Insect cuticle consists of numerous structural proteins, which could interact with polysaccharide, chitin, and alter properly mechanical property of the cuticle. Cuticular Protein Analogous to Peritrophins (CPAPs) are characterized by presence of one (CPAP1s) or three (CPAP3s) chitin-binding domain belong to CBM14/ChtBD2 family. In this study, we investigated physiological functions of TcCPAP1-H and TcCPAP3-C in Tribolium castaneum. RNAi for either TcCPAP1-H or TcCPAP3-C at late instar larvae had no effect on larval-pupal molt nor pupal development. However, the resulting pharate adults failed to shed their old pupal cuticle and died entrapped in it without undergoing eclosion. TEM analysis, in addition, revealed disorganized chitinous horizontal laminae and/or vertical pore canals of rigid cuticle from TcCPAP1-H- and TcCPAP3-C-deficient adults. Desiccation-induced death produced by injection of dsTcCPAP1-H into young instar larvae is also discussed.

Cuticular proteins (CPs) and the polysaccharide chitin are the major components of the exo- and endocuticular layers or procuticle. CPs contain a conserved sequence known as the Rebers & Riddiford (R&R) motif, which may function as a chitin-binding domain that helps to coordinate the interaction between chitin fibers and the protein network. We identified two highly abundant RR-2 CPs, TcCPR18 and TcCPR27, in protein samples extracted from elytra (rigid cuticle) of Tribolium castaneum adults and determined that these two CPs are required for rigid cuticle morphology. In this study, we identified the third most abundant protein (TcCP30) extracted from the elytra, and cloned a full-length cDNA. It encodes a very unusual 171 amino acid residue protein of which 36% of the residues of the mature protein are Glu, 21% are His, 19% are Arg, and 16% are Gly, organized in a regular pattern but not R&R consensus motif. TcCPR18 and TcCPR27 genes are expressed at 4 d-old pupae, while TcCP30 is highly expressed at 5 d-old pupae (last pupal stage) and 0 d-old adults. Immunohistochemical studies revealed the presence of TcCP30 in rigid adult cuticle (e.g. elytron, pronotum and ventral abdomen) but not soft cuticle (e.g. hindwing and dorsal abdomen). Injection of dsRNA for TcCP30 into late instar larvae had no affect on larval and pupal growth and development. The subsequent pupal-adult molt, however, more than 50% adults were unable to shed their exuvium and died. In addition, the resulting adults exhibited wrinkled, warped and split elytra. TcCP30-deficient adults could not fold their hindwings properly. These results indicate that TcCP30 may play critical roles in rigid adult cuticle formation, development and insect growth and survival. This work was supported by NRF (NRF-2012R1A2A1A01006467).