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 <i>Cherax quadricarinatus</i>, 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. <i>Crusticuls</i> knock-down <i>via</i> RNAi resulted in over 95% reduced relative expression in treated <i>versus</i> 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.