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. Author manuscript; available in PMC: 2016 Jul 1.
Published in final edited form as: Insect Biochem Mol Biol. 2015 Jan 8;62:100–113. doi: 10.1016/j.ibmb.2014.12.010

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

Neal T Dittmer a,*, Guillaume Tetreau b, Xiaolong Cao c, Haobo Jiang d, Ping Wang b, Michael R Kanost a
PMCID: PMC4476932  NIHMSID: NIHMS654268  PMID: 25576653

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.

Keywords: cuticular proteins, Rebers & Riddiford, RNAseq, orthology, Manduca sexta

1. Introduction

The insect exoskeleton (cuticle) is a remarkable extracellular structure secreted by epidermal cells that serves as the outer body covering. It helps to protect the insect from environmental stresses such as predators, parasites, abrasions, desiccation, and UV radiation. It also functions as an attachment site for internal muscles and organs, and is instrumental to locomotion and flight. The bulk of the cuticle is made primarily of a network of chitin embedded in a proteinaceous matrix, with water, catecholamines, and some lipids (Moussian, 2013). Despite what may seem like a limited pallet of materials, insects are able to synthesize cuticles that differ widely with respect to physical properties. Measurements of hardness have differed by more than 30 fold, while the stiffness of cuticle has been shown to vary by more than seven orders of magnitude (Klocke and Schmitz (2011) and references within; Vincent and Wegst, 2004). Investigations of the reasons behind these differences have focused on the role of dehydration and chemical cross-linking (Andersen, 2010; Sugumaran, 2010; Vincent 2009). However, it has been apparent for many years that differences exist between proteins extracted from different types of cuticle (hard versus soft), different developmental stages (i.e. larva, pupa, adult), as well as different time points (pre-molt versus post-molt) (Andersen et al., 1986, 1987, 1995a; Cox and Willis, 1985; Dittmer et al., 2012; Jensen et al., 1997; Kiely and Riddiford, 1985; Missios et al., 2000). Obtaining sequence information of these proteins was limited as it required either solubilization, purification, digestion, and sequencing of peptide fragments, or screening of cDNA expression libraries with antibodies raised against cuticle extracts in order to identify the proteins involved. Now, the emergence of large scale genomic, proteomic, and transcriptomic analyses has allowed for a renewed look at the importance of the protein content.

The first insect genome sequenced was that of Drosophila melanogaster (Adams et al., 2000); there are now 112 insect genome sequences available at NCBI. An analysis of 12 genomes by Ioannidou et al. (2014) suggested that genes encoding structural cuticular proteins (CP) represent on average 1% of the total protein-coding genes in insects. The total number of genes varied from 63 for Pediculus humanus to 301 for Aedes aegypti. These numbers, as well as those given for other insects, likely represent a minimum as most genomes have not gone through a rigorous manual annotation and rely on homology to other known CPs. Combining proteomics with genomics has been used to identify cuticular proteins for several insect species (Bae et al., 2011; Carrasco et al., 2011; Dittmer et al., 2012; Fu et al., 2011; He et al., 2007). Similarly, genomics and transcriptomics have contributed valuable information on CP gene expression, discerning variations in the timing (pre- or post-molt), developmental stage (larval, pupal, adult), and relative expression levels of these genes (Cornman and Willis, 2009; Dittmer et al., 2012; Futahashi et al., 2008; Gallot et al., 2010; Liang et al., 2010; Okamoto et al., 2008; Suetsugu et al., 2013; Togawa et al., 2007, 2008).

Many CPs can be classified by the conserved sequence motifs they contain (Ioannidou et al., 2014; Willis et al., 2012). The largest group is known as CPs with the Rebers and Riddiford motif (CPR) that contain a core 28 amino acid sequence that is now recognized as part of a larger 63 amino acid consensus sequence (pfam00379) (Rebers and Riddiford, 1988; Willis et al., 2012). Variations in the extended consensus have been recognized, and CPs are often classified into one of three sub types: RR-1, RR-2, and RR-3. The number of CPR genes can vary greatly among species but many insects have more than 100 (Ioannidou et al., 2014). Additional groups having conserved sequence motifs include CPs analogous to peritrophins (CPAP) (Jasrapuria et al., 2010), CPs with a 44 amino acid motif (CPF) (Andersen et al., 1997; Togawa et al., 2007), CPF-like (CPFL) (Togawa et al., 2007), CPs with a Tweedle motif (CPT) (Guan et al., 2006), CPs with two or three repeats of C-X5-C motif (CPCFC) (Jensen et al., 1997; Willis et al., 2012), and CPs with an 18 amino acid motif (Andersen, 2000; Nakato et al., 1990). Additionally, low-complexity proteins can be found in the cuticle that are rich in glycine or contain repeats of AAP(A/V), P(V/Y), GYGL, or GLLG (Willis et al., 2012). However, since these proteins lack any other distinctive sequences, presence of these repeats alone is not proof enough of their location in the cuticle. Excellent reviews on CPs can be found in Willis (2010) and Willis et al, (2012).

The goal of this research was to annotate the CP genes in the genome of the tobacco hornworm, Manduca sexta, with respect to the groups described above. These seven groups (CPR, CPAP, CPF, CPFL, CPT, CPCFC, and 18 aa motif) were chosen as they all contain conserved sequences previously shown to be present in known cuticular structural proteins and, therefore, can serve as a diagnostic feature. We compared the CP genes in M. sexta with those of Bombyx mori (Futahashi et al., 2008) in order to identify orthologs. Finally, we utilized 52 RNAseq libraries prepared from various tissues and developmental stages to look for patterns of coordinated expression among the CP genes. This analysis offers new insights into the CPs present in cuticle synthesized at different times and developmental stages.

2. Materials and Methods

2.1 CP gene annotation

To identify putative CP genes, the M. sexta genome and first official gene set (OGS1), available from the Agricultural Pest Genomics Resource Database (www.agripestbase.org), were searched by tBLASTn (Altschul et al., 1997) with the following sequences: for the CPR family, the consensus sequence GxFxYxxPDGxxxxVxYxADENGYQPxGAHLP was used to identify the RR-1 subtype, and EYDAxPxYxFxYxVxDxHTGDxKSQxExRDGDVVxGxYSLxExDGxxRTVxYTADxxNGFNAVVxxE was used to identify the RR-2 subtype (Figure 3 in Willis et al., 2005); classification into the appropriate subfamily was confirmed by the use of a profile hidden Markov model that discriminates between the two subtypes, available at the cuticleDB website (biophysics.biol.uoa.gr/cuticleDB/, Karouzou et al., 2007). For the CPAP family, the Tribolium castaneum CPAP1-D (GenBank ACY95469) and CPAP3-A1 (GenBank ACY95475) sequences were used to identify CPAP family members. CPF genes were identified using the most highly conserved portion of the 44 amino acid motif, VSxYSKAVDTPFSSVRKxDxRIVNxA, (derived from Figure 1B in Togawa et al., 2007). CPFL genes were identified with the sequence LxYSAAPAVSHVAYxGxGxxYGW (derived from Figure 3 in Togawa et al., 2007). For Tweedle genes, the 100 amino acid weblogo sequence from Figure 3A in Willis (2010) was used to identify homologs. The sequence YPAGVNPAACPNYPYCD was used to identify members of the CPCFC family, and PVDTPEVAAAKAAHFAAH was used to identify genes encoding CPs with the 18 amino acid motif (Figure 4C and 4B in Willis, 2010).

For all genes except those of the CPAP family, names were assigned based on putative orthology to B. mori homologs. Reciprocal BLAST was performed to confirm that the B. mori protein identified as the top hit to a M. sexta query identified the same M. sexta protein as the top hit when it was used as the query sequence. Orthology was further established through the use of microsynteny. When the genes flanking a CP gene were homologous between B. mori and M. sexta, the CP genes were considered to be orthologous; identification of genes surrounding B. mori CP genes was inferred from the Gene Report page at NCBI associated with that particular CP gene. Because the CPAP genes in B. mori have not been annotated yet, naming of the M. sexta CPAP genes was based on a phylogenetic analysis of homologous proteins from several insect species; a detailed description of this analysis can be found in Tetreau et al. (2014, this issue).

2.2 Phylogenetic analysis

Phylogenetic analysis was performed using the corresponding protein sequence of selected genes from the CPR, CPAP, and CPFL groups. Details of the CPAP analysis can be found in a companion paper (Tetreau et al., 2014, this issue). Analysis was performed using MEGA software (v5.2.1; Tamura et al., 2011). Sequences were aligned globally using the ClustalW program in MEGA and then adjusted manually by eye. For the CPR group, the extended RR domain (pfam00379) was used; RR-1 and RR-2 subgroups were treated separately. For the CPFL group, the entire sequence of the M. sexta and B. mori proteins were used. Trees were constructed by the neighbor-joining method with a Poisson correction model. Gaps were treated by the pairwise deletion method and statistical analysis was performed by the bootstrap method using 1000 repetitions.

2.3 CP gene expression

Fifty-two RNAseq libraries had been prepared from various tissues and developmental stages as part of the Manduca Genome Sequencing Project (GenBank assembly accession number GCA_000262585.1). The libraries were sequenced by Illumina technology either at the Weill Cornel Medical College or the Baylor College of Medicine Human Genome Sequencing Center. The RNAseq reads were trimmed to 50 base pair and mapped onto the predicted open reading frame of the CP genes. Transcript abundance was determined by the FPKM method (fragments per kilobase of transcript per million fragments mapped). Read mapping and determination of FPKM values were performed using the RSEM software package (v1.2.12) (Li and Dewey, 2011). (The expression data for all genes in the M. sexta OGS2 can be downloaded at ftp://ftp.bioinformatics.ksu.edu/pub/Manduca/OGS2/OSU_files/, file 20140508RSEM_OGS2_Gene_FPKM.xlsx.) The FPKM values ranged from 0 to greater than 10,000 and gene expression was grouped as follows: less than 1, no expression; 1 – 10, very low expression; 10 – 100, low expression; 100 – 1,000, moderate expression; 1,000 – 10,000, high expression; greater than 10,000, very high expression. Hierarchical clustering of CP gene expression was performed using MultiExperiment Viewer (v4.9) (Saeed et al., 2003) with the Pearson correlation-based metric and average linkage clustering method.

3. Results and Discussion

3.1.1 CP gene annotation

Our analysis of OGS1 identified 206 gene models that belong to seven different CP groups (CPR, CPAP, CPF, CPFL, CPT, CPCFC, 18 aa motif). However, upon annotation of these models it became apparent that 28 of them likely represented 44 genes that had been incorrectly spliced. An additional 26 genes, for which no OGS1 gene model was predicted, were identified by searching the M. sexta genome sequence. These findings were incorporated during the development of OGS2 and increased the total number of gene models to 276 (Table 1). Some of these models are likely allelic variants of the same gene. For example, Msex2.14790 encodes a partial CPR gene that is 98% identical at the nucleotide level, and 100% identical at the amino acid level, to Msex2.11885 (MsCPR38). Furthermore, Msex2.14790 is on a small scaffold of ∼2000 nucleotides which is 94% identical at the nucleotide level to position 14,274 - 16,027 of scaffold00529, where Msex2.11885 resides. Thus it was judged to be an allelic variant of Msex2.11885.

Table 1. Manduca structural cuticle protein genes.
OGS2 Gene ID Gene Name a Family Scaffold No. b
Msex2.07923 MsCPR1 CPR-RR1 scaffold00208
Msex2.00114 MsCPR154 (LCP14) CPR-RR1 scaffold00003
Msex2.00115 MsCPR155 CPR-RR1 scaffold00003
Msex2.00116 MsCPR156 CPR-RR1 scaffold00003
Msex2.00117 MsCPR2 CPR-RR1 scaffold00003
Msex2.00118 MsCPR3 CPR-RR1 scaffold00003
Msex2.09274 MsCPR4 (CP14.6) CPR-RR1 scaffold00267
Msex2.09273 MsCPR5 CPR-RR1 scaffold00267
Msex2.11639 MsCPR6 CPR-RR1 scaffold00482
Msex2.05318 MsCPR7 CPR-RR1 scaffold00107
Msex2.05319 MsCPR8 CPR-RR1 scaffold00107
Msex2.05320 MsCPR9 (CP20) CPR-RR1 scaffold00107
Msex2.05189 MsCPR10 CPR-RR1 scaffold00106
Msex2.08400 MsCPR11 CPR-RR1 scaffold00224
Msex2.08401 MsCPR12 CPR-RR1 scaffold00224
Msex2.08402 MsCPR13 CPR-RR1 scaffold00224
Msex2.08403 MsCPR174 CPR-RR1 scaffold00224
Msex2.08404 MsCPR175 CPR-RR1 scaffold00224
Msex2.08405 MsCPR176 CPR-RR1 scaffold00224
Msex2.08406 MsCPR177 CPR-RR1 scaffold00224
Msex2.08407 MsCPR14 CPR-RR1 scaffold00224
Msex2.08408 MsCPR15 (CP36) CPR-RR1 scaffold00224
Msex2.08409 MsCPR16 (CP27) CPR-RR1 scaffold00224
Msex2.08410 MsCPR17 CPR-RR1 scaffold00224
Msex2.08411 MsCPR18 CPR-RR1 scaffold00224
Msex2.08412 MsCPR19 CPR-RR1 scaffold00224
Msex2.08413 MsCPR52 CPR-RR1 scaffold00224
Msex2.00741 MsCPR20 CPR-RR1 scaffold00009
Msex2.00742 MsCPR21 CPR-RR1 scaffold00009
Msex2.04086 MsCPR157 CPR-RR1 scaffold00073
Msex2.04415 MsCPR158 CPR-RR1 scaffold00081
Msex2.04416 MsCPR22 CPR-RR1 scaffold00081
Msex2.04417 MsCPR23 CPR-RR1 scaffold00081
Msex2.04418 MsCPR24 CPR-RR1 scaffold00081
Msex2.04419 MsCPR25 CPR-RR1 scaffold00081
Msex2.04420 MsCPR159 CPR-RR1 scaffold00081
Msex2.04424 MsCPR26 CPR-RR1 scaffold00081
Msex2.04425 MsCPR30 CPR-RR1 scaffold00081
Msex2.04427 MsCPR160 CPR-RR1 scaffold00081
Msex2.04428 MsCPR27 CPR-RR1 scaffold00081
Msex2.04429, Msex2.14415 MsCPR28 CPR-RR1 scaffold00081, scaffold01804
Msex2.11897 MsCPR182 CPR-RR1 scaffold00529
Msex2.11896 MsCPR181 CPR-RR1 scaffold00529
Msex2.11895 MsCPR180 CPR-RR1 scaffold00529
Msex2.11894 MsCPR179 CPR-RR1 scaffold00529
Msex2.11892 MsCPR178 CPR-RR1 scaffold00529
Msex2.11891 MsCPR32 CPR-RR1 scaffold00529
Msex2.11890 MsCPR33 CPR-RR1 scaffold00529
Msex2.11889 MsCPR34 CPR-RR1 scaffold00529
Msex2.11888 MsCPR35 CPR-RR1 scaffold00529
Msex2.11887 MsCPR36 CPR-RR1 scaffold00529
Msex2.11886 MsCPR37 CPR-RR1 scaffold00529
Msex2.11885, Msex2.14790 MsCPR38 CPR-RR1 scaffold00529, scaffold03863
Msex2.11884, Msex2.14482 MsCPR39 CPR-RR1 scaffold00529, scaffold02011
Msex2.14550, Msex2.11883 MsCPR40 CPR-RR1 scaffold02427, scaffold0529
Msex2.06320 MsCPR161 CPR-RR1 scaffold00136
Msex2.06321 graphic file with name nihms654268ig1.jpg CPR-RR1 scaffold00136
Msex2.06322 MsCPR42 CPR-RR1 scaffold00136
Msex2.06323 MsCPR43 CPR-RR1 scaffold00136
Msex2.06324 MsCPR44 CPR-RR1 scaffold00136
Msex2.06325 MsCPR45 CPR-RR1 scaffold00136
Msex2.06326 MsCPR162 CPR-RR1 scaffold00136
Msex2.06327 MsCPR163 CPR-RR1 scaffold00136
Msex2.06328 MsCPR164 CPR-RR1 scaffold00136
Msex2.06329 MsCPR165 CPR-RR1 scaffold00136
Msex2.06330 MsCPR166 CPR-RR1 scaffold00136
Msex2.06331 MsCPR167 (LCP16/17) CPR-RR1 scaffold00136
Msex2.06332 MsCPR168 CPR-RR1 scaffold00136
Msex2.06333 MsCPR169 CPR-RR1 scaffold00136
Msex2.06334 MsCPR170 CPR-RR1 scaffold00136
Msex2.06335 MsCPR171 CPR-RR1 scaffold00136
Msex2.06336 MsCPR46 CPR-RR1 scaffold00136
Msex2.13066 MsCPR47 CPR-RR1 scaffold00793
Msex2.08425 MsCPR48 CPR-RR1 scaffold00221
Msex2.11013 MsCPR49 CPR-RR1 scaffold00396
Msex2.13464 MsCPR51 CPR-RR1 scaffold01003
Msex2.06407 MsCPR54 CPR-RR1 scaffold00137
Msex2.06406 MsCPR55 CPR-RR1 scaffold00137
Msex2.04351 MsCPR56 CPR-RR1 scaffold00079
Msex2.00132 MsCPH5 CPR-RR2 scaffold00003
Msex2.02008 MsCPR57 CPR-RR2 scaffold00029
Msex2.00295 MsCPR58 CPR-RR2 scaffold00006
Msex2.00296 MsCPR59 CPR-RR2 scaffold00006
Msex2.00297 MsCPR61 CPR-RR2 scaffold00006
Msex2.00298 MsCPR62 CPR-RR2 scaffold00006
Msex2.00299 MsCPR63 CPR-RR2 scaffold00006
Msex2.00301 MsCPR64 CPR-RR2 scaffold00006
Msex2.00302 MsCPR65 CPR-RR2 scaffold00006
Msex2.00303 MsCPR134 CPR-RR2 scaffold00006
Msex2.09232 MsCPR66 CPR-RR2 scaffold00266
Msex2.08273 MsCPR67 CPR-RR2 scaffold00227
Msex2.08276 MsCPR172 CPR-RR2 scaffold00227
Msex2.08279 MsCPR173 CPR-RR2 scaffold00227
Msex2.08274 MsCPR68 CPR-RR2 scaffold00227
Msex2.08275 MsCPR69 CPR-RR2 scaffold00227
Msex2.08278 MsCPR70 CPR-RR2 scaffold00227
Msex2.08280 MsCPR71 CPR-RR2 scaffold00227
Msex2.08282 MsCPR72 CPR-RR2 scaffold00227
Msex2.08283 MsCPR73 CPR-RR2 scaffold00227
Msex2.05356 MsCPR74 CPR-RR2 scaffold00108
Msex2.04619 MsCPR76 CPR-RR2 scaffold00086
Msex2.04620 MsCPR77 CPR-RR2 scaffold00086
Msex2.03062 MsCPR79 CPR-RR2 scaffold00054
Msex2.03061 MsCPR80 CPR-RR2 scaffold00054
Msex2.15511 MsCPR153 c CPR-RR2 scaffold00054
Msex2.03060 MsCPR152 c CPR-RR2 scaffold00054
Msex2.12550 MsCPR82 CPR-RR2 scaffold00685
Msex2.12551 MsCPR83 CPR-RR2 scaffold00685
Msex2.12552 MsCPR84 CPR-RR2 scaffold00685
Msex2.12553 MsCPR183 CPR-RR2 scaffold00685
Msex2.12554 MsCPR184 CPR-RR2 scaffold00685
Msex2.12555, Msex2.14741 MsCPR185 CPR-RR2 scaffold00685, scaffold03458
Msex2.15538 MsCPR186 CPR-RR2 scaffold00685
Msex2.15539 MsCPR187 CPR-RR2 scaffold00685
Msex2.12556 MsCPR188 CPR-RR2 scaffold00685
Msex2.12557, Msex2.14630 MsCPR189 CPR-RR2 scaffold00685, scaffold02706
Msex2.12558 MsCPR190 CPR-RR2 scaffold00685
Msex2.12559 MsCPR191 CPR-RR2 scaffold00685
Msex2.12560 MsCPR192 CPR-RR2 scaffold00685
Msex2.12561 MsCPR193 CPR-RR2 scaffold00685
Msex2.12562 MsCPR90 CPR-RR2 scaffold00685
Msex2.12563 MsCPR91 CPR-RR2 scaffold00685
Msex2.12837 MsCPR214 CPR-RR2 scaffold00717
Msex2.15541 MsCPR213 CPR-RR2 scaffold00717
Msex2.12836 MsCPR212 CPR-RR2 scaffold00717
Msex2.12835 MsCPR211 CPR-RR2 scaffold00717
Msex2.12834 MsCPR210 CPR-RR2 scaffold00717
Msex2.12833 MsCPR209 CPR-RR2 scaffold00717
Msex2.12832, Msex2.14053 MsCPR208 CPR-RR2 scaffold00717, scaffold01268
Msex2.12831, Msex2.14054 MsCPR207 CPR-RR2 scaffold00717, scaffold01268
Msex2.12830, Msex2.14055 MsCPR206 CPR-RR2 scaffold00717, scaffold01268
Msex2.12829, Msex2.14056 MsCPR205 CPR-RR2 scaffold00717, scaffold01268
Msex2.12828, Msex2.14057 MsCPR204 CPR-RR2 scaffold00717, scaffold01268
Msex2.12827, Msex2.14058, Msex2.14910 MsCPR203 CPR-RR2 scaffold00717, scaffold01268, scaffold04887
Msex2.12826 MsCPR202 CPR-RR2 scaffold00717
Msex2.12825 MsCPR201 CPR-RR2 scaffold00717
Msex2.12824 MsCPR200 CPR-RR2 scaffold00717
Msex2.12823 MsCPR199 CPR-RR2 scaffold00717
Msex2.12822 MsCPR198 CPR-RR2 scaffold00717
Msex2.12821 MsCPR197 CPR-RR2 scaffold00717
Msex2.15540 MsCPR196 CPR-RR2 scaffold00717
Msex2.12820 MsCPR195 CPR-RR2 scaffold00717
Msex2.12819 MsCPR194 CPR-RR2 scaffold00717
Msex2.12818 MsCPR121 CPR-RR2 scaffold00717
Msex2.12816 MsCPR122 CPR-RR2 scaffold00717
Msex2.12817 MsCPR123 CPR-RR2 scaffold00717
Msex2.12815 MsCPR124 CPR-RR2 scaffold00717
Msex2.12814 MsCPR150 CPR-RR2 scaffold00717
Msex2.12194 MsCPR125 CPR-RR2 scaffold00562
Msex2.12193 MsCPR126 CPR-RR2 scaffold00562
Msex2.11250 MsCPR128 CPR-RR2 scaffold00476
Msex2.11252 MsCPR129 CPR-RR2 scaffold00476
Msex2.11256 MsCPR130 CPR-RR2 scaffold00476
Msex2.11351 MsCPR131 CPR-RR2 scaffold00447
Msex2.13191, Msex2.14515 MsCPR132 CPR-RR2 scaffold00893, scaffold02588
Msex2.01496 MsCPR133 CPR-RR2 scaffold00024
Msex2.08525 MsCPR135 CPR-RR2 scaffold00228
Msex2.08526 MsCPR136 CPR-RR2 scaffold00228
Msex2.08527 MsCPR137 CPR-RR2 scaffold00228
Msex2.08354 MsCPR140 (Pro-resilin) CPR-RR2 scaffold00240
Msex2.04385 MsCPR141 CPR-RR2 scaffold00081
Msex2.09926 MsCPR142 CPR-RR2 scaffold00325
Msex2.11692 MsCPR143 CPR-RR2 scaffold00565
Msex2.12993 MsCPR144 CPR-RR2 scaffold00779
Msex2.12994 MsCPR145 CPR-RR2 scaffold00779
Msex2.09922 MsCPR151 CPR-RR2 scaffold00325
Msex2.13636, Msex2.14049 MsCPR215 CPR-RR2 scaffold01004, scaffold01304
Msex2.13637, Msex2.14048 MsCPR216 CPR-RR2 scaffold01004, scaffold01304
Msex2.13638 MsCPR217 CPR-RR2 scaffold01004
Msex2.13639, Msex2.15551 MsCPR218 CPR-RR2 scaffold01004, scaffold01304
Msex2.13640, Msex2.14047 MsCPR219 CPR-RR2 scaffold01004, scaffold01304
Msex2.13641, Msex2.14046 MsCPR220 CPR-RR2 scaffold01004, scaffold01304
Msex2.13643 MsCPR221 CPR-RR2 scaffold01004
Msex2.13644 MsCPR222 CPR-RR2 scaffold01004
Msex2.13645A MsCPR223 CPR-RR2 scaffold01004
Msex2.13645C MsCPR224 CPR-RR2 scaffold01004
Msex2.13656, Msex2.14579 MsCPR225 CPR-RR2 scaffold01013, scaffold02441
Msex2.13657, Msex2.15550 MsCPR226 CPR-RR2 scaffold01013, scaffold02441
Msex2.13659 MsCPR227 CPR-RR2 scaffold01013
Msex2.13660 MsCPR228 CPR-RR2 scaffold01013
Msex2.13661 MsCPR229 CPR-RR2 scaffold01013
Msex2.13662 MsCPR230 CPR-RR2 scaffold01013
Msex2.13663 MsCPR231 CPR-RR2 scaffold01013
Msex2.13664 MsCPR232 CPR-RR2 scaffold01013
Msex2.13665 MsCPR233 CPR-RR2 scaffold01013
Msex2.13666 MsCPR234 CPR-RR2 scaffold01013
Msex2.15544 MsCPR235 CPR-RR2 scaffold01013
Msex2.13667 MsCPR236 CPR-RR2 scaffold01013
Msex2.13668 MsCPR237 CPR-RR2 scaffold01013
Msex2.13669 MsCPR238 CPR-RR2 scaffold01013
Msex2.13670 MsCPR239 CPR-RR2 scaffold01013
Msex2.13671 MsCPR240 CPR-RR2 scaffold01013
Msex2.13672 MsCPR241 CPR-RR2 scaffold01013
Msex2.13814 MsCPR242 CPR-RR2 scaffold01084
Msex2.14031 MsCPR243 CPR-RR2 scaffold01333
Msex2.14032, Msex2.14432 MsCPR244 CPR-RR2 scaffold01333, scaffold01931
Msex2.14033, Msex2.15549 MsCPR245 CPR-RR2 scaffold01333, scaffold01931
Msex2.14385 MsCPR246 CPR-RR2 scaffold01713
Msex2.14386 MsCPR247 CPR-RR2 scaffold01713
Msex2.14960 MsCPR248 CPR-RR2 scaffold05463
Msex2.15340 MsCPR249 CPR-RR2 scaffold12390
Msex2.15463 MsCPR250 CPR-RR2 scaffold17868
Msex2.06360 MsCPR146 CPR-RR3 scaffold00136
Msex2.11251 MsCPR147 CPR-RR3 scaffold00476
Msex2.07106 MsCPR148 CPR-RR3 scaffold00167
Msex2.04414 MsCPR149 CPR-RR3 scaffold00081
Msex2.13160, Msex2.11877 MsCPAP1-A CPAP1 scaffold00817, scaffold00519
Msex2.00613 MsCPAP1-B CPAP1 scaffold00007
Msex2.00614 MsCPAP1-B2 CPAP1 scaffold00007
Msex2.00381 MsCPAP1-C CPAP1 scaffold00004
Msex2.03859 MsCPAP1-D CPAP1 scaffold00064
Msex2.02135 MsCPAP1-F CPAP1 scaffold00032
Msex2.00269 MsCPAP1-G CPAP1 scaffold00006
Msex2.02134 MsCPAP1-H CPAP1 scaffold00032
Msex2.03076 MsCPAP1-I CPAP1 scaffold00054
Msex2.03236 MsCPAP1-J CPAP1 scaffold00052
Msex2.01703 MsCPAP1-K CPAP1 scaffold00025
Msex2.09867 MsCPAP1-L CPAP1 scaffold00311
Msex2.03103 MsCPAP1-M CPAP1 scaffold00054
Msex2.02137 MsCPAP1-N CPAP1 scaffold00032
Msex2.08722 MsCPAP1-O CPAP1 scaffold00236
Msex2.08805 MsCPAP3-A1 CPAP3 scaffold00255
Msex2.08806 MsCPAP3-A2 CPAP3 scaffold00255
Msex2.08808, Msex2.15015, Msex2.14185 MsCPAP3-B CPAP3 scaffold00255, scaffold06133, scaffold01361
Msex2.08810 MsCPAP3-C CPAP3 scaffold00255
Msex2.08807, Msex2.14226 MsCPAP3-D1 CPAP3 scaffold00255, scaffold01383
Msex2.04890 MsCPAP3-D2 CPAP3 scaffold00101
Msex2.03293 MsCPAP3-E CPAP3 scaffold00068
Msex2.03294, Msex2.15120 MsCPAP3-E2 CPAP3 scaffold00068, scaffold07708
Msex2.03295 MsCPAP3-E3 CPAP3 scaffold00068
Msex2.14886 MsCPAP3-E4 CPAP3 scaffold04686
Msex2.06560 MsCPT1 Tweedle scaffold00143
Msex2.04779 MsCPT2 Tweedle scaffold00091
Msex2.00101 MsCPT3 Tweedle scaffold00003
Msex2.09186 MsCPT4 Tweedle scaffold00268
Msex2.03833 MsCPH1 CPCFC scaffold00064
Msex2.05601 MsCPF1 CPF scaffold00116
Msex2.02569 MsCPFL1 CPFL scaffold00039
Msex2.04027 MsCPFL2 CPFL scaffold00070
Msex2.04028 MsCPFL3 CPFL scaffold00070
Msex2.04036 MsCPFL4 CPFL scaffold00070
Msex2.04037 MsCPFL5 CPFL scaffold00070
Msex2.04035 MsCPFL6 CPFL scaffold00070
Msex2.01072 MsCPH15 18 aa scaffold00013
Msex2.01073 MsCPH16 18 aa scaffold00013
Msex2.03612 MsCPH30 18 aa scaffold00059
Msex2.03635 MsCPH31 18 aa scaffold00059
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a

Gene names were assigned based on putative orthology to B. mori. Red type indicates genes in which one to one orthology could not be clearly established and, therefore, new gene numbers were assigned (MsCPR154-250). Blue type indicates genes previously deposited in GenBank (names given in parentheses). Genes boxed in gray identify expansion in M. sexta (putative paralogs) for which only a single ortholog occurs in B. mori.

b

Scaffolds boxed in yellow or orange contain clusters of five or more CP genes.

c

Genes MsCPR152 and 153 are orthologous to B. mori LOC101743422.

Additionally, some CP genes were also split between multiple gene models due to gaps in the genome assembly (supplementary file 1). This was evident for MsCPAP3-B. Both Msex2.08808 and 15015 show high sequence identity at the amino acid level (80% and 96% respectively) with obstructor-B (i.e. CPAP3-B) from Papilio xuthus (GenBank BAM17933). Interestingly, Msex2.08808's shared identity with PxObst-B covers amino acids 1-110 and 245-291, while Msex2.15015 aligns with residues 113-242. An examination of the genomic sequence for Msex2.08808 reveals a gap between exons 3 and 4 which likely contains the missing exons encoding residues 111-244 present in Msex2.15015. Thus, the correct sequence for MsCPAP3-B is a composite of Msex2.08808 and Msex2.15015. This conclusion is supported by the RNAseq data assembled by the Trinity software package which predicts a 1.24 kb transcript encoding a 296 amino acid protein that is the same as the composite sequence and 87% identical to PxObst-B (data not shown). (Msex2.14185 encodes for a protein that is 99% identical at both the amino acid and nucleotide levels with Msex2.15015 and therefore was judged to be an allele.) After accounting for multiple gene models representing the same gene, we identified 248 distinct CP genes in M. sexta.

3.1.2 CPR genes

Two-hundred and seven of the annotated genes belong to the CPR family: 79 RR-1, 124 RR-2, and 4 RR-3 (Table 1). This represents the largest number of CPR genes in any insect genome annotated to date and is 36% greater than the number of CPR genes in the silkworm B. mori: 56 RR-1, 93 RR-2, and 4 RR-3; 149 were originally annotated by Futahashi et al. (2008) including the misclassified CPH5, three more were added by Liang et al. (2010), and one additional gene, LOC101743422, was discovered in our analysis. Similar to observations for other insects (Cornman et al., 2008; Dittmer et al., 2012; Gallot et al., 2010; Liang et al., 2010), many of the genes occurred in clusters: 55 of the RR-1 genes were found in clusters of 11-17 genes on four scaffolds (00081, 00136, 00224, 00529), while 86 of the RR-2 genes were found in clusters of 8-26 genes on six scaffolds (00006, 00227, 00685, 00717, 01004, 01013) (Table 1).

As automated annotation is being carried out on several sequenced lepidopteran genomes, B. mori genes are frequently the top hit resulting in some continuity in gene naming among the lepidoptera. We endeavored to continue this practice by naming the M. sexta CP genes based on their deduced orthology to B. mori as determined by sequence identity, phylogenetic analysis, and microsynteny. For some of the M. sexta genes no clear orthology with B. mori could be established. For others, gene duplication resulted in multiple M. sexta genes being orthologous to a single B. mori gene. In these cases, new CPR numbers were assigned that begin with CPR152. Therefore, the CPR gene numbering went as high as CPR250 even though there are only 207 genes. Nevertheless, we believe that those numbered CPR1 - 151 are orthologous to the B. mori gene with the same name.

We were able to assign orthology to 52 of the 56 B. mori RR-1 genes; only BmCPR29, 31, 50, and 53 did not have clear orthologs in M. sexta. (BmCPR50 and 53 may be orthologs of Msex2.04420 and 06326 but this could not be confirmed by synteny, therefore, the M. sexta genes were given the names CPR159 and 162). Sixteen of the additional RR-1 genes in M. sexta likely arose through duplication of just four genes: MsCPR2, 13, 41, and 46 (boxed in grey in Table 1). For example, in B. mori the five genes CPR41-46 are flanked by CPG21 on one end and ORC3 and T-related protein-like on the other (Figure 1). These same three genes in M. sexta flank a cluster of 17 CPR genes on scaffold00136. BLAST results and phylogenetic analysis indicate that Msex2.06320 and 06321 are co-orthologs of BmCPR41, Msex2.06322 – 06325 are orthologous to BmCPR42-45, and Msex2.06327 – 06336 are all most closely related to BmCPR46. As no ortholog could be identified by sequence similarity or phylogenetic analysis, Msex2.06336 was given the name MsCPR46 because of its location and orientation with respect to ORC3 and T-related protein-like. Similar analysis indicates that Msex2.00115 and 00116 are paralogs of Msex2.00117 (MsCPR2), and Msex2.08403 – 08406 are paralogs of Msex2.08402 (MsCPR13).

Figure 1. Evidence of gene duplication in M. sexta.

Figure 1

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(A) Alignment of M. sexta genes (green arrows) on scaffold00136 with the corresponding region in B. mori chromosome 22 (blue arrows). Filled arrows represent CPR genes while open arrows represent non-CPR genes; the arrows are only meant to show gene order and orientation and are not representative of gene size or distances between genes. The dashed red line denotes putative orthologs. (B) Phylogenetic analysis of the CPR genes shown in A. All branches with a bootstrap value less than 50 were collapsed.

Determining orthology among the RR-2 genes was more difficult as only 51 of the 93 RR-2 genes in B. mori had clear one- to-one orthologs in M. sexta. Interestingly, orthology could be established to most of the B. mori genes from CPR57 – 84 and 121 – 145; it was only for CPR85 – 120 (with the exception of 90 and 91) that direct orthology could not be conclusively resolved, although groups of relatedness were apparent. Phylogenetic analysis showed that 11 genes clustered on scaffold00685 (Msex2.12553 – 12561, 15538, 15539, which we have named MsCPR183 – 193) were most similar to BmCPR85 – 89 (Figure S1). The common node for these two groups suggest that they arose from a common ancestral gene and that duplication after speciation gave rise to additional genes. This conclusion is further supported by the location of MsCPR183 – 193 between genes MsCPR84 (Msex2.12552) and MsCPR90 (Msex2.12562) on scaffold00685, thus making it likely that they would be related to BmCPR85 – 89. Similar homologous groupings are shown in Table 2 and Figure S2.

Table 2. Orthologous groups of CPR RR-2 genes between B. mori and M. sexta a.
Group No. B. mori M. sexta b
1 CPR85, 86, 87, 88, 89 CPR183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 246, 247
2 CPR95, 96, 97, 98, 99, 100 CPR215, 216, 217, 218, 219, 220, 244, 245, 248
3 CPR101, 102, 103, 104, 105 CPR221, 222, 223, 224, 240, 241
4 CPR107, 109, 111, 120 CPR195, 196, 197, 199, 201, 207, 209, 225, 227, 229, 232
5 CPR106, 108, 110 CPR211, 213
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a

See supplementary figure S2 for phylogenetic analysis

b

See Table 1 for OGS2 Gene ID

Four RR-3 genes were identified, Msex2.06360, 11251, 07106, and 04414, that all have orthologs in B.mori (CPR146 – 149). BmCPR149 was originally annotated as encoding an RR-2 protein (GenBank ACY06906), however, we have classified the M.sexta gene as an RR-3 based on greater sequence identity with the RR-3 consensus sequence (Andersen, 2000; data not shown). MsCPR149 is unique in that it encodes a protein with two RR domains, one each at the N- and C-termini of the protein; this model is supported by the RNAseq data suggesting that it is not an error in the gene assembly resulting from the fusion of two adjacent genes. It is similar to CPR58 of Nansonia vitripennis (GenBank XP_001601627) which has a comparable domain architecture (Willis, 2010).

3.1.3 CPAP genes

The second largest group of CP genes annotated was the CPAPs. CPAPs are classified according to the number of peritrophin A-type chitin binding domains they contain, with CPAP1 proteins containing one domain and CPAP3 proteins containing three domains (Jasrapuria et al., 2010). The CPAP3 genes were first recognized in D. melanogaster and alternately named gasp (gene analogous to small peritrophins) and obstructor (Barry et al., 1999; Behr and Hoch, 2005). This family of genes was further divided into two groups consisting of obstructor-A – E, and F – J. Genes orthologous to obst-A – E have been identified in other insects while obst-F – J have only been described for D. melanogaster. The related CPAP1 genes were identified and characterized by Jasrapuria et al. (2010) and contained ten family members named CPAP1-A – J.

We identified 15 putative CPAP1 genes and 10 CPAP3 genes (Table 1). This is greater than the 10 CPAP1 and 7 CPAP3 genes described from T. castaneum for which the most extensive annotation of this gene family has been carried out (Jasrapuria et al., 2010). Since similar work had not yet been performed for B. mori, we named the M. sexta genes based on orthology to T. castaneum and homologous genes in other insects. This analysis is described in a companion paper detailing the different groups of proteins containing peritrophin A-type chitin-binding domains in M. sexta (Tetreau et al., 2014, this issue) but will be summarized here. M. sexta has orthologs to 9 of the 10 CPAP1 genes in T. castaneum with 1-E being the exception. The six additional CPAP1 genes in M. sexta all have orthologs in other insect species and we have named these CPAP1-B2 and 1-K – O. M. sexta has orthologs to all seven of the CPAP3 genes in T. castaneum, and phylogenetic analysis shows that the three additional genes cluster with CPAP3-E and, therefore, we have named them E2 – E4; it should be noted that MsCPAP3-E2 (Msex2.03294) is not orthologous to Obst-E2 of D. melanogaster as the latter is a spliced isoform of the Obst-E gene while the former is a separate gene distinct from MsCPAP3-E1 (Msex2.03293). Similar to what Jasrapuria et al. (2010) reported for T. castaneum, the CPAP3-C ortholog of M. sexta (Msex2.08810) has the potential for alternative splicing of exon 5, giving rise to two different proteins. Eight of the ten CPAP3 genes are present in tandem arrays on two scaffolds: A1, A2, D1, B, and C on scaffold00255 (with one gene between B and C), and E1 – E3 on scaffold00068. The CPAP1 genes don't exhibit the same clustering characteristic although B1 and B2 are adjacent on scaffold00007, while H, F, and N are grouped on scaffold00032 (with one gene between F and N) (Table 1).

In general, very little annotation of these genes has been carried out for insect genomes. Recently, Ioannidou and co-workers (2014) developed profile hidden Markov models to identify CPAP and other cuticle structural proteins from sequenced genomes and applied this analysis to 14 arthropod genomes (12 insect and 2 crustacean). The number of putative CPAP genes identified ranged from 10 to 20 for CPAP1 and from 5 to 12 for CPAP3 (supplementary file 2 in Ioannidou et al., 2014). Combining our analysis of M. sexta with their analysis of several insect genomes allowed us to assign orthology to the CPAP genes from eight insect species (Table 3). The CPAP3 genes had the highest degree of conservation, with Acyrthosiphon pisum, Apis mellifera, B. mori, M. sexta, and T. castaneum all having orthologs to the seven described genes. Anopheles gambiae and P. humanus had orthologs to six of these genes with both lacking an ortholog to CPAP3-A2, although A. gambiae appears to have two duplications of CPAP3-A1 and P. humanus a duplication of CPAP3-D1 (Table 3). Interestingly, we observed that the gene model PHUM434540 appears to be a fusion of two genes with the 5-prime half orthologous to CPAP3-B and the 3-prime half orthologs to CPAP3-A1. As detailed by Behr and Hoch (2005), D. melanogaster has five CPAP3 (a.k.a. obstructor) genes, lacking orthologs to CPAP3-A2 and CPAP3-D2.

Table 3. CPAP gene orthologs from eight insect species.
Gene A. pisum A. gambiae A. melifera B. mori D. melanogaster M. sexta P. humanus T. castaneum
CPAP1-A ACYPI45536 AGAP001203 GB51442 LOC101737637 CG32036 Msex2.13160 PHUM600940 TC004733
CPAP1-B ACYPI001105 AGAP009480 GB44831 LOC101741926 CG14301 Msex2.00613 TC000587
CPAP1-B2 ACYPI007439 AGAP009479 GB44832 LOC101743233 CG14304 Msex2.00614 PHUM376120 TC000588
CPAP1-C AGAP003751 GB42318 LOC101740411 CG14880 Msex2.00381 TC000316
CPAP1-D LOC101744246 Msex2.03859 TC009263
CPAP1-E GB41618 CG14959 PHUM034260 TC009887
CPAP1-F AGAP006435 GB41624 LOC101746039 CG13675 Msex2.02135 PHUM034480 TC009893
CPAP1-G AGAP007613 GB49021 LOC101737536 CG8192 Msex2.00269 PHUM071270 TC008877
CPAP1-H ACYPI004632 AGAP005489 GB41625 LOC101740962 CG13676 Msex2.02134 PHUM263920 TC009894
CPAP1-I ACYPI009675 AGAP002052 GB41792 LOC101742705 CG13643 Msex2.03076 PHUM575010 TC012766
CPAP1-J ACYPI000845 AGAP010302 GB54921 LOC101747065 CG14608 Msex2.03236 PHUM355660 TC011101
CPAP1-K ACYPI008601 AGAP001597 GB49734 LOC101736353 CG7549 (mtg) Msex2.01703 PHUM601170 TC013568
CPAP1-L ACYPI009002 LOC101746263 CG14607 Msex2.09867 PHUM355640 LOC657301
CPAP1-M ACYPI002996 AGAP028105 GB48858 LOC101738097 Msex2.03103 PHUM135070 TC011724
CPAP1-N AGAP005586 GB41623 LOC101740538 CG12009 Msex2.02137 TC009890
CPAP1-O AGAP007089 LOC101735358 CG5756 Msex2.08722
CPAP1 Unclassified ACYPI30472 GB51439, GB53319 PHUM106940
CPAP3-A1 ACYPI007911 AGAP000989 GB50636 LOC101739926 CG17052 (Obst-A) Msex2.08805 PHUM434540 TC011140
CPAP3-A1-like AGAP000987, AGAP000988
CPAP3-A2 ACYPI006031 GB41945 LOC101740062 Msex2.08806 TC011141
CPAP3-B ACYPI004093 AGAP009790 GB41227 LOC101740330 CG4778 (Obst-B) Msex2.08808 + Msex2.15015 PHUM434540 TC011139
CPAP3-C ACYPI007860 AGAP003308 GB41203 LOC733010 CG10287 (Obst-C; gasp) Msex2.08810 PHUM179210 TC001169
CPAP3-D1 ACYPI009786 AGAP000986 GB41946 LOC101740197 CG17058 (Obst-D) Msex2.08807 PHUM434550, PHUM434570 TC011142
CPAP3-D2 ACYPI001579 AGAP002909 GB41270 LOC101742643 Msex2.04890 PHUM434580 TC001350
CPAP3-E ACYPI000583 AGAP009405 GB52854 LOC101743739 CG11142 (Obst-E1) Msex2.03293 PHUM291220 TC011349
CPAP3-E2 LOC101743600 CG11142 (Obst-E2) Msex2.03294
CPAP3-E3 Msex2.03295
CPAP3-E4 LOC101743460 Msex2.14886
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Gene IDs were derived from Jasrapuria (2011) and Ioannidou et al. (2014) except for B. mori and D. melanogaster which were from GenBank or Behr and Hoch (2005), and M. sexta from this report.

More interspecies variability was observed with the CPAP1 genes. Of the original ten genes described by Jasrapuria et al. (2010), the number of genes present in the eight species analyzed varied from five (A. pisum) to ten (T. castaneum), with most having eight or nine (Table 3). All eight species had orthologs to the newly described CPAP1-B2 branch, and at least three orthologs to CPAP1-K – O groups. A few genes from A. pisum, A. mellifera, and P. humanus could not be clearly grouped within any of the 16 CPAP1 branches and were left unclassified. It should be noted that although both our analysis (this report and Tetreau et al., 2014, this issue) and that of Ioannidou et al. (2014) independently verified the close relationship of the new members of the CPAP1 group to the original ten genes described previously for T. castaneum, the presence of the encoded proteins in the cuticle must still be established (and hence are they truly cuticle proteins). The D. melanogaster ortholog to CPAP1-K (CG7549), alternately known as mind-the-gap (mtg), is expressed in neuronal cells and encodes a protein critical for the organization of the synaptic extracelluar matrix of neuromuscular junctions (Rohrbough et al., 2007; Rushton et al., 2012). Interestingly, mtg null mutants, which die during the embryonic stage, exhibit a weakened cuticle and herniated hindgut phenotype (Rohrbough et al., 2007). This phenotype can be rescued by ubiquitous expression of a wildtype transgene (Rushton et al., 2009). According to FlyBase, moderate levels of mtg expression was also detected in the larval hindgut, tracheae, and carcass, all cuticle expressing tissues. Thus, it seems that mtg, and possibly other CPAPs, may have multiple roles.

3.1.4 Other CP genes

All of the remaining CP genes we annotated had identifiable orthologs in B. mori (Table 1). M. sexta and B. mori each have four CPT genes, one CPCFC gene, and one CPF gene. B. mori has five genes with the 18 amino acid motif (BmCPH14, 15, 16, 30, and 31) whereas M. sexta has only four. An examination of the protein sequences reveals that BmCPH14 and 15 are 92% identical and likely resulted from a gene duplication event that did not occur in M. sexta. The M. sexta ortholog (Msex2.01072) had a slightly higher sequence identity to BmCPH15 and, therefore, was given that name. Whereas six CPFL genes were identified in M. sexta, only four were in B. mori. The corresponding proteins of genes Msex2.04035, 04036, and 04037 were all similar to BmCPFL4. Msex2.04036 had the highest sequence similarity and was designated the ortholog; the remaining two genes were named MsCPFL5 and MsCPFL6.

3.2 CP gene expression

Fifty-two RNAseq libraries were created to provide transcript information to aid in the annotation of the genome. We sought to take advantage of this information to examine the expression profiles of the CP genes across multiple tissues and developmental stages. One caveat is that a majority of the libraries were created from tissues collected during the post-molt period. Thus, expression data of genes expressed primarily or exclusively during pre-molt (pharate) stages is limited. A second complication concerns the potential (if not likely) contamination of non-cuticle synthesizing samples with cuticle expressing tissues such as trachea or epidermis during the collection process. For example, we would not expect CP genes to be expressed in fat body, midgut, Malpighian tubules, or muscle. However, these samples may also contain pieces of epidermis or trachea and, therefore, CP gene expression was observed in some of these libraries. Finally, most libraries were sequenced only once, thus caution is advised when interpreting expression levels. (Four libraries were sequenced by both paired-end reads and single-end reads: midgut 5th instar wandering, and abdominal muscle (proleg) 5th instar at 12 h, pre-wandering, and wandering stages.)

The average number of CP genes expressed across all libraries was 88, with a minimum of 6, a maximum of 216, and a median of 72 (Figure 2A). The libraries with the highest number of CP genes expressed were the whole head and abdominal muscle (proleg) libraries of 4th instar larva after head capsule slippage (HCS, i.e. pharate 5th instar), each with 216 genes, while the library with the fewest CP genes expressed was prepared from adult midguts 3 -5 days post eclosion (6 genes). In total, 21 libraries had “above average” (> 88 genes) expression. This includes the 11 whole head libraries, the 5 libraries prepared form eggs or whole larvae (1st through 3rd instar), 3 of the abdominal muscle (proleg) libraries (4th instar HCS and 5th instar 12 h), midgut from 3rd instar HCS, and fat body from 4th instar HCS and pupa days 15-18; the latter three libraries would not be predicted to have abundant CP gene expression but this may be an indication of contamination from trachea or epidermis.

Figure 2. CP gene expression in the RNAseq libraries.

Figure 2

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(A) Total number of CP genes expressed in each library. (B) Total FPKM values for CPR RR-1 and RR-2 genes in each library; the pattern looks the same when the total FPKM value for all CP genes is displayed. Red type indicates libraries prepared from pre-molt (pharate) stages.

The libraries having the highest levels of CP transcripts (as judged by total FPKM values) are predictably from tissues enriched for epidermal cells; namely whole heads, whole larvae, or muscle scraped from the abdominal body wall either just prior to or just after molting (Figure 2B). CPR RR-1 transcripts dominated most libraries, having the highest total FPKM values in all but nine libraries (Table 4). Genes from RR-1, RR-2, CPH, and CPFL all had high levels of expression in the whole head libraries (supplementary file 1) with RR-2 having the highest transcript levels in the 4th instar HCS library and CPH the highest in adult day 1. On average, the top ten expressed genes in each library accounted for nearly 77% of the CP transcripts, and we view them as representing the major CPs expressed in that library. We compared the top ten expressed genes in various libraries to identify differences and commonalities between them. Transcripts for RR-1 genes CPR154 and 156, the RR-2 gene CPR63, and genes CPH1 and CPH15 (CPCFC and 18 aa groups, respectively) are among the top ten in most post-molt larval head libraries but not adult. Conversely, CPR19, 23, 37, CPH30, and CPH31 are more highly expressed in the adult head than in the larva (Table 5). In contrast, CPFL1 and Msex2.02570 were commonly found in both the larval and adult whole head libraries. (Msex2.02570 encodes a low complexity protein that is not a member of the CP groups discussed here but it is annotated in GenBank as a pupal cuticle protein; accession number AY585211.) Only two of the whole head libraries were made from tissue collected pre-molt. For the 4th instar HCS (pharate 5th instar) library, six of the ten most highly expressed genes were RR-2, as compared to only two in the post-molt larval whole head libraries. A different pattern was observed when comparing the pre-molt adult head library (pupa days 21-22) with post-molt adult head libraries in which many of the top expressed genes were the same (CPR19, 23, 37, 67, CPH30, 31, and CPFL1) (Table 5).

Table 4. Most abundant CP group expressed per library.

CP Group Library FPKMa %b
RR2 Head 4th instar HCS, 250,848 56,
Fat body pupa days 15-18, 3,669 59,
Testes pupa days 15-18 827 48
CPAP1 Fat body pupa days 1-3, 1,899 42,
Midgut pupa day 1, 91 37,
Ovaries adult day 1 135 40
CPAP3 Eggs 3 h 8,116 86
CPH Head adult day 1 25,587 41
CPT Testes pupa day 3 247 32
RR1 All others 32-402,401 30-90
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a

Total FPKM value for indicated CP group.

b

Relative to total FPKM value for all CP genes.

Table 5. Top ten expressing CP genes in whole head librariesa.

2nd instar day 1 3rd instar day 1 4th instar day 1 4th instar HCS 5th instar 12 h 5th instar day 2 5th instar pre-wandering Pupa days 21-22 Adult day 1 Adult day 3 Adult day 7
CPR134 CPH15 CPR134 CPR216 CPR46 CPR169 CPR46 CPFL1 CPH31 CPR19 CPR19
CPH15 CPH1 CPR154 CPR241 CPR163 CPR164 CPR134 CPH31 CPH30 CPR56 Msex2.02570
CPH1 CPR154 CPH15 CPFL1 CPR169 CPR165 CPR63 CPR37 CPFL1 CPR37 CPR56
CPR170 CPR134 CPR156 CPR3 CPR63 CPR46 CPR163 CPFL3 CPFL5 CPH30 CPH30
CPR42 CPR156 CPR3 CPR43 CPR5 CPR166 CPR4 CPR73 CPR19 CPH31 CPR23
CPR154 CPR170 CPH1 CPR83 CPR4 CPR167 CPR169 CPR19 CPR37 Msex2.02570 CPR37
CPR156 CPR42 Msex2.02570 CPH31 CPR134 CPR168 Msex2.02570 CPR67 CPR63 CPR23 CPR67
CPR3 CPR63 CPFL1 CPR184 CPR154 CPR63 CPR3 CPH30 CPR10 CPFL1 CPR191
CPFL1 CPR3 CPH31 CPR215 CPR156 CPR42 CPR5 CPR32 CPR23 CPR67 CPR141
CPR63 Msex2.02570 CPR63 CPR248 CPH15 CPR6 CPR156 CPR74 CPAP3-Ca CPR41 CPH31
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a

As determined by FPKM values; red type indicates RR-1 genes and blue type indicates RR-2 genes.

Differences between pre-molt and post-molt CP expression were also observed with the 3 day old eggs (pharate 1st instar) and the whole larva libraries (1st through 3rd instars 1 day post-molt). The whole larva libraries shared seven genes in common among the top ten (RR-1 genes CPR2, 3, 4, 5, 154, 156, and 170) of which only two (CPR3 and 4) were also highly expressed in 3 day old eggs. Top CP expressing genes in 3 day old eggs include CPT2, CPT3, CPAP3-A1 and CPAP3-Ca (supplementary file 1). Differences between pre-molt and post molt CP expression was not seen with the three libraries from abdominal muscle (proleg) having high CP transcript levels (4th instar HCS and 5th instar 12 h both single and paired end reads); CPR RR-1 transcripts dominated the list with six of the top ten genes the same between the three libraries (CPR3, 4, 46, 154, 156, and 163; supplementary file 1). Common top expressed genes among the larval libraries (whole head, whole larvae, abdominal muscle) include the RR-1 genes CPR3, 4, 5, 154, and 156 (supplementary file 1 and Fig S3). In a similar fashion, the corresponding genes in B. mori (BmCPR2-5) were among the top expressing genes during the pre- and post-molt 5th instar larval stage (Okamoto et al., 2008). Recently, Qiao and coworkers (2014) identified BmCPR2 as the gene responsible for the stony mutant in B. mori. This mutation lead to significant defects in cuticle extension, tensile strength, larval mobility, and body shape. It was also observed for B. mori that CPR46 was highly expressed post-molt in larval epidermis. As noted in section 3.1.2, the M. sexta ortholog to CPR46 has undergone gene expansion that encompasses 10 genes, CPR46 and CPR163 – 171 (Table 1). Members of this co-orthologous group appear as top expressed genes in many of the post-molt larval libraries in M. sexta as well (supplementary file 1).

Twenty of the annotated genes showed no expression in any of the libraries; 19 of these belong to the CPR RR-2 group and one belonged to the CPR RR-1 group (see supplementary file 1). Furthermore, 10 of these 20 genes belonged to the same cluster of related genes (group 4 in Table 2). An additional 29 genes were not detected in at least 47 of the 52 libraries (90%), with 22 of these belonging to the CPR RR-2 group; 8 of these had established orthology with B. mori genes (with CPR numbers between 121 – 145) while 7 belonged to a cluster whose orthology could not be established. The lone CPF gene in M. sexta, Msex2.05601, was not detected in 48 of the libraries, but had high expression in the whole head library from pupa day 21 – 22 (pharate adult) and then decreased thereafter (low expression in adult day 1 whole head and very low expression in adult days 3 and 7 whole head libraries). Similarly, transcripts for CPAP3-E4 (Msex2.14886) were detected at moderate levels in 3 h old eggs but no other libraries. These results suggest that genes showing very low or no expression in most (or all) libraries may have a restricted tissue or temporal expression, or that they may be pseudogenes.

Clustering analysis was performed to identify genes that had similar expression patterns across all 52 libraries. Figure 3 shows selected profiles from this analysis. (A heat map depicting the grouping and relative expression levels of all the CP genes is shown in supplementary Figure S3.) One of the most striking observation was of eight genes that had low to moderate levels of expression in nearly all of the libraries, producing an “expression band” that extended the width of the heat map (Figure 3A and supplementary Figure S3). Five of the eight genes in this cluster belonged to the CPAP group (CPAP1-C, 1-H, 1-M, and CPAP3-D2 and 3-Cb) and two were RR-3 (CPR146 and 149). The nearly ubiquitous expression pattern of these genes suggests that they may be important for general cuticle synthesis or synthesis of tracheal cuticle. Another six genes were nearly exclusively expressed only in pre-molt libraries during larval development (the exception being expression in fat body from pupa days 15 – 18) (Figure 3B). Remarkably, three of the genes belonged to the Tweedle family (of which only four were found in M. sexta) and the other three are CPR RR-2 genes that are clustered together in the genome. Similar observations were reported for B. mori in that CPT2-4 shared the same expression pattern with the highest expression occurring at the larval stage during the molt (Liang et al., 2010).

Figure 3. Selected expression patterns from cluster analysis.

Figure 3

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The expression pattern of each CP gene across all 52 RNAseq libraries were grouped by cluster analysis as described in section 2.3. A – G highlight some of the unique expression patterns observed; a full heat map of all of the genes is shown in supplemental Figure S3. Red type indicates libraries prepared from pre-molt (pharate) stages.

Several genes showed expression in just a few libraries such as whole heads 4th instar HCS and 3 day old eggs (Figure 3C). What these two libraries have in common is that they were prepared from tissue synthesizing larval head capsule cuticle pre-molt. The fact that those genes were not expressed in other larval pre-molt libraries that may contain tracheal or epidermis contamination such as fat body 4th instar HCS, midgut 3rd and 4th instars HCS, or abdominal muscle (proleg) 4th instar HCS, suggests that those genes are not involved in synthesis of tracheal or body wall cuticle. Similarly, several genes were expressed almost exclusively in 4th instar whole heads and abdominal muscle (proleg) HCS libraries, indicating likely function in larval head capsule and body wall synthesis pre-molt (Figure 3D). Additional interesting patterns observed include restriction of expression to whole heads of 5th instar day 2 and abdominal muscle (proleg) 5th instar post-molt (Figure 3E), 3 day old eggs and midguts of 2nd and 3rd instar larvae (Figure 3F), and eggs 3 hours old (Figure 3G). Three of the four genes in the last example are the additional members of the CPAP3-E group found in B. mori and M. sexta but not the other species presented in Table 3. This time point is too early for embryonic or 1st instar cuticle synthesis (Konopova and Zrzavy, 2005; Ziese and Dorn, 2003) but the transcripts may be maternally loaded.

4. Conclusions

Combining reciprocal BLAST, phylogenetic analysis, and microsynteny proved an effective method for identifying orthologous CP genes between M. sexta and B. mori. The most challenging identification was for the CPR RR-2 group which appears to be undergoing duplication after speciation more often than for other CP genes. For the remaining groups examined, one to one orthology was more easily established. The 207 CPR genes identified are currently the most in any insect for which this type of annotation has been carried out (although recent analysis by Ioannidou et al. (2014) indicates that the yellow fever mosquito, Aedes aegypti, may have as many as 240), and is 37% more than the number of CPR genes identified in B. mori; it remains to be determined which number is more typical for the Lepidoptera. Our analysis has also expanded the number of genes in the CPAP1 group from 10 to 16 but the veracity of the new genes as true CPs must be confirmed.

The RNAseq libraries provided valuable data on gene expression. In most libraries, CPR RR-1 genes accounted for most of the CP gene transcripts but this may have been influenced by the number of libraries prepared from larval tissues (32 of 52) or post-molt tissues (40 of 52). RR-1 genes have been proposed to be more prevalent in soft cuticle than hard cuticle (Andersen, 2000). As larva are mostly soft bodied and cuticle synthesized post-molt is thought to undergo less (or no) sclerotization than cuticle synthesized pre-molt (Andersen et al., 1995b), this may account for the abundance of RR-1 transcripts detected. Additionally, trachea is a likely contamination of the libraries prepared from midguts, Malpighian tubules, and fat body. The flexibility needed may necessitate a less sclerotized tracheal cuticle and, therefore, it is reasonable to anticipate a greater number of RR-1 proteins. In B. mori, Okamoto and co-workers (2008) also found that the majority of CP transcripts from two larval EST libraries (a pharate 5th instar and 5th instar day 3) were from RR-1 genes. Nevertheless, several distinct patterns of CP gene expression were observed, with differences noted between larval and adult cuticle or pre-molt and post-molt cuticle. As the purpose of the RNAseq libraries was to aid in the genome annotation, it was desirable to sample multiple tissues at several time points and all developmental stages. As a consequence, careful selection of a single tissue (i.e. epidermis, trachea, or hard cuticle or soft cuticle) or more refined time points was not a necessity. A more meticulous collection on both accounts in future experiments may yield a better characterization of CP gene expression.

Supplementary Material

1

Figure S1. Comparison ofM. sexta CP genes on scaffold00685 withBmCPR82–91. . (A) Alignment of M. sexta genes (green arrows) on scaffold00685 with the corresponding region in B. mori chromosome 22 (blue arrows); the arrows are only meant to show gene order and orientation and are not representative of gene size or distances between genes. The dashed red line denotes putative orthologs as determined by reciprocal BLAST and phylogenetic analysis. Genes boxed in yellow likely arose through duplication of a shared gene that occurred after separation of M. sexta and B. mori from a common ancestor. (B) Phylogenetic analysis of the CPR genes shown in A. All branches with a bootstrap value less than 50 were collapsed.

Figure S2. Identification of putative orthologous CPR RR-2 groups betweenM. sexta and B. mori by phylogenetic analysis. Phylogenetic analysis was used to determine the relationship between M. sexta proteins MsCPR183 – 250 and B. mori proteins BmCPR85 – 89 and 92 – 120. Branches with bootstrap values less than 50 were collapsed. Red diamonds (◆) indicate branch points between putative orthologous groups that presumable arose through gene duplication that occurred after separation of M. sexta and B. mori from a common ancestor.

Figure S3. Cluster analysis of the annotated CP genes. The expression pattern of each CP gene across all 52 RNAseq libraries were grouped by cluster analysis as described in section 2.3. Letters A – G identify the expression patterns shown in Figure 3. Red type indicates libraries prepared from pre-molt (pharate) stages.

2
3
4

  • 248 cuticular protein (CP) genes were annotated in Manduca sexta

  • Orthology was established between M. sexta and Bombyx mori CP genes

  • Gene expression was analyzed via 52 RNAseq libraries

  • Diverse expression was observed across various tissues and developmental stages

Acknowledgments

This research was supported by grants from the National Institutes of Health (GM41247) and the National Science Foundation (IOS1257961) to M. Kanost, from the NIH (GM58634) to H. Jiang, and by the Cornell University Agricultural Experiment Station federal formula funds received from the USDA Cooperative State Research, Education, and Extension Service to P. Wang. This is contribution number 15-142-J from the Kansas Agricultural Experiment Station.

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1

Figure S1. Comparison ofM. sexta CP genes on scaffold00685 withBmCPR82–91. . (A) Alignment of M. sexta genes (green arrows) on scaffold00685 with the corresponding region in B. mori chromosome 22 (blue arrows); the arrows are only meant to show gene order and orientation and are not representative of gene size or distances between genes. The dashed red line denotes putative orthologs as determined by reciprocal BLAST and phylogenetic analysis. Genes boxed in yellow likely arose through duplication of a shared gene that occurred after separation of M. sexta and B. mori from a common ancestor. (B) Phylogenetic analysis of the CPR genes shown in A. All branches with a bootstrap value less than 50 were collapsed.

Figure S2. Identification of putative orthologous CPR RR-2 groups betweenM. sexta and B. mori by phylogenetic analysis. Phylogenetic analysis was used to determine the relationship between M. sexta proteins MsCPR183 – 250 and B. mori proteins BmCPR85 – 89 and 92 – 120. Branches with bootstrap values less than 50 were collapsed. Red diamonds (◆) indicate branch points between putative orthologous groups that presumable arose through gene duplication that occurred after separation of M. sexta and B. mori from a common ancestor.

Figure S3. Cluster analysis of the annotated CP genes. The expression pattern of each CP gene across all 52 RNAseq libraries were grouped by cluster analysis as described in section 2.3. Letters A – G identify the expression patterns shown in Figure 3. Red type indicates libraries prepared from pre-molt (pharate) stages.

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