Two Major Structural Proteins Are Required for Rigid Adult Cuticle Formation in the Red Flour Beetle, Tribolium castaneum

Abstract

The insect exoskeleton or cuticle is a complex extracellular matrix consisting of several functional layers. Cuticular proteins (CPs) and the polysaccharide chitin are the major components of the exo‐ and endocuticular layers or procuticle. During cuticle tanning (sclerotization and pigmentation), CPs are cross‐linked by quinones or quinone methides produced by oxidation of catechols catalyzed by the phenoloxidase, laccase. 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. More than 100 CP‐like genes have been identified in Drosophila melanogaster, with a similar number present in the red flour beetle, Tribolium castaneum. Different CPs have been found in the different developmental stages and in different regions of the exoskeleton, indicating that specific CPs are required to produce a cuticle with the proper morphology and mechanical properties.To study the function(s) of insect CPs, we focused on adult elytra of the T. castaneum. In protein samples extracted from elytra of newly emerged adults, yielding two highly abundant proteins with apparent sizes of 10 and 20 kDa. To characterize these major proteins further, each was digested with trypsin, and the resulting peptides were analyzed by MALDI‐TOF mass spectrometry. Comparison of this result with conceptual trypsinization of the computed proteome of Tribolium identified two candidate genes, which we call TcCP10 and TcCP20. We cloned the full‐length cDNAs corresponding to these genes. Both proteins belong to the RR‐2 family (cuticle DB, http://biophysics.biol.uoa.gr/cuticleDB/) and possess a putative signal peptide sequence. TcCP20 is an ortholog of the Tenebrio adult cuticle protein, acp22. Both TcCP10 and TcCP20 were mapped to linkage group 3. The expression patterns revealed by real‐time PCR showed that the transcript levels of both genes dramatically increase at the pharate adult stage (1 ‐2 days before molting), and then decline after adult eclosion.RNA interference (RNAi) was used to investigate the functions of TcCP10 and TcCP20. Specific dsRNAs for each gene were injected into last‐instar larvae. Following dsRNA injections, expression of TcCP10 and TcCP20 genes was analyzed by real‐time PCR and SDS PAGE to evaluate transcript and protein levels. Both genes were substantially and specifically down‐regulated at the mRNA and protein levels after RNAi treatment. Injection of dsTcCP10 or dsTcCP20 had no effect on larval‐larval, larval‐pupal or pupal‐adult molts. The elytra of the resulting adults, however, were malformed and abnormal. Elytra of dsTcCP20 adults did not fully elongate and did not extend far enough to cover the abdomen. These elytra were slightly separated and not smooth as compared to those of control insects. Like the dsTcCP20 phenotype, the resulting adults obtained after injection dsTcCP10 also exhibited elytral defects, but they were more severe. These elytra were short, wrinkled, bumpy, warped and fenestrated. CP10‐deficient insects could not fold their hind wings properly and eventually died approximately 7 days after eclosion, probably because of dehydration. TcCP10 and TcCP20 were also extracted from the pronotum cuticle of Tribolium. These results demonstrate that two cuticle proteins, TcCP10 and TcCP20, play critical roles in adult cuticle formation of T. castaneum.