The outer layer of the shell of members of the genus Mytilus is made of long, slender fibres of calcite (some 1-2 mm wide and hundreds of mm long), which reach the internal surface of the shell at an angle. This microstructure has been called anvil-type fibrous calcitic and its organization, crystallography and relationships to the organic phase are poorly known. We have studied the outer calcitic layer of the Mediterranean mussel M. galloprovincialis by means of optical and scanning and transmission electron microscopy (SEM and TEM), X-ray diffraction (XRD) and atomic-force microscopy (AFM). SEM data from other species have also been gathered. All data together imply that the material is extremely well ordered both from the morphological and crystallographic viewpoints. The XRD pole figures show that there are discrete 001 and 104 maxima; therefore, the material has a well-defined sheet texture. In living animals there is an organic membrane (surface membrane) that coats the inner surface of the shell. TEM sections of the decalcified material show that this mainly proteinaceous surface layer is internally laminated and fills all the spaces left between the growing fibres. Every fibre is a monocrystal with three well developed {104} rhombohedral faces at its growth end. One of these faces is directly in contact with, and strictly parallel, to the sublayers of the surface membrane and thus to the inner shell surface. AFM experiments consisting on growing calcite onto shell pieces in which the surface membranes are preserved, show that the calcitic fibres of the shell easily regrow across the membrane, demonstrating that it is permeable to ions. In this way, prisms are able to grow despite the existence of the intermediate membrane in the living animal. Additional experiments of calcite growth onto the inner side of the surface membrane show that crystals grow onto their {104} surfaces. The surface membrane is responsible for the high degree of ordering of the fibrous calcitic layer, because it stabilizes the orientation of a rhombohedral surface, once this is parallel to the protein sublayers. This is one of the very few cases in which the influence of the organic matter on the organization of microstructures can be demonstrated.
The current model for the ultrastructure of the interlamellar membranes of molluscan nacre imply that they consist of a core of aligned chitin fibers surrounded on both sides by acidic proteins. This model was based on observations taken on previously demineralized shells, where the original structure had disappeared. Despite other earlier claims, no direct observations exist in which the different components can be unequivocally discriminated. We have applied different labeling protocols on non-demineralized nacreous shells of the bivalve Pteria. With this method, we have revealed the disposition and nature of the different fibers of the interlamellar membranes that can be observed on the surface of the nacreous shell of the bivalve Pteria hirundo by high resolution scanning electron microscopy (SEM). The minor chitin component consists of very thin fibers with a high aspect ratio and which are seemingly disoriented. Each fiber has a protein coat, which probably forms a complex with the chitin. The chitin-protein-complex fibers are embedded in an additional proteinaceous matrix. This is the first time in which the sizes, positions and distribution of the chitin fibers have been observed in situ.
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