Abstract
The functional characterization of an entire class of 17 genes from the red flour beetle, Tribolium castaneum, which encode two families of Cuticular Proteins Analogous to Peritrophins (CPAPs) has been carried out. CPAP genes in T. castaneum are expressed exclusively in cuticle-forming tissues and have been classified into two families, CPAP1 and CPAP3, based on whether the proteins contain either one (CPAP1), or three copies (CPAP3) of the chitin-binding domain, ChtBD2, with its six characteristically spaced cysteine residues. Individual members of the TcCPAP1 and TcCPAP3 gene families have distinct developmental patterns of expression. Many of these proteins serve essential and non-redundant functions in maintaining the structural integrity of the cuticle in different parts of the insect anatomy. Three genes of the TcCPAP1 family and five genes of the TcCPAP3 family are essential for insect development, molting, cuticle integrity, proper locomotion or fecundity. RNA interference (RNAi) targeting TcCPAP1-C, TcCPAP1-H, TcCPAP1-J or TcCPAP3-C transcripts resulted in death at the pharate adult stage of development. RNAi for TcCPAP3-A1, TcCPAP3-B, TcCPAP3-D1 or TcCPAP3-D2 genes resulted in different developmental defects, including adult/embryonic mortality, abnormal elytra or hindwings, or an abnormal ‘stiff-jointed’ gait. These results provide experimental support for specialization in the functions of CPAP proteins in T. castaneum and a biological rationale for the conservation of CPAP orthologs in other orders of insects. This is the first comprehensive functional analysis of an entire class of cuticular proteins with one or more ChtBD2 domains in any insect species.
Highlights
Chitin, a linear polymer of ß-1-4 linked N-acetyl-D-glucosamine, is an important component of the insect exoskeleton
As a first step in the functional analysis of individual members of the Cuticular Proteins Analogous to Peritrophins (CPAPs) gene families and optimization of RNA interference experiments, the expression profiles of the genes belonging to the two CPAP families of T. castaneum were determined by RT-PCR using cDNA prepared from RNA isolated at different stages of development
CPAP Proteins are Involved in Cuticle Organization The main focus of this investigation has been the elucidation of the functions of members of the CPAP1 and CPAP3 families of chitin-binding proteins in T. castaneum
Summary
A linear polymer of ß-1-4 linked N-acetyl-D-glucosamine, is an important component of the insect exoskeleton. It is present in epidermal and tracheal cuticles and in the peritrophic matrix (PM) that lines the midgut. Insect procuticle is composed mainly of chitin complexed with proteins that contain chitinbinding domains. Two major groups of chitin-binding domains found in insect cuticular proteins have either the Rebers & Riddiford Consensus sequence (R&R Consensus; pfam00379; [1]) that lacks cysteine residues, or the peritrophin-A motif with six distinctly spaced cysteine residues (ChtBD2 domain, pfam 01607). Additional genes encoding proteins with the ChtBD2 domain were found to be expressed in cuticle-forming tissues from Drosophila melanogaster and Ctenocephalides felis [3,4,5], suggesting that the peritrophin-A motif is not restricted to proteins associated only with the PM
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