Formation of Rigid, Non-Flight Forewings (Elytra) of a Beetle Requires Two Major Cuticular Proteins

Abstract

Insect cuticle is composed primarily of chitin and structural proteins. To study the function of structural cuticular proteins, we focused on the proteins present in elytra (modified forewings that become highly sclerotized and pigmented covers for the hindwings) of the red flour beetle, Tribolium castaneum. We identified two highly abundant proteins, TcCPR27 (10 kDa) and TcCPR18 (20 kDa), which are also present in pronotum and ventral abdominal cuticles. Both are members of the Rebers and Riddiford family of cuticular proteins and contain RR2 motifs. Transcripts for both genes dramatically increase in abundance at the pharate adult stage and then decline quickly thereafter. Injection of specific double-stranded RNAs for each gene into penultimate or last instar larvae had no effect on larval–larval, larval–pupal, or pupal–adult molting. The elytra of the resulting adults, however, were shorter, wrinkled, warped, fenestrated, and less rigid than those from control insects. TcCPR27-deficient insects could not fold their hindwings properly and died prematurely approximately one week after eclosion, probably because of dehydration. TcCPR18-deficient insects exhibited a similar but less dramatic phenotype. Immunolocalization studies confirmed the presence of TcCPR27 in the elytral cuticle. These results demonstrate that TcCPR27 and TcCPR18 are major structural proteins in the rigid elytral, dorsal thoracic, and ventral abdominal cuticles of the red flour beetle, and that both proteins are required for morphogenesis of the beetle's elytra.

Author Summary

Primitive insects have two pairs of membranous flight wings, but during the evolution of the beetle lineage the forewings lost their flight function and became modified as hard, rigid covers called elytra for protection of soft body parts of the abdomen and also the delicate flexible hindwings, which retained their flight function. This transformation is manifested by a greatly thickened and rigid (sclerotized) exoskeletal cuticle secreted by the forewing epidermis. We demonstrate that this evolutionary modification is accompanied by the incorporation of two highly abundant structural proteins into the elytral cuticle, namely TcCPR18 and TcCPR27. Depletion of these proteins by RNA interference results in malformation and weakening of the elytra, culminating in insect death. These proteins are also abundant in hard cuticle from other regions such as the pronotum and ventral abdomen, but are absent in soft cuticles, and therefore may function as key determinants of rigid cuticle. Expression of such proteins at high levels in the modified forewing appears to have been a fundamental evolutionary step in the transformation of the membranous wing into a thickened and rigid elytron in the Coleoptera.