Vesicles play a key role in biomineralization pathways by mediating ion concentration and transport while maintaining cellular homeostasis. Whereas calcium-loaded vesicles have been extensively studied in organisms such as coccolithophores and vertebrates, comparatively little is known about those involved in mollusc shell mineralization. Using electron microscopy and electron energy loss spectroscopy (EELS), we analyzed the ultrastructure and composition of three vesicle types predominant near the shell’s growth surface in cryofixed juvenile Mytilus galloprovincialis : bilayered vesicles, ribosome-associated vesicles (RAVs), and mitochondria containing electron-dense granules. Calcium associated with organic compounds was detected in all of them. Mitochondrial granules were shown to be composed of an amorphous mixed phase containing substantial amounts of calcium and phosphorus, in addition to organic compounds. Identical Ca- and P-rich granules have been widely reported in the mitochondria of bone-related cells, where their presence is associated with the regulation of intracellular calcium and the initiation of hydroxyapatite formation. Their presence in calcifying cells of both molluscs and vertebrates, together with increasing evidence of phosphate involvement in the regulation of transient calcium carbonate precursors, leads us to propose a conserved pathway for calcium transport, concentration and storage across phyla, regardless of the final mineral phase.

The periostracum is the outermost shell layer and the first produced during shell formation in molluscs. This organic layer isolates the extrapallial space from the external environment and provides a scaffold for subsequent calcification. In cephalopods with an internal shell, some organic shell structures are putatively homologous to the periostracum of other molluscan groups. However, neither their detailed structure nor their mode of formation has been described, leaving the extent of this homology unresolved. To address this issue, we investigated the morphology and formation of the organic layer of the dorsal shield and the gladius in embryos of the cuttlefish Sepia officinalis Linnaeus, 1758, and the squid Loligo vulgaris Lamarck, 1798, respectively, using light microscopy and transmission electron microscopy. In both species, the periostracum forms within a periostracal groove located along the lateral and anterior margins of the shell sac. As in other molluscs, secretions from columnar cells at the bottom of the groove produce a dense layer, while a translucent layer is subsequently added beneath it through secretions from cuboidal cells. The main difference is the absence of both a pellicle and of the specialized glandular cells that typically secrete it at the bottom of the periostracal groove.