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TEM SAED and HRTEM Investigation of the Morphology and Structure of the Opisthobranch Gastropod Shell Aplysia punctata

This work is a continuation of the previous work reported at the 16th Croatian-Slovenian Crystallographic Meeting. Transmission electron microscopy (TEM), SAED and HRTEM were used in order to find and locate starting biominerals in the early embryos of the sea hare species Aplysia punctata. The former results obtained by scanning electron microscopy (ESEM) with energy dispersive X-ray analysis (EDS) showed morphological changes during development of numerous (about 40 in each capsule) embryos incorporated in a jelly-like egg-string immediately after deposition on the substratum ( in a form of a spaghetti-like tissue ) until hatch of the veliger larvae. Diffraction patterns of A. punctata embryos confirmed that the very first aragonite mineral appeared 12 days after egg-strings deposition. Fully formed aragonite crystals were recorded in more developed embryos even before hatching, which occurred 23 days after deposition. The same series of powdered samples of egg-strings and embryos were also used for TEM, SAED and HRTEM investigation. A special attention was paid to the sample of 9 days old embryos that appeared amorphous for XRD. During observation of the amorphous area by TEM, the appearance/precipitation of first grains of a nanocrystalline phase was marked showing a strong reflection in SAED. This precipitation of the first nucleated phase was induced by the electron beam in the amorphous area. Three separate regions were analyzed in order to give confidence of distribution of crystallographic phases. At higher magnification (area of 100x100 nm2), after disappearance of the amorphous area, layers of monocrystalline dolomite in [010] orientation were seen by SAED. In all regions nanocrystalline diffraction rings of calcite or aragonite were observed. It could be presumed that in A. punctata biomineralization process starts by formation of the dolomite crystals which serve as centers of crystallization for further aragonite deposition in the larval shell.

TEM SAED; HRTEM; Opisthobranchia; Aplysia punctata; veliger; shell; biomineralization

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