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.     

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.     

Interaction between herbivore defense and microbial signaling: bacterial quorum-sensing compounds weaken JA-mediated herbivore resistance in Nicotiana attenuata

Bacteria play important roles in plant–herbivore interactions and communicate with each other with chemical signals, often N-acylhomoserine lactones (AHL). Plant responses to these signals may influence resistance to microbial attack, but the effects of these signals on herbivore defense are unstudied. To determine whether AHL influence jasmonate (JA)-mediated herbivore resistance in Nicotiana attenuata, we treated wild-type (WT) and JA-deficient genotypes (antisense expression of NaLOX3) with N-hexanoyl-dl-homoserine lactone (C6-HSL) and measured the performance of Manduca sexta larvae. Larval mass gain on C6-HSL-treated WT plants was equivalent to that on non-treated NaLOX3-silenced plants, but significantly 4.1-fold larger than on untreated WT plants. Mass gain was unaffected by C6-HSL treatment of NaLOX3-silenced plants. Microarray analysis of the plants elicited with C6-HSL and JA inducing fatty acid–amino acid conjugates revealed a down-regulation of a proteinase inhibitor in the C6-HSL-treated WT plants. The results therefore suggest that the increased performance of M. sexta was due to direct or indirect effect of C6-HSL on JA-mediated defenses.     

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.     

Sediment-induced reduction in the pumping rate of the tropical sponge Verongia lacunosa

The pumping rate of Verongia lacunosa (Lamarck), a tropical marine sponge, varied between 1 and 6 l h^-1 in clear seawater for sponges with a volume of about 500 ml. Sponges were exposed to seawater containing suspensions of clay maintained at a constant level for 4 h; concentrations of 11 mg l^-1 or greater significantly reduced the pumping rate, while concentrations of 3 mg l^-1 did not. Other sponges were exposed to suspensions of clay for 4 days; a concentration of 95 mg l^-1 caused a continuing decline in the pumping rate. These sponges were more sensitive to sediment than some other suspension-feeding organisms. Such sensitivity may limit the distribution of V. lacunosa and other sponge species.Please address requests for reprints to A.O. Flechsig at the address shown above.     

Oxygen dynamics and transport in the Mediterranean sponge Aplysina aerophoba

The Mediterranean sponge Aplysina aerophoba kept in aquaria or cultivation tanks can stop pumping for several hours or even days. To investigate changes in the chemical microenvironments, we measured oxygen profiles over the surface and into the tissue of pumping and non-pumping A. aerophoba specimens with Clark-type oxygen microelectrodes (tip diameters 18–30 μm). Total oxygen consumption rates of whole sponges were measured in closed chambers. These rates were used to back-calculate the oxygen distribution in a finite-element model. Combining direct measurements with calculations of diffusive flux and modeling revealed that the tissue of non-pumping sponges turns anoxic within 15 min, with the exception of a 1 mm surface layer where oxygen intrudes due to molecular diffusion over the sponge surface. Molecular diffusion is the only transport mechanism for oxygen into non-pumping sponges, which allows total oxygen consumption rates of 6–12 μmol cm⁻³ sponge day⁻¹. Sponges of different sizes had similar diffusional uptake rates, which is explained by their similar surface/volume ratios. In pumping sponges, oxygen consumption rates were between 22 and 37 μmol cm⁻³ sponge day⁻¹, and the entire tissue was oxygenated. Combining different approaches of direct oxygen measurement in living sponges with a dynamic model, we can show that tissue anoxia is a direct function of the pumping behavior. The sponge-microbe system of A. aerophoba thus has the possibility to switch actively between aerobic and anaerobic metabolism by stopping the water flow for more than 15 min. These periods of anoxia will greatly influence physiological variety and activity of the sponge microbes. Detailed knowledge about the varying chemical microenvironments in sponges will help to develop protocols to cultivate sponge-associated microbial lineages and improve our understanding of the sponge-microbe-system.     

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.     

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.     

Host specificity in sponge-encrusting zoanthidea (Anthozoa: Zoantharia) of Barbados,West Indies

Six species of common Caribbean Zoanthidea, Parazoanthus swiftii, P. parasiticus, P. catenularis, P. puertoricense, Epizoanthus cutressi, and Epizoanthus sp., are virtually restricted to living on surfaces of reef-dwelling sponges. Quantitative surveys on Barbados reefs indicate that substrate specificity is relatively high among these zoanthids with three restricted to a single primary host sponge species and three restricted to three closely related sponges. One species, P. swiftii, exhibits a broad range of acceptable secondary substrates, due to its unique ability to execute migrational spread in the adult polyp stage. Variations in substrate specificity have been noted between island populations within the extensive Caribbean range and appear to be due to different species compositions of local sponge communities and slight differences in zoanthid larval settling specificities.     

Investigation of the relationship between invertebrate predation and biochemical composition,energy content,spicule armament and toxicity of benthic sponges at McMurdo Sound,Antarctica

The biochemical and energetic composition, spicule content, and toxicity of benthic sponges was investigated in McMurdo Sound, Antarctica from October through December 1984. The predominant organic constituent of sponges was soluble and insoluble protein. Levels of total protein ranged from 17.0 to 55.9% dr. wt. Levels of lipid and carbohydrate were low, ranging from 2.1 to 9.6 and 0.6 to 3.5% dr. wt, respectively. Levels of ash were high and variable (32 t0 79% dr. wt), reflecting species-specific differences in spicule contents. Calculated energy contents of sponges were low, with a mean of 9.8±3.5 kJ g^-1 dr. wt; ranging from 5.1 kJ g^-1 dr. wt in Sphaerotylus antarcticus to 17.4 kJ g^-1 dr. wt in Dendrilla membranosa. Insoluble protein accounted for the greatest contribution to the energetic composition of the sponges, while lipid and carbohydrate combined contributed to less than 25% of the overall energy. Normalized spicule volumes of sponges ranged from 0.15 to 0.38 cm³ g^-1 dr. wt. Ichthyotoxicity assays indicated that 9 (56%) of 16 antarctic sponge species were toxic. The most highly toxic species were Mycale acerata and Leucetta leptorhapsis. The high incidence of toxicity in antarctic sponges indicates that the current hypothesis suggesting a simple inverse relationship between toxicity and latitude in marine sponges is invalid. There was little correspondence between the energetic composition or spicule contents of the sponges and feeding patterns (electivity indices) of sponge-eating predators. Although the asteroid Perknaster fuscus antarcticus specializes on the highly toxic, fast-growing M. acerata, most antarctic sponge-eating predators appear to be generalists which feed on the more abundant, non- to mildly-toxic, sponge species. This feeding strategy is based on exploitation of low energy, sedentary prey, which require a minimal energy output to harvest.     

Unravelling host and symbiont phylogenies of halichondrid sponges (Demospongiae, Porifera) using a mitochondrial marker

We present the first comparative phylogenetic analysis of a selected set of marine sponges and their bacterial associates. The Halichondrida form an important order in demosponge systematics and are of a particular interest due to the production of secondary metabolites. We sequenced a fragment of the cytochrome oxidase subunit 1 (CO1) gene of the sponges and their bacterial associates, compared the reconstructed phylogenies and found evidence for radiation in coevolution. The tree of six host-species associations showed four supported cospeciation events between the sponges and the bacteria. In addition, we present the first gene tree of sponges based on a mitochondrial marker. The tree shows major congruences with previous morphological studies and suggests the applicability of a mitochondrial marker in sponge molecular systematics.     

Biodiversity and rockfish recruitment in sponge gardens and bioherms of southern British Columbia,Canada

In the Strait of Georgia and Howe Sound, British Columbia, colonies of individual cloud sponges, growing on rock (known as sponge gardens) receive resource subsidies from the high biodiversity of epifauna on adjacent rock habitats. Bioherms are reefs of glass sponges living on layers of dead sponges. In the same area as the sponge gardens, newly discovered bioherms in Howe Sound, BC (49.34.67 N, 123.16.26 W) at depths of 28- to 35-m are constructed exclusively by Aphrocallistes vastus, the cloud sponge. The sponge gardens had much higher taxon richness than the bioherms. The sponge garden had 106 species from 10 phyla, whereas the bioherm had only 15 species from 5 phyla. For recruiting juvenile rockfish (quillback, Sebastes maliger), the food subsidy of sponge gardens appears to be missing on bioherms of cloud sponge, where biodiversity is relatively low. While adult and subadult rockfishes (S. maliger, S. ruberrimus, S. proriger, and S. elongatus) were present on bioherms, no evidence for nursery recruitment of inshore rockfishes to bioherms was observed, whereas the sponge gardens supported high densities of newly recruited S. maliger, perhaps owing to the combination of both refuge and feeding opportunities. These results indicate that sponge gardens form a habitat for early stages of inshore S. maliger, whereas A. vastus bioherms are associated only with older juvenile and adult rockfishes.     

Synthesis of some novel (2-oxo-3-(arylimino) indolin-1-yl)-N-aryl acetamides and evaluation as antimicrobial agents

A series of sixteen 2-oxo-3-(arylimino) indolin-1-yl)-N-aryl acetamide derivatives were synthesized, characterized by physical and spectral data (IR, ¹H nuclear magnetic resonance (NMR), and mass spectrometry), and evaluated for their antibacterial and antifungal activities against various pathogenic microorganisms. Some of the synthesized compounds showed promising antibacterial and antifungal activities, the best being 2-(3-(4-chlorophenylimino)-2-oxoindolin-1-yl)-N-(3-methoxyphenyl) acetamide.     

Responses of bacterial strains isolated from drinking water environments to N-acyl-L-homoserine lactones and their analogs during biofilm formation

Often as a result ofbiofilm formation, drinking water distribution systems （DWDS） are regularly faced with the problem of microbial contamination. Quorum sensing （QS） systems play a marked role in the regulation of microbial biofilm formation; thus, inhibition of QS systems may provide a promising approach to biofilm formation control in DWDS. In the present study, 22 bacterial strains were isolated from drinking water-related environments. The following properties of the strains were investigated： bacterial biofilm formation capacity, QS signal molecule N-acyl-L-homoserine lactones （AHLs） production ability, and responses to AHLs and AHL analogs, 3-chloro-4-（dichloromethyl）-5-hydroxy-2（5H）- furanone （MX） and 2（5H）-furanone. Four AHLs were added to developed biofilms at dosages ranging from 0.1 nmol.L J to 100nmol.L1. As a result, the biofilm growth of more than 1/4 of the isolates, which included AHL producers and non-producers, were significantly promoted. Further, the biofilm biomasses were closely associated with respective AHLs concentrations. These results provided evidence to support the idea that AHLs play a definitive role in biofilm formation in many of the studied bacteria. Meanwhile, two AHLs analogs demon- strated unexpectedly minimal negative effects on biofilm formation. This suggested that, in order to find an applicable QS inhibition approach for biofilm control in DWDS, the testing and analysis of more analogs is needed.     

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.     

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.     

Dispersal and use of sponges and ascidians as camouflage by Cryptodromia hilgendorfi (Brachyura: Dromiacea)

From May 1977 to February 1979, the use of sponges and ascidians by Cryptodromia hilgendorfi was studied in Moreton Bay, Queensland, Australia. The aim was to investigate patterns of seasonal use, cap making behaviour, cap turnover, the effect of intraspecific interactions on cap life and the effect of movement of crabs between hosts on background matching. C. hilgendorfi uses 12 (of 16 available) species of sponge and 3 species of ascidians to construct caps, which are carried by the crabs using their last two pairs of legs. Cap area increases non-linearly with crab size, and caps are normally two to three times as large as the crabs. Cap making behaviour is described. It occurs during intermoult periods, with females making most of their caps at night. Caps decrease in size with time, but conceal the crabs which commonly occupy exposed sites on sponges. Cap life is independent of crab size, differs between different cap species and is influenced by the presence of other crabs who can dislodge caps through aggressive behaviour. Caps are made from the sponge Suberites carnosus more often than from other available sponges. S. carnosus caps also decay less rapidly than caps made from other sponges. Use of sponge and ascidian species varies seasonally, with Halichondria sp. and S. carnosus being used in all months. C. hilgendorfi exhibits a preference for certain sponges. The majority of crabs carried caps which matched their host sponge or ascidian, but mis-matches varied seasonally with a winter peak following the breeding season. Young C. hilgendorfi settle only on S. carnosus sponges and disperse from this host to other species in the environment. Males and females differ in their rate of discovery of new hosts. Males, despite their greater mobility, find new hosts slower than females. It is hypothesized that males occupy “home ranges” which females do not. Crabs frequently move between sponges, mostly at night. Sponges and ascidian species grow in intimate association with each other, and sponge crabs act a selective asexual propagation mechanism. Depending upon the nature of the interactions between sponge and ascidian species (co-operative or competitive) and whether competitive hierarchies or networks are involved, the sponge crabs may have either stabilizing or destabilizing effects on the sponge community.     

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     

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     

Habitat use by sponge-dwelling brittlestars

Cryptic organisms often associate with sessile invertebrates for refuge in space-limited environments. To examine interspecific habitat associations on coral reefs, tube- and vase-shaped sponges were surveyed for associated brittlestars at six sites on the coral reefs off Key Largo, Florida. Of 179 sponges encountered, Callyspongia vaginalis was the most abundant (43.0%), followed by Niphates digitalis (39.7%), and Callyspongia plicifera (4.5%). Three of eight sponge species surveyed did not differ from C. vaginalis in two physical refuge characteristics: oscular diameter and inner tube surface area. Brittlestars (416 total), all of the genus Ophiothrix, were only found in C. vaginalis, N. digitalis, and C. plicifera. The most abundant brittlestar, O. lineata (326), occurred on C. vaginalis (99.0%) and N. digitalis (1.0%), while O. suensonii (67) occurred on C. vaginalis (79.1%), N. digitalis (19.4%), and C. plicifera (1.5%). There was no pattern of co-occurrence of O. lineata and O. suensonii on C. vaginalis. The abundance of O. lineata increased with surface area of C. vaginalis. Differential habitat use was observed in O. lineata, with small individuals (<5 mm disk diameter) located inside and on the surface of sponge tubes and large individuals (5 mm) solely inside tubes. The number of large O. lineata in C. vaginalis never exceeded the number of tubes per sponge, and tagged O. lineata remained in the same sponge for at least 3 weeks. In density manipulations, no pattern of intraspecific competition among large O. lineata was observed; however, there was evidence for interaction between size-classes. Brittlestars selected live sponge habitat over a non-living refuge, suggesting a mechanism for sponge habitat recognition. Sponge-dwelling brittle stars prefer some tube- and vase-shaped sponge species despite similar oscular diameters and surface areas. Surprisingly, these preferred sponge species are known from previous studies to be chemically undefended against generalist fish predators; therefore, brittlestars that inhabit these sponges do not gain an associational chemical defense. Sponge habitat use by O. lineata may be governed by intraspecific interactions to maintain habitat and access to food. While past studies have suggested that O. lineata is an obligate sponge commensal, the present study suggests that O. lineata has a species-specific association with the tube-sponge C. vaginalis.Communicated by O. Kinne, Oldendorf/Luhe