Abstract

Austromegabalanus psittacus is a large (normally up to 30 cm high) sessile balanomorph barnacle from the coast of Chile and South Peru. Its hard shell is composed of twelve calcareous side plates, six parietes and six radii, joined in the form of a truncated cone opened at the top. Plates rest on a basal disk firmly cemented to the substratum. Although the crystalline microstructure of barnacle's shell has been studied to some extent, its organic composition and the mechanisms governing the biomineralization of such highly ordered nanocomposite have remained obscure. By using X-ray diffraction, infrared spectrometry, SEM and TEM electron microscopy, histochemistry, immuno-histochemistry and -ultrastructure, biochemistry and a crystallization assay, we have studied the cell-shell interactions, the crystalline microstructure of the inorganic moiety and the localization of particular macromolecules, and tested their influence on crystallization. The mineral of the plates and basal disk was calcite showing a (104) preferential orientation. Plates were not solid but porous. While parietes have longitudinal canals (from the base to the apex), radii have transversal canals arranged parallel to the base. These canals are not in the center of the plates but displaced to the outside of the shell delimiting a thinner solid outer lamina and a thicker inner one. The inner lamina consisted of parallel calcified layers separated by organic sheets. These sheets showed autofluorescence and consisted of chitin surrounded by proteoglycans and other minor proteins, which seems to be responsible for the fluorescent behaviour. These organic sheets were also organized as several concentric rings around the canals. The shell matrix obtained after decalcification, which surrounded the crystals, also contained a loose net of such proteoglycans. Mantle epithelial cells covered the entire surface of the inner side of the inner lamina and extend to the plate canals. While isolated chitin did not promote or alter calcite crystallization, the proteoglycan-rich fraction dramatically modified crystal morphology and size. As we have demonstrated in another model of biomineralization, such as the eggshell, hereby we suggest that these structured polyanionic proteoglycan moieties could also be part of the regulatory mechanisms of the barnacle shell mineralization.