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Mollusk shells have unique microstructures and mechanical properties such as hardness and flexibility. Calcite in the prismatic layer of is extremely tough due to small crystal defects and localized organic networks inside calcites. Electron microscopic observations have suggested that such crystal defects are caused by the organic networks during calcite formation. Our previous work reported that the chitin which is the main component of organic networks and chitinolytic enzymes that bind to chitin were identified. In this article, to investigate the effects of chitin and chitinolytic enzymes on the formation of calcites, calcites were synthesized in chitin gel after treatment with chitinolytic enzymes. Chitin fibers seemed to become smooth and loosened after degradation. The crystal defects became larger as the chitin fibers became more degraded by chitinolytic enzymes in a dose-dependent manner. These results suggest that the shape of chitin fiber, which is regulated by the degradation of chitinolytic enzymes, contributes to the formation of small crystal defects.

Many genes have been identified to participate in the shell formation so far. Nevertheless, the whole picture of the molecular mechanisms underlying the shell formation has remained unknown. In our previous study, we analyzed comprehensively genes expressed in the shell-producing tissues and identified 14 genes to be involved in the shell formation by the RNA interference (RNAi) method. In the present study, we performed further screening to find additional novel genes involved in the formation of the nacreous and prismatic layers. We here selected 80 genes from the EST data as candidates to function in the shell formation, conducted knockdown experiments by the RNAi method, and observed surface appearances on the nacreous and prismatic layers. We newly identified 64 genes that could participate in the shell formation. Taken together with our previous study, 78 genes were supposed to function in the shell formation. These findings indicate that the combination of transcriptome and knockdown analyses is a powerful tool to screen novel genes involved in the shell formation.

The shell of pearl oysters consists of two distinct layers, nacre and prismatic. Mantle is the tissue involved in the shell formation, and its ventral part (mantle edge) forms the prismatic layers, whereas the dorsal part (pallium) forms the nacre. In pearl culture, mantle grafts from the pallium of donor are transplanted into the recipient. Then pearl sac is formed by proliferation of epithelial cells from the grafted mantle to form pearls. It has been reported that gene expression patterns are different between mantle edge and pallium in accordance with their distinct functions in the shell formation. However, it is not well addressed whether gene expression is identical or not between two nacre-forming tissues, pallium and pearl sac. Here, we examined expression patterns of known genes related to nacre and prismatic layer formation in mantle edge, pallium, and pearl sac of . Although the pallium and pearl sac have the same function in terms of nacre formation, various genes were not expressed identically to the respective tissues, suggesting that shell matrix proteins differently function in the formation of shell nacre and pearls.

The following SEM and TEM images were taken by late Dr. Hiroshi Nakahara many years ago, left unpublished, and shown on the screen during lunchtimes in the symposium. He graduated from the course of zoology of the Faculty of Sciences, University of Hokkaido, and studied abroad in the University of New York and School of Dentistry, University of Texas. After returning home, he taught oral anatomy at Meikai University, School of Dentistry (former Josai Dental University). Using electron microscopes both SEM and TEM, he studied the mineralization processes of a variety of shellfish as well as vertebral hard tissues such as tooth enamel, dentin, and bone.