The novel Craymin gene regulates calcium carbonate crystallization in the crustacean exoskeleton

Crustaceans undergo periodic exoskeleton replacement during a rapid and highly regulated molt cycle to allow growth and/or morphological changes. The new exoskeleton is hardened in a biomineralization process involving various minerals, mainly amorphous calcium carbonate and calcite, its main crystalline polymorph. Biomineralization also requires the involvement of proteins, but knowledge of the protein-encoding genes in crustaceans remains scarce. To address this knowledge gap, we utilized binary expression pattern analysis of a molt-related transcriptomic library generated from the cuticle-forming epithelium of the crayfish Cherax quadricarinatus. We thereby mined a gene encoding a glycine-rich protein that was found to exhibit exoskeleton-mineralization-related molt expression; we named this gene Craymin (crayfish mineralization). The Craymin protein was found in the exoskeleton cuticle and in exuvia, and RNA interference knockdown of its encoding gene reduced its relative expression in epithelium-forming cells by 90 %. Following knockdown, newly formed cuticles were largely depleted of Craymin protein molecules, resulting in a 47 % reduction in exoskeleton calcium content, together with larger CaCO₃ crystallites, compared to the control. Concomitantly with the reduction in calcium content, more than 80 % reduction in confined high-density regions (representing highly mineralized cuticular areas) was measured following knockdown, resulting in an overall reduction of cuticular density. These alterations were phenotypically translated into a 50 % reduction in cuticular width, accompanied by decreased structural integrity. Craymin thus seems to be a key protein in the crustacean exoskeleton, being involved in cuticular mineralization by mediating the deposition of calcium and the control of its crystalline polymorphs.