Proteomic and Transcriptomic Analyses of Rigid and Membranous Cuticles and Epidermis from the Elytra and Hindwings of the Red Flour Beetle, Tribolium castaneum

Abstract

The insect cuticle is a composite biomaterial made up primarily of chitin and proteins. The physical properties of the cuticle can vary greatly from hard and rigid to soft and flexible. Understanding how different cuticle types are assembled can aid in the development of novel biomimetic materials for use in medicine and technology. Toward this goal, we have taken a combined proteomics and transcriptomics approach with the red flour beetle, Tribolium castaneum, to examine the protein and gene expression profiles of the elytra and hindwings, appendages that contain rigid and soft cuticles, respectively. Two-dimensional gel electrophoresis analysis revealed distinct differences in the protein profiles between elytra and hindwings, with four highly abundant proteins dominating the elytral cuticle extract. MALDI/TOF mass spectrometry identified 19 proteins homologous to known or hypothesized cuticular proteins (CPs), including a novel low complexity protein enriched in charged residues. Microarray analysis identified 372 genes with a 10-fold or greater difference in transcript levels between elytra and hindwings. CP genes with higher expression in the elytra belonged to the Rebers and Riddiford family (CPR) type 2, or cuticular proteins of low complexity (CPLC) enriched in glycine or proline. In contrast, a majority of the CP genes with higher expression in hindwings were classified as CPR type 1, cuticular proteins analogous to peritrophins (CPAP), or members of the Tweedle family. This research shows that the elyra and hindwings, representatives of rigid and soft cuticles, have different protein and gene expression profiles for structural proteins that may influence the mechanical properties of these cuticles.

Two major cuticular proteins are required for assembly of horizontal laminae and vertical pore canals in rigid cuticle of Tribolium castaneum

“…Bioinformatics searches of fully sequenced and annotated genomes of several insect species such as the honey bee, Apis melifera (Honeybee Genome Sequencing Consortium, 2006), the fruit fly, Drosophila melanogaster (Karouzou et al, 2007) and the red flour beetle, Tribolium castaneum (Dittmer et al, 2012;Richards et al, 2008) indicate that there is a large number of genes encoding CP-like proteins in insect genomes. Indeed, more than 200 putative CP genes have been identified in the malaria mosquito, Anopheles gambiae (Cornman et al, 2008) and the silkworm, Bombyx mori (Futahashi et al, 2008).…”

“…Indeed, more than 200 putative CP genes have been identified in the malaria mosquito, Anopheles gambiae (Cornman et al, 2008) and the silkworm, Bombyx mori (Futahashi et al, 2008). T. castaneum has 108 genes encoding CP-like proteins (Dittmer et al, 2012;Richards et al, 2008). …”

“…These two proteins are members of the CPR family that contains the RR-2 motif. TcCPR27 and TcCPR18 proteins are abundant in rigid cuticles found in the elytron, pronotum and ventral abdomen but are absent or very minor in soft and flexible cuticles present in the dorsal abdomen and hindwing of T. castaneum (Arakane et al, 2012;Dittmer et al, 2012). dsRNA-mediated gene silencing (RNAi) of these proteins resulted in phenotypes with malformed and weakened elytra.…”

Characterization of the Secondary Structure of CP30, a Highly Repetitive Ampholytic Protein in Beetle Elytral Cuticle

“…[8][9][10][11] Our research team recently identified and sequenced several abundant proteins within the elytron (rigid forewing) of Tribolium castaneum. [12] The third most abundant protein in the elytron is CP30 (or GLEAN no. 14462).…”

“…14462). [12] CP stands for cuticle protein and 30 reflects the fact that it tracks with the 30 kDa standard protein in SDS-PAGE. It has a molar mass of 19 kDa with an isoelectric point of pH 5.82.…”