ULTRASTRUCTURE AND EARLY EMBRYONIC SHELL FORMATION IN THE TERRESTRIAL PULMONATE SNAIL, EUHADRA HICKONIS

The distribution of calcium ion (Ca-ion) was demonstrated in the mantle epithelial cells by means of two-step chemical precipitation of Ca-ion in the pulmonate snail, Euhadra hickonis. K-oxalate/ K-antimonate chemical replacement was done using a simultaneous computerized microwavestimulated fixation method. These precipitates in the mantle epithelial cells were studied by electronprobe X-ray microanalysis (EDX) and electron energy-filtered imaging (EFI). The calculated values of the essential-elemental X-ray pulse ratios, which were obtained by computerized EDX analysis, agreed with those of the standard samples and theoretical values of Ca-antimonate. Typical EFI images and spectra energy of Ca and Sb were obtained from the surface mantle epithelial cells in the same tissue block as that used for EDX analysis where the elemental distribution of Ca and Sb was the same. The Ca-antimonate precipitates were formed in the intercellular space, vesicles and microvilli of mantle cells. In day 13 embryos, the columella and peripheral area were almost calcified, but in the dorsal area, Ca was still in the ionic state; the binding ratio of the former case was Ca:Sb = 1:0, that of the latter was Ca:Sb = 1:2, and that of the small intermediate area was Ca:Sb = 1:1.4. After day 17, the embryonal periostracum was almost calcified. Between days 15 and 20, strong Ca 2+ -ATPase and carbonic anhydrase (CAHase) enzymatic activities were observed at the sites where Ca-ion was observed; intercellular spaces, vacuoles in the mantle cells, periostracum and homogeneous layer between the periostracum and mantle cell, and in the mitochondria. Ca 2+ -ATPase may play important roles in the transportation of Ca-ion from the basal cell layer to the surface layer of the mantle cells and CAHase may play important roles in the chemical binding of Ca-ion and carbonate ion to produce calcium carbonate crystals in the newly developing embryonal shell.