Embryonic development of the freshwater snail Biomphalaria glabrata under microgravity conditions (STS-89 mission).

The embryonic development of the fresh-water snail Biomphalaria glabrata was examined under microgravity-conditions and compared with the ground control and standard embryos, putting special emphasis on the shell formation. The process of shell formation may be particularly sensitive to the change of gravitational forces. The project aimed at determining whether the processes of mineralization during the formation of the exoskeleton in the growing snail embryo take place normally under microgravity conditions. Twenty-four adult individuals of the tropical freshwater snail B. glabrata were maintained 9 days in the Closed Equilibrated Biological Aquatic System (CEBAS Minimodule) on Space Shuttle flight STS-89. The animals produced spawning packs throughout the duration of the mission so that embryos of all developmental stages were achieved. The embryos developed slightly slower in the CEBAS than under standard conditions, and in older embryos a decreased mineralization of the shell was detected. These phenomena, however, were observed in the flight module as well as in the ground control specimens and was not an effect caused by the microgravity conditions. Embryos of B. glabrata showed a correct morphogenesis under microgravity, no teratological effects were noticed, and the shell formation proceeded normally.

Calcium carbonate modifications in the mineralized shell of the freshwater snail Biomphalaria glabrata.

The mineralized shell (consisting of calcium carbonate) of the tropical freshwater snail Biomphalaria glabrata was investigated with high resolution synchrotron X-ray powder diffractometry and X-ray absorption spectroscopy (EXAFS). Parts from different locations of the snail shell were taken from animals of different age grown under various keeping conditions. Additionally, eggs with ages of 60, 72, 120, and 140 hours were examined. Traces of aragonite were found as first crystalline phase in 120 h old eggs, however, Ca K-edge EXAFS indicated the presence of aragonitic structures already in the X-ray amorphous sample of 72 h age. The main component of the shell of adult animals was aragonite in all cases, but in some cases minor amounts of vaterite (below 1.5%) are formed. The content of vaterite is generally low in the oldest part of the shell (the center) and increases towards the mineralizing zone (the shell margin). In juvenile snails, almost no vaterite was detectable in any part of the shell.

Calcium binding constituents of the organic shell matrix from the freshwater snail Biomphalaria glabrata.

The Ca2+ binding of an EDTA-free water-soluble (SM) and -insoluble (IM) organic matrix of the freshwater snail Biomphalaria glabrata was investigated, using a 45Ca2+ autoradiography after SDS-electrophoretical separation and a calcium binding assay. Electrophoresis of the SM showed a considerable amount of Alcian blue and Stains all positive material, regarded as glycosaminoglycans (GAGs) or proteoglycans (PGs). This part of the SM was slightly positive after 45Ca2+ autoradiography at pH 6.8. The Ca2+ binding increased, raising the pH to 7.4 and 8.0 and was especially strong when simulating the real conditions of the extrapallial space with a carbonate buffer of pH 7.4. The Ca2+ binding assay of the IM showed the same pH-dependency that was observed in the SM. The titration of the IM with Ca2+ at pH 8.0 lead to a dissociation constant of 7.5 x 10(-5) M. While Mg2+ displaced 45Ca2+ in the same way as nonradioactive Ca2+, an approximately 400-fold amount of Na+ was necessary to reduce the binding of 45Ca2+ to 50%. The Ca2+ binding of the organic matrix from the B. glabrata shell appears to be a process of low specificity, medium affinity and high pH-dependency. Apparently, acidic carbohydrate-rich PGs are the only calcium binding constituents of the organic shell matrix.

Carbohydrates of the Organic Shell Matrix and the Shell-Forming Tissue of the Snail Biomphalaria glabrata (Say).

Sulfated carbohydrates may play a role in the biomineralization of the molluscan shell. The carbohydrates of the extracted water-insoluble organic shell matrix (IM) of the freshwater snail Biomphalaria glabrata were identified as glucose, mannose, galactose, and N-acetyl-glucosamine, whereas the water-soluble organic matrix (SM) additionally contained N-acetyl-galactosamine. A specific lectin binding pattern of the matrix was obtained. One prominent protein of the SM, with a size of 19.6 kDa and a pI of 7.4, was shown to be a glycoprotein with terminal glucosyl or mannosyl moieties. The acidic constitutents of the matrix showed a variety of possible terminal sugars, indicating a heterogenous mixture of proteoglycans or glycosaminoglycans (GAGs) and glycoproteins. At the shell-forming mantle edge, an alcian-blue-positive material was observed in the periostracum groove (PG), the belt, and apically in the cells of the outer mantle epithelium (OME). With the help of lectins, all sugars in question were detected in the PG and the belt, whereas the OME was bound by glucose/mannose- and GlcNac-specific lectins only. Although the complete set of GAGs will be produced in the PG and the belt, a very acidic fraction of GAGs and the 19.6-kDa protein can also be delivered by the OME.