Cuticular proteins (crusticuls) affect 3D chitin bundle nanostructure

The crustacean exoskeleton features a micrometric, three-dimensional chitin scaffold. The intricate organization of this structure makes it an ideal model for investigating scaffold proteins at the nanoscale. Periodic exoskeleton replacement during a rapid and punctual molt cycle involves proteins that govern exoskeleton formation. Relying on binary expression pattern analysis of a molt-related transcriptomic library generated from the cuticle-forming epithelium of the crayfish Cherax quadricarinatus, a family of crustacean cuticle structural proteins termed ‘crusticuls’ was discovered and shown to present an exoskeleton formation-related expression pattern. All nine crusticuls include a chitin-binding domain bordered by two acidic residue-rich regions, putative functional domains related to exoskeletal formation and biomineralization. Crusticuls knock-down via RNAi resulted in over 95% reduced relative expression in treated versus control crayfish, with phenotypic effects ranging from prolonged molt cycles to lethality. Crusticuls were largely absent from newly formed cuticles following knockdown, resulting in exoskeletal deformities in the three-dimensional organization of chitinous bundles at the micro- and nanometric scales. These structural alterations were phenotypically translated into changes in cuticular hardness and elasticity. The identification of crusticuls as being key for proper nanometric three-dimensional organization of cuticular chitinous scaffolds opens new avenues for synthetic scaffold bio-mimetic applications.