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Structural Studies of Biomineralization in the Sea Hare Aplysia Punctata by Electron Microscopy and Diffraction

X-ray diffraction (XRD), transmission electron microscopy (TEM), selected area electron diffraction (SAED), high resolution transmission electron microscopy (HRTEM), environmental scanning electron microscopy (ESEM) and energy dispersive X-ray analysis (EDS) were used in study of starting biomineralization processes in embryos of the sea hare species Aplysia punctata. 10 days old embryos (sample C4) appeared amorphous according to XRD patterns. TEM of the same sample showed that first grains of nanocrystalline dolomite began to form in the amorphous area. The identification of dolomite was confirmed according to TEM dark field images and SAED (Fig.1). SAED was taken with (104) diffraction spot of dolomite, D. Grains of dolomite have sizes about 10 nm. In Fig. 1. corresponding SAED pattern shows that the strongest diffraction spots of dolomite D(104) form nanocrystalline diffraction ring. In HRTEM (Fig. 2) the lattice fringes of dolomite D(104), spacing d104 =0.289 nm, and aragonite A(110), spacing d110 = 0.421 nm, are marked. The corresponding SAED pattern in [010] D orientation of dolomite from an area 100x100 nm2 (Fig. 2). In further development stages of the embryos very faint aragonite rings became visible by SAED [1]. It was shown that the biomineralization process in Aplysia punctata started by formation of the dolomite nanograins which served as centers of crystallization for further aragonite deposition in the larval shell. The accepted theory dealing with the starting biomineralization in marine invertebrates is based on the fact that, during the embryonic development, amorphous calcium carbonate (ACC) precedes the first crystal formation, thus ACC being the precursor phase for further mineralization of the skeleton [2]. In the present study we have used the combination of biological and instrumental methods with an aim to register and describe the formation of the first biominerals in the marine gastropod Aplysia punctata (Cuvier, 1803). ESEM with energy dispersive X-ray analysis (EDS) was used in order to find appearance of crystallographic phases in the particular periods of growth and development of Aplysia punctata, found in adult shells by X-ray diffraction [1]. The idea of the combined ESEM and EDS investigation was to observe the beginning of appearance of calcium and magnesium as constituents of crystallographic phases observed in embryo in the early phases of shell morphology formation. Magnesium, the constitutive element of dolomite, was observed in a very small amount in sample C4 (Fig.3 7b) before the appearance of calcium. That confirms the results obtained by HRTEM and SAED that dolomite is the first crystalline phase in Aplysia punctata embryos precipitated from amorphous calcium carbonate. Also, these results are in accordance with the general model of initial biomineralization processes referred to in the relevant literature [2, 3].

HRTEM; SAED; ESEM EDS; biomineralization

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