Silica transport in the demosponge Suberites domuncula: fluorescence emission analysis using the PDMPO probe and cloning of a potential transporter.

Silicon is, besides oxygen, the most abundant element on earth. Only two taxa use this element as a major constituent of their skeleton, namely sponges (phylum Porifera) and unicellular diatoms. Results from combined cytobiological and molecularbiological techniques suggest that, in the demosponge Suberites domuncula, silicic acid is taken up by a transporter. Incubation of cells with the fluorescent silica tracer PDMPO [2-(4-pyridyl)-5-[[4-(2-dimethylaminoethylaminocarbamoyl)methoxy]phenyl]-oxazole] showed a response to silicic acid by an increase in fluorescence; this process is temperature-dependent and can be blocked by DIDS (4,4-di-isothiocyanatostilbene-2,2-disulphonic acid). The putative NBC (Na+/HCO3-) transporter was identified, cloned and analysed. The deduced protein comprises all signatures characteristic of those molecules, and phylogenetic analysis also classifies it to the NBC transporter family. This cDNA was used to demonstrate that the expression of the gene is strongly up-regulated after treatment of cells with silicic acid. In situ hybridization demonstrated that the expression of the sponge transporter occurs in those cells that are located adjacent to the spicules (the skeletal element of the animal) or in areas in which spicule formation occurs. We conclude that this transporter is involved in silica uptake and have therefore termed it the NBCSA [Na+/HCO3-[Si(OH)4]] co-transporter.

Magnetic resonance imaging of the siliceous skeleton of the demosponge Lubomirskia baicalensis.

The skeletal elements (spicules) of the demosponge Lubomirskia baicalensis were analyzed; they are composed of amorphous, non-crystalline silica, and contain in a central axial canal the axial filament which consists of the enzyme silicatein. The axial filament, that orients the spicule in its longitudinal axis exists also in the center of the spines which decorate the spicule. During growth of the sponge, new serially arranged modules which are formed from longitudinally arranged spicule bundles are added at the tip of the branches. X-ray analysis revealed that these serial modules are separated from each other by septate zones (annuli). We describe that the longitudinal bundles of spicules of a new module originate from the apex of the earlier module from where they protrude. A cross section through the oscular/apical-basal axis shows that the bundle rays are organized in a concentric and radiate pattern. High resolution magnetic resonance microimaging studies showed that the silica spheres of the spicules in the cone region contain high amounts of 'mobile' water. We conclude that the radiate accretive growth pattern of sponges is initiated in the apical region (cones) by newly growing spicules which are characterized by high amounts of 'mobile' water; subsequently spicule bundles are formed laterally around the cones.

Formation of siliceous spicules in the marine demosponge Suberites domuncula.

The siliceous skeleton of demosponges is constructed of spicules. We have studied the formation of spicules in primmorphs from Suberites domuncula. Scanning electron microscopy and transmission electron-microscopical (TEM) analyses have revealed, in the center of the spicules, an axial canal that is 0.3-1.6 microm wide and filled with an axial filament. This filament is composed of the enzyme silicatein, which synthesizes the spicules. TEM analysis has shown that spicule formation starts intracellularly and ends extracellularly in the mesohyl. At the initial stage, the axial canal is composed only of silicatein, whereas membranous structures and fibrils (10-15 nm in width) can later also be identified, suggesting that intracellular components protrude into the axial canal. Antibodies against silicatein have been applied for Western blotting; intracellularly, silicatein is processed to the mature form (24 kDa), whereas the pro-enzyme with the propeptide (33 kDa) is detected extracellularly. Silicatein undergoes phosphorylation at five sites. Immunohistological analysis has shown that silicatein exists in the axial canal (axial filament) and on the surface of the spicules, suggesting that they grow by apposition. Finally, we have demonstrated that the enzymic reaction of silicatein is inhibited by anti-silicatein antibodies. These data provide, for the first time, a comprehensive outline of spicule formation.

Dynamics of skeleton formation in the Lake Baikal sponge Lubomirskia baicalensis. Part I. Biological and biochemical studies.

In ancient Lake Baikal (East Siberia), freshwater sponges have diversified to an extraordinary degree. The skeleton of Lubomirskia baicalensis, which attains a size of up to 1 m, is constructed from spicules, which are cemented into longitudinal bundles. Our X-ray analysis revealed that the architecture of the specimens follows a highly ordered radiate accretive growth pattern. The spicules have a central axial canal with an axial filament inside. This organic filament is composed of silicatein, the major enzyme involved in silica formation of the spicules. We found that the specific activity of silicatein in samples from the non-growing (basal) zone is much lower than in those from the growth zone (tips) and that even the composition of this molecule differs in these regions. The present study shows for the first time that the turnover of silicatein, the major element of the axial canal of sponge spicules, changes within a sponge specimen depending on the region in which it is found.