-shaped colony of Z. niveum generates a unidirectional flow of seawater through the colony with no recirculation. The source of the feeding current was the free-flowing water although the size of the colonies suggests that they live partly submerged in the diffusive boundary layer. We showed that the filtered volume allows Z. niveum to assimilate sufficient sulfide to sustain the symbiosis at a few micromoles per liter in ambient concentration. Numerical modeling shows that sulfide oxidizing bacteria on the surfaces of Z. niveum can sustain 100-times higher sulfide uptake than bacteria on flat surfaces, such as microbial mats. The study demonstrates that the filter feeding zooids of Z. niveum are preadapted to be prime habitats for sulfide oxidizing bacteria due to Z. niveum’s habitat preference and due to the feeding current. Z. niveum is capable of exploiting low concentrations of sulfide in near norm-oxic seawater. This links its otherwise dissimilar habitats and makes it functionally similar to invertebrates with thiotrophic symbionts in filtering organs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Bacterial colonization of the phyllosphere of nineteen plant species and antimicrobial activity of their leaf secondary metabolites against leaf associated bacteria

Summary. The scope of this work was to examine whether leaf constitutive secondary metabolites play a role in determining bacterial colonization of the phyllosphere. To this aim, we surveyed nineteen native or cultivated plant species that share a common bacterial pool in a North Mediterranean area, and estimated the size of total and ice nucleation active (INA) bacterial populations on their leaves. Large differences in the colonization of their phyllosphere were found; the population size of epiphytic bacteria ranged from 7.5 × 10² to 1 × 10⁶ CFU/g fresh weight, in eucalypt and celery, respectively. Species native in Mediterranean-type climate areas, particularly those belonging to the group of aromatic plants, are characterized by scarce presence of INA bacteria. The antibacterial activity of essential oils, surface phenolics and leaf tissue extracts was also estimated against the INA strains P. syringae and E. herbicola, isolated from two of these plant species. E. herbicola proved more sensitive than P. syringae. Of the species examined, oregano [Origanum vulgare L. subsp. hirtum (Link.) Ietswaart], an aromatic plant, had the highest antimicrobial activity, whereas six species showed no activity at all. Further experiments were performed with oregano and bean (Phaseolus vulgaris L.) that represent two extremes in their secondary metabolite content. Both plants were inoculated with P. syringae. By the end of incubation, the bacterial population on bean plants was about 100 times higher than that on oregano leaves. Scanning electron micrographs showed that bacterial growth on oregano leaves was confined to sites away from glandular hairs. Results from the bacterial colonization survey together with those from the toxicity tests showed that all species rich in antibacterial secondary metabolites harbored low leaf bacterial populations. These results provide substantial evidence that leaf secondary metabolites function as constitutive defense chemicals against microbial invasions. However, the fact that species with non- or moderately active leaf secondary metabolites are not always highly colonized suggests mediation of other unknown factors, the contribution of which requires further investigation.     

Culturable actinobacteria isolated from marine sponge <Emphasis Type="Italic">Iotrochota</Emphasis> sp.

The study describes the diversity of actinobacteria isolated from the marine sponge Iotrochota sp. collected in the South China Sea. Species and natural product diversity of isolates were analyzed, including screening for genes encoding polyketide synthases (PKS) and nonribosomal peptide synthetase (NRPS), and 16S rRNA gene restriction fragment length polymorphism (RFLP). PKS and NRPS sequences were detected in more than half of the isolates and the different “PKS-I–PKS-II–NRPS” combinations in different isolates belonging to the same species indicated a potential natural product diversity and divergent genetic evolution. The phylogenetic analysis based on 16S rRNA gene sequencing showed that the isolates belonged to genera Streptomyces, Cellulosimicrobium, and Nocardiopsis. The majority of the strains tested belonged to the genus Streptomyces and one of them may be a new species. To our knowledge, this is the first report of a bacterium classified as Cellulosimicrobium sp. isolated from a marine sponge. Key Laboratory of Marine Bio-recourses Sustainable Utilization (LMB-CAS), Guangdong Key Laboratory of Marine Materia Medica (LMMM-GD), South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, People’s Republic of China.     

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 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     

Rapid tissue reduction and recovery in the sponge Aplysinella sp.

We observed a pronounced, yet reversible tissue reduction in the tropical sponge Aplysinella sp. under non-experimental conditions in its natural habitat, after transfer into seawater tanks, as well as after transplantation from deep to shallow water in the field. Tissue reduction resulted in the formation of small “reduction bodies” tightly attached to the sponge skeleton. Although volume loss and gain were substantial, both tissue reduction and regeneration were often remarkably rapid, occurring within few hours. Microscopic analysis of the reduction bodies revealed morphological similarities to previously described sponge primmorphs, with densely packed archaeocytes and spherulous cells enclosed by a thin layer of epithelial-like cells. Denaturing gradient gel electrophoresis (DGGE) revealed pronounced changes in the sponge-associated microbial community upon tissue reduction during laboratory and field experiments and following changes in ambient conditions after transplantation in the field. Generally, the microbial community associated with this sponge proved less stable, less abundant, and less diverse than those of other, previously investigated Verongid sponges. However, one single phylotype was consistently present in DGGE profiles of Aplysinella sp. This phylotype clustered with γ-proteobacterial sequences found previously in other sponge species of different taxonomic affiliations and geographic provenances, as well as in sponge larvae. No apparent changes in the total secondary metabolite content (per dry weight) occurred in Aplysinella sp. upon tissue reduction; however, comparative analysis of intact and reduced tissue suggested changes in the concentrations of two minor compounds. Besides being ecologically interesting, the tissue reduction phenomenon in Aplysinella sp. provides an experimentally manipulable system for studies on sponge/microbe symbioses. Moreover, it may prove useful as a model system to investigate molecular mechanisms of basic Metazoan traits in vivo, complementing the in vitro sponge primmorph system currently used in this context.     

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.