Analysis of functions of the cuticular protein analogous to Peritrophins (CPAP) gene family in Tribolium castaneum

Insect cuticle is composed mainly of chitin complexed with proteins, that contain chitin-binding domains. The two major groups of chitin-binding domains found in cuticular proteins have either the cysteine-rich peritrophin-A motif or the histidine-rich Rebers & Riddiford motif. This study is focused on the functional characterization of genes encoding Cuticular Protein Analogous to Peritrophins (CPAP) proteins with cysteine-containing ChtBD2 domains (Peritrophin A motif=CBM_14=Pfam 01607) in Tribolium castaneum. The CPAP family of genes is expressed in cuticle-forming tissue and according to the number of ChtBD2 domains is divided into two subgroups. The CPAP1 group of genes encodes an assortment of 10 proteins, each with only one ChtBD2 domain with divergent developmental and tissue-specific expression. The CPAP3 group of genes encodes 8 proteins with three ChtBD2 domains and a domain organization similar to the proteins encoded by the gasp/obstructor family of genes of Drosophila melanogaster. All the genes of the CPAP family are expressed predominantly in epidermal tissues including the elytra and hindwings. RNAi for these genes results in lethal phenotypes that are manifested at different developmental stages, including embryonic, pupal and adult stages. RNAi for CPAP1-C, CPAP1-H, CPAP1-J, and CPAP3-C results in death at the pharate adult stage. RNAi for other CPAP3 genes results in abnormal elytra and wings, walking defects, and/or adult mortality. Scanning electron microscopic analysis of pharate adults following RNAi for the CPAP3-D gene shows that elytral cuticle is affected. These results provide experimental support for specialization in the functions of individual CPAP proteins of T. castaneum.