Annotation and expression analysis of cuticular proteins from the tobacco hornworm, Manduca sexta

Abstract

The insect cuticle is a unique material that covers the exterior of the animal as well as lining the foregut, hindgut, and tracheae. It offers protection from predators and desiccation, defines body shape, and serves as an attachment site for internal organs and muscle. It has demonstrated remarkable variations in hardness, flexibility and elasticity, all the while being light weight, which allows for ease of movement and flight. It is composed primarily of chitin, proteins, catecholamines, and lipids. Proteomic analyses of cuticle from different life stages and species of insects has allowed for a more detailed examination of the protein content and how it relates to cuticle mechanical properties. It is now recognized that several groups of cuticular proteins exist and that they can be classified according to conserved amino acid sequence motifs. We have annotated the genome of the tobacco hornworm, Manduca sexta, for genes that encode putative cuticular proteins that belong to seven different groups: proteins with a Rebers and Riddiford motif (CPR), proteins analogous to peritrophins (CPAP), proteins with a tweedle motif (CPT), proteins with a 44 amino acid motif (CPF), proteins that are CPF-like (CPFL), proteins with an 18 amino acid motif (18 aa), and proteins with two to three copies of a C-X5-C motif (CPCFC). In total we annotated 248 genes, of which 207 belong to the CPR family, the most for any insect genome annotated to date. Additionally, we discovered new members of the CPAP family and determined that orthologous genes are present in other insects. We established orthology between the M. sexta and Bombyx mori genes and identified duplication events that occurred after separation of the two species. Finally, we utilized 52 RNAseq libraries to ascertain gene expression profiles that revealed commonalities and differences between different tissues and developmental stages.