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.

Identification and expression of cuticular protein genes based on Locusta migratoria transcriptome

“…However, there are still some cuticular protein genes with unexpected expression in the tissues not derived from ectoderm such as the goad, Malpighian tubule, and fat body (Figs 5, 6, 7, 8). Similar results were found in other species, such as B. mori 20 and Manduca sexta 39. It has been speculated that these proteins are formed and transported to the cuticle-forming tissues from the internal tissues, or its transcripts of these protein genes originated in the attached trachea that possess the epidermic structure6.…”

Multifaceted biological insights from a draft genome sequence of the tobacco hornworm moth, Manduca sexta

“…The largest family is CPR with gene numbers ranging from as few as 32 in A. mellifera to as many as 156 in Anopheles gambiae . Astonishingly, 207 CPR genes have now been identified in M. sexta (79 RR-1,124 RR-2, 4 RR-3), indicating extensive gene duplication, thus making M. sexta the insect with the greatest number of CPR genes described to date (Dittmer et al, 2015). In comparison, B. mori has 148 CPR genes (56 RR-1, 93 RR-2, 4 RR-3) (Futahashi et al, 2008).…”

“…In comparison, B. mori has 148 CPR genes (56 RR-1, 93 RR-2, 4 RR-3) (Futahashi et al, 2008). Comparison of the M. sexta and B. mori CPR genes (Dittmer et al, 2015) indicated that the greatest difference was evident with the RR-2 genes, for which only 51 orthologous pairs could be established. However, five orthologous groups could be identified, in which 3–13 M. sexta RR-2 genes (42 in total) were clearly related to 3–6 B. mori RR-2 genes (23 in total), although within these groups the M. sexta genes were more closely related to each other than any one of them was to a B. mori gene.…”

“…Only 11 of the M. sexta RR-1 genes did not have an identifiable ortholog in B. mori . In contrast to the CPR genes, M. sexta has similar numbers of CPF, CPFL, TWDL, and CPAP genes in comparison to other insects, with one-to-one orthology identifiable for many of them (Table 3) (Dittmer et al, 2015). A further finding from this annotation is the identification of five additional CPAP1 genes, expanding on the 10 genes originally identified (Jasrapuria et al, 2010).…”

“…Another group of 6 genes (the RR-2 genes CPR68 - 70 and TWDL2–4 ) were near exclusively expressed in 7 of the 16 libraries prepared from pre-molt tissues (head, fat body, midgut, and abdominal muscle from 4th instar larvae after head capsule slippage, as well as 3 day old eggs, midgut from 3rd instar larvae after head capsule slippage, and fat body of 15e18 day old pupae); this is similar to B. mori where the TWDL genes were shown to be coordinately expressed, with the highest expression occurring at the larval stage during the molt (Liang et al, 2010). A more comprehensive description of the expression analysis can be found in Dittmer et al (2015).…”