Apoptosis in marine sponges: a biomarker for environmental stress (cadmium and bacteria)

The marine demosponge Suberites domuncula is abundantly present on muddy sand bottoms, both in the open sea and in harbors. In the present study it is shown that exposure of S. domuncula to cadmium (CdCl₂) in concentrations ranging from 0.01 to 5.0 g ml⁻¹ for up to 5 d results in apoptotic fragmentation of DNA. Kinetics experiments revealed that after 24 h a significant increase of DNA fragmentation already occurred. Besides cadmium a second stimulus was identified to also cause apoptosis in this species, namely exposure to heat-treated Escherichia coli. In order to support the finding that both cadmium and E. coli induce apoptosis in the sponge, expression of the apoptotic gene MA-3 was studied. The cDNA, SDMA3, was isolated and found to be 2247 nucleotides long. The deduced amino acid sequence (M_r 50 765) shares high similarity with the corresponding mouse molecule. Like the mouse gene, the sponge MA-3 gene undergoes increased expression in response to apoptotic stimuli. While the specimens remained alive after treatment with cadmium, the sponges treated with E. coli died after approximately 12 d. The E. coli-treated animals started to form gemmules 10 to 12 d after addition of the bacteria. Hence, the process of apoptosis in sponges is triggered by two different pathways, one which is initiated by exogenous factors, e.g. heavy metals, and a second one, caused by endogenous factors, which leads to gemmule formation and a shift of the presumably immortal cells to mortal cells. The latter assumption is supported by the finding that during the process of bacteria-induced apoptosis, which results in the death of the specimens, the activity of the telomerase drops. It is concluded that the cells which appear to be immortal and telomerase-positive undergo apoptosis during the process of gemmule formation. In consequence cells not involved in the production of gemmules become mortal. Based on these data, it is proposed that apoptosis is a suitable biomarker in the bioindicator organism S. domuncula to monitor unfavorable environmental conditions, at least in this animal phylum. Received: 10 November 1997 / Accepted: 6 March 1998     

N-acyl homoserine lactone production by bacteria within the sponge Suberites domuncula (Olivi, 1792) (Porifera, Demospongiae)

Many bacteria live in close association with sponges. Within these consortia, molecules of communication such as quorum-sensing and hormone-like molecules may occur in order to regulate the partnership. Of particular interest, bacterial N-acyl-l-homoserine lactones (AHLs) were screened in supernatants from Suberites domuncula-associated bacteria using an E. coli bioluminescent reporter system. These sponge-associated bacteria were beforehand isolated on several media supplemented or not with a sponge extract to attempt to isolate sponge-specific bacteria. Out of 81 AHL-producing bacteria, three strains requiring sponge extract to grow were selected for AHL characterization. The in vitro produced AHLs, that is, in bacterial culture supernatants, were identified as N-(3-butanoyl)-l-homoserine lactone and N-(3-oxododecanoyl)-l-homoserine lactone and quantified using LC–ESI–MS/MS. The in vivo production of AHLs by sponge-associated bacteria has also been demonstrated in a healthy host for the first time: N-(3-oxododecanoyl)-l-homoserine lactone, N-(3-hexanoyl)-l-homoserine lactone, and N-(3-heptanoyl)-l-homoserine lactone. This AHL production in sponges may suggest a potential role of these molecules between sponge-associated bacteria and/or between sponge-associated bacteria and the sponge.     

Cloning and expression of a tauropine dehydrogenase from the marine sponge <Emphasis Type="Italic">Suberites </Emphasis><Emphasis Type="Italic">domuncula</Emphasis>

The cDNA sequence coding for tauropine dehydrogenase (TaDH) [belonging to the family of opine dehydrogenases] has been determined. Using the demosponge Suberites domuncula, we describe for the first time the tauropine dehydrogenase gene (of length 2,992 kb) from a eukaryote, consisting of two introns flanked by three exons. Moreover, two allelic variants have been identified, which are present in the different specimens either in a homozygotic or in a heterozygotic way; the data suggest an intermediary type of heritance. Phylogenetic analyses indicate that S. domuncula TaDH is only distantly related to the opine dehydrogenases from marine invertebrates; rather it comprises high sequence similarity to bacterial ornithine cyclodeaminases (OCD). In addition, expression studies revealed that the steady-state level of TaDH dropped drastically in animals, which had been exposed to elevated aeration. Antibodies raised against the recombinant sponge TaDH were used to demonstrate that S. domuncula expresses high levels of this enzyme in almost all cells. If tissue samples were kept under additional aeration no immuno-signals could be identified. A strong accumulation of the enzyme was seen around the bacteria, existing in bacteriocytes, indicating that under aerobic conditions the bacteria might produce taurine. These data suggest involvement of the sponge TaDH in the final step of the glycolytic pathway, more specifically, in regeneration of NAD(+) under anaerobic conditions. Furthermore, potential mutual influences between bacteria and sponge are discussed, claiming a horizontal gene transfer of the gene from a bacterium to the sponge. The following sequences from Suberites domuncula have been deposited (EMBL/GenBank): the cDNA encoding the tauropine [(carboxyethyl)-taurine/derived from sulfhydryl-amino acids] dehydrogenase (SD_TaDH) under AM712888; the corresponding gene under AM712889; and the fragments of the sponge-associated bacteria comprising the opine dehydrogenase domain: SUBDO_BAC1 (AM712890), SUBDO_BAC2 (AM712891), SUBDO_BAC3 (AM712892), SUBDO_BAC5 (AM712894), SUBDO_BAC6 (AM712895), SUBDO_BAC7 (AM712896), SUBDO_BAC8 (AM712897) and SUBDO_BAC9 (AM712898).     

Okadaic acid: a potential defense molecule for the sponge <Emphasis Type="Italic">Suberites domuncula</Emphasis>

Sponges are sessile filter feeders that are exposed to toxic compounds/xenobiotics and a huge number of diverse pro- and eukaryotic organisms occurring in the aqueous milieu. To cope with these threats, sponges have developed a variety of chemical and immunological defense systems. Among them are potent secondary metabolites or toxins. The demosponge Suberites domuncula contains the bioactive compound okadaic acid; concentrations of ᅢ ng/g (ᅬ nM) of wet tissue have been quantified by LC/MS. In the present study, we sought to determine whether okadaic acid is beneficial for this sponge. Antibodies were raised against okadaic acid and used to localize this compound in situ. It could be demonstrated that the polyclonal antibodies/antiserum stained the sponge cells (endopinacocytes) which form the epithelium of the lacunae and the water channels. They also reacted with bacteria present in sponge tissue. Okadaic acid was found to augment the immune response of S. domuncula against the bacterial endotoxin lipopolysaccharide (LPS). If primmorphs, a special form of 3-D aggregates, are incubated with LPS at a concentration of 3 µg/ml, a reduction in the incorporation rate of [³H] phenylalanine is measured. In the presence of okadaic acid, this effect is measured already at a concentration of 1 µg/ml, suggesting that okadaic acid sensitizes this (immune) response. Okadaic acid is a known inhibitor of protein phosphatases which, as shown here, cause an increased phosphorylation of p38, a central kinase of the MAP kinase pathway; this may explain the sensitive response of the sponge cells to LPS. At higher concentrations, okadaic acid induces apoptosis, as was demonstrated here by a differential expression of the Bcl-2 homolog and the caspase gene. It is concluded that okadaic acid has two functions in the sponge; first, at low concentrations (<100 nM) a stimulation of the defense system against bacteria, and second, at concentrations above 500 nM, a differential effect on the pro-/anti-apoptotic genes.     

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.     

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.     

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.     

Molecular/chemical ecology in sponges: evidence for an adaptive antibacterial response in<Emphasis Type="Italic"> Suberites domuncula</Emphasis>

Sponges (Porifera) represent the evolutionary oldest metazoan phylum still extant today. They have developed a complex Bauplan, based on the existence of structural and regulatory molecules; many of these have been cloned and analyzed in the past years. The demosponge Suberites domuncula has been used as a suitable model to demonstrate that these animals not only possess an adaptive immune response on the level of cytokines, but also, as pointed out here, on the level of synthesis of bioactive alkyl-lipid derivatives. From specimens of S. domuncula the two lyso-PAF (platelet-activating factor) compounds, 1-O-hexadecyl-sn-glycero-3-phosphocholine and 1-O-octadecyl-sn-glycero-3-phosphocholine, have been identified and characterized. These two lyso-PAFs showed pronounced anti-bacterial activity. In the central part of this paper it is shown that the level of synthesis of the lyso-PAF congeners increased in response to the model compound, the endotoxin lipopolysaccharide (LPS). Treatment of the tissue with LPS for 72 h substantially increased the synthesis. In order to prove that the lyso-PAFs are really synthesized by the sponge, the key enzyme of the alkyl-dihydroxyacetonephosphate pathway, i.e. alkyl-dihydroxyacetonephosphate synthase (ADS), was cloned from S. domuncula. This sponge enzyme comprises the characteristic features of metazoan ADS enzymes; it is increasingly expressed in the tissue and in the in vitro cell culture system after exposure to LPS. These data are taken as a strong indication that bioactive, low-molecular weight, non-proteinaceous lipid derivatives function in an adaptive manner in response to the endotoxin.Communicated by R. Cattaneo-Vietti, Genova     

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.     

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.     

Trends in space occupation by the encrusting sponge Crambe crambe: variation in shape as a function of size and environment

The relationship between sponge size, habitat and shape was studied in the encrusting sponge Crambe crambe (Schmidt, 1862), which is distributed widely throughout the shallow Mediterranean littoral. Examination of sponge patches in shaded and well-illuminated habitats showed that the degree of peripheral irregularity of the edges of a patch is directly related to patch size. This relationship is valid only for sponges of >100 mm² in area. Photophilic and sciaphilous sponges display different growth forms. The pattern of growth is interpreted in terms of competition for space. The directional growth of sciaphilous sponges may be due to the presence of dominant neighbours that are good space competitors, and the irregular growth of photophilic sponges to the absence of such neighbours.     

Evidence for morphology-induced sediment settlement prevention on the tubular sponge<Emphasis Type="Italic"> Haliclona urceolus</Emphasis>

Several mechanisms are known to assist the survival of sponges in highly sedimented environments. This study considers the potential of sponge morphology and the positioning of exhalant water jets (through the osculum) in the adaptation of Haliclona urceolus to highly sedimented habitats. This sponge is cylindrical with an apical osculum, which is common in sedimented subtidal habitats at Lough Hyne Marine Nature Reserve, Cork, Ireland. Fifteen sponges were collected, preserved (killed with the structure and morphology maintained) and then replaced in a high sediment environment next to a living specimen (at 24 m). After 5 days, the sediment settled on both living and preserved sponges was collected and dried. No sediment was collected from living sponges, while preserved specimens had considerable amounts of settled sediment on their surfaces. The amount of sediment collected on these preserved specimens was significantly linearly correlated with sponge dry weight, maximum diameter and oscula width (R²>0.70, P<0.001, df=14). Observations of flow direction (using coloured dye) through H. urceolus showed that water is drawn into the sponge on its underside and exits via a large vertically pointing osculum. Sponge morphologies (shape) have often been considered as a means of passive adaptation to a number of different environmental parameters with oscula position enabling entrained flow through the sponge in high flow conditions. However, this study shows how the combination of sponge morphology (tubular shape) and positioning of the osculum may enable H. urceolus to survive in highly sedimented environments. Similar mechanisms may also aid the survival of some deep-water sponge species with similar morphologies.Communicated by J.P. Thorpe, Port Erin     

Redwood of the reef: growth and age of the giant barrel sponge Xestospongia muta in the Florida Keys

The growth of animals in most taxa has long been well described, but the phylum Porifera has remained a notable exception. The giant barrel sponge Xestospongia muta dominates Caribbean coral reef communities, where it is an important spatial competitor, increases habitat complexity, and filters seawater. It has been called the ‘redwood of the reef’ because of its size (often >1 m height and diameter) and presumed long life, but very little is known about its demography. Since 1997, we have established and monitored 12 permanent 16 m diameter circular transects on the reef slope off Key Largo, Florida, to study this important species. Over a 4.5-year interval, we measured the volume of 104 tagged sponges using digital images to determine growth rates of X. muta. Five models were fit to the cubed root of initial and final volume estimates to determine which best described growth. Additional measurements of 33 sponges were taken over 6-month intervals to examine the relationship between the spongocoel, or inner-osculum space, and sponge size, and to examine short-term growth dynamics. Sponge volumes ranged from 24.05 to 80,281.67 cm³. Growth was variable, and specific growth rates decreased with increasing sponge size. The mean specific growth rate was 0.52 ± 0.65 year⁻¹, but sponges grew as fast or slow as 404 or 2% year⁻¹. Negative growth rates occurred over short temporal scales and growth varied seasonally, significantly faster during the summer. No differences in specific growth rate were found between transects at three different depths (15, 20, 30 m) or at two different reef sites. Spongocoel volume was positively allometric with increasing sponge size and scaling between the vertical and horizontal dimensions of the sponge indicated that morphology changes from a frustum of a cone to cylindrical as volume increases. Growth of X. muta was best described by the general von Bertalanffy and Tanaka growth curves. The largest sponge within our transects (1.23 × 0.98 m height × diameter) was estimated to be 127 years old. Although age extrapolations for very large sponges are subject to more error, the largest sponges on Caribbean reefs may be in excess of 2,300 years, placing X. muta among the longest-lived animals on earth.     

Effect of iron and dissolved silica on primmorphs of Petrosia ficiformis (Poiret, 1789)

The suitability of the primmorphs system as a good model for biotechnological applications led researchers on primmorphs to look for a medium to stimulate cell proliferation and therefore growth of aggregates. Recent efforts have focused on the use of Fe⁺³ and Si that, supplemented to cell culture medium, were found to be promising for growth and morphogenesis of the sponge Suberites domuncula. In this work, we analysed the effect of iron and dissolved silica on primmorphs of Petrosia ficiformis, by testing them at different concentrations in successive experiments. The purpose of these experiments was to test their effect on primmorphs and individuate their optimal concentration for this species. Our results suggest a negative effect of iron on primmorphs of P. ficiformis and a positive effect of silica on primmorphs size and spiculogenesis at a concentration of 120 μM.     

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.     

Parrotfish predation on cryptic sponges of Caribbean coral reefs

Some sponge species that live in crevices in the reef frame appear to be restricted to their cryptic habitat by predation. When cryptic sponges were excavated, on Guigalotupo reef, San Blas, Panama, exposing them to potential predators, they were eaten by fishes that are generally considered to be herbivores, primarily parrotfishes of the genus Sparisoma: S.aurofrenatum (Cuvier & Valenciennes), S.viride (Bonnaterre), and S.chrysopterum (Bloch & Schneider). Of the 9150 bites observed to be taken by these species during paired (i.e., with sponges versus without sponges) trials conducted in defined feeding areas during 1986, 1987, and 1988, 72% (i.e. 6581 bites) were on cryptic sponges, even though these were only offered during half of the total observation time and never constituted more than 7% of the cover of the feeding observation areas. Individual parrotfish returned over and over to take bites of the exposed cryptic sponges until they were entirely consumed. They vigorously chased each other away from the sponges, but exhibited no such defense of their usual algal foods. A total of 18 sponge species were tested. Of the cryptic and semi-cryptic sponge species tested, only one of six was rejected by the parrotfish. Two of these six sponge species were consistently consumed entirely, and two were consumed entirely whenever their surfaces were sliced off with a razor blade, demonstrating that these sponges concentrate defenses against predators in their surfaces. One semi-cryptic species and one semi-exposed species were fed upon, but not entirely consumed. By contrast, 11 of 12 of the exposed and semi-exposed species were rejected. Cryptic sponges grew out of their cavities in the reef only when protected by seasonally thick mats of macroalgae or by cages that excluded fish. Received: 10 January 1997 / Accepted: 4 February 1997     

Induction and reversion of multixenobiotic resistance in the marine snail Monodonta turbinata

Many marine organisms inherently express the presence and function of a new defence mechanism, termed multixenobiotic resistance mechanism (MXRM) because of its similarity to multidrug resistance (MDR) found in tumor cell lines resistant to chemotherapeutic drugs. However, previously no information was available on a possible induction of the activity of MXRM in organisms living at polluted sites. The purpose of the present study was to demonstrate the inducibility of this defence mechanism in the marine snail Monodonta turbinata, an organism known to inhabit a wide range of environmental conditions existing along a pollution gradient. Specimens of M. turbinata used in the present study were collected from March 1993 through March 1994 near Rovinj, Croatia. The accumulation of generally labelled ³H-vincristine (³H-VCR) in the gills of snails living at an unpolluted site was 67% higher and very sensitive to verapamil, an inhibitor of P-glycoprotein activity in comparison to snails living at a polluted site. The accumulation of vincristine (VCR) in snails from a polluted site was reduced and insensitive to verapamil due to the induced state of P-glycoprotein, as was demonstrated immunochemically by the enhanced concentration of a 140 kDa protein cross-reacting with the polyclonal antibodies raised against mammalian P-glycoprotein. MXRM could be induced in snails from the unpolluted site to the level found in snails living at a polluted site either by transplantation for 3 d to a polluted site, or by exposure for 3 d to sea water polluted with Diesel-2 oil (hydrocarbon concentration equivalent to 50 ppb of Kuwait oil). Discovery of the inducibility of this new defence mechanism in aquatic organisms may have important implications in ecotoxicology, as was demonstrated by a 104% enhanced accumulation of VCR in the presence of the MXRM-inhibitor, the so-called chemosensitizer. Besides, a striking difference in the levels of MXRM-elements (the sensitivity of the accumulation rate of xenobiotics to verapamil, immunochemical expression of P-glycoprotein, and the sensitivity of the binding of xenobiotics on membrane vesicles to verapamil) found between specimens living at the polluted and unpolluted sites offers a new molecular biomarker for exposure to pollutants.     

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.     

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.     

Linking abundance and diversity of sponge-associated microbial communities to metabolic differences in host sponges

Many sponge species contain large and diverse communities of microorganisms. Some of these microbes are suggested to be in a mutualistic interaction with their host sponges, but there is little evidence to support these hypotheses. Stable nitrogen isotope ratios of sponges in the Key Largo, Florida (USA) area grouped sponges into species with relatively low δ¹⁵N ratios and species with relatively high δ¹⁵N ratios. Using samples collected in June 2002 from Three Sisters Reef and Conch Reef in the Key Largo, Florida area, transmission electron microscopy (TEM) and denaturing gradient gel electrophoresis were performed on tissues of the sponges Ircinia felix and Aplysina cauliformis, which are in the low δ¹⁵N group, and on tissue of the sponge Niphates erecta, which is in the high δ¹⁵N group. Results showed that I. felix and A. cauliformis have large and diverse microbial communities, while N. erecta has a low biomass of one bacterial strain. As the low δ¹⁵N ratios indicated a microbial input of nitrogen, these results suggested that I. felix and A. cauliformis were receiving nitrogen from their associated microbial community, while N. erecta was obtaining nitrogen solely from external sources. Sequence analysis of the microbial communities showed a diversity of metabolic capabilities among the microbes of the low δ¹⁵N group, which are lacking in the high δ¹⁵N group, further supporting metabolic differences between the two groups. This research provides support for hypotheses of mutualisms between sponges and their associated microbial communities.