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Background The mollusk shells present distinctive microstructures that are formed by small amounts of organic matrices controlling the crystal growth of calcium carbonate. These microstructures show superior mechanical properties such as strength or flexibility. The shell of Pinctada fucata has the prismatic layer consisting of prisms of single calcite crystals. These crystals contain small angle grain boundaries caused by a dense intracrystalline organic matrix network to improve mechanical strength. Previously, we identified chitin and chitinolytic enzymes as components of this intracrystalline organic matrix. In this study, we analyzed the function of those organic matrices in calcium carbonate crystallization by in vitro and in vivo experiments. Results We analyzed calcites synthesized in chitin gel with or without chitinolytic enzymes by using transmission electron microscope (TEM) and atom probe tomography (APT). TEM observations showed that grain boundary was more induced as concentration of chitinolytic enzymes increased and thus, chitin became thinner. In an optimal concentration of chitinolytic enzymes, small angle grain boundaries were observed. APT analysis showed that ion clusters derived from chitin were detected. In order to clarify the importance of chitinolytic enzymes on the formation of the prismatic layer in vivo, we performed the experiment in which chitinase inhibitor was injected into a living Pinctada fucata and then analyzed the change of mechanical properties of the prismatic layer. The hardness and elastic modulus increased after injection of chitinase inhibitor. Electron back scattered diffraction (EBSD) mapping data showed that the spread of crystal orientations in whole single crystal also increased by the effect of inhibitor injections. Conclusion Our results suggested that chitinolytic enzymes may function cooperatively with chitin to regulate the crystal growth and mechanical properties of the prismatic layer, and chitinolytic enzymes are essential for the formation of the normal prismatic layer of P. fucata.

The fine structure of the calcite prism in the outer layer of a pearl oyster, Pinctada fucata, has been investigated using various electron beam techniques, in order to understand its characteristics and growth mechanism including the role of intracrystalline organic substances. As the calcite prismatic layer grows thicker, sinuous boundaries develop to divide the prism into a number of domains. The crystal misorientation between the adjacent domains is several to more than ten degrees. The component of the misorientation is mainly the rotation about the c-axis. There is no continuous organic membrane at the boundaries. Furthermore, the crystal orientation inside the domains changes gradually, as indicated by the electron back-scattered diffraction (EBSD) in a scanning electron microscope (SEM). Transmission electron microscopy (TEM) examination revealed that the domain consists of sub-grains of a few hundred nanometers divided by small-angle grain boundaries, which are probably the origin of the gradual change of the crystal orientation inside the domains. Spherular Fresnel contrasts were often observed at the small-angle grain boundaries, in defocused TEM images. Electron energy-loss spectroscopy (EELS) indicated the spherules are organic macromolecules, suggesting that incorporation of organic macromolecules during the crystal growth forms the sub-grain structure of the calcite prism.