Functional study of cuticular proteins with a low complexity proline-rich sequence in the red flour beetle, Tribolium castaneum

Structural cuticular proteins (CPs) play important roles in determining the diverse physical properties of the insect exoskeleton as a result of their interactions and/or cross-linking among themselves and possibly with the extracellular matrix polysaccharide, chitin. Insect CPs have been classified into several families based on specific amino acid sequence motifs. In this study, we focused on the functions of one of the CP families, Cuticular Proteins of Low Complexity, Proline-rich (CPLCPs), in the red flour beetle, Tribolium castaneum. Eleven genes encoding TcCPLCP-like proteins have been identified by searching the T. castaneum genome database. Phylogenetic analysis indicates that all of the TcCPLCPs except for TcCPLCP1 fall into two distinct clades. The expression profiles of these genes during development were analyzed by real-time PCR. RNAi for TcCPLCP1-6 did not affect insect growth and development. Similarly, injection of dsRNA specific for TcCPLCP7-11 genes into larvae had no effect on larval growth, larval-pupal molting or pupal development. However, the subsequent pupal-adult molting was adversely affected. TcCPLCP7- and TcCPLCP10-deficient insects did not shed their pupal cuticles and the adults died entrapped in their pupal exuviae. The resulting adults that had been treated with dsRNA for TcCPLCP8, -9 or -11 exhibited abnormally expanded elytra. In addition, TEM analysis revealed ultrastructural abnormalities/defects in the adult cuticles after RNAi for TcCPLCP7-11. These results support the hypothesis that TcCPLCP7-11 proteins are required for development and maturation of the adult cuticle, but not the larval and pupal cuticles.