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Functional genomics of TcCPR4 belongs to RR-1 CP family in the red flour beetle, Tribolium castaneum
Insects have a protective exoskeleton consisted with cuticle to adapt various environments and pathogens. Insect cuticle mainly composed of the polysaccharide chitin and numerous of cuticular proteins (CPs). CPs are important for insect cuticle formation, development, and growth because it produces proper combination of mechanical and physical properties of cuticle depend on the regions of an exoskeleton. The largest family of CPs contains a 28-residue motif known as the Rebers-Riddiford (R&R) consensus sequence. When sequences containing the R&R consensus are aligned, they fall into three groups based on sequence similarity, and these groups tend to correlate with the type of cuticle (soft or hard) from which the proteins are derived. Proteins with the RR-1 motif have been found primarily in soft cuticle, whereas many proteins from rigid cuticle have an extended region of similarity called RR-2.
We recently reportedthat two major CPs, TcCPR18 and TcCPR27 belong to RR-2, are essential for formation of highly sclerotized modified-forewings (elytra) of a beetle. In this study, we performed functional genomics of TcCPR4, which encodes RR-1 motif. The transcript levels of TcCPR4 drastically increased in 3 d-old pupae at when adult cuticle synthesis appears to be begun. Immunohistochemical studies revealed that TcCPR4 protein was detected in the rigid cuticle of elyton and ventral abdomen but not in the flexible cuticle of hindwing and dorsal abdomen of T. castaneum adult. Furthermore, TcCPR4 protein was specifically present at basal side of the procuticle (near the epidermal cells) and vertical canals, whereas TcCPR27 protein was found entire procuticle. Injection of double-stranded RNA of TcCPR4 (dsTcCPR4) into late instar larvae had no effect on development and any types of molting such as larval-larval, larval-pupal or pupal-adult. Interestingly, depletion of both TcCPR4 and TcCPR27 transcripts could rescue the elytral cuticle defect and mortality produced by injection of dsTcCPR27 alone. Transmission electron microscopy analysis revealed that depletion of TcCPR4 had abnormal vertical canals in rigid adult cuticle while dsTcCPR27 injection showed less electron-dense-horizontal laminae and vertical canals. Surprisingly, co-injection of dsRNA for TcCPR4 and TcCPR27 exhibited more severe cuticle defect with thinner elytral cuticle and abnormal vertical canals and chtin laminae compared to those from insects treated with dsRNA for each gene. These results suggest that TcCPR4 as a RR-1 is essential structural component in the rigid cuticle of T. castaneum adult.
