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The present research aimed to investigate the concentrations and patterns of six potentially toxic elements (PTEs) in three common sponge species collected along the Moroccan Mediterranean coast, as well as their levels in ambient seawater and sediments. Distinct inter-species variability in PTEs bioaccumulation was observed among the three species, suggesting that sponges have distinct selectivity for assimilating PTEs from the surrounding environment. C. crambe had a higher enrichment capacity for Cu, As, Cr and Ni, while P. ficiformis and C. reniformis exhibited the highest concentration of Cd and Pb, respectively. Interestingly, a similar spatial distribution patterns of PTEs was observed in the three media, with high values occurring in Tangier and Al-Hoceima locations. Overall, our results confirm that sponges reliably reflect the bioavailability of PTEs in their immediate environment, especially C. crambe, whose PTE tissue contents were highly and positively correlated with the contents of all PTEs in the sediments.

Two co-occurring, enigmatic aspects of bivalve reproduction were investigated in the common cockle Cerastoderma edule: the oocyte coat and oocyte atresia. Qualitative histology and transmission electron microscopy (TEM) of cockles collected on the French Atlantic coast revealed not only the fine structure of the oocyte coat, but also confirmed that it is secreted by the oocyte itself and composed of acid mucopolysaccharides (AMPS), known to be viscous and adhesive. Quantitative histology showed that at the peak of oogenesis, oocyte coats occupy the largest fraction (approx. 40%) of the gonad acinal volume, representing both a significant sacrifice of female gamete capacity, and a non-gamete energetic investment. Potential benefits of the coat include protection from mechanical abrasion, predation, and opportunistic microbes. Atresia (oocyte degeneration) was a known second source of reduced fecundity, with a minimum impact of approximately 50% of the total oocyte volume. It is suggested that this high proportion of atresic oocytes is related to the previously-documented genetic inviability of early post-fertilization life stages. The qualitative histological and TEM observations revealed atresic debris adhering to the exterior surface of the oocyte coats. Such an arrangement would isolate adjacent oocyte coats, enabling the oocytes to be spawned individually, rather than as an egg mass, and therefore to undergo planktonic development and dispersion. Oocyte atresia and the oocyte coat of Cerastoderma edule therefore appear to be linked in the first indication of an adaptive function in bivalves.

High quality of sperm is essential to a high fertilization rate, especially post- cryopreservation. Assessment of sperm integrity, motility and energy reserves before cryopreservation is necessary for selection of milt with optimal fertilizing potential. We describe the effect of cryopreservation on the quality of black-lip pearl oyster, Pinctada margaritifera var. cumingii sperm. Evaluated quality indices of fresh and frozen/thawed P. margaritifera spermatozoa, included morphology, ultrastructure and motility characteristics relative to the energy content (ATP) and its capacity to be sustained by mitochondrial respiration. Morphology and ultrastructure were quantitatively evaluated using images obtained by optical microscopy assisted by the Image J software and TEM, respectively. Sperm motility was assessed using Image J software combined with a computer assisted sperm analysis plugin adapted for assessing P. margaritifera spermatozoa. Other sperm quality parameters evaluated included O2 consumption, ATP content, and creatine kinase activity. Frozen/thawed spermatozoa exhibited damage to the head but retained a compact spherical shape. Sperm motility indicators showed a significant decrease in quality resulting from the freeze/thaw process. The percent of motile cells was 54% compared to 84% in fresh sperm, O2 consumption was 4.8 compared to 44 nanomol min−1, ATP content was 0.72 nmol/109 spermatozoa in the activating medium compared to 4.54 nmol/109 spermatozoa, and creatine kinase activity was 9.06 × 10−5 IU mg−1 protein compared to 12.5 × 10−5 IU mg−1 protein. The cryopreservation protocol allowed obtaining an acceptable motility rate after thawing, confirming the predictive value of sperm motility measurements before cryopreservation in terms of their ability to withstand freezing process.

Nowadays, there is a huge lack of information available about the distribution and diversity of marine sponge in the Mediterranean coast of Morocco. In the present study, we identified the most abundant and common marine sponges of the western Mediterranean coast of Morocco and establish an inventory of the marine sponges described for this region. Sponge samples were collected from intertidal regions and shallow coastal waters from June 2017 to May 2018 during spring low tides. A total of 18 sponge species were identified which belonging to 13 different genera, 12 families and 9 orders. Maximum species diversity of sponges is recorded at Belyounech coast, followed by an equal number of species at Jebha and M’diaq coasts (8 species). Five species of sponges were recorded at Hoceima and only one at Merkala. The present study has indicated that the stations Belyounech, M’diaq and Jebha are rich in diversity and abundance of sponges, compared to Hoceima and Merkala. The demosponge Petrosia ficiformis was the common species, present in all sampling locations.

WOS:000492408400001; Codium tomentosum, as all organisms, hosts transiently and permanently numerous microorganisms. These holobionts can undergo environmental pressures influencing both partners creating modifications/imbalances within the associations, which may directly influence their physiological status by selecting tolerant bacteria. Furthermore, the capability of remediation of the associated bacterioflora, in particular of metallic trace elements, may provide the host with survival potential in polluted environments. In this context, we incubated C. tomentosum thalli in the presence of copper and studied its influence on the reference bacteriome. Whatever the concentration of copper, no shift was evidenced on the bacteriome at the phylum level. However, a high copper concentration enriched the bacteriome of C. tomentosum in both the genera Clostridium and Pseudolteromonas.

Sponges, which are in close contact with numerous bacteria in prey/predator, symbiotic and pathogenic relationships, must provide an appropriate response in such situations. This starts with a discriminating recognition of the partner either by a physical contact or through secreted molecules or both. We investigated the expression of the Toll-like receptor, Caspase 3/7, Tumor Necrosis Factor receptor-associated factor 6, Bcl-2 homology protein-2 and macrophage expressed genes of axenic sponge cells in the presence of a symbiotic bacterium (Endozoicomonas sp. Hex311), a pathogen bacterium (Pseudoalteromonas sp. 1A1), their exoproducts and lipopolysaccharides. The vast majority of answers are in line with what could be observed with the symbiotic bacterium. The pathogenic bacterium seems to profit from the eukaryotic cell: suppression of the production of the antibacterial compound, inhibition of the apoptosis caspase-dependent pathway, deregulation of bacterial recognition. This work contributes new scientific knowledge in the field of immunology and apoptosis in early branching metazoan harboring within its tissue and cells a large number of symbiotic bacteria.

Sponges together with associated bacteria form complex holobionts governed by various relationships established between the partners. Molecules of communication may participate in the homeostasis of these biological associations. Bacteria produce N-acyl homoserine lactones to communicate. In the present study, we characterized 102 cultivable bacteria isolated from the sponge Suberites domuncula at the genus level and described their production of AHLs during 96 h of culture in a Marine Broth medium. The presence of AHLs was monitored using the Escherichia coli pSB406 reporter strain. Sixty-seven bacteria were AHLs positive. Among them, 46 were related to the Gammaproteobacteria, eight to the Alphaproteobacteria, seven to the Firmicutes and six to the Flavobacteria. The kinetic production of AHLs was determined and compared with a reference bacterium, Pseudomonas aeruginosa PAO1 (pDA224). We established three profiles of AHLs production with no definitive pattern related to an order, a family or a genus of bacteria.

Endozoicomonas sp. HEX311 is a Gram-negative bacterium known to establish a commensal interaction with the marine demosponge Suberites domuncula. The molecular bases of the sponge-microbe interaction events are still poorly defined. Nevertheless, it has been proved that S. domuncula possesses an innate immune system with similarities to the mammalian one and is able to recognize the main component of the Gram-negative bacteria cell wall: the lipopolysaccharide. Whether this recognition occurs in a structure-dependent manner, which is typical for mammalian immune system receptors, is still under investigation. Herein, we report the Endozoicomonas sp. HEX311 lipid A structure obtained by a combination of data attained from chemical, MALDI MS, and MS2 approaches. The lipid A is a complex family of species decorated by pyrophosphate and phosphate units and carrying (R)-3-hydroxydodecanoic acid, (R)-3-hydroxytetradecanonic acid, iso-2-hydroxyundecanoic acid, iso-(R)-3-hydroxyundecanoic acid, and iso-nonanoic acid as acyl chains.

International audience; Marine sponges harbor a rich bacterioflora with which they maintain close relationships. However, the way these animals make the distinction between bacteria which are consumed to meet their metabolic needs and opportunistic and commensal bacteria which are hosted is not elucidated. Among the elements participating in this discrimination, bacterial cell wall components such as lipopolysaccharides (LPS) could play a role. In the present study, we investigated the LPS chemical structure of two bacteria associated with the sponge Suberites domuncula: a commensal Endozoicomonas sp. and an opportunistic Pseudoalteromonas sp. Electrophoretic patterns indicated different LPS structures for these bacteria. The immunomodulatory lipid A was isolated after mild acetic acid hydrolysis. The electrospray ionization ion-trap mass spectra revealed monophosphorylated molecules corresponding to tetra-and pentaacylated structures with common structural features between the two strains. Despite peculiar structural characteristics, none of these two LPS influenced the expression of the macrophage-expressed gene S. domuncula unlike the Escherichia coli ones. Further research will have to include a larger number of genes to understand how this animal can distinguish between LPS with resembling structures and discriminate between bacteria associated with it.

Marine sponges (Porifera) are producers of the largest variety of bioactive compounds among benthic marine organisms. In vitro culture of marine sponge cells has been proposed for the sustainable production of these pharmacologically interesting compounds from marine sponges but with limited success. The development of a suitable growth medium is an essential prerequisite for sponge cells grown in vitro. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was adapted to screen for potential nutritional factors in formulating a growth medium for primary cell culture of Suberites domuncula. In 96-well plates, the effects of nutritional factors including glutamine, pyruvate, iron citrate, silicon, RPMI 1640, and Marine Broth 2216 on the viable cell density were examined in primary cell culture of S. domuncula 36 h after inoculation. Ferric iron (Fe(3+)) and pyruvate were found to significantly improve cell viability in a dose-dependent manner. Silicon and glutamine showed limited improvements at certain concentrations. The supplement of RPMI 1640 and Marine Broth 2216 did not increase cell viability. As a result, several improved media able to maintain higher cell viability in a short-term culture of primary sponge cells could be formulated.

The calyptraeid gastropod Crepidula fornicata is the object of considerable research attention, due to its invasive status in the North-Eastern Atlantic, its introduction to habitats throughout the Northern hemisphere, and its scientific interest as a model organism for the study of developmental and reproductive processes in the Metazoa. Since the knowledge concerning the structural foundations for its reproductive processes is surprisingly weak, we investigated the seminal receptacle, a key structure in the reproductive biology of other metazoans, using histology, scanning electron and transmission electron microscopy. The seminal receptacle consists of 9-11 lobes, each subdivided into small, narrow lobules. The inner epithelium of the lobules appears to be highly dynamic, characterised by the perforation and attachment of received spermatozoa, the progressive degeneration of this epithelium, and the concomitant detachment of the spermatozoa. The allocation of spermatozoa to many different lobules, in different phases, may explain the extended reproductive season of C. fornicata, and thereby contribute to its colonizing and invasive success. The same compartmentalisation, as well as the complete covering of the inner epithelium of the lobules by spermatozoa and the large amount of spermatozoan debris in the lumina, suggest that the C. fornicata seminal receptacle may be a site of sperm competition in this polyandrous species.

Gonad of the Mediterranean pectinid Flexopecten glaber was studied in order to describe the morphology and the ultrastructural characteristics of the mature spermatozoa. The spermatozoon has a 2.5 μm pyriform head and a 40–45 μm flagellum. The four mitochondria of the mid-piece are 0.6 μm in diameter. The nucleus contains dense chromatin fibres and possesses two depressions: a wide (0.19 μm) and deep (0.58 μm) sub-acrosomal one, and the other at the base of the head, less deep (0.36 μm) and less wide (0.12 μm). The objective was to situate this scallop in the phylogenetic diagrams found in the literature concerning the commercial pectinid species.

The main inorganic material forming the skeletal elements in Demospongiae as well as in Hexactinellida, the spicules, is amorphous silica. The spicules occur in the cytoplasm and the extracellular space and also in the nucleus (as silicate crystals) of some sponge cells; the function in the latter compartment is unknown. Recent evidence shows that the formation of spicules is mediated by the enzyme silicatein. The cDNA as well as the gene encoding this enzyme was cloned from Suberites domuncula. The recombinant silicatein catalyzes the syn- thesis of amorphous silicate using tetraethoxysilane as substrate. The enzyme is dependent on ferric iron. Silicatein also has proteolytic (cathepsin-like) activity. Incubation of primmorphs, a special form of 3D-cell aggregates, with silicon resulted in a strong increase of their size from 1-7 mm to 10 mm. The morphogenetic activity of silicate is underscored by the finding that this ion increases gene expression of silicatein and collagen. Based on these findings, it is concluded that both iron and silicate stimulate the activity of silicatein. Furthermore, it is proposed that the growing spicules are surrounded by the scavenger receptor which might be considered as a docking molecule for the collagen matrix into which the spicules are embedded. Microsc. Res. Tech. 62:368 -377, 2003. © 2003 Wiley-Liss, Inc.

The mechanism of nutrient transfer from the digestive system to the gonad acini and developing oocytes was investigated in the gonad-intestinal loop system of the queen scallop Pecten maximus L. Ferritin was injected directly into the purged intestine of specimens from the wild. Subsequently, a histochemical reaction and transmission electron microscopy were used to localize ferritin in various cell types. Ferritin was rapidly absorbed by the intestinal epithelium, and then appeared in hemocytes in the surrounding connective tissue. In the hemocytes, ferritin was stored in variously sized inclusions, as well as in the general cytoplasm. In all sections examined for the 12 experimental individuals, hemocytes were always found in association with connective tissue fibers extending from the base of the intestinal epithelium to gonad acini. After 30-min incubation, ferritin appeared inside the acini of all individuals. Ferritin-bearing cells were rarely found in association with male acini or gametes, nor with mature female gametes, but often with developing female gametes. Not all individuals showed the same temporal dynamics of ferritin transport, suggesting that nutrient transfer to oocytes is either not a continuous process, or that among individuals, transfer is not synchronized on short time scales. This is the first demonstration of a pathway of nutrient transfer from the intestine, and more generally the digestive system, to developing oocytes in the Bivalvia.

In the last three decades, marine invertebrates have been used to monitor environmental health conditions and potential pollution, e.g. in the Mussel Watch Program. The whole animal or specific organs are used to determine contamination levels and disturbances. In the present study, a new in vitro cell culture model was validated for pollution monitoring. A commercial species, the scallop Pecten maximus, was tested for the presence and induction of phase II glutathione-S-transferase (GST) enzymes. These activities were monitored for a year, and the results were found to be consistent with those in the literature. Tributyltin, ethylmethane sulfonate and the water-soluble fraction of crude oil were assayed in, in vitro induction studies. A rapid increase of GST activities was observed within 24 h with all compounds tested, and a time- as well as a dose-response was established. This in vitro cell culture model seems suitable for routine use to predict the effects of pollutants on whole organisms within an ecosystem and in fisheries.

Summary Gametogenesis in the Chinese pen-shell bivalve, Atrina maura (Sowerby, 1835) was provoked under artificial conditions in a running water system maintained under constant environmental conditions in semi-closed tanks. The specimens were fed with a diet commonly used in experimental hatcheries. Gametogenesis was described using stereological techniques (based on the oocyte diameters), classical and semi-thin histology procedures, and electron microscopy. Four reproductive stages were recognized based on oocyte size and cytological characteristics of the gonad: early gametogenesis (4–15 μm), growing (16–30 μm), mature (31–45 μm), and degenerating (46–65 μm). During gametogenesis, the auxiliary cells surrounded the oocyte basal region in the growing stage, afterward disappearing. A process of auto- and heterosynthesis characterize the mature stage, and nuclear deterioration and cytoplasmic membrane disintegration define the degenerating stage. The ultrastructural study of gonads showed that the cytolo...

The feeding biology of Bathypecten vulcani, a peri-hydrothermal vent pectinacean, was investigated using histological and scanning electron microscope studies of the gills and stomach contents of specimens sampled from the 9° and 13°N sites of the East Pacific Rise. Salient characteristics were compared with those of Bathymodiolus thermophilus from the same and similar habitats. The gills of Bathypecten vulcani displayed heterogeneous organic and mineral particles on their frontal surface. The digestive tract possessed well-developed structures (whose anatomical and cytological characteristics indicated full functionality), typically found in bivalves from littoral aerobic environments, as well as in Bathymodiolus thermophilus, which is capable of suspension-feeding: oesophagus, stomach, crystalline style, digestive gland, and intestine. Observations of stomach contents revealed diverse particles from the photic zone, including debris from diatoms and coccolithophorans. In contrast to Bathymodiolus thermophilus, bacteria were rarely observed in the digestive tract, indicating a more complete reliance on surface-originating particles.

Summary Bathypecten vulcani is considered a relict species from the Paleozoic, based on shell characteristics such as the presence of calcite prisms. To date, it is the only pectinid species reported from hydrothermal ecosystems. Histological and ultrastructural studies show that spermatogenesis is identical to that of littoral pectinids. The spermatozoon has a 2.7 μm long pyriform head and a 40 μm flagellum. The four mitochondria of the mid-piece are about 1.2 μm in diameter. The nucleus contains dense chromatin fibres and possesses a wide, shallow (0.1 μm) anterior fossa and a narrow, deeper (0.2 μm) posterior nuclear fossa. Comparison of the ultrastructural characteristics of the spermatozoon of B. vulcani with those of littoral pectinids shows that they can be used as a diagnostic feature of this species. In particular, its acrosome characters will be a useful complement to the shell characters in the study of the phylogenetic position of this species in relation to other pectinids.

International audience; The aim of this paper is to study the impact of the substrate stiffness on the bacterial adhesion. For this purpose, agarose hydrogels are used as substrates with controlled mechanical properties. Indeed, the elastic modulus of these hydrogels, more precisely the shear storage moduli G', evolves with the agarose concentration (in this study from 0.75% to 3%). Other physico-chemical characteristics of the surface, known to be involved in bacterial adhesion, as hydrophobicity, were confirmed to remain constant. Two marine bacterial strains, a positive Gram Bacillus sp. 4J6 and a negative Gram Pseudoalteromonas sp. D41 were selected. Their retention on the substrates was analysed by confocal laser scanning microscopy and by counting of viable adhered bacteria. It was demonstrated that surface elastic modulus correlated with bacterial retention. Bacteria 041 adhered in higher numbers to rigid surfaces. For 416, bacterial adhesion patterns were changed: clusterings were observed on surfaces with lower elastic modulus. Furthermore, a proteomic study, based on the total soluble proteome of D41 strain, highlights an impact of elastic modulus on proteins synthesis. These data demonstrated an adapted response of adhering bacteria on hydrogels of varying mechanical properties

Cytological and biochemical changes in the digestive gland of Pecten maximus throughout a one-year period were investigated in the Bay of Brest (France) relative to lipid storage and glycogen and α-amylase concentrations in tubuloacinar terminations. Seasonal variations in both cell structure and biochemical composition were observed. These changes were independent of the tidal cycle, but correspond to phytoplankton abundance. From November to January, cells of digestive acini were hypertrophied due to the high lipid storage. From February to May, these reserves were markedly reduced, cell size decreased, and the acinar lumen was clearly apparent. From May to September, the RNAs transcribing for α-amylase increased, and a correlation was found between digestion events and lipid storage in acinar cells. The relationships between metabolite transfers from digestive gland to gonad and other tissues are described. Distinct lipid storage sites appear to be associated with maintenance energy and acute demand energy, such as gametogenesis when adductor muscle reserves have been depleted. The digestive gland may thus function as a relay organ during periods of energetic stress, notably during the first gametogenesis in February and March and for shell growth in early spring.

The digestive gland of bivalve molluscs is a model of choice for experiments in ecotoxicology because of its implication in detoxification processes moreover of its classical functions in digestive phenomena. All physiological deteriorations of this organ, related or not to pollution, can lead to animal death. The recent development of a method allowing digestive acini of Pecten maximus to be maintained alive in vitro for 96 h opens up new research prospects in ecotoxicology. The action of contaminants considered to be cytotoxic or genotoxic in the literature were tested on this model. The results show the high cytotoxicity of ethylmethane sulphonate 80 and 5 mM after 2 h of contact with acini. Other compounds such as 4-nitroquinoline-N-oxide 0.1 mM, cadmium chloride 10−5 M and atrazine 10−4 M, which were weakly toxic after 2 h, became highly toxic after 48 h of contact. Compounds such as 4-nitroquinoline-N-oxide 1 mM, which were not cytotoxic after 2 h, proved to be the most genotoxic of all those tested. Others, such as MMS 1 mM, cadmium chloride 10−4 and 10−5 M, and atrazine 10−5 M, showed an unconfirmed tendency to be genotoxic. The results obtained with this ‘acinus’ model seem more readily transposable to the whole organism than those obtained with ‘isolated cell’ models, in that acini can be considered as digestive glands ‘in miniature’.

International audience; Sponges and bacteria have lived together in complex consortia for 700 million years. As filter feeders, sponges prey on bacteria. Nevertheless, some bacteria are associated with sponges in symbiotic relationships. To enable this association, sponges and bacteria are likely to have developed molecular communication systems. These may include molecules such as N-acyl-L-homoserine lactones, produced by Gram-negative bacteria also within sponges. In this study, we examined the role of N-3-oxododecanoyl-L-homoserine lactone (3-oxo-C12-HSL) on the expression of immune and apoptotic genes of the host sponge Suberites domuncula. This molecule seemed to inhibit the sponge innate immune system through a decrease of the expression of genes coding for proteins sensing the bacterial membrane: a Toll-Like Receptor and a Toll-like Receptor Associated Factor 6 and for an anti-bacterial perforin-like molecule. The expression of the pro-apoptotic caspase-like 3/7 gene decreased as well, whereas the level of mRNA of anti-apoptotic genes Bcl-2 Homolog Proteins did not change. Then, we demonstrated the differential expression of proteins in presence of this 3-oxo-C12-HSL using 3D sponge cell cultures. Proteins involved in the first steps of the endocytosis process were highlighted using the 2D electrophoresis protein separation and the MALDI-TOF/TOF protein characterization: α and β subunits of the lysosomal ATPase, a cognin, cofilins-related proteins and cytoskeleton proteins actin, α tubulin and α actinin. The genetic expression of some of these proteins was subsequently followed. We propose that the 3-oxo-C12-HSL may participate in the tolerance of the sponge apoptotic and immune systems towards the presence of bacteria. Besides, the sponge may sense the 3-oxo-C12-HSL as a molecular evidence of the bacterial presence and/or density in order to regulate the populations of symbiotic bacteria in the sponge. This study is the first report of a bacterial secreted molecule acting on sponge cells and regulating the symbiotic relationship.

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.

International audience; Bacterial biofilm development is conditioned by complex processes involving bacterial attachment to surfaces, growth, mobility, and exoproduct production. The marine bacterium Pseudoalteromonas sp. strain D41 is able to attach strongly onto a wide variety of substrates, which promotes subsequent biofilm development. Study of the outer-membrane and total soluble proteomes showed ten spots with significant intensity variations when this bacterium was grown in biofilm compared to planktonic cultures. MS/MS de novo sequencing analysis allowed the identification of four outer-membrane proteins of particular interest since they were strongly induced in biofilms. These proteins are homologous to a TonB-dependent receptor (TBDR), to the OmpW and OmpA porins, and to a type IV pilus biogenesis protein (PilF). Gene expression assays by quantitative RT-PCR showed that the four corresponding genes were upregulated during biofilm development on hydrophobic and hydrophilic surfaces. The Pseudomonas aeruginosa mutants unable to produce any of the OmpW, OmpA, and PilF homologues yielded biofilms with lower biovolumes and altered architectures, confirming the involvement of these proteins in the biofilm formation process. Our results indicate that Pseudoalteromonas sp. D41 shares biofilm formation mechanisms with human pathogenic bacteria, but also relies on TBDR, which might be more specific to the marine environment.

This study is the first work on the reproductive biology of lessepsian bivalve Fulvia fragilis. Monthly and bimonthly samplings were made in the period from April 2006 to October 2007, at the Bay of Tunis (northern Tunisia). From histological preparations of gonad, a gametogenic scale was drawn up based on eight stages: sexual rest; initiation of gametogenesis; advanced gametogenesis; ripe; partial spawning; advanced spawning; restoration; and spent. After examination of gonad preparations, F. fragilis appeared to be a simultaneous hermaphrodite species. Spawning was continuous all along the year, scarce in winter and with peaks during the rest of the year. The spawning peaks were positively correlated with the rise of the sea surface temperature and the nutrient availability. Furthermore, the presence of oocytes surrounded by spermatozoa in female and male follicles and in male gonoducts was observed. Self-fertilization may occur in this species. High summer mortalities probably due to the combination of vulnerability of the bivalve in response to the reproductive effort and high temperature stress were recorded. The F. fragilis reproduction strategy explains the success of its installation on the Tunisian coast.

Sponges (Porifera), represent the phylogenetically oldest metazoan phylum still extant today. Recently, molecular biological studies provided compelling evidence that these animals share basic receptor/ligand systems, especially those involved in bodyplan formation and in immune recognition, with the higher metazoan phyla. An in vitro cell/organ-like culture system, the primmorphs, has been established that consists of proliferating and differentiating cells, but no canals of the aquiferous system. We show that after the transfer of primmorphs from the demosponge Suberites domuncula to a homologous matrix (galectin), canal-like structures are formed in these 3D-cell aggregates. In parallel with the formation of these structures a gene is expressed whose deduced protein falls into the CD36/LIMPII receptor family. The receptor was cloned and found to be strongly expressed after adhesion to the galectin matrix. This process was suppressed if primmorphs were co-incubated with a homologous polypeptide containing the CSVTCG domain, as found in thrombospondin-1 (and related) molecules of vertebrates. In situ hybridization studies revealed that the S. domuncula CD36/LIMPII receptor is localized in the pinacocytes that surround the canals of the sponge. Furthermore, a secondary metabolite from a sponge-associated bacterium was isolated and characterized, the 2-methylthio-1,4-naphthoquinone (MTN). MTN causes inhibition of cell proliferation of vertebrate tumor cells at concentrations of >80 ng/ml. However, doses of only 2 ng are required to potently inhibit angiogenesis in the chick chorio-allantoic membrane assay. At concentrations of 10 ng/ml this compound was also found to suppress the expression of the S. domuncula CD36/LIMPII; this result is a first indication that this secondary metabolite has a conserved functional activity: the suppression of the formation of the circulation system, from sponges to vertebrates.

Sponges (phylum Porifera) are sessile marine filter feeders that have developed efficient defense mechanisms against foreign attackers such as viruses, bacteria, or eukaryotic organisms. Protected by a highly complex immune system, as well as by the capacity to produce efficient antiviral compounds (e.g., nucleoside analogues), antimicrobial compounds (e.g., polyketides), and cytostatic compounds (e.g., avarol), they have not become extinct during the last 600 million years. It can be assumed that during this long period of time, bacteria and microorganisms coevolved with sponges, and thus acquired a complex common metabolism. It is suggested that (at least) some of the bioactive secondary metabolites isolated from sponges are produced by functional enzyme clusters, which originated from the sponges and their associated microorganisms. As a consequence, both the host cells and the microorganisms lost the ability to grow independently from each other. Therefore, it was--until recently--impossible to culture sponge cells in vitro. Also the predominant number of "symbiotic bacteria" proved to be nonculturable. In order to exploit the bioactive potential of both the sponge and the "symbionts," a 3D-aggregate primmorph culture system was established; also it was proved that one bioactive compound, avarol/avarone, is produced by the sponge Dysidea avara. Another promising way to utilize the bioactive potential of the microorganisms is the cloning and heterologous expression of enzymes involved in secondary metabolism, such as the polyketide synthases.

The main inorganic material forming the skeletal elements in Demospongiae as well as in Hexactinellida, the spicules, is amorphous silica. The spicules occur in the cytoplasm and the extracellular space and also in the nucleus (as silicate crystals) of some sponge cells; the function in the latter compartment is unknown. Recent evidence shows that the formation of spicules is mediated by the enzyme silicatein. The cDNA as well as the gene encoding this enzyme was cloned from Suberites domuncula. The recombinant silicatein catalyzes the synthesis of amorphous silicate using tetraethoxysilane as substrate. The enzyme is dependent on ferric iron. Silicatein also has proteolytic (cathepsin-like) activity. Incubation of primmorphs, a special form of 3D-cell aggregates, with silicon resulted in a strong increase of their size from 1-7 mm to approximately 10 mm. The morphogenetic activity of silicate is underscored by the finding that this ion increases gene expression of silicatein and collagen. Based on these findings, it is concluded that both iron and silicate stimulate the activity of silicatein. Furthermore, it is proposed that the growing spicules are surrounded by the scavenger receptor which might be considered as a docking molecule for the collagen matrix into which the spicules are embedded.

Two classes of the phylum Porifera, the Demospongiae and the Hexactinellida,produce hydrated, amorphous, and noncrystalline silica which they deposit in their spicules. In contrast, the third class of Porifera, the Calcarea, have a skeleton which is composed exclusively of calcite spicules. In Demospongiae the amorphous hydrated silica, similar to opal, is formed primarily intracellularly in a special type of cells, the sclerocytes (see Uriz et al. 2000).

In living organisms four major groups of biominerals exist: (1) iron compounds, which are restricted primarily to Prokaryota; (2) calcium phosphates, found in Metazoa; (3) calcium carbonates, used by Prokaryota, Protozoa, Plantae, Fungi and Metazoa and (4) silica (opal) present in sponges and diatoms (reviewed in: Bengtson 1994; Baeuerlein 2000). It is surprising that the occurrence of silica as a major skeletal element is restricted to some Protozoa and to sponges (Porifera). The element silicon (Si) contributes to 28% of the earth crust and is - after oxygen - the second most abundant element on earth (Windholz 1983).

Digestive gland cells of Pecten maximus accumulate and release lipid storage according to a seasonal cycle. For the first time in molluscs, molecular probes were developed and applied to monitor the lipid accumulation and consumption cycle related to phytoplankton blooms and phenomena of cellular proliferation and apoptosis. The molecular probes consisted of glyceraldehyde-3-phosphate dehydrogenase (GPD), which is involved in the acetylation of fatty acids; aldolase, which favours the formation of pyruvate and dihydroxyacetone; actin, an essential element of the cytoskeleton that disappears during adipocyte cell transformation; and cycline B, an ubiquitous cell cycle protein. α-Amylase, provided by IFREMER-Brest (France), was used to relate these different events to the animal's food supply. A positive relation between GPD and aldolase gene expressions was inversely correlated with that of actin, confirming results in mammals. In P. maximus, mRNA transcripts of GPD and aldolase decrease rapidly before gamete emissions whereas those of actin increase rapidly. After gamete emission, the mRNA levels of aldolase, GPD and α-amylase increase, while those of actin decrease. Cycline B mRNA transcripts indicate that the period of digestive cell proliferation is initiated during winter, prior to the release of lipids into the digestive tract and apoptosis.

Marine demosponges (phylum Porifera) are rich sources for potent bioactive compounds. With the establishment of the primmorph system from sponges, especially from Suberites domuncula , the technology to cultivate sponge cells in vitro improved considerably. This progress was possible after the elucidation that sponges are provided with characteristic metazoan cell adhesion receptors and extracellular matrix molecules which allow their cells a positioning in a complex organization pattern. This review summarizes recent data on the cultivation of sponges in aquaria and—with main emphasis—of primmorphs in vitro. It is outlined that silicon and Fe(+++) contribute substantially to the formation of larger primmorphs (size of 10 mm) as well as of a canal system in primmorphs; canals are probably required for an improved oxygen and food supply. We conclude that the primmorph system will facilitate a sustainable use of sponges in the production of bioactive compounds; it may furthermore allow new and hitherto not feasible insights into basic questions on the origin of Metazoa.

We studied the marine pollution of the sediments and coastal waters of the Cape Verde peninsula and the Petite Côte. The results show that the perception of the population is not always in line with scientific observations. The population reports a mainly moderate level of pollution, which has worsened over the last ten years. The majority of the sites studied do not comply with the microbiological quality of bathing water from a microbiological point of view. The quantities of microplastics at some sites are high. Macroplastics at sea are not significantly present at all the sites sampled. For TMEs only chromium and nickel are above the probable effect concentration (PEC) on marine organisms for the fraction ≤ 100 μm. The first trophic levels of the marine food chain do not seem to be affected by sediment toxicity, whereas the middle and upper levels are more affected. The majority (81%) of the sites studied revealed a level of ecotoxicity greater than 20%, via the Magallana gigas embryotoxicity test, which is known to be more sensitive than other bioassays. Consequently, the assessment of the toxicity of marine sediments shows that the majority of the selected sites appear to be in a poor ecotoxicological state. The inter-site variability of the results is due to the characteristics of the sites.; Nous avons étudié la pollution marine des sédiments et des eaux côtières de la presqu’ile du Cap-Vert et de la Petite côte. Les résultats montrent que la perception des populations n’est pas toujours en phase avec les observations scientifiques. La population signale un niveau de pollution principalement modérée mais qui s’est aggravé ces dix dernières années. La majorité des sites étudiés n’est pas conforme aux qualités microbiologiques de l’eau de baignade d’un point de vu microbiologique. Les quantités de microplastiques de certains sites sont élevées. Les macroplastiques en mer, ne sont pas significativement présents sur tous les sites échantillonnés. Pour les ETM seuls le chrome et le nickel sont au-dessus de la concentration d'effet probable (CEP) sur les organismes marins en ce qui concerne la fraction ≤ 100 μm. Les premiers niveaux trophiques de la chaine alimentaire du milieu marin ne semblent pas être affectés par la toxicité des sédiments alors que les niveaux moyens et supérieurs le sont davantage. La majorité (81%) des sites étudiés ont révélé un niveau d’écotoxicité supérieur à 20%, via le test d’embryotoxicité de Magallana gigas reconnu pour sa plus grande sensibilité par rapport aux autres bioessais. Par conséquent, l'évaluation de la toxicité des sédiments marins montre que la majorité des sites sélectionnés apparaissent dans un mauvais état écotoxicologique. La variabilité inter-sites des résultats sont dues aux caractéristiques des sites.